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Sample records for voltage-activated ion channels

  1. Voltage-dependent gating in a "voltage sensor-less" ion channel.

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    Harley T Kurata

    2010-02-01

    Full Text Available The voltage sensitivity of voltage-gated cation channels is primarily attributed to conformational changes of a four transmembrane segment voltage-sensing domain, conserved across many levels of biological complexity. We have identified a remarkable point mutation that confers significant voltage dependence to Kir6.2, a ligand-gated channel that lacks any canonical voltage-sensing domain. Similar to voltage-dependent Kv channels, the Kir6.2[L157E] mutant exhibits time-dependent activation upon membrane depolarization, resulting in an outwardly rectifying current-voltage relationship. This voltage dependence is convergent with the intrinsic ligand-dependent gating mechanisms of Kir6.2, since increasing the membrane PIP2 content saturates Po and eliminates voltage dependence, whereas voltage activation is more dramatic when channel Po is reduced by application of ATP or poly-lysine. These experiments thus demonstrate an inherent voltage dependence of gating in a "ligand-gated" K+ channel, and thereby provide a new view of voltage-dependent gating mechanisms in ion channels. Most interestingly, the voltage- and ligand-dependent gating of Kir6.2[L157E] is highly sensitive to intracellular [K+], indicating an interaction between ion permeation and gating. While these two key features of channel function are classically dealt with separately, the results provide a framework for understanding their interaction, which is likely to be a general, if latent, feature of the superfamily of cation channels.

  2. Cnidarian Toxins Acting on Voltage-Gated Ion Channels

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    Robert M. Greenberg

    2006-04-01

    Full Text Available Abstract: Voltage-gated ion channels generate electrical activity in excitable cells. As such, they are essential components of neuromuscular and neuronal systems, and are targeted by toxins from a wide variety of phyla, including the cnidarians. Here, we review cnidarian toxins known to target voltage-gated ion channels, the specific channel types targeted, and, where known, the sites of action of cnidarian toxins on different channels.

  3. Dual Regulation of Voltage-Sensitive Ion Channels by PIP2

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    Aldo A Rodríguez Menchaca

    2012-09-01

    Full Text Available Over the past 16 years, there has been an impressive number of ion channels shown to be sensitive to the major phosphoinositide in the plasma membrane, phosphatidilinositol 4,5-bisphosphate (PIP2. Among them are voltage-gated channels, which are crucial for both neuronal and cardiac excitability. Voltage-gated calcium (Cav channels were shown to be regulated bidirectionally by PIP2. On one hand, PIP2 stabilized their activity by reducing current rundown but on the other hand it produced a voltage-dependent inhibition by shifting the activation curve to more positive voltages. For voltage-gated potassium (Kv channels PIP2 was first shown to prevent N-type inactivation. Careful examination of the effects of PIP2 on the activation mechanism of Kv1.2 has shown a similar bidirectional regulation as in the Cav channels. The two effects could be distinguished kinetically, in terms of their sensitivities to PIP2 and by distinct molecular determinants. The rightward shift of the Kv1.2 voltage dependence implicated basic residues in the S4-S5 linker and was consistent with stabilization of the inactive state of the voltage sensor. A third type of a voltage-gated ion channel modulated by PIP2 is the hyperpolarization-activated cyclic nucleotide-gated (HCN channel. PIP2 has been shown to enhance the opening of HCN channels by shifting their voltage-dependent activation toward depolarized potentials. The sea urchin HCN channel, SpIH, showed again a PIP2-mediated bidirectional effect but in reverse order than the depolarization-activated Cav and Kv channels: a voltage-dependent potentiation, like the mammalian HCN channels, but also an inhibition of the cGMP-induced current activation. Just like the Kv1.2 channels, distinct molecular determinants underlied the PIP2 dual effects on SpIH channels. The dual regulation of these very different ion channels, all of which are voltage dependent, points to conserved mechanisms of regulation of these channels by PIP2.

  4. Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes.

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    Sheyda R Frolova

    Full Text Available The ability of azobenzene trimethylammonium bromide (azoTAB to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav, calcium (ICav, and potassium (IKv currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na+ and calcium (Ca2+ currents and potentiation of net potassium (K+ currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.

  5. Free-energy relationships in ion channels activated by voltage and ligand

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    Chowdhury, Sandipan

    2013-01-01

    Many ion channels are modulated by multiple stimuli, which allow them to integrate a variety of cellular signals and precisely respond to physiological needs. Understanding how these different signaling pathways interact has been a challenge in part because of the complexity of underlying models. In this study, we analyzed the energetic relationships in polymodal ion channels using linkage principles. We first show that in proteins dually modulated by voltage and ligand, the net free-energy change can be obtained by measuring the charge-voltage (Q-V) relationship in zero ligand condition and the ligand binding curve at highly depolarizing membrane voltages. Next, we show that the voltage-dependent changes in ligand occupancy of the protein can be directly obtained by measuring the Q-V curves at multiple ligand concentrations. When a single reference ligand binding curve is available, this relationship allows us to reconstruct ligand binding curves at different voltages. More significantly, we establish that the shift of the Q-V curve between zero and saturating ligand concentration is a direct estimate of the interaction energy between the ligand- and voltage-dependent pathway. These free-energy relationships were tested by numerical simulations of a detailed gating model of the BK channel. Furthermore, as a proof of principle, we estimate the interaction energy between the ligand binding and voltage-dependent pathways for HCN2 channels whose ligand binding curves at various voltages are available. These emerging principles will be useful for high-throughput mutagenesis studies aimed at identifying interaction pathways between various regulatory domains in a polymodal ion channel. PMID:23250866

  6. Voltage-gated lipid ion channels

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    Blicher, Andreas; Heimburg, Thomas Rainer

    2013-01-01

    Synthetic lipid membranes can display channel-like ion conduction events even in the absence of proteins. We show here that these events are voltage-gated with a quadratic voltage dependence as expected from electrostatic theory of capacitors. To this end, we recorded channel traces and current...... histograms in patch-experiments on lipid membranes. We derived a theoretical current-voltage relationship for pores in lipid membranes that describes the experimental data very well when assuming an asymmetric membrane. We determined the equilibrium constant between closed and open state and the open...... probability as a function of voltage. The voltage-dependence of the lipid pores is found comparable to that of protein channels. Lifetime distributions of open and closed events indicate that the channel open distribution does not follow exponential statistics but rather power law behavior for long open times...

  7. Beyond voltage-gated ion channels: Voltage-operated membrane proteins and cellular processes.

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    Zhang, Jianping; Chen, Xingjuan; Xue, Yucong; Gamper, Nikita; Zhang, Xuan

    2018-04-18

    Voltage-gated ion channels were believed to be the only voltage-sensitive proteins in excitable (and some non-excitable) cells for a long time. Emerging evidence indicates that the voltage-operated model is shared by some other transmembrane proteins expressed in both excitable and non-excitable cells. In this review, we summarize current knowledge about voltage-operated proteins, which are not classic voltage-gated ion channels as well as the voltage-dependent processes in cells for which single voltage-sensitive proteins have yet to be identified. Particularly, we will focus on the following. (1) Voltage-sensitive phosphoinositide phosphatases (VSP) with four transmembrane segments homologous to the voltage sensor domain (VSD) of voltage-gated ion channels; VSPs are the first family of proteins, other than the voltage-gated ion channels, for which there is sufficient evidence for the existence of the VSD domain; (2) Voltage-gated proton channels comprising of a single voltage-sensing domain and lacking an identified pore domain; (3) G protein coupled receptors (GPCRs) that mediate the depolarization-evoked potentiation of Ca 2+ mobilization; (4) Plasma membrane (PM) depolarization-induced but Ca 2+ -independent exocytosis in neurons. (5) Voltage-dependent metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P 2 , PIP 2 ) in the PM. These recent discoveries expand our understanding of voltage-operated processes within cellular membranes. © 2018 Wiley Periodicals, Inc.

  8. CONTRIBUTIONS OF INTRACELLULAR IONS TO Kv CHANNEL VOLTAGE SENSOR DYNAMICS.

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    Samuel eGoodchild

    2012-06-01

    Full Text Available Voltage sensing domains of Kv channels control ionic conductance through coupling of the movement of charged residues in the S4 segment to conformational changes at the cytoplasmic region of the pore domain, that allow K+ ions to flow. Conformational transitions within the voltage sensing domain caused by changes in the applied voltage across the membrane field are coupled to the conducting pore region and the gating of ionic conductance. However, several other factors not directly linked to the voltage dependent movement of charged residues within the voltage sensor impact the dynamics of the voltage sensor, such as inactivation, ionic conductance, intracellular ion identity and block of the channel by intracellular ligands. The effect of intracellular ions on voltage sensor dynamics is of importance in the interpretation of gating current measurements and the physiology of pore/voltage sensor coupling. There is a significant amount of variability in the reported kinetics of voltage sensor deactivation kinetics of Kv channels attributed to different mechanisms such as open state stabilization, immobilization and relaxation processes of the voltage sensor. Here we separate these factors and focus on the causal role that intracellular ions can play in allosterically modulating the dynamics of Kv voltage sensor deactivation kinetics. These considerations are of critical importance in understanding the molecular determinants of the complete channel gating cycle from activation to deactivation.

  9. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons.

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    Lazcano-Pérez, Fernando; Castro, Héctor; Arenas, Isabel; García, David E; González-Muñoz, Ricardo; Arreguín-Espinosa, Roberto

    2016-05-05

    The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels.

  10. Expression and distribution of voltage-gated ion channels in ferret sinoatrial node.

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    Brahmajothi, Mulugu V; Morales, Michael J; Campbell, Donald L; Steenbergen, Charles; Strauss, Harold C

    2010-10-01

    Spontaneous diastolic depolarization in the sinoatrial (SA) node enables it to serve as pacemaker of the heart. The variable cell morphology within the SA node predicts that ion channel expression would be heterogeneous and different from that in the atrium. To evaluate ion channel heterogeneity within the SA node, we used fluorescent in situ hybridization to examine ion channel expression in the ferret SA node region and atrial appendage. SA nodal cells were distinguished from surrounding cardiac myocytes by expression of the slow (SA node) and cardiac (surrounding tissue) forms of troponin I. Nerve cells in the sections were identified by detection of GAP-43 and cytoskeletal middle neurofilament. Transcript expression was characterized for the 4 hyperpolarization-activated cation channels, 6 voltage-gated Na(+) channels, 3 voltage-gated Ca(2+) channels, 24 voltage-gated K(+) channel α-subunits, and 3 ancillary subunits. To ensure that transcript expression was representative of protein expression, immunofluorescence was used to verify localization patterns of voltage-dependent K(+) channels. Colocalizations were performed to observe any preferential patterns. Some overlapping and nonoverlapping binding patterns were observed. Measurement of different cation channel transcripts showed heterogeneous expression with many different patterns of expression, attesting to the complexity of electrical activity in the SA node. This study provides insight into the possible role ion channel heterogeneity plays in SA node pacemaker activity.

  11. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons

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    Fernando Lazcano-Pérez

    2016-05-01

    Full Text Available The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7, voltage-gated calcium channel (CaV2.2, the A-type transient outward (IA and delayed rectifier (IDR currents of KV channels of the superior cervical ganglion (SCG neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels.

  12. Investigating ion channel conformational changes using voltage clamp fluorometry.

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    Talwar, Sahil; Lynch, Joseph W

    2015-11-01

    Ion channels are membrane proteins whose functions are governed by conformational changes. The widespread distribution of ion channels, coupled with their involvement in most physiological and pathological processes and their importance as therapeutic targets, renders the elucidation of these conformational mechanisms highly compelling from a drug discovery perspective. Thanks to recent advances in structural biology techniques, we now have high-resolution static molecular structures for members of the major ion channel families. However, major questions remain to be resolved about the conformational states that ion channels adopt during activation, drug modulation and desensitization. Patch-clamp electrophysiology has long been used to define ion channel conformational states based on functional criteria. It achieves this by monitoring conformational changes at the channel gate and cannot detect conformational changes occurring in regions distant from the gate. Voltage clamp fluorometry involves labelling cysteines introduced into domains of interest with environmentally sensitive fluorophores and inferring structural rearrangements from voltage or ligand-induced fluorescence changes. Ion channel currents are monitored simultaneously to verify the conformational status. By defining real time conformational changes in domains distant from the gate, this technique provides unexpected new insights into ion channel structure and function. This review aims to summarise the methodology and highlight recent innovative applications of this powerful technique. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Atomistic Modeling of Ion Conduction through the Voltage-Sensing Domain of the Shaker K+ Ion Channel.

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    Wood, Mona L; Freites, J Alfredo; Tombola, Francesco; Tobias, Douglas J

    2017-04-20

    Voltage-sensing domains (VSDs) sense changes in the membrane electrostatic potential and, through conformational changes, regulate a specific function. The VSDs of wild-type voltage-dependent K + , Na + , and Ca 2+ channels do not conduct ions, but they can become ion-permeable through pathological mutations in the VSD. Relatively little is known about the underlying mechanisms of conduction through VSDs. The most detailed studies have been performed on Shaker K + channel variants in which ion conduction through the VSD is manifested in electrophysiology experiments as a voltage-dependent inward current, the so-called omega current, which appears when the VSDs are in their resting state conformation. Only monovalent cations appear to permeate the Shaker VSD via a pathway that is believed to be, at least in part, the same as that followed by the S4 basic side chains during voltage-dependent activation. We performed μs-time scale atomistic molecular dynamics simulations of a cation-conducting variant of the Shaker VSD under applied electric fields in an experimentally validated resting-state conformation, embedded in a lipid bilayer surrounded by solutions containing guanidinium chloride or potassium chloride. Our simulations provide insights into the Shaker VSD permeation pathway, the protein-ion interactions that control permeation kinetics, and the mechanism of voltage-dependent activation of voltage-gated ion channels.

  14. Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening.

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    Zaydman, Mark A; Silva, Jonathan R; Delaloye, Kelli; Li, Yang; Liang, Hongwu; Larsson, H Peter; Shi, Jingyi; Cui, Jianmin

    2013-08-06

    Voltage-gated ion channels generate dynamic ionic currents that are vital to the physiological functions of many tissues. These proteins contain separate voltage-sensing domains, which detect changes in transmembrane voltage, and pore domains, which conduct ions. Coupling of voltage sensing and pore opening is critical to the channel function and has been modeled as a protein-protein interaction between the two domains. Here, we show that coupling in Kv7.1 channels requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP2. This result is due to loss of coupling because PIP2 was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP2-dependent coupling at the interface between the voltage-sensing domain and the pore domain. This site is actually a conserved lipid-binding site among different K(+) channels, suggesting that lipids play an important role in coupling in many ion channels.

  15. Ion Concentration- and Voltage-Dependent Push and Pull Mechanisms of Potassium Channel Ion Conduction.

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    Kota Kasahara

    Full Text Available The mechanism of ion conduction by potassium channels is one of the central issues in physiology. In particular, it is still unclear how the ion concentration and the membrane voltage drive ion conduction. We have investigated the dynamics of the ion conduction processes in the Kv1.2 pore domain, by molecular dynamics (MD simulations with several different voltages and ion concentrations. By focusing on the detailed ion movements through the pore including selectivity filter (SF and cavity, we found two major conduction mechanisms, called the III-IV-III and III-II-III mechanisms, and the balance between the ion concentration and the voltage determines the mechanism preference. In the III-IV-III mechanism, the outermost ion in the pore is pushed out by a new ion coming from the intracellular fluid, and four-ion states were transiently observed. In the III-II-III mechanism, the outermost ion is pulled out first, without pushing by incoming ions. Increases in the ion concentration and voltage accelerated ion conductions, but their mechanisms were different. The increase in the ion concentrations facilitated the III-IV-III conductions, while the higher voltages increased the III-II-III conductions, indicating that the pore domain of potassium channels permeates ions by using two different driving forces: a push by intracellular ions and a pull by voltage.

  16. Grafting voltage and pharmacological sensitivity in potassium channels.

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    Lan, Xi; Fan, Chunyan; Ji, Wei; Tian, Fuyun; Xu, Tao; Gao, Zhaobing

    2016-08-01

    A classical voltage-gated ion channel consists of four voltage-sensing domains (VSDs). However, the roles of each VSD in the channels remain elusive. We developed a GVTDT (Graft VSD To Dimeric TASK3 channels that lack endogenous VSDs) strategy to produce voltage-gated channels with a reduced number of VSDs. TASK3 channels exhibit a high host tolerance to VSDs of various voltage-gated ion channels without interfering with the intrinsic properties of the TASK3 selectivity filter. The constructed channels, exemplified by the channels grafted with one or two VSDs from Kv7.1 channels, exhibit classical voltage sensitivity, including voltage-dependent opening and closing. Furthermore, the grafted Kv7.1 VSD transfers the potentiation activity of benzbromarone, an activator that acts on the VSDs of the donor channels, to the constructed channels. Our study indicates that one VSD is sufficient to voltage-dependently gate the pore and provides new insight into the roles of VSDs.

  17. Axonal voltage-gated ion channels as pharmacological targets for pain

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    Moldovan, Mihai; Alvarez, Susana; Romer Rosberg, Mette

    2013-01-01

    Upon peripheral nerve injury (caused by trauma or disease process) axons of the dorsal root ganglion (DRG) somatosensory neurons have the ability to sprout and regrow/remyelinate to reinnervate distant target tissue or form a tangled scar mass called a neuroma. This regenerative response can become...... maladaptive leading to a persistent and debilitating pain state referred to as chronic pain corresponding to the clinical description of neuropathic/chronic inflammatory pain. There is little agreement to what causes peripheral chronic pain other than hyperactivity of the nociceptive DRG neurons which...... ultimately depends on the function of voltage-gated ion channels. This review focuses on the pharmacological modulators of voltage-gated ion channels known to be present on axonal membrane which represents by far the largest surface of DRG neurons. Blockers of voltage-gated Na(+) channels, openers of voltage...

  18. Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics.

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    Beattie, Kylie A; Hill, Adam P; Bardenet, Rémi; Cui, Yi; Vandenberg, Jamie I; Gavaghan, David J; de Boer, Teun P; Mirams, Gary R

    2018-03-24

    Ion current kinetics are commonly represented by current-voltage relationships, time constant-voltage relationships and subsequently mathematical models fitted to these. These experiments take substantial time, which means they are rarely performed in the same cell. Rather than traditional square-wave voltage clamps, we fitted a model to the current evoked by a novel sum-of-sinusoids voltage clamp that was only 8 s long. Short protocols that can be performed multiple times within a single cell will offer many new opportunities to measure how ion current kinetics are affected by changing conditions. The new model predicts the current under traditional square-wave protocols well, with better predictions of underlying currents than literature models. The current under a novel physiologically relevant series of action potential clamps is predicted extremely well. The short sinusoidal protocols allow a model to be fully fitted to individual cells, allowing us to examine cell-cell variability in current kinetics for the first time. Understanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics - the voltage-dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an example, with the aim of generating a quantitatively predictive representation of the ion current. We fitted a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp in CHO cells overexpressing hERG1a. The model was then used to predict over 5 minutes of recordings in the same cell in response to

  19. The NH2 terminus regulates voltage-dependent gating of CALHM ion channels.

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    Tanis, Jessica E; Ma, Zhongming; Foskett, J Kevin

    2017-08-01

    Calcium homeostasis modulator protein-1 (CALHM1) and its Caenorhabditis elegans (ce) homolog, CLHM-1, belong to a new family of physiologically important ion channels that are regulated by voltage and extracellular Ca 2+ (Ca 2+ o ) but lack a canonical voltage-sensing domain. Consequently, the intrinsic voltage-dependent gating mechanisms for CALHM channels are unknown. Here, we performed voltage-clamp experiments on ceCLHM-1 chimeric, deletion, insertion, and point mutants to assess the role of the NH 2 terminus (NT) in CALHM channel gating. Analyses of chimeric channels in which the ceCLHM-1 and human (h)CALHM1 NH 2 termini were interchanged showed that the hCALHM1 NT destabilized channel-closed states, whereas the ceCLHM-1 NT had a stabilizing effect. In the absence of Ca 2+ o , deletion of up to eight amino acids from the ceCLHM-1 NT caused a hyperpolarizing shift in the conductance-voltage relationship with little effect on voltage-dependent slope. However, deletion of nine or more amino acids decreased voltage dependence and induced a residual conductance at hyperpolarized voltages. Insertion of amino acids into the NH 2 -terminal helix also decreased voltage dependence but did not prevent channel closure. Mutation of ceCLHM-1 valine 9 and glutamine 13 altered half-maximal activation and voltage dependence, respectively, in 0 Ca 2+ In 2 mM Ca 2+ o , ceCLHM-1 NH 2 -terminal deletion and point mutant channels closed completely at hyperpolarized voltages with apparent affinity for Ca 2+ o indistinguishable from wild-type ceCLHM-1, although the ceCLHM-1 valine 9 mutant exhibited an altered conductance-voltage relationship and kinetics. We conclude that the NT plays critical roles modulating voltage dependence and stabilizing the closed states of CALHM channels. Copyright © 2017 the American Physiological Society.

  20. Voltage-Sensitive Ion Channels Biophysics of Molecular Excitability

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    Leuchtag, H. Richard

    2008-01-01

    Voltage-sensitive ion channels are macromolecules embedded in the membranes of nerve and muscle fibers of animals. Because of their physiological functions, biochemical structures and electrical switching properties, they are at an intersection of biology, chemistry and physics. Despite decades of intensive research under the traditional approach of gated structural pores, the relation between the structure of these molecules and their function remains enigmatic. This book critically examines physically oriented approaches not covered in other ion-channel books. It looks at optical and thermal as well as electrical data, and at studies in the frequency domain as well as in the time domain. Rather than presenting the reader with only an option of mechanistic models at an inappropriate pseudo-macroscopic scale, it emphasizes concepts established in organic chemistry and condensed state physics. The book’s approach to the understanding of these unique structures breaks with the unproven view of ion channels as...

  1. Surface dynamics of voltage-gated ion channels

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    Heine, Martin; Ciuraszkiewicz, Anna; Voigt, Andreas; Heck, Jennifer; Bikbaev, Arthur

    2016-01-01

    ABSTRACT Neurons encode information in fast changes of the membrane potential, and thus electrical membrane properties are critically important for the integration and processing of synaptic inputs by a neuron. These electrical properties are largely determined by ion channels embedded in the membrane. The distribution of most ion channels in the membrane is not spatially uniform: they undergo activity-driven changes in the range of minutes to days. Even in the range of milliseconds, the composition and topology of ion channels are not static but engage in highly dynamic processes including stochastic or activity-dependent transient association of the pore-forming and auxiliary subunits, lateral diffusion, as well as clustering of different channels. In this review we briefly discuss the potential impact of mobile sodium, calcium and potassium ion channels and the functional significance of this for individual neurons and neuronal networks. PMID:26891382

  2. Interaction of a dinoflagellate neurotoxin with voltage-activated ion channels in a marine diatom.

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    Kitchen, Sheila A; Bourdelais, Andrea J; Taylor, Alison R

    2018-01-01

    The potent neurotoxins produced by the harmful algal bloom species Karenia brevis are activators of sodium voltage-gated channels (VGC) in animals, resulting in altered channel kinetics and membrane hyperexcitability. Recent biophysical and genomic evidence supports widespread presence of homologous sodium (Na + ) and calcium (Ca 2+ ) permeable VGCs in unicellular algae, including marine phytoplankton. We therefore hypothesized that VGCs of these phytoplankton may be an allelopathic target for waterborne neurotoxins produced by K. brevis blooms that could lead to ion channel dysfunction and disruption of signaling in a similar manner to animal Na + VGCs. We examined the interaction of brevetoxin-3 (PbTx-3), a K. brevis neurotoxin, with the Na + /Ca 2+ VGC of the non-toxic diatom Odontella sinensi s using electrophysiology. Single electrode current- and voltage- clamp recordings from O. sinensis in the presence of PbTx-3 were used to examine the toxin's effect on voltage gated Na + /Ca 2+ currents. In silico analysis was used to identify the putative PbTx binding site in the diatoms. We identified Na + /Ca 2+ VCG homologs from the transcriptomes and genomes of 12 diatoms, including three transcripts from O. sinensis and aligned them with site-5 of Na + VGCs, previously identified as the PbTx binding site in animals. Up to 1 µM PbTx had no effect on diatom resting membrane potential or membrane excitability. The kinetics of fast inward Na + /Ca 2+ currents that underlie diatom action potentials were also unaffected. However, the peak inward current was inhibited by 33%, delayed outward current was inhibited by 25%, and reversal potential of the currents shifted positive, indicating a change in permeability of the underlying channels. Sequence analysis showed a lack of conservation of the PbTx binding site in diatom VGC homologs, many of which share molecular features more similar to single-domain bacterial Na + /Ca 2+ VGCs than the 4-domain eukaryote channels

  3. A complicated complex: Ion channels, voltage sensing, cell membranes and peptide inhibitors.

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    Zhang, Alan H; Sharma, Gagan; Undheim, Eivind A B; Jia, Xinying; Mobli, Mehdi

    2018-04-21

    Voltage-gated ion channels (VGICs) are specialised ion channels that have a voltage dependent mode of action, where ion conduction, or gating, is controlled by a voltage-sensing mechanism. VGICs are critical for electrical signalling and are therefore important pharmacological targets. Among these, voltage-gated sodium channels (Na V s) have attracted particular attention as potential analgesic targets. Na V s, however, comprise several structurally similar subtypes with unique localisations and distinct functions, ranging from amplification of action potentials in nociception (e.g. Na V 1.7) to controlling electrical signalling in cardiac function (Na V 1.5). Understanding the structural basis of Na V function is therefore of great significance, both to our knowledge of electrical signalling and in development of subtype and state selective drugs. An important tool in this pursuit has been the use of peptides from animal venoms as selective Na V modulators. In this review, we look at peptides, particularly from spider venoms, that inhibit Na V s by binding to the voltage sensing domain (VSD) of this channel, known as gating modifier toxins (GMT). In the first part of the review, we look at the structural determinants of voltage sensing in VGICs, the gating cycle and the conformational changes that accompany VSD movement. Next, the modulation of the analgesic target Na V 1.7 by GMTs is reviewed to develop bioinformatic tools that, based on sequence information alone, can identify toxins that are likely to inhibit this channel. The same approach is also used to define VSD sequences, other than that from Na V 1.7, which are likely to be sensitive to this class of toxins. The final section of the review focuses on the important role of the cellular membrane in channel modulation and also how the lipid composition affects measurements of peptide-channel interactions both in binding kinetics measurements in solution and in cell-based functional assays. Copyright © 2018

  4. Voltage gating of mechanosensitive PIEZO channels.

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    Moroni, Mirko; Servin-Vences, M Rocio; Fleischer, Raluca; Sánchez-Carranza, Oscar; Lewin, Gary R

    2018-03-15

    Mechanosensitive PIEZO ion channels are evolutionarily conserved proteins whose presence is critical for normal physiology in multicellular organisms. Here we show that, in addition to mechanical stimuli, PIEZO channels are also powerfully modulated by voltage and can even switch to a purely voltage-gated mode. Mutations that cause human diseases, such as xerocytosis, profoundly shift voltage sensitivity of PIEZO1 channels toward the resting membrane potential and strongly promote voltage gating. Voltage modulation may be explained by the presence of an inactivation gate in the pore, the opening of which is promoted by outward permeation. Older invertebrate (fly) and vertebrate (fish) PIEZO proteins are also voltage sensitive, but voltage gating is a much more prominent feature of these older channels. We propose that the voltage sensitivity of PIEZO channels is a deep property co-opted to add a regulatory mechanism for PIEZO activation in widely different cellular contexts.

  5. Voltage-dependent ion channels in the mouse RPE: comparison with Norrie disease mice.

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    Wollmann, Guido; Lenzner, Steffen; Berger, Wolfgang; Rosenthal, Rita; Karl, Mike O; Strauss, Olaf

    2006-03-01

    We studied electrophysiological properties of cultured retinal pigment epithelial (RPE) cells from mouse and a mouse model for Norrie disease. Wild-type RPE cells revealed the expression of ion channels known from other species: delayed-rectifier K(+) channels composed of Kv1.3 subunits, inward rectifier K(+) channels, Ca(V)1.3 L-type Ca(2+) channels and outwardly rectifying Cl(-) channels. Expression pattern and the ion channel characteristics current density, blocker sensitivity, kinetics and voltage-dependence were compared in cells from wild-type and Norrie mice. Although no significant differences were observed, our study provides a base for future studies on ion channel function and dysfunction in transgenic mouse models.

  6. Voltage-Dependent Gating of hERG Potassium Channels

    Science.gov (United States)

    Cheng, Yen May; Claydon, Tom W.

    2012-01-01

    The mechanisms by which voltage-gated channels sense changes in membrane voltage and energetically couple this with opening of the ion conducting pore has been the source of significant interest. In voltage-gated potassium (Kv) channels, much of our knowledge in this area comes from Shaker-type channels, for which voltage-dependent gating is quite rapid. In these channels, activation and deactivation are associated with rapid reconfiguration of the voltage-sensing domain unit that is electromechanically coupled, via the S4–S5 linker helix, to the rate-limiting opening of an intracellular pore gate. However, fast voltage-dependent gating kinetics are not typical of all Kv channels, such as Kv11.1 (human ether-à-go-go related gene, hERG), which activates and deactivates very slowly. Compared to Shaker channels, our understanding of the mechanisms underlying slow hERG gating is much poorer. Here, we present a comparative review of the structure–function relationships underlying activation and deactivation gating in Shaker and hERG channels, with a focus on the roles of the voltage-sensing domain and the S4–S5 linker that couples voltage sensor movements to the pore. Measurements of gating current kinetics and fluorimetric analysis of voltage sensor movement are consistent with models suggesting that the hERG activation pathway contains a voltage independent step, which limits voltage sensor transitions. Constraints upon hERG voltage sensor movement may result from loose packing of the S4 helices and additional intra-voltage sensor counter-charge interactions. More recent data suggest that key amino acid differences in the hERG voltage-sensing unit and S4–S5 linker, relative to fast activating Shaker-type Kv channels, may also contribute to the increased stability of the resting state of the voltage sensor. PMID:22586397

  7. Fragile X mental retardation protein controls ion channel expression and activity.

    Science.gov (United States)

    Ferron, Laurent

    2016-10-15

    Fragile X-associated disorders are a family of genetic conditions resulting from the partial or complete loss of fragile X mental retardation protein (FMRP). Among these disorders is fragile X syndrome, the most common cause of inherited intellectual disability and autism. FMRP is an RNA-binding protein involved in the control of local translation, which has pleiotropic effects, in particular on synaptic function. Analysis of the brain FMRP transcriptome has revealed hundreds of potential mRNA targets encoding postsynaptic and presynaptic proteins, including a number of ion channels. FMRP has been confirmed to bind voltage-gated potassium channels (K v 3.1 and K v 4.2) mRNAs and regulates their expression in somatodendritic compartments of neurons. Recent studies have uncovered a number of additional roles for FMRP besides RNA regulation. FMRP was shown to directly interact with, and modulate, a number of ion channel complexes. The sodium-activated potassium (Slack) channel was the first ion channel shown to directly interact with FMRP; this interaction alters the single-channel properties of the Slack channel. FMRP was also shown to interact with the auxiliary β4 subunit of the calcium-activated potassium (BK) channel; this interaction increases calcium-dependent activation of the BK channel. More recently, FMRP was shown to directly interact with the voltage-gated calcium channel, Ca v 2.2, and reduce its trafficking to the plasma membrane. Studies performed on animal models of fragile X syndrome have revealed links between modifications of ion channel activity and changes in neuronal excitability, suggesting that these modifications could contribute to the phenotypes observed in patients with fragile X-associated disorders. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  8. Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation.

    Science.gov (United States)

    Arrigoni, Cristina; Rohaim, Ahmed; Shaya, David; Findeisen, Felix; Stein, Richard A; Nurva, Shailika Reddy; Mishra, Smriti; Mchaourab, Hassane S; Minor, Daniel L

    2016-02-25

    Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Voltage-dependent gating of hERG potassium channels

    Directory of Open Access Journals (Sweden)

    Yen May eCheng

    2012-05-01

    Full Text Available The mechanisms by which voltage-gated channels sense changes in membrane voltage and energetically couple this with opening of the ion conducting pore has been the source of significant interest. In voltage-gated potassium (Kv channels, much of our knowledge in this area comes from Shaker-type channels, for which voltage-dependent gating is quite rapid. In these channels, activation and deactivation are associated with rapid reconfiguration of the voltage-sensing domain unit that is electromechanically coupled, via the S4-S5 linker helix, to the rate-limiting opening of an intracellular pore gate. However, fast voltage-dependent gating kinetics are not typical of all Kv channels, such as Kv11.1 (human ether-a-go-go related gene, hERG, which activates and deactivates very slowly. Compared to Shaker channels, our understanding of the mechanisms underlying slow hERG gating is much poorer. Here, we present a comparative review of the structure-function relationships underlying voltage-dependent gating in Shaker and hERG channels, with a focus on the roles of the voltage sensing domain and the S4-S5 linker that couples voltage sensor movements to the pore. Measurements of gating current kinetics and fluorimetric analysis of voltage sensor movement are consistent with models suggesting that the hERG activation pathway contains a voltage independent step, which limits voltage sensor transitions. Constraints upon hERG voltage sensor movement may result from loose packing of the S4 helices and additional intra-voltage sensor counter charge interactions. More recent data suggest that key amino acid differences in the hERG voltage sensing unit and S4-S5 linker, relative to fast activating Shaker-type Kv channels, may also contribute to the increased stability of the resting state of the voltage sensor.

  10. Mutations in the voltage-sensing domain affect the alternative ion permeation pathway in the TRPM3 channel.

    Science.gov (United States)

    Held, Katharina; Gruss, Fabian; Aloi, Vincenzo Davide; Janssens, Annelies; Ulens, Chris; Voets, Thomas; Vriens, Joris

    2018-03-31

    Mutagenesis at positively charged amino acids (arginines and lysines) (R1-R4) in the voltage-sensor domain (transmembrane segment (S) 4) of voltage-gated Na + , K + and Ca 2+ channels can lead to an alternative ion permeation pathway distinct from the central pore. Recently, a non-canonical ion permeation pathway was described in TRPM3, a member of the transient receptor potential (TRP) superfamily. The non-canonical pore exists in the native TRPM3 channel and can be activated by co-stimulation of the endogenous agonist pregnenolone sulphate and the antifungal drug clotrimazole or by stimulation of the synthetic agonist CIM0216. Alignment of the voltage sensor of Shaker K + channels with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3, and suggested that only one single gating charge arginine (R2) in the putative S4 region is conserved. Mutagenesis studies in the voltage-sensing domain of TRPM3 revealed several residues in the voltage sensor (S4) as well as in S1 and S3 that are crucial for the occurrence of the non-canonical inward currents. In conclusion, this study provides evidence for the involvement of the voltage-sensing domain of TRPM3 in the formation of an alternative ion permeation pathway. Transient receptor potential (TRP) channels are cationic channels involved in a broad array of functions, including homeostasis, motility and sensory functions. TRP channel subunits consist of six transmembrane segments (S1-S6), and form tetrameric channels with a central pore formed by the region encompassing S5 and S6. Recently, evidence was provided for the existence of an alternative ion permeation pathway in TRPM3, which allows large inward currents upon hyperpolarization independently of the central pore. However, very little knowledge is available concerning the localization of this alternative pathway in the native TRPM3 channel protein. Guided by sequence homology with Shaker K + channels, in which

  11. The role of entropic potential in voltage activation and K+ transport through Kv 1.2 channels

    Science.gov (United States)

    Wawrzkiewicz-Jałowiecka, Agata; Grzywna, Zbigniew J.

    2018-03-01

    We analyze the entropic effects of inner pore geometry changes of Kv 1.2 channel during membrane depolarization and their implications for the rate of transmembrane transport of potassium ions. We base this on the idea that spatial confinements within the channel pore give rise to entropic barriers which can both effectively affect the stability of open macroconformation and influence channel's ability to conduct the potassium ions through the membrane. First, we calculate the differences in entropy between voltage-activated and resting states of the channel. As a template, we take a set of structures of channel pore in an open state at different membrane potentials generated in our previous research. The obtained results indicate that tendency to occupy open states at membrane depolarization is entropy facilitated. Second, we describe the differences in rates of K+ transport through the channel pore at different voltages based on the results of appropriate random walk simulations in entropic and electric potentials. The simulated single channel currents (I) suggest that the geometry changes during membrane depolarization are an important factor contributing to the observed flow of potassium ions through the channel. Nevertheless, the charge distribution within the channel pore (especially at the extracellular entrance) seems most prominent for the observed I/Imax relation at a qualitative level at analyzed voltages.

  12. Molecular pathophysiology and pharmacology of the voltage-sensing module of neuronal ion channels.

    Science.gov (United States)

    Miceli, Francesco; Soldovieri, Maria Virginia; Ambrosino, Paolo; De Maria, Michela; Manocchio, Laura; Medoro, Alessandro; Taglialatela, Maurizio

    2015-01-01

    Voltage-gated ion channels (VGICs) are membrane proteins that switch from a closed to open state in response to changes in membrane potential, thus enabling ion fluxes across the cell membranes. The mechanism that regulate the structural rearrangements occurring in VGICs in response to changes in membrane potential still remains one of the most challenging topic of modern biophysics. Na(+), Ca(2+) and K(+) voltage-gated channels are structurally formed by the assembly of four similar domains, each comprising six transmembrane segments. Each domain can be divided into two main regions: the Pore Module (PM) and the Voltage-Sensing Module (VSM). The PM (helices S5 and S6 and intervening linker) is responsible for gate opening and ion selectivity; by contrast, the VSM, comprising the first four transmembrane helices (S1-S4), undergoes the first conformational changes in response to membrane voltage variations. In particular, the S4 segment of each domain, which contains several positively charged residues interspersed with hydrophobic amino acids, is located within the membrane electric field and plays an essential role in voltage sensing. In neurons, specific gating properties of each channel subtype underlie a variety of biological events, ranging from the generation and propagation of electrical impulses, to the secretion of neurotransmitters and to the regulation of gene expression. Given the important functional role played by the VSM in neuronal VGICs, it is not surprising that various VSM mutations affecting the gating process of these channels are responsible for human diseases, and that compounds acting on the VSM have emerged as important investigational tools with great therapeutic potential. In the present review we will briefly describe the most recent discoveries concerning how the VSM exerts its function, how genetically inherited diseases caused by mutations occurring in the VSM affects gating in VGICs, and how several classes of drugs and toxins

  13. Mechanisms of Gain Control by Voltage-Gated Channels in Intrinsically-Firing Neurons

    Science.gov (United States)

    Patel, Ameera X.; Burdakov, Denis

    2015-01-01

    Gain modulation is a key feature of neural information processing, but underlying mechanisms remain unclear. In single neurons, gain can be measured as the slope of the current-frequency (input-output) relationship over any given range of inputs. While much work has focused on the control of basal firing rates and spike rate adaptation, gain control has been relatively unstudied. Of the limited studies on gain control, some have examined the roles of synaptic noise and passive somatic currents, but the roles of voltage-gated channels present ubiquitously in neurons have been less explored. Here, we systematically examined the relationship between gain and voltage-gated ion channels in a conductance-based, tonically-active, model neuron. Changes in expression (conductance density) of voltage-gated channels increased (Ca2+ channel), reduced (K+ channels), or produced little effect (h-type channel) on gain. We found that the gain-controlling ability of channels increased exponentially with the steepness of their activation within the dynamic voltage window (voltage range associated with firing). For depolarization-activated channels, this produced a greater channel current per action potential at higher firing rates. This allowed these channels to modulate gain by contributing to firing preferentially at states of higher excitation. A finer analysis of the current-voltage relationship during tonic firing identified narrow voltage windows at which the gain-modulating channels exerted their effects. As a proof of concept, we show that h-type channels can be tuned to modulate gain by changing the steepness of their activation within the dynamic voltage window. These results show how the impact of an ion channel on gain can be predicted from the relationship between channel kinetics and the membrane potential during firing. This is potentially relevant to understanding input-output scaling in a wide class of neurons found throughout the brain and other nervous systems

  14. Mapping of Residues Forming the Voltage Sensor of the Voltage-Dependent Anion-Selective Channel

    Science.gov (United States)

    Thomas, Lorie; Blachly-Dyson, Elizabeth; Colombini, Marco; Forte, Michael

    1993-06-01

    Voltage-gated ion-channel proteins contain "voltage-sensing" domains that drive the conformational transitions between open and closed states in response to changes in transmembrane voltage. We have used site-directed mutagenesis to identify residues affecting the voltage sensitivity of a mitochondrial channel, the voltage-dependent anion-selective channel (VDAC). Although charge changes at many sites had no effect, at other sites substitutions that increased positive charge also increased the steepness of voltage dependance and substitutions that decreased positive charge decreased voltage dependance by an appropriate amount. In contrast to the plasma membrane K^+ and Na^+ channels, these residues are distributed over large parts of the VDAC protein. These results have been used to define the conformational transitions that accompany voltage gating of an ion channel. This gating mechanism requires the movement of large portions of the VDAC protein through the membrane.

  15. Functional diversity of potassium channel voltage-sensing domains.

    Science.gov (United States)

    Islas, León D

    2016-01-01

    Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology.

  16. Guanidinium Toxins and Their Interactions with Voltage-Gated Sodium Ion Channels

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    Lorena M. Durán-Riveroll

    2017-10-01

    Full Text Available Guanidinium toxins, such as saxitoxin (STX, tetrodotoxin (TTX and their analogs, are naturally occurring alkaloids with divergent evolutionary origins and biogeographical distribution, but which share the common chemical feature of guanidinium moieties. These guanidinium groups confer high biological activity with high affinity and ion flux blockage capacity for voltage-gated sodium channels (NaV. Members of the STX group, known collectively as paralytic shellfish toxins (PSTs, are produced among three genera of marine dinoflagellates and about a dozen genera of primarily freshwater or brackish water cyanobacteria. In contrast, toxins of the TTX group occur mainly in macrozoa, particularly among puffer fish, several species of marine invertebrates and a few terrestrial amphibians. In the case of TTX and analogs, most evidence suggests that symbiotic bacteria are the origin of the toxins, although endogenous biosynthesis independent from bacteria has not been excluded. The evolutionary origin of the biosynthetic genes for STX and analogs in dinoflagellates and cyanobacteria remains elusive. These highly potent molecules have been the subject of intensive research since the latter half of the past century; first to study the mode of action of their toxigenicity, and later as tools to characterize the role and structure of NaV channels, and finally as therapeutics. Their pharmacological activities have provided encouragement for their use as therapeutants for ion channel-related pathologies, such as pain control. The functional role in aquatic and terrestrial ecosystems for both groups of toxins is unproven, although plausible mechanisms of ion channel regulation and chemical defense are often invoked. Molecular approaches and the development of improved detection methods will yield deeper understanding of their physiological and ecological roles. This knowledge will facilitate their further biotechnological exploitation and point the way towards

  17. Regulation of KV channel voltage-dependent activation by transmembrane β subunits

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    Xiaohui eSun

    2012-04-01

    Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

  18. Large conductance Ca2+-activated K+ (BK channel: Activation by Ca2+ and voltage

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    RAMÓN LATORRE

    2006-01-01

    Full Text Available Large conductance Ca2+-activated K+ (BK channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv channels characterized by having six (S1-S6 transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0 transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 μM-100 μM in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.

  19. 24-channel dual microcontroller-based voltage controller for ion optics remote control

    Science.gov (United States)

    Bengtsson, L.

    2018-05-01

    The design of a 24-channel voltage control instrument for Wenzel Elektronik N1130 NIM modules is described. This instrument is remote controlled from a LabVIEW GUI on a host Windows computer and is intended for ion optics control in electron affinity measurements on negative ions at the CERN-ISOLDE facility. Each channel has a resolution of 12 bits and has a normally distributed noise with a standard deviation of <1 mV. The instrument is designed as a standard 2-unit NIM module where the electronic hardware consists of a printed circuit board with two asynchronously operating microcontrollers.

  20. MOLECULAR PATHOPHYSIOLOGY AND PHARMACOLOGY OF THE VOLTAGE-SENSING DOMAIN OF NEURONAL ION CHANNELS

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    Francesco eMiceli

    2015-07-01

    Full Text Available Voltage-gated ion channels (VGIC are membrane proteins that switch from a closed to open state in response to changes in membrane potential, thus enabling ion fluxes across the cell membranes. The mechanism that regulate the structural rearrangements occurring in VGIC in response to changes in membrane potential still remains one of the most challenging topic of modern biophysics. Na+, Ca2+ and K+ voltage-gated channels are structurally formed by the assembly of four similar domains, each comprising six transmembrane segments. Each domain can be divided in two main regions: the Pore Module (PM and the Voltage-Sensing Module (VSM. The PM (helices S5 and S6 and intervening linker is responsible for gate opening and ion selectivity; by contrast, the VSM, comprising the first four transmembrane helices (S1-S4, undergoes the first conformational changes in response to membrane voltage. In particular, the S4 segment of each domain, which contains several positively charged residues interspersed with hydrophobic amino acids, is located within the membrane electric field and plays an essential role in voltage sensing. In neurons, specific gating properties of each channel subtype underlie a variety of biological events, ranging from the generation and propagation of electrical impulses, to the secretion of neurotransmitters, to the regulation of gene expression. Given the important functional role played by the VSM in neuronal VGICs, it is not surprising that various VSM mutations affecting the gating process of these channels are responsible for human diseases, and that compounds acting on the VSM have emerged as important investigational tools with great therapeutic potential. In the present review we will briefly describe the most recent discoveries concerning how the VSM exerts its function, how genetically inherited diseases caused by mutations occurring in the VSM affects gating in VGICs, and how several classes of drugs and toxins selectively

  1. Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels

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    Mohamed-Yassine eAMAROUCH

    2015-02-01

    Full Text Available Voltage-gated sodium channels (Nav are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the voltage-gated sodium channels. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the voltage-gated sodium channels by shifting the voltage-dependence of steady state activation towards more negative potentials.

  2. A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels.

    Science.gov (United States)

    Schewe, Marcus; Nematian-Ardestani, Ehsan; Sun, Han; Musinszki, Marianne; Cordeiro, Sönke; Bucci, Giovanna; de Groot, Bert L; Tucker, Stephen J; Rapedius, Markus; Baukrowitz, Thomas

    2016-02-25

    Two-pore domain (K2P) K(+) channels are major regulators of excitability that endow cells with an outwardly rectifying background "leak" conductance. In some K2P channels, strong voltage-dependent activation has been observed, but the mechanism remains unresolved because they lack a canonical voltage-sensing domain. Here, we show voltage-dependent gating is common to most K2P channels and that this voltage sensitivity originates from the movement of three to four ions into the high electric field of an inactive selectivity filter. Overall, this ion-flux gating mechanism generates a one-way "check valve" within the filter because outward movement of K(+) induces filter opening, whereas inward movement promotes inactivation. Furthermore, many physiological stimuli switch off this flux gating mode to convert K2P channels into a leak conductance. These findings provide insight into the functional plasticity of a K(+)-selective filter and also refine our understanding of K2P channels and the mechanisms by which ion channels can sense voltage. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Mechanism of voltage-gated channel formation in lipid membranes.

    Science.gov (United States)

    Guidelli, Rolando; Becucci, Lucia

    2016-04-01

    Although several molecular models for voltage-gated ion channels in lipid membranes have been proposed, a detailed mechanism accounting for the salient features of experimental data is lacking. A general treatment accounting for peptide dipole orientation in the electric field and their nucleation and growth kinetics with ion channel formation is provided. This is the first treatment that explains all the main features of the experimental current-voltage curves of peptides forming voltage-gated channels available in the literature. It predicts a regime of weakly voltage-dependent conductance, followed by one of strong voltage-dependent conductance at higher voltages. It also predicts values of the parameters expressing the exponential dependence of conductance upon voltage and peptide bulk concentration for both regimes, in good agreement with those reported in the literature. Most importantly, the only two adjustable parameters involved in the kinetics of nucleation and growth of ion channels can be varied over broad ranges without affecting the above predictions to a significant extent. Thus, the fitting of experimental current-voltage curves stems naturally from the treatment and depends only slightly upon the choice of the kinetic parameters. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Mechanism of electromechanical coupling in voltage-gated potassium channels

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    Rikard eBlunck

    2012-09-01

    Full Text Available Voltage-gated ion channels play a central role in the generation of action potentials in the nervous system. They are selective for one type of ion – sodium, calcium or potassium. Voltage-gated ion channels are composed of a central pore that allows ions to pass through the membrane and four peripheral voltage sensing domains that respond to changes in the membrane potential. Upon depolarization, voltage sensors in voltage-gated potassium channels (Kv undergo conformational changes driven by positive charges in the S4 segment and aided by pairwise electrostatic interactions with the surrounding voltage sensor. Structure-function relations of Kv channels have been investigated in detail, and the resulting models on the movement of the voltage sensors now converge to a consensus; the S4 segment undergoes a combined movement of rotation, tilt and vertical displacement in order to bring 3-4 e+ each through the electric field focused in this region. Nevertheless, the mechanism by which the voltage sensor movement leads to pore opening, the electromechanical coupling, is still not fully understood. Thus, recently, electromechanical coupling in different Kv channels has been investigated with a multitude of techniques including electrophysiology, 3D crystal structures, fluorescence spectroscopy and molecular dynamics simulations. Evidently, the S4-S5 linker, the covalent link between the voltage sensor and pore, plays a crucial role. The linker transfers the energy from the voltage sensor movement to the pore domain via an interaction with the S6 C-termini, which are pulled open during gating. In addition, other contact regions have been proposed. This review aims to provide (i an in-depth comparison of the molecular mechanisms of electromechanical coupling in different Kv channels; (ii insight as to how the voltage sensor and pore domain influence one another; and (iii theoretical predictions on the movement of the cytosolic face of the KV channels

  5. Opposite effects of the S4-S5 linker and PIP2 on voltage-gated channel function: KCNQ1/KCNE1 and other channels

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    Frank S Choveau

    2012-07-01

    Full Text Available Voltage-gated potassium (Kv channels are tetramers, each subunit presenting six transmembrane segments (S1-S6, with each S1-S4 segments forming a voltage-sensing domain (VSD and the four S5-S6 forming both the conduction pathway and its gate. S4 segments control the opening of the intracellular activation gate in response to changes in membrane potential. Crystal structures of several voltage-gated ion channels in combination with biophysical and mutagenesis studies highlighted the critical role of the S4-S5 linker (S4S5L and of the S6 C-terminal part (S6T in the coupling between the VSD and the activation gate. Several mechanisms have been proposed to describe the coupling at a molecular scale. This review summarizes the mechanisms suggested for various voltage-gated ion channels, including a mechanism that we described for KCNQ1, in which S4S5L is acting like a ligand binding to S6T to stabilize the channel in a closed state. As discussed in this review, this mechanism may explain the reverse response to depolarization in HCN-like channels. As opposed to S4S5L, the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2, stabilizes KCNQ1 channel in an open state. Many other ion channels (not only voltage-gated require PIP2 to function properly, confirming its crucial importance as an ion channel co-factor. This is highlighted in cases in which an altered regulation of ion channels by PIP2 leads to channelopathies, as observed for KCNQ1. This review summarizes the state of the art on the two regulatory mechanisms that are critical for KCNQ1 and other voltage-gated channels function (PIP2 and S4-S5L, and assesses their potential physiological and pathophysiological roles.

  6. VKCDB: Voltage-gated potassium channel database

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    Gallin Warren J

    2004-01-01

    Full Text Available Abstract Background The family of voltage-gated potassium channels comprises a functionally diverse group of membrane proteins. They help maintain and regulate the potassium ion-based component of the membrane potential and are thus central to many critical physiological processes. VKCDB (Voltage-gated potassium [K] Channel DataBase is a database of structural and functional data on these channels. It is designed as a resource for research on the molecular basis of voltage-gated potassium channel function. Description Voltage-gated potassium channel sequences were identified by using BLASTP to search GENBANK and SWISSPROT. Annotations for all voltage-gated potassium channels were selectively parsed and integrated into VKCDB. Electrophysiological and pharmacological data for the channels were collected from published journal articles. Transmembrane domain predictions by TMHMM and PHD are included for each VKCDB entry. Multiple sequence alignments of conserved domains of channels of the four Kv families and the KCNQ family are also included. Currently VKCDB contains 346 channel entries. It can be browsed and searched using a set of functionally relevant categories. Protein sequences can also be searched using a local BLAST engine. Conclusions VKCDB is a resource for comparative studies of voltage-gated potassium channels. The methods used to construct VKCDB are general; they can be used to create specialized databases for other protein families. VKCDB is accessible at http://vkcdb.biology.ualberta.ca.

  7. Highly selective water channel activity measured by voltage clamp: analysis of planar lipid bilayers reconstituted with purified AqpZ.

    Science.gov (United States)

    Pohl, P; Saparov, S M; Borgnia, M J; Agre, P

    2001-08-14

    Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 10(9) transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins.

  8. Voltage-sensing domain of voltage-gated proton channel Hv1 shares mechanism of block with pore domains.

    Science.gov (United States)

    Hong, Liang; Pathak, Medha M; Kim, Iris H; Ta, Dennis; Tombola, Francesco

    2013-01-23

    Voltage-gated sodium, potassium, and calcium channels are made of a pore domain (PD) controlled by four voltage-sensing domains (VSDs). The PD contains the ion permeation pathway and the activation gate located on the intracellular side of the membrane. A large number of small molecules are known to inhibit the PD by acting as open channel blockers. The voltage-gated proton channel Hv1 is made of two VSDs and lacks the PD. The location of the activation gate in the VSD is unknown and open channel blockers for VSDs have not yet been identified. Here, we describe a class of small molecules which act as open channel blockers on the Hv1 VSD and find that a highly conserved phenylalanine in the charge transfer center of the VSD plays a key role in blocker binding. We then use one of the blockers to show that Hv1 contains two intracellular and allosterically coupled gates. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

    Science.gov (United States)

    Martín, Pedro; Enrique, Nicolás; Palomo, Ana R. Roldán; Rebolledo, Alejandro; Milesi, Veronica

    2012-01-01

    Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K+ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca2+-activated K+ channels (BKCa). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K+ currents carried by BKCa channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC50 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K+ currents (~45% blockage) in which, under our working conditions, BKCa is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BKCa channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account. PMID:22688134

  10. Monitoring voltage-dependent charge displacement of Shaker B-IR K+ ion channels using radio frequency interrogation.

    Directory of Open Access Journals (Sweden)

    Sameera Dharia

    2011-02-01

    Full Text Available Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR K(+ ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents. Simultaneously, RF electric fields were applied to perturb the membrane potential about the TEVC level and to measure voltage-dependent RF displacement currents. ShB-IR expressing oocytes showed significantly larger changes in RF displacement currents upon membrane depolarization than control oocytes. Voltage-dependent changes in RF displacement currents further increased in ShB-IR expressing oocytes after ∼120 µM Cu(2+ addition to the external bath. Cu(2+ is known to bind to the ShB-IR ion channel and inhibit Shaker K(+ conductance, indicating that changes in the RF displacement current reported here were associated with RF vibration of the Cu(2+-linked mobile domain of the ShB-IR protein. Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains--capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug-protein interactions.

  11. Monitoring voltage-dependent charge displacement of Shaker B-IR K+ ion channels using radio frequency interrogation.

    Science.gov (United States)

    Dharia, Sameera; Rabbitt, Richard D

    2011-02-28

    Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz) electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K(+) ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents. Simultaneously, RF electric fields were applied to perturb the membrane potential about the TEVC level and to measure voltage-dependent RF displacement currents. ShB-IR expressing oocytes showed significantly larger changes in RF displacement currents upon membrane depolarization than control oocytes. Voltage-dependent changes in RF displacement currents further increased in ShB-IR expressing oocytes after ∼120 µM Cu(2+) addition to the external bath. Cu(2+) is known to bind to the ShB-IR ion channel and inhibit Shaker K(+) conductance, indicating that changes in the RF displacement current reported here were associated with RF vibration of the Cu(2+)-linked mobile domain of the ShB-IR protein. Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains--capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug-protein interactions.

  12. Functionalized Fullerene Targeting Human Voltage-Gated Sodium Channel, hNav1.7.

    Science.gov (United States)

    Hilder, Tamsyn A; Robinson, Anna; Chung, Shin-Ho

    2017-08-16

    Mutations of hNa v 1.7 that cause its activities to be enhanced contribute to severe neuropathic pain. Only a small number of hNa v 1.7 specific inhibitors have been identified, most of which interact with the voltage-sensing domain of the voltage-activated sodium ion channel. In our previous computational study, we demonstrated that a [Lys 6 ]-C 84 fullerene binds tightly (affinity of 46 nM) to Na v Ab, the voltage-gated sodium channel from the bacterium Arcobacter butzleri. Here, we extend this work and, using molecular dynamics simulations, demonstrate that the same [Lys 6 ]-C 84 fullerene binds strongly (2.7 nM) to the pore of a modeled human sodium ion channel hNa v 1.7. In contrast, the fullerene binds only weakly to a mutated model of hNa v 1.7 (I1399D) (14.5 mM) and a model of the skeletal muscle hNa v 1.4 (3.7 mM). Comparison of one representative sequence from each of the nine human sodium channel isoforms shows that only hNa v 1.7 possesses residues that are critical for binding the fullerene derivative and blocking the channel pore.

  13. Molecular mechanism of voltage sensing in voltage-gated proton channels

    Science.gov (United States)

    Rebolledo, Santiago; Perez, Marta E.

    2013-01-01

    Voltage-gated proton (Hv) channels play an essential role in phagocytic cells by generating a hyperpolarizing proton current that electrically compensates for the depolarizing current generated by the NADPH oxidase during the respiratory burst, thereby ensuring a sustained production of reactive oxygen species by the NADPH oxidase in phagocytes to neutralize engulfed bacteria. Despite the importance of the voltage-dependent Hv current, it is at present unclear which residues in Hv channels are responsible for the voltage activation. Here we show that individual neutralizations of three charged residues in the fourth transmembrane domain, S4, all reduce the voltage dependence of activation. In addition, we show that the middle S4 charged residue moves from a position accessible from the cytosolic solution to a position accessible from the extracellular solution, suggesting that this residue moves across most of the membrane electric field during voltage activation of Hv channels. Our results show for the first time that the charge movement of these three S4 charges accounts for almost all of the measured gating charge in Hv channels. PMID:23401575

  14. The twisted ion-permeation pathway of a resting voltage-sensing domain.

    Science.gov (United States)

    Tombola, Francesco; Pathak, Medha M; Gorostiza, Pau; Isacoff, Ehud Y

    2007-02-01

    Proteins containing voltage-sensing domains (VSDs) translate changes in membrane potential into changes in ion permeability or enzymatic activity. In channels, voltage change triggers a switch in conformation of the VSD, which drives gating in a separate pore domain, or, in channels lacking a pore domain, directly gates an ion pathway within the VSD. Neither mechanism is well understood. In the Shaker potassium channel, mutation of the first arginine residue of the S4 helix to a smaller uncharged residue makes the VSD permeable to ions ('omega current') in the resting conformation ('S4 down'). Here we perform a structure-guided perturbation analysis of the omega conductance to map its VSD permeation pathway. We find that there are four omega pores per channel, which is consistent with one conduction path per VSD. Permeating ions from the extracellular medium enter the VSD at its peripheral junction with the pore domain, and then plunge into the core of the VSD in a curved conduction pathway. Our results provide a model of the resting conformation of the VSD.

  15. Ion channel recordings on an injection-molded polymer chip

    DEFF Research Database (Denmark)

    Tanzi, Simone; Matteucci, Marco; Christiansen, Thomas Lehrmann

    2013-01-01

    state-of-the-art system for automated ion channel recordings. These experiments considered current–voltage (IV) relationships for activation and inactivation of the Nav1.7 channels and their sensitivity to a local anesthetic, lidocaine. Both IVs and lidocaine dose–response curves obtained from...

  16. Tarantula toxins use common surfaces for interacting with Kv and ASIC ion channels.

    Science.gov (United States)

    Gupta, Kanchan; Zamanian, Maryam; Bae, Chanhyung; Milescu, Mirela; Krepkiy, Dmitriy; Tilley, Drew C; Sack, Jon T; Yarov-Yarovoy, Vladimir; Kim, Jae Il; Swartz, Kenton J

    2015-05-07

    Tarantula toxins that bind to voltage-sensing domains of voltage-activated ion channels are thought to partition into the membrane and bind to the channel within the bilayer. While no structures of a voltage-sensor toxin bound to a channel have been solved, a structural homolog, psalmotoxin (PcTx1), was recently crystalized in complex with the extracellular domain of an acid sensing ion channel (ASIC). In the present study we use spectroscopic, biophysical and computational approaches to compare membrane interaction properties and channel binding surfaces of PcTx1 with the voltage-sensor toxin guangxitoxin (GxTx-1E). Our results show that both types of tarantula toxins interact with membranes, but that voltage-sensor toxins partition deeper into the bilayer. In addition, our results suggest that tarantula toxins have evolved a similar concave surface for clamping onto α-helices that is effective in aqueous or lipidic physical environments.

  17. Cholesterol influences voltage-gated calcium channels and BK-type potassium channels in auditory hair cells.

    Directory of Open Access Journals (Sweden)

    Erin K Purcell

    Full Text Available The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitability are unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type potassium current by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (~30%, ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.

  18. Ciguatoxins: Cyclic Polyether Modulators of Voltage-gated Iion Channel Function

    Science.gov (United States)

    Nicholson, Graham M.; Lewis, Richard J.

    2006-01-01

    Ciguatoxins are cyclic polyether toxins, derived from marine dinoflagellates, which are responsible for the symptoms of ciguatera poisoning. Ingestion of tropical and subtropical fin fish contaminated by ciguatoxins results in an illness characterised by neurological, cardiovascular and gastrointestinal disorders. The pharmacology of ciguatoxins is characterised by their ability to cause persistent activation of voltage-gated sodium channels, to increase neuronal excitability and neurotransmitter release, to impair synaptic vesicle recycling, and to cause cell swelling. It is these effects, in combination with an action to block voltage-gated potassium channels at high doses, which are believed to underlie the complex of symptoms associated with ciguatera. This review examines the sources, structures and pharmacology of ciguatoxins. In particular, attention is placed on their cellular modes of actions to modulate voltage-gated ion channels and other Na+-dependent mechanisms in numerous cell types and to current approaches for detection and treatment of ciguatera.

  19. Voltage-Dependent Gating: Novel Insights from KCNQ1 Channels

    Science.gov (United States)

    Cui, Jianmin

    2016-01-01

    Gating of voltage-dependent cation channels involves three general molecular processes: voltage sensor activation, sensor-pore coupling, and pore opening. KCNQ1 is a voltage-gated potassium (Kv) channel whose distinctive properties have provided novel insights on fundamental principles of voltage-dependent gating. 1) Similar to other Kv channels, KCNQ1 voltage sensor activation undergoes two resolvable steps; but, unique to KCNQ1, the pore opens at both the intermediate and activated state of voltage sensor activation. The voltage sensor-pore coupling differs in the intermediate-open and the activated-open states, resulting in changes of open pore properties during voltage sensor activation. 2) The voltage sensor-pore coupling and pore opening require the membrane lipid PIP2 and intracellular ATP, respectively, as cofactors, thus voltage-dependent gating is dependent on multiple stimuli, including the binding of intracellular signaling molecules. These mechanisms underlie the extraordinary KCNE1 subunit modification of the KCNQ1 channel and have significant physiological implications. PMID:26745405

  20. Calcium homeostasis modulator (CALHM) ion channels.

    Science.gov (United States)

    Ma, Zhongming; Tanis, Jessica E; Taruno, Akiyuki; Foskett, J Kevin

    2016-03-01

    Calcium homeostasis modulator 1 (CALHM1), formerly known as FAM26C, was recently identified as a physiologically important plasma membrane ion channel. CALHM1 and its Caenorhabditis elegans homolog, CLHM-1, are regulated by membrane voltage and extracellular Ca(2+) concentration ([Ca(2+)]o). In the presence of physiological [Ca(2+)]o (∼1.5 mM), CALHM1 and CLHM-1 are closed at resting membrane potentials but can be opened by strong depolarizations. Reducing [Ca(2+)]o increases channel open probability, enabling channel activation at negative membrane potentials. Together, voltage and Ca(2+) o allosterically regulate CALHM channel gating. Through convergent evolution, CALHM has structural features that are reminiscent of connexins and pannexins/innexins/LRRC8 (volume-regulated anion channel (VRAC)) gene families, including four transmembrane helices with cytoplasmic amino and carboxyl termini. A CALHM1 channel is a hexamer of CALHM1 monomers with a functional pore diameter of ∼14 Å. CALHM channels discriminate poorly among cations and anions, with signaling molecules including Ca(2+) and ATP able to permeate through its pore. CALHM1 is expressed in the brain where it plays an important role in cortical neuron excitability induced by low [Ca(2+)]o and in type II taste bud cells in the tongue that sense sweet, bitter, and umami tastes where it functions as an essential ATP release channel to mediate nonsynaptic neurotransmitter release. CLHM-1 is expressed in C. elegans sensory neurons and body wall muscles, and its genetic deletion causes locomotion defects. Thus, CALHM is a voltage- and Ca(2+) o-gated ion channel, permeable to large cations and anions, that plays important roles in physiology.

  1. Conductance of Ion Channels - Theory vs. Experiment

    Science.gov (United States)

    Pohorille, Andrew; Wilson, Michael; Mijajlovic, Milan

    2013-01-01

    Transmembrane ion channels mediate a number of essential physiological processes in a cell ranging from regulating osmotic pressure to transmission of neural signals. Kinetics and selectivity of ion transport is of critical importance to a cell and, not surprisingly, it is a subject of numerous experimental and theoretical studies. In this presentation we will analyze in detail computer simulations of two simple channels from fungi - antiamoebin and trichotoxin. Each of these channels is made of an alpha-helical bundle of small, nongenomically synthesized peptides containing a number of rare amino acids and exhibits strong antimicrobial activity. We will focus on calculating ionic conductance defined as the ratio of ionic current through the channel to applied voltage. From molecular dynamics simulations, conductance can be calculated in at least two ways, each involving different approximations. Specifically, the current, given as the number of charges transferred through the channel per unit of time, can be obtained from the number of events in which ions cross the channel during the simulation. This method works well for large currents (high conductance values and/or applied voltages). If the number of crossing events is small, reliable estimates of current are difficult to achieve. Alternatively, conductance can be estimated assuming that ion transport can be well approximated as diffusion in the external potential given by the free energy profile. Then, the current can be calculated by solving the one-dimensional diffusion equation in this external potential and applied voltage (the generalized Nernst-Planck equation). To do so three ingredients are needed: the free energy profile, the position-dependent diffusion coefficient and the diffusive flux of ions into the channel. All these quantities can be obtained from molecular dynamics simulations. An important advantage of this method is that it can be used equally well to estimating large and small currents

  2. Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains

    Science.gov (United States)

    Lörinczi, Éva; Gómez-Posada, Juan Camilo; de La Peña, Pilar; Tomczak, Adam P.; Fernández-Trillo, Jorge; Leipscher, Ulrike; Stühmer, Walter; Barros, Francisco; Pardo, Luis A.

    2015-03-01

    Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4-S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4-S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules.

  3. Voltage-gated sodium channels in taste bud cells

    Directory of Open Access Journals (Sweden)

    Williams Mark E

    2009-03-01

    Full Text Available Abstract Background Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. Results We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. Conclusion SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.

  4. Voltage-gated sodium channels in taste bud cells.

    Science.gov (United States)

    Gao, Na; Lu, Min; Echeverri, Fernando; Laita, Bianca; Kalabat, Dalia; Williams, Mark E; Hevezi, Peter; Zlotnik, Albert; Moyer, Bryan D

    2009-03-12

    Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.

  5. Ion channel signaling influences cellular proliferation and phagocyte activity during axolotl tail regeneration.

    Science.gov (United States)

    Franklin, Brandon M; Voss, S Randal; Osborn, Jeffrey L

    2017-08-01

    Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, K V 2.1, K V 2.2, L-type Ca V channels and H/K ATPases) or completely (GlyR, GABA A R, K V 1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K + channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H + pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore.

    Science.gov (United States)

    Tomczak, Adam P; Fernández-Trillo, Jorge; Bharill, Shashank; Papp, Ferenc; Panyi, Gyorgy; Stühmer, Walter; Isacoff, Ehud Y; Pardo, Luis A

    2017-05-01

    Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4-S5 linker). However, our recent work on channels disrupted in the S4-S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of K V 10.1 revealed that the S4-S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use "split" channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in K V 10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4-S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4-S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism. © 2017 Tomczak et al.

  7. Voltage-gated proton channel is expressed on phagosomes

    International Nuclear Information System (INIS)

    Okochi, Yoshifumi; Sasaki, Mari; Iwasaki, Hirohide; Okamura, Yasushi

    2009-01-01

    Voltage-gated proton channel has been suggested to help NADPH oxidase activity during respiratory burst of phagocytes through its activities of compensating charge imbalance and regulation of pH. In phagocytes, robust production of reactive oxygen species occurs in closed membrane compartments, which are called phagosomes. However, direct evidence for the presence of voltage-gated proton channels in phagosome has been lacking. In this study, the expression of voltage-gated proton channels was studied by Western blot with the antibody specific to the voltage-sensor domain protein, VSOP/Hv1, that has recently been identified as the molecular correlate for the voltage-gated proton channel. Phagosomal membranes of neutrophils contain VSOP/Hv1 in accordance with subunits of NADPH oxidases, gp91, p22, p47 and p67. Superoxide anion production upon PMA activation was significantly reduced in neutrophils from VSOP/Hv1 knockout mice. These are consistent with the idea that voltage-gated proton channels help NADPH oxidase in phagocytes to produce reactive oxygen species.

  8. Ciguatoxins: Cyclic Polyether Modulators of Voltage-gated Iion Channel Function

    Directory of Open Access Journals (Sweden)

    Richard J. Lewis

    2006-04-01

    Full Text Available Ciguatoxins are cyclic polyether toxins, derived from marine dinoflagellates, which are responsible for the symptoms of ciguatera poisoning. Ingestion of tropical and subtropical fin fish contaminated by ciguatoxins results in an illness characterised by neurological, cardiovascular and gastrointestinal disorders. The pharmacology of ciguatoxins is characterised by their ability to cause persistent activation of voltage-gated sodium channels, to increase neuronal excitability and neurotransmitter release, to impair synaptic vesicle recycling, and to cause cell swelling. It is these effects, in combination with an action to block voltage-gated potassium channels at high doses, which are believed to underlie the complex of symptoms associated with ciguatera. This review examines the sources, structures and pharmacology of ciguatoxins. In particular, attention is placed on their cellular modes of actions to modulate voltage-gated ion channels and other Na+-dependent mechanisms in numerous cell types and to current approaches for detection and treatment of ciguatera.

  9. Monitoring Voltage-Dependent Charge Displacement of Shaker B-IR K+ Ion Channels Using Radio Frequency Interrogation

    OpenAIRE

    Dharia, Sameera; Rabbitt, Richard D.

    2011-01-01

    Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz) electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K(+) ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential a...

  10. Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

    International Nuclear Information System (INIS)

    Schulz, Joseph R.; Vega-Beltran, Jose L. de la; Beltran, Carmen; Vacquier, Victor D.; Darszon, Alberto

    2004-01-01

    The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca 2+ and Na + influx and K + and H + efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba 2+ , and has a PK + /PNa + selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization

  11. Distribution and function of voltage-gated sodium channels in the nervous system.

    Science.gov (United States)

    Wang, Jun; Ou, Shao-Wu; Wang, Yun-Jie

    2017-11-02

    Voltage-gated sodium channels (VGSCs) are the basic ion channels for neuronal excitability, which are crucial for the resting potential and the generation and propagation of action potentials in neurons. To date, at least nine distinct sodium channel isoforms have been detected in the nervous system. Recent studies have identified that voltage-gated sodium channels not only play an essential role in the normal electrophysiological activities of neurons but also have a close relationship with neurological diseases. In this study, the latest research findings regarding the structure, type, distribution, and function of VGSCs in the nervous system and their relationship to neurological diseases, such as epilepsy, neuropathic pain, brain tumors, neural trauma, and multiple sclerosis, are reviewed in detail.

  12. Contributions of counter-charge in a potassium channel voltage-sensor domain

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Galpin, Jason D; Niciforovic, Ana P

    2011-01-01

    Voltage-sensor domains couple membrane potential to conformational changes in voltage-gated ion channels and phosphatases. Highly coevolved acidic and aromatic side chains assist the transfer of cationic side chains across the transmembrane electric field during voltage sensing. We investigated...... the functional contribution of negative electrostatic potentials from these residues to channel gating and voltage sensing with unnatural amino acid mutagenesis, electrophysiology, voltage-clamp fluorometry and ab initio calculations. The data show that neutralization of two conserved acidic side chains...

  13. Coupling between the voltage-sensing and pore domains in a voltage-gated potassium channel.

    Science.gov (United States)

    Schow, Eric V; Freites, J Alfredo; Nizkorodov, Alex; White, Stephen H; Tobias, Douglas J

    2012-07-01

    Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4-S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4-S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.

  14. Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels.

    Science.gov (United States)

    Tuluc, Petronel; Benedetti, Bruno; Coste de Bagneaux, Pierre; Grabner, Manfred; Flucher, Bernhard E

    2016-06-01

    Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3-S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3-S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3-S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3-S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3-S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms

  15. Meet me on the other side: trans-bilayer modulation of a model voltage-gated ion channel activity by membrane electrostatics asymmetry.

    Directory of Open Access Journals (Sweden)

    Loredana Mereuta

    Full Text Available While it is accepted that biomembrane asymmetry is generated by proteins and phospholipids distribution, little is known about how electric changes manifested in a monolayer influence functional properties of proteins localized on the opposite leaflet. Herein we used single-molecule electrophysiology and investigated how asymmetric changes in the electrostatics of an artificial lipid membrane monolayer, generated oppositely from where alamethicin--a model voltage-gated ion channel--was added, altered peptide activity. We found that phlorizin, a membrane dipole potential lowering amphiphile, augmented alamethicin activity and transport features, whereas the opposite occurred with RH-421, which enhances the monolayer dipole potential. Further, the monolayer surface potential was decreased via adsorption of sodium dodecyl sulfate, and demonstrated that vectorial modification of it also affected the alamethicin activity in a predictive manner. A new paradigm is suggested according to which asymmetric changes in the monolayer dipole and surface potential extend their effects spatially by altering the intramembrane potential, whose gradient is sensed by distantly located peptides.

  16. Signature and Pathophysiology of Non-canonical Pores in Voltage-Dependent Cation Channels.

    Science.gov (United States)

    Held, Katharina; Voets, Thomas; Vriens, Joris

    2016-01-01

    Opening and closing of voltage-gated cation channels allows the regulated flow of cations such as Na(+), K(+), and Ca(2+) across cell membranes, which steers essential physiological processes including shaping of action potentials and triggering Ca(2+)-dependent processes. Classical textbooks describe the voltage-gated cation channels as membrane proteins with a single, central aqueous pore. In recent years, however, evidence has accumulated for the existence of additional ion permeation pathways in this group of cation channels, distinct from the central pore, which here we collectively name non-canonical pores. Whereas the first non-canonical pores were unveiled only after making specific point mutations in the voltage-sensor region of voltage-gated Na(+) and K(+) channels, recent evidence indicates that they may also be functional in non-mutated channels. Moreover, several channelopathies have been linked to mutations that cause the appearance of a non-canonical ion permeation pathway as a new pathological mechanism. This review provides an integrated overview of the biophysical properties of non-canonical pores described in voltage-dependent cation channels (KV, NaV, Cav, Hv1, and TRPM3) and of the (patho)physiological impact of opening of such pores.

  17. The Voltage-Sensing Domain of Kv7.2 Channels as a Molecular Target for Epilepsy-Causing Mutations and Anticonvulsants

    Science.gov (United States)

    Miceli, Francesco; Soldovieri, Maria Virginia; Iannotti, Fabio Arturo; Barrese, Vincenzo; Ambrosino, Paolo; Martire, Maria; Cilio, Maria Roberta; Taglialatela, Maurizio

    2010-01-01

    Understanding the molecular mechanisms underlying voltage-dependent gating in voltage-gated ion channels (VGICs) has been a major effort over the last decades. In recent years, changes in the gating process have emerged as common denominators for several genetically determined channelopathies affecting heart rhythm (arrhythmias), neuronal excitability (epilepsy, pain), or skeletal muscle contraction (periodic paralysis). Moreover, gating changes appear as the main molecular mechanism by which several natural toxins from a variety of species affect ion channel function. In this work, we describe the pathophysiological and pharmacological relevance of the gating process in voltage-gated K+ channels encoded by the Kv7 gene family. After reviewing the current knowledge on the molecular mechanisms and on the structural models of voltage-dependent gating in VGICs, we describe the physiological relevance of these channels, with particular emphasis on those formed by Kv7.2–Kv7.5 subunits having a well-established role in controlling neuronal excitability in humans. In fact, genetically determined alterations in Kv7.2 and Kv7.3 genes are responsible for benign familial neonatal convulsions, a rare seizure disorder affecting newborns, and the pharmacological activation of Kv7.2/3 channels can exert antiepileptic activity in humans. Both mutation-triggered channel dysfunction and drug-induced channel activation can occur by impeding or facilitating, respectively, channel sensitivity to membrane voltage and can affect overlapping molecular sites within the voltage-sensing domain of these channels. Thus, understanding the molecular steps involved in voltage-sensing in Kv7 channels will allow to better define the pathogenesis of rare human epilepsy, and to design innovative pharmacological strategies for the treatment of epilepsies and, possibly, other human diseases characterized by neuronal hyperexcitability. PMID:21687499

  18. The voltage-sensing domain of kv7.2 channels as a molecular target for epilepsy-causing mutations and anticonvulsants

    Directory of Open Access Journals (Sweden)

    Francesco eMiceli

    2011-02-01

    Full Text Available Understanding the molecular mechanisms underlying voltage-dependent gating in voltage-gated ion channels (VGICs has been a major effort over the last decades. In recent years, changes in the gating process have emerged as common denominators for several genetically-determined channelopathies affecting heart rhythm (arrhythmias, neuronal excitability (epilepsy, pain or skeletal muscle contraction (periodic paralysis. Moreover, gating changes appear as the main molecular mechanism by which several natural toxins from a variety of species affect ion channel function.In this work, we describe the pathophysiological and pharmacological relevance of the gating process in voltage-gated K+ channels encoded by the Kv7 gene family. After reviewing the current knowledge on the molecular mechanisms and on the structural models of voltage-dependent gating in VGICs, we describe the physiological relevance of these channels, with particular emphasis on those formed by Kv7.2-5 subunits having a well-established role in controlling neuronal excitability in humans. In fact, genetically-determined alterations in Kv7.2 and Kv7.3 genes are responsible for benign familial neonatal convulsions, a rare seizure disorder affecting newborns, and the pharmacological activation of Kv7.2/3 channels can exert antiepileptic activity in humans. Both mutation-triggered channel dysfunction and drug-induced channel activation can occur by impeding or facilitating, respectively, channel sensitivity to membrane voltage and can affect overlapping molecular sites within the voltage-sensing domain of these channels. Thus, understanding the molecular steps involved in voltage-sensing in Kv7 channels will allow to better define the pathogenesis of rare human epilepsy, and to design innovative pharmacological strategies for the treatment of epilepsies and, possibly, other human diseases characterized by neuronal hyperexcitability.

  19. SGK3 Sensitivity of Voltage Gated K+ Channel Kv1.5 (KCNA5

    Directory of Open Access Journals (Sweden)

    Musaab Ahmed

    2016-01-01

    Full Text Available Background: The serum & glucocorticoid inducible kinase isoform SGK3 is a powerful regulator of several transporters, ion channels and the Na+/K+ ATPase. Targets of SGK3 include the ubiquitin ligase Nedd4-2, which is in turn a known regulator of the voltage gated K+ channel Kv1.5 (KCNA5. The present study thus explored whether SGK3 modifies the activity of the voltage gated K+ channel KCNA5, which participates in the regulation of diverse functions including atrial cardiac action potential, activity of vascular smooth muscle cells, insulin release and tumour cell proliferation. Methods: cRNA encoding KCNA5 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild-type SGK3, constitutively active S419DSGK3, inactive K191NSGK3 and/or wild type Nedd4-2. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp. Results: Voltage gated current in KCNA5 expressing Xenopus oocytes was significantly enhanced by wild-type SGK3 and S419DSGK3, but not by K191NSGK3. SGK3 was effective in the presence of ouabain (1 mM and thus did not require Na+/K+ ATPase activity. Coexpression of Nedd4-2 decreased the voltage gated current in KCNA5 expressing Xenopus oocytes, an effect largely reversed by additional coexpression of SGK3. Conclusion: SGK3 is a positive regulator of KCNA5, which is at least partially effective by abrogating the effect of Nedd4-2.

  20. Trans-Channel Interactions in Batrachotoxin-Modified Skeletal Muscle Sodium Channels: Voltage-Dependent Block by Cytoplasmic Amines, and the Influence of μ-Conotoxin GIIIA Derivatives and Permeant Ions

    Science.gov (United States)

    Pavlov, Evgeny; Britvina, Tatiana; McArthur, Jeff R.; Ma, Quanli; Sierralta, Iván; Zamponi, Gerald W.; French, Robert J.

    2008-01-01

    External μ-conotoxins and internal amine blockers inhibit each other's block of voltage-gated sodium channels. We explore the basis of this interaction by measuring the shifts in voltage-dependence of channel inhibition by internal amines induced by two μ-conotoxin derivatives with different charge distributions and net charges. Charge changes on the toxin were made at residue 13, which is thought to penetrate most deeply into the channel, making it likely to have the strongest individual interaction with an internal charged ligand. When an R13Q or R13E molecule was bound to the channel, the voltage dependence of diethylammonium (DEA)-block shifted toward more depolarized potentials (23 mV for R13Q, and 16 mV for R13E). An electrostatic model of the repulsion between DEA and the toxin simulated these data, with a distance between residue 13 of the μ-conotoxin and the DEA-binding site of ∼15 Å. Surprisingly, for tetrapropylammonium, the shifts were only 9 mV for R13Q, and 7 mV for R13E. The smaller shifts associated with R13E, the toxin with a smaller net charge, are generally consistent with an electrostatic interaction. However, the smaller shifts observed for tetrapropylammonium than for DEA suggest that other factors must be involved. Two observations indicate that the coupling of permeant ion occupancy of the channel to blocker binding may contribute to the overall amine-toxin interaction: 1), R13Q binding decreases the apparent affinity of sodium for the conducting pore by ∼4-fold; and 2), increasing external [Na+] decreases block by DEA at constant voltage. Thus, even though a number of studies suggest that sodium channels are occupied by no more than one ion most of the time, measurable coupling occurs between permeant ions and toxin or amine blockers. Such interactions likely determine, in part, the strength of trans-channel, amine-conotoxin interactions. PMID:18658222

  1. Trans-channel interactions in batrachotoxin-modified skeletal muscle sodium channels: voltage-dependent block by cytoplasmic amines, and the influence of mu-conotoxin GIIIA derivatives and permeant ions.

    Science.gov (United States)

    Pavlov, Evgeny; Britvina, Tatiana; McArthur, Jeff R; Ma, Quanli; Sierralta, Iván; Zamponi, Gerald W; French, Robert J

    2008-11-01

    External mu-conotoxins and internal amine blockers inhibit each other's block of voltage-gated sodium channels. We explore the basis of this interaction by measuring the shifts in voltage-dependence of channel inhibition by internal amines induced by two mu-conotoxin derivatives with different charge distributions and net charges. Charge changes on the toxin were made at residue 13, which is thought to penetrate most deeply into the channel, making it likely to have the strongest individual interaction with an internal charged ligand. When an R13Q or R13E molecule was bound to the channel, the voltage dependence of diethylammonium (DEA)-block shifted toward more depolarized potentials (23 mV for R13Q, and 16 mV for R13E). An electrostatic model of the repulsion between DEA and the toxin simulated these data, with a distance between residue 13 of the mu-conotoxin and the DEA-binding site of approximately 15 A. Surprisingly, for tetrapropylammonium, the shifts were only 9 mV for R13Q, and 7 mV for R13E. The smaller shifts associated with R13E, the toxin with a smaller net charge, are generally consistent with an electrostatic interaction. However, the smaller shifts observed for tetrapropylammonium than for DEA suggest that other factors must be involved. Two observations indicate that the coupling of permeant ion occupancy of the channel to blocker binding may contribute to the overall amine-toxin interaction: 1), R13Q binding decreases the apparent affinity of sodium for the conducting pore by approximately 4-fold; and 2), increasing external [Na(+)] decreases block by DEA at constant voltage. Thus, even though a number of studies suggest that sodium channels are occupied by no more than one ion most of the time, measurable coupling occurs between permeant ions and toxin or amine blockers. Such interactions likely determine, in part, the strength of trans-channel, amine-conotoxin interactions.

  2. Ion channels in plants.

    Science.gov (United States)

    Hedrich, Rainer

    2012-10-01

    Since the first recordings of single potassium channel activities in the plasma membrane of guard cells more than 25 years ago, patch-clamp studies discovered a variety of ion channels in all cell types and plant species under inspection. Their properties differed in a cell type- and cell membrane-dependent manner. Guard cells, for which the existence of plant potassium channels was initially documented, advanced to a versatile model system for studying plant ion channel structure, function, and physiology. Interestingly, one of the first identified potassium-channel genes encoding the Shaker-type channel KAT1 was shown to be highly expressed in guard cells. KAT1-type channels from Arabidopsis thaliana and its homologs from other species were found to encode the K(+)-selective inward rectifiers that had already been recorded in early patch-clamp studies with guard cells. Within the genome era, additional Arabidopsis Shaker-type channels appeared. All nine members of the Arabidopsis Shaker family are localized at the plasma membrane, where they either operate as inward rectifiers, outward rectifiers, weak voltage-dependent channels, or electrically silent, but modulatory subunits. The vacuole membrane, in contrast, harbors a set of two-pore K(+) channels. Just very recently, two plant anion channel families of the SLAC/SLAH and ALMT/QUAC type were identified. SLAC1/SLAH3 and QUAC1 are expressed in guard cells and mediate Slow- and Rapid-type anion currents, respectively, that are involved in volume and turgor regulation. Anion channels in guard cells and other plant cells are key targets within often complex signaling networks. Here, the present knowledge is reviewed for the plant ion channel biology. Special emphasis is drawn to the molecular mechanisms of channel regulation, in the context of model systems and in the light of evolution.

  3. Single amino acids in the carboxyl terminal domain of aquaporin-1 contribute to cGMP-dependent ion channel activation

    Directory of Open Access Journals (Sweden)

    Yool Andrea J

    2003-10-01

    Full Text Available Abstract Background Aquaporin-1 (AQP1 functions as an osmotic water channel and a gated cation channel. Activation of the AQP1 ion conductance by intracellular cGMP was hypothesized to involve the carboxyl (C- terminus, based on amino acid sequence alignments with cyclic-nucleotide-gated channels and cGMP-selective phosphodiesterases. Results Voltage clamp analyses of human AQP1 channels expressed in Xenopus oocytes demonstrated that the nitric oxide donor, sodium nitroprusside (SNP; 3–14 mM activated the ionic conductance response in a dose-dependent manner. Block of soluble guanylate cyclase prevented the response. Enzyme immunoassays confirmed a linear dose-dependent relationship between SNP and the resulting intracellular cGMP levels (up to 1700 fmol cGMP /oocyte at 14 mM SNP. Results here are the first to show that the efficacy of ion channel activation is decreased by mutations of AQP1 at conserved residues in the C-terminal domain (aspartate D237 and lysine K243. Conclusions These data support the idea that the limited amino acid sequence similarities found between three diverse classes of cGMP-binding proteins are significant to the function of AQP1 as a cGMP-gated ion channel, and provide direct evidence for the involvement of the AQP1 C-terminal domain in cGMP-mediated ion channel activation.

  4. Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

    DEFF Research Database (Denmark)

    Grunnet, Morten; Kaufmann, Walter A

    2004-01-01

    Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration...

  5. The Voltage-Sensing Domain of K(v)7.2 Channels as a Molecular Target for Epilepsy-Causing Mutations and Anticonvulsants.

    Science.gov (United States)

    Miceli, Francesco; Soldovieri, Maria Virginia; Iannotti, Fabio Arturo; Barrese, Vincenzo; Ambrosino, Paolo; Martire, Maria; Cilio, Maria Roberta; Taglialatela, Maurizio

    2011-01-01

    Understanding the molecular mechanisms underlying voltage-dependent gating in voltage-gated ion channels (VGICs) has been a major effort over the last decades. In recent years, changes in the gating process have emerged as common denominators for several genetically determined channelopathies affecting heart rhythm (arrhythmias), neuronal excitability (epilepsy, pain), or skeletal muscle contraction (periodic paralysis). Moreover, gating changes appear as the main molecular mechanism by which several natural toxins from a variety of species affect ion channel function. In this work, we describe the pathophysiological and pharmacological relevance of the gating process in voltage-gated K(+) channels encoded by the K(v)7 gene family. After reviewing the current knowledge on the molecular mechanisms and on the structural models of voltage-dependent gating in VGICs, we describe the physiological relevance of these channels, with particular emphasis on those formed by K(v)7.2-K(v)7.5 subunits having a well-established role in controlling neuronal excitability in humans. In fact, genetically determined alterations in K(v)7.2 and K(v)7.3 genes are responsible for benign familial neonatal convulsions, a rare seizure disorder affecting newborns, and the pharmacological activation of K(v)7.2/3 channels can exert antiepileptic activity in humans. Both mutation-triggered channel dysfunction and drug-induced channel activation can occur by impeding or facilitating, respectively, channel sensitivity to membrane voltage and can affect overlapping molecular sites within the voltage-sensing domain of these channels. Thus, understanding the molecular steps involved in voltage-sensing in K(v)7 channels will allow to better define the pathogenesis of rare human epilepsy, and to design innovative pharmacological strategies for the treatment of epilepsies and, possibly, other human diseases characterized by neuronal hyperexcitability.

  6. Intracellular calcium modulation of voltage-gated sodium channels in ventricular myocytes

    NARCIS (Netherlands)

    Casini, Simona; Verkerk, Arie O.; van Borren, Marcel M. G. J.; van Ginneken, Antoni C. G.; Veldkamp, Marieke W.; de Bakker, Jacques M. T.; Tan, Hanno L.

    2009-01-01

    AIMS: Cardiac voltage-gated sodium channels control action potential (AP) upstroke and cell excitability. Intracellular calcium (Ca(i)(2+)) regulates AP properties by modulating various ion channels. Whether Ca(i)(2+) modulates sodium channels in ventricular myocytes, is unresolved. We studied

  7. Ion channel recordings on an injection-molded polymer chip.

    Science.gov (United States)

    Tanzi, Simone; Matteucci, Marco; Christiansen, Thomas Lehrmann; Friis, Søren; Christensen, Mette Thylstrup; Garnaes, Joergen; Wilson, Sandra; Kutchinsky, Jonatan; Taboryski, Rafael

    2013-12-21

    In this paper, we demonstrate recordings of the ion channel activity across the cell membrane in a biological cell by employing the so-called patch clamping technique on an injection-molded polymer microfluidic device. The findings will allow direct recordings of ion channel activity to be made using the cheapest materials and production platform to date and with the potential for very high throughput. The employment of cornered apertures for cell capture allowed the fabrication of devices without through holes and via a scheme comprising master origination by dry etching in a silicon substrate, electroplating in nickel and injection molding of the final part. The most critical device parameters were identified as the length of the patching capillary and the very low surface roughness on the inside of the capillary. The cross-sectional shape of the orifice was found to be less critical, as both rectangular and semicircular profiles seemed to have almost the same ability to form tight seals with cells with negligible leak currents. The devices were functionally tested using human embryonic kidney cells expressing voltage-gated sodium channels (Nav1.7) and benchmarked against a commercial state-of-the-art system for automated ion channel recordings. These experiments considered current-voltage (IV) relationships for activation and inactivation of the Nav1.7 channels and their sensitivity to a local anesthetic, lidocaine. Both IVs and lidocaine dose-response curves obtained from the injection-molded polymer device were in good agreement with data obtained from the commercial system.

  8. Sterol Regulation of Voltage-Gated K+ Channels.

    Science.gov (United States)

    Balajthy, Andras; Hajdu, Peter; Panyi, Gyorgy; Varga, Zoltan

    2017-01-01

    Cholesterol is an essential lipid building block of the cellular plasma membrane. In addition to its structural role, it regulates the fluidity and raft structure of the membrane and influences the course of numerous membrane-linked signaling pathways and the function of transmembrane proteins, including ion channels. This is supported by a vast body of scientific data, which demonstrates the modulation of ion channels with a great variety of ion selectivity, gating, and tissue distribution by changes in membrane cholesterol. Here, we review what is currently known about the modulation of voltage-gated K + (Kv) channels by changes in membrane cholesterol content, considering raft association of the channels, the roles of cholesterol recognition sites, and those of adaptor proteins in cholesterol-Kv channel interactions. We specifically focus on Kv1.3, the dominant K + channel of human T cells. Effects of cholesterol depletion and enrichment and 7-dehydrocholesterol enrichment on Kv1.3 gating are discussed in the context of the immunological synapse and the comparison of the in vitro effects of sterol modifications on Kv1.3 function with ex vivo effects on cells from hypercholesterolemic and Smith-Lemli-Opitz patients. © 2017 Elsevier Inc. All rights reserved.

  9. Global versus local mechanisms of temperature sensing in ion channels.

    Science.gov (United States)

    Arrigoni, Cristina; Minor, Daniel L

    2018-05-01

    Ion channels turn diverse types of inputs, ranging from neurotransmitters to physical forces, into electrical signals. Channel responses to ligands generally rely on binding to discrete sensor domains that are coupled to the portion of the channel responsible for ion permeation. By contrast, sensing physical cues such as voltage, pressure, and temperature arises from more varied mechanisms. Voltage is commonly sensed by a local, domain-based strategy, whereas the predominant paradigm for pressure sensing employs a global response in channel structure to membrane tension changes. Temperature sensing has been the most challenging response to understand and whether discrete sensor domains exist for pressure and temperature has been the subject of much investigation and debate. Recent exciting advances have uncovered discrete sensor modules for pressure and temperature in force-sensitive and thermal-sensitive ion channels, respectively. In particular, characterization of bacterial voltage-gated sodium channel (BacNa V ) thermal responses has identified a coiled-coil thermosensor that controls channel function through a temperature-dependent unfolding event. This coiled-coil thermosensor blueprint recurs in other temperature sensitive ion channels and thermosensitive proteins. Together with the identification of ion channel pressure sensing domains, these examples demonstrate that "local" domain-based solutions for sensing force and temperature exist and highlight the diversity of both global and local strategies that channels use to sense physical inputs. The modular nature of these newly discovered physical signal sensors provides opportunities to engineer novel pressure-sensitive and thermosensitive proteins and raises new questions about how such modular sensors may have evolved and empowered ion channel pores with new sensibilities.

  10. The hitchhiker’s guide to the voltage-gated sodium channel galaxy

    Science.gov (United States)

    2016-01-01

    Eukaryotic voltage-gated sodium (Nav) channels contribute to the rising phase of action potentials and served as an early muse for biophysicists laying the foundation for our current understanding of electrical signaling. Given their central role in electrical excitability, it is not surprising that (a) inherited mutations in genes encoding for Nav channels and their accessory subunits have been linked to excitability disorders in brain, muscle, and heart; and (b) Nav channels are targeted by various drugs and naturally occurring toxins. Although the overall architecture and behavior of these channels are likely to be similar to the more well-studied voltage-gated potassium channels, eukaryotic Nav channels lack structural and functional symmetry, a notable difference that has implications for gating and selectivity. Activation of voltage-sensing modules of the first three domains in Nav channels is sufficient to open the channel pore, whereas movement of the domain IV voltage sensor is correlated with inactivation. Also, structure–function studies of eukaryotic Nav channels show that a set of amino acids in the selectivity filter, referred to as DEKA locus, is essential for Na+ selectivity. Structures of prokaryotic Nav channels have also shed new light on mechanisms of drug block. These structures exhibit lateral fenestrations that are large enough to allow drugs or lipophilic molecules to gain access into the inner vestibule, suggesting that this might be the passage for drug entry into a closed channel. In this Review, we will synthesize our current understanding of Nav channel gating mechanisms, ion selectivity and permeation, and modulation by therapeutics and toxins in light of the new structures of the prokaryotic Nav channels that, for the time being, serve as structural models of their eukaryotic counterparts. PMID:26712848

  11. Voltage-gated ion channels in the axon initial segment of human cortical pyramidal cells and their relationship with chandelier cells.

    Science.gov (United States)

    Inda, Maria Carmen; DeFelipe, Javier; Muñoz, Alberto

    2006-02-21

    The axon initial segment (AIS) of pyramidal cells is a critical region for the generation of action potentials and for the control of pyramidal cell activity. Here we show that Na+ and K+ voltage-gated channels, together with other molecules involved in the localization of ion channels, are distributed asymmetrically in the AIS of pyramidal cells situated in the human temporal neocortex. There is a high density of Na+ channels distributed along the length of the AIS together with the associated proteins spectrin betaIV and ankyrin G. In contrast, Kv1.2 channels are associated with the adhesion molecule Caspr2, and they are mostly localized to the distal region of the AIS. In general, the distal region of the AIS is targeted by the GABAergic axon terminals of chandelier cells, whereas the proximal region is innervated, mostly by other types of GABAergic interneurons. We suggest that this molecular segregation and the consequent regional specialization of the GABAergic input to the AIS of pyramidal cells may have important functional implications for the control of pyramidal cell activity.

  12. [3H]PN200-110 and [3H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes

    International Nuclear Information System (INIS)

    Hamilton, S.L.; Alvarez, R.M.; Fill, M.; Hawkes, M.J.; Brush, K.L.; Schilling, W.P.; Stefani, E.

    1989-01-01

    Skeletal muscle membranes derived either from the tubular (T) network or from the sarcoplasmic reticulum (SR) were characterized with respect to the binding of the dihydropyridine, [ 3 H]PN200-110, and the alkaloid, [ 3 H]ryanodine; polypeptide composition; and ion channel activity. Conditions for optimizing the binding of these radioligands are discussed. A bilayer pulsing technique is described and is used to examine the channels present in these membranes. Fusion of T-tubule membranes into bilayers revealed the presence of chloride channels and dihydropyridine-sensitive calcium channels with three distinct conductances. The dihydropyridine-sensitive channels were further characterized with respect to their voltage dependence. Pulsing experiments indicated that two different populations of dihydropyridine-sensitive channels existed. Fusion of heavy SR vesicles revealed three different ion channels; the putative calcium release channel, a potassium channel, and a chloride channel. Thus, this fractionation procedure provides T-tubules and SR membranes which, with radioligand binding and single channel recording techniques, provide a useful tool to study the characteristics of skeletal muscle ion channels and their possible role in excitation-contraction coupling

  13. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

    Science.gov (United States)

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-09-15

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system.

  14. Conotoxins as Tools to Understand the Physiological Function of Voltage-Gated Calcium (CaV Channels

    Directory of Open Access Journals (Sweden)

    David Ramírez

    2017-10-01

    Full Text Available Voltage-gated calcium (CaV channels are widely expressed and are essential for the completion of multiple physiological processes. Close regulation of their activity by specific inhibitors and agonists become fundamental to understand their role in cellular homeostasis as well as in human tissues and organs. CaV channels are divided into two groups depending on the membrane potential required to activate them: High-voltage activated (HVA, CaV1.1–1.4; CaV2.1–2.3 and Low-voltage activated (LVA, CaV3.1–3.3. HVA channels are highly expressed in brain (neurons, heart, and adrenal medulla (chromaffin cells, among others, and are also classified into subtypes which can be distinguished using pharmacological approaches. Cone snails are marine gastropods that capture their prey by injecting venom, “conopeptides”, which cause paralysis in a few seconds. A subset of conopeptides called conotoxins are relatively small polypeptides, rich in disulfide bonds, that target ion channels, transporters and receptors localized at the neuromuscular system of the animal target. In this review, we describe the structure and properties of conotoxins that selectively block HVA calcium channels. We compare their potency on several HVA channel subtypes, emphasizing neuronal calcium channels. Lastly, we analyze recent advances in the therapeutic use of conotoxins for medical treatments.

  15. Gating of Connexin Channels by transjunctional-voltage: Conformations and models of open and closed states.

    Science.gov (United States)

    Bargiello, Thaddeus A; Oh, Seunghoon; Tang, Qingxiu; Bargiello, Nicholas K; Dowd, Terry L; Kwon, Taekyung

    2018-01-01

    Voltage is an important physiologic regulator of channels formed by the connexin gene family. Connexins are unique among ion channels in that both plasma membrane inserted hemichannels (undocked hemichannels) and intercellular channels (aggregates of which form gap junctions) have important physiological roles. The hemichannel is the fundamental unit of gap junction voltage-gating. Each hemichannel displays two distinct voltage-gating mechanisms that are primarily sensitive to a voltage gradient formed along the length of the channel pore (the transjunctional voltage) rather than sensitivity to the absolute membrane potential (V m or V i-o ). These transjunctional voltage dependent processes have been termed V j - or fast-gating and loop- or slow-gating. Understanding the mechanism of voltage-gating, defined as the sequence of voltage-driven transitions that connect open and closed states, first and foremost requires atomic resolution models of the end states. Although ion channels formed by connexins were among the first to be characterized structurally by electron microscopy and x-ray diffraction in the early 1980's, subsequent progress has been slow. Much of the current understanding of the structure-function relations of connexin channels is based on two crystal structures of Cx26 gap junction channels. Refinement of crystal structure by all-atom molecular dynamics and incorporation of charge changing protein modifications has resulted in an atomic model of the open state that arguably corresponds to the physiologic open state. Obtaining validated atomic models of voltage-dependent closed states is more challenging, as there are currently no methods to solve protein structure while a stable voltage gradient is applied across the length of an oriented channel. It is widely believed that the best approach to solve the atomic structure of a voltage-gated closed ion channel is to apply different but complementary experimental and computational methods and to use

  16. Inactivation of Mechanically Activated Piezo1 Ion Channels Is Determined by the C-Terminal Extracellular Domain and the Inner Pore Helix

    Directory of Open Access Journals (Sweden)

    Jason Wu

    2017-11-01

    Full Text Available Piezo proteins form mechanically activated ion channels that are responsible for our sense of light touch, proprioception, and vascular blood flow. Upon activation by mechanical stimuli, Piezo channels rapidly inactivate in a voltage-dependent manner through an unknown mechanism. Inactivation of Piezo channels is physiologically important, as it modulates overall mechanical sensitivity, gives rise to frequency filtering of repetitive mechanical stimuli, and is itself the target of numerous human disease-related channelopathies that are not well understood mechanistically. Here, we identify the globular C-terminal extracellular domain as a structure that is sufficient to confer the time course of inactivation and a single positively charged lysine residue at the adjacent inner pore helix as being required for its voltage dependence. Our results are consistent with a mechanism for inactivation that is mediated through voltage-dependent conformations of the inner pore helix and allosteric coupling with the C-terminal extracellular domain.

  17. Chloride ions in the pore of glycine and GABA channels shape the time course and voltage dependence of agonist currents

    Science.gov (United States)

    Moroni, Mirko; Biro, Istvan; Giugliano, Michele; Vijayan, Ranjit; Biggin, Philip C.; Beato, Marco; Sivilotti, Lucia G.

    2011-01-01

    In the vertebrate CNS, fast synaptic inhibition is mediated by GABA and glycine receptors. We recently reported that the time course of these synaptic currents is slower when intracellular chloride is high. Here we extend these findings to measure the effects of both extracellular and intracellular chloride on the deactivation of glycine and GABA currents at both negative and positive holding potentials. Currents were elicited by fast agonist application to outside-out patches from HEK293 cells expressing rat glycine or GABA receptors. The slowing effect of high extracellular chloride on current decay was detectable only in low intracellular chloride (4 mM). Our main finding is that glycine and GABA receptors “sense” chloride concentrations because of interactions between the M2 pore-lining domain and the permeating ions. This hypothesis is supported by the observation that the sensitivity of channel gating to intracellular chloride is abolished if the channel is engineered to become cation-selective, or if positive charges in the external pore vestibule are eliminated by mutagenesis. The appropriate interaction between permeating ions and channel pore is also necessary to maintain the channel voltage sensitivity of gating, which prolongs current decay at depolarized potentials. Voltage-dependence is abolished by the same mutations that suppress the effect of intracellular chloride and also by replacing chloride with another permeant ion, thiocyanate. These observations suggest that permeant chloride affects gating by a foot-in-the-door effect, binding to a channel site with asymmetrical access from the intracellular and extracellular sides of the membrane. PMID:21976494

  18. Anti-addiction drug ibogaine inhibits voltage-gated ionic currents: A study to assess the drug's cardiac ion channel profile

    International Nuclear Information System (INIS)

    Koenig, Xaver; Kovar, Michael; Rubi, Lena; Mike, Agnes K.; Lukacs, Peter; Gawali, Vaibhavkumar S.; Todt, Hannes; Hilber, Karlheinz; Sandtner, Walter

    2013-01-01

    The plant alkaloid ibogaine has promising anti-addictive properties. Albeit not licenced as a therapeutic drug, and despite hints that ibogaine may perturb the heart rhythm, this alkaloid is used to treat drug addicts. We have recently reported that ibogaine inhibits human ERG (hERG) potassium channels at concentrations similar to the drugs affinity for several of its known brain targets. Thereby the drug may disturb the heart's electrophysiology. Here, to assess the drug's cardiac ion channel profile in more detail, we studied the effects of ibogaine and its congener 18-Methoxycoronaridine (18-MC) on various cardiac voltage-gated ion channels. We confirmed that heterologously expressed hERG currents are reduced by ibogaine in low micromolar concentrations. Moreover, at higher concentrations, the drug also reduced human Na v 1.5 sodium and Ca v 1.2 calcium currents. Ion currents were as well reduced by 18-MC, yet with diminished potency. Unexpectedly, although blocking hERG channels, ibogaine did not prolong the action potential (AP) in guinea pig cardiomyocytes at low micromolar concentrations. Higher concentrations (≥ 10 μM) even shortened the AP. These findings can be explained by the drug's calcium channel inhibition, which counteracts the AP-prolonging effect generated by hERG blockade. Implementation of ibogaine's inhibitory effects on human ion channels in a computer model of a ventricular cardiomyocyte, on the other hand, suggested that ibogaine does prolong the AP in the human heart. We conclude that therapeutic concentrations of ibogaine have the propensity to prolong the QT interval of the electrocardiogram in humans. In some cases this may lead to cardiac arrhythmias. - Highlights: • We study effects of anti-addiction drug ibogaine on ionic currents in cardiomyocytes. • We assess the cardiac ion channel profile of ibogaine. • Ibogaine inhibits hERG potassium, sodium and calcium channels. • Ibogaine’s effects on ion channels are a potential

  19. Hydrogen bonds as molecular timers for slow inactivation in voltage-gated potassium channels

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Galpin, Jason D; Niciforovic, Ana P

    2013-01-01

    Voltage-gated potassium (Kv) channels enable potassium efflux and membrane repolarization in excitable tissues. Many Kv channels undergo a progressive loss of ion conductance in the presence of a prolonged voltage stimulus, termed slow inactivation, but the atomic determinants that regulate the k...... subunit(s). DOI: http://dx.doi.org/10.7554/eLife.01289.001....

  20. Multiple-channel detection of cellular activities by ion-sensitive transistors

    Science.gov (United States)

    Machida, Satoru; Shimada, Hideto; Motoyama, Yumi

    2018-04-01

    An ion-sensitive field-effect transistor to record cellular activities was demonstrated. This field-effect transistor (bio transistor) includes cultured cells on the gate insulator instead of gate electrode. The bio transistor converts a change in potential underneath the cells into variation of the drain current when ion channels open. The bio transistor has high detection sensitivity to even minute variations in potential utilizing a subthreshold swing region. To open ion channels, a reagent solution (acetylcholine) was added to a human-originating cell cultured on the bio transistor. The drain current was successfully decreased with the addition of acetylcholine. Moreover, we attempted to detect the opening of ion channels using a multiple-channel measurement circuit containing several bio transistors. As a consequence, the drain current distinctly decreased only after the addition of acetylcholine. We confirmed that this measurement system including bio transistors enables to observation of cellular activities sensitively and simultaneously.

  1. Expression, purification and functional reconstitution of slack sodium-activated potassium channels.

    Science.gov (United States)

    Yan, Yangyang; Yang, Youshan; Bian, Shumin; Sigworth, Fred J

    2012-11-01

    The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na(+)-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

  2. History-dependent dynamics in a generic model of ion channels - an analytic study

    Directory of Open Access Journals (Sweden)

    Daniel Soudry

    2010-04-01

    Full Text Available Recent experiments have demonstrated that the timescale of adaptation of single neurons and ion channel populations to stimuli slows down as the length of stimulation increases; in fact, no upper bound on temporal time-scales seems to exist in such systems. Furthermore, patch clamp experiments on single ion channels have hinted at the existence of large, mostly unobservable, inactivation state spaces within a single ion channel. This raises the question of the relation between this multitude of inactivation states and the observed behavior. In this work we propose a minimal model for ion channel dynamics which does not assume any specific structure of the inactivation state space. The model is simple enough to render an analytical study possible. This leads to a clear and concise explanation of the experimentally observed exponential history-dependent relaxation in sodium channels in a voltage clamp setting, and shows that their recovery rate from slow inactivation must be voltage dependent. Furthermore, we predict that history-dependent relaxation cannot be created by overly sparse spiking activity. While the model was created with ion channel populations in mind, its simplicity and genericalness render it a good starting point for modeling similar effects in other systems, and for scaling up to higher levels such as single neurons which are also known to exhibit multiple time scales.

  3. Functional role of voltage gated Ca2+ channels in heart automaticity

    Directory of Open Access Journals (Sweden)

    Pietro eMesirca

    2015-02-01

    Full Text Available Pacemaker activity of automatic cardiac myocytes controls the heartbeat in everyday life. Cardiac automaticity is under the control of several neurotransmitters and hormones and is constantly regulated by the autonomic nervous system to match the physiological needs of the organism. Several classes of ion channels and proteins involved in intracellular Ca2+ dynamics contribute to pacemaker activity. The functional role of voltage-gated calcium channels (VGCCs in heart automaticity and impulse conduction has been matter of debate for 30 years. However, growing evidence shows that VGCCs are important regulators of the pacemaker mechanisms and play also a major role in atrio-ventricular impulse conduction. Incidentally, studies performed in genetically modified mice lacking L-type Cav1.3 (Cav1.3-/- or T-type Cav3.1 (Cav3.1-/- channels show that genetic inactivation of these channels strongly impacts pacemaking. In cardiac pacemaker cells, VGCCs activate at negative voltages at the beginning of the diastolic depolarization and importantly contribute to this phase by supplying inward current. Loss-of-function of these channels also impairs atrio-ventricular conduction. Furthermore, inactivation of Cav1.3 channels promotes also atrial fibrillation and flutter in knockout mice suggesting that these channels can play a role in stabilizing atrial rhythm. Genomic analysis demonstrated that Cav1.3 and Cav3.1 channels are widely expressed in pacemaker tissue of mice, rabbits and humans. Importantly, human diseases of pacemaker activity such as congenital bradycardia and heart block have been attributed to loss-of-function of Cav1.3 and Cav3.1 channels. In this article, we will review the current knowledge on the role of VGCCs in the generation and regulation of heart rate and rhythm. We will discuss also how loss of Ca2+ entry through VGCCs could influence intracellular Ca2+ handling and promote atrial arrhythmias.

  4. Electromagnetic fields (UHF) increase voltage sensitivity of membrane ion channels; possible indication of cell phone effect on living cells.

    Science.gov (United States)

    Ketabi, N; Mobasheri, H; Faraji-Dana, R

    2015-03-01

    The effects of ultra high frequency (UHF) nonionizing electromagnetic fields (EMF) on the channel activities of nanopore forming protein, OmpF porin, were investigated. The voltage clamp technique was used to study the single channel activity of the pore in an artificial bilayer in the presence and absence of the electromagnetic fields at 910 to 990 MHz in real time. Channel activity patterns were used to address the effect of EMF on the dynamic, arrangement and dielectric properties of water molecules, as well as on the hydration state and arrangements of side chains lining the channel barrel. Based on the varied voltage sensitivity of the channel at different temperatures in the presence and absence of EMF, the amount of energy transferred to nano-environments of accessible groups was estimated to address the possible thermal effects of EMF. Our results show that the effects of EMF on channel activities are frequency dependent, with a maximum effect at 930 MHz. The frequency of channel gating and the voltage sensitivity is increased when the channel is exposed to EMF, while its conductance remains unchanged at all frequencies applied. We have not identified any changes in the capacitance and permeability of membrane in the presence of EMF. The effect of the EMF irradiated by cell phones is measured by Specific Absorption Rate (SAR) in artificial model of human head, Phantom. Thus, current approach applied to biological molecules and electrolytes might be considered as complement to evaluate safety of irradiating sources on biological matter at molecular level.

  5. Functional modifications of acid-sensing ion channels by ligand-gated chloride channels.

    Directory of Open Access Journals (Sweden)

    Xuanmao Chen

    Full Text Available Together, acid-sensing ion channels (ASICs and epithelial sodium channels (ENaC constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR. Here we show that ASICs were reversibly inhibited by activation of GABA(A receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABA(A receptor-mediated currents. Moreover, activation of the GABA(A receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABA(A receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABA(A receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABA(A receptors, also modified ASICs in spinal neurons. We conclude that GABA(A receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels.

  6. Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels.

    Science.gov (United States)

    Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal; Arrigoni, Cristina; Wong, Stephanie; Nurva, Shailika Reddy; Loussouarn, Gildas; Minor, Daniel L

    2014-01-23

    Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail "neck", are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the "outer ion" site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of CaV high-affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. © 2013. Published by Elsevier Ltd. All rights reserved.

  7. Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains.

    Science.gov (United States)

    de la Peña, Pilar; Domínguez, Pedro; Barros, Francisco

    2018-03-01

    Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4-S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4-S5 linker induces an increasing negative shift in activation voltage dependence, a reduced z g value and a more negative ΔG 0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4-S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4-S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.

  8. Nonsensing residues in S3-S4 linker's C terminus affect the voltage sensor set point in K+ channels.

    Science.gov (United States)

    Carvalho-de-Souza, Joao L; Bezanilla, Francisco

    2018-02-05

    Voltage sensitivity in ion channels is a function of highly conserved arginine residues in their voltage-sensing domains (VSDs), but this conservation does not explain the diversity in voltage dependence among different K + channels. Here we study the non-voltage-sensing residues 353 to 361 in Shaker K + channels and find that residues 358 and 361 strongly modulate the voltage dependence of the channel. We mutate these two residues into all possible remaining amino acids (AAs) and obtain Q-V and G-V curves. We introduced the nonconducting W434F mutation to record sensing currents in all mutants except L361R, which requires K + depletion because it is affected by W434F. By fitting Q-Vs with a sequential three-state model for two voltage dependence-related parameters ( V 0 , the voltage-dependent transition from the resting to intermediate state and V 1 , from the latter to the active state) and G-Vs with a two-state model for the voltage dependence of the pore domain parameter ( V 1/2 ), Spearman's coefficients denoting variable relationships with hydrophobicity, available area, length, width, and volume of the AAs in 358 and 361 positions could be calculated. We find that mutations in residue 358 shift Q-Vs and G-Vs along the voltage axis by affecting V 0 , V 1 , and V 1/2 according to the hydrophobicity of the AA. Mutations in residue 361 also shift both curves, but V 0 is affected by the hydrophobicity of the AA in position 361, whereas V 1 and V 1/2 are affected by size-related AA indices. Small-to-tiny AAs have opposite effects on V 1 and V 1/2 in position 358 compared with 361. We hypothesize possible coordination points in the protein that residues 358 and 361 would temporarily and differently interact with in an intermediate state of VSD activation. Our data contribute to the accumulating knowledge of voltage-dependent ion channel activation by adding functional information about the effects of so-called non-voltage-sensing residues on VSD dynamics. © 2018

  9. Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

    Science.gov (United States)

    Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

    2012-01-01

    The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

  10. Phosphorylation of rat brain purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal kinase-3 modifies open-channel noise.

    Science.gov (United States)

    Gupta, Rajeev

    2017-09-02

    The drift kinetic energy of ionic flow through single ion channels cause vibrations of the pore walls which are observed as open-state current fluctuations (open-channel noise) during single-channel recordings. Vibration of the pore wall leads to transitions among different conformational sub-states of the channel protein in the open-state. Open-channel noise analysis can provide important information about the different conformational sub-state transitions and how biochemical modifications of ion channels would affect their transport properties. It has been shown that c-Jun N-terminal kinase-3 (JNK3) becomes activated by phosphorylation in various neurodegenerative diseases and phosphorylates outer mitochondrion associated proteins leading to neuronal apoptosis. In our earlier work, JNK3 has been reported to phosphorylate purified rat brain mitochondrial voltage-dependent anion channel (VDAC) in vitro and modify its conductance and opening probability. In this article we have compared the open-state noise profile of the native and the JNK3 phosphorylated VDAC using Power Spectral Density vs frequency plots. Power spectral density analysis of open-state noise indicated power law with average slope value α ≈1 for native VDAC at both positive and negative voltage whereas average α value open-state noise arises due to coupling of ionic transport and conformational sub-states transitions in open-state and this coupling is perturbed as a result of channel phosphorylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Porous Materials to Support Bilayer Lipid Membranes for Ion Channel Biosensors

    Directory of Open Access Journals (Sweden)

    Thai Phung

    2011-01-01

    Full Text Available To identify materials suitable as membrane supports for ion channel biosensors, six filter materials of varying hydrophobicity, tortuosity, and thickness were examined for their ability to support bilayer lipid membranes as determined by electrical impedance spectroscopy. Bilayers supported by hydrophobic materials (PTFE, polycarbonate, nylon, and silanised silver had optimal resistance (14–19 GΩ and capacitance (0.8–1.6 μF values whereas those with low hydrophobicity did not form BLMs (PVDF or were short-lived (unsilanised silver. The ability of ion channels to function in BLMs was assessed using a method recently reported to improve the efficiency of proteoliposome incorporation into PTFE-supported bilayers. Voltage-gated sodium channel activation by veratridine and inhibition by saxitoxin showed activity for PTFE, nylon, and silanised silver, but not polycarbonate. Bilayers on thicker, more tortuous, and hydrophobic materials produced higher current levels. Bilayers that self-assembled on PTFE filters were the longest lived and produced the most channel activity using this method.

  12. Mining Protein Evolution for Insights into Mechanisms of Voltage-Dependent Sodium Channel Auxiliary Subunits.

    Science.gov (United States)

    Molinarolo, Steven; Granata, Daniele; Carnevale, Vincenzo; Ahern, Christopher A

    2018-02-21

    Voltage-gated sodium channel (VGSC) beta (β) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC β-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC β1-β4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC β-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern β-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and β eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for β-subunit interactions with voltage-sensor containing ion channels and membrane proteins.

  13. Structure-function of proteins interacting with the alpha1 pore-forming subunit of high voltage-activated calcium channel

    Directory of Open Access Journals (Sweden)

    Alan eNeely

    2014-06-01

    Full Text Available Openings of high-voltage-activated calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, high-voltage-activated calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1 associated with four additional polypeptide chains β, α2, δ and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of high-voltage-activated calcium channels.

  14. Low voltage-activated calcium channels gate transmitter release at the dorsal root ganglion sandwich synapse.

    Science.gov (United States)

    Rozanski, Gabriela M; Nath, Arup R; Adams, Michael E; Stanley, Elise F

    2013-11-15

    A subpopulation of dorsal root ganglion (DRG) neurons are intimately attached in pairs and separated solely by thin satellite glial cell membrane septa. Stimulation of one neuron leads to transglial activation of its pair by a bi-, purinergic/glutamatergic synaptic pathway, a transmission mechanism that we term sandwich synapse (SS) transmission. Release of ATP from the stimulated neuron can be attributed to a classical mechanism involving Ca(2+) entry via voltage-gated calcium channels (CaV) but via an unknown channel type. Specific blockers and toxins ruled out CaV1, 2.1 and 2.2. Transmission was, however, blocked by a moderate depolarization (-50 mV) or low-concentration Ni(2+) (0.1 mM). Transmission persisted using a voltage pulse to -40 mV from a holding potential of -80 mV, confirming the involvement of a low voltage-activated channel type and limiting the candidate channel type to either CaV3.2 or a subpopulation of inactivation- and Ni(2+)-sensitive CaV2.3 channels. Resistance of the neuron calcium current and SS transmission to SNX482 argue against the latter. Hence, we conclude that inter-somatic transmission at the DRG SS is gated by CaV3.2 type calcium channels. The use of CaV3 family channels to gate transmission has important implications for the biological function of the DRG SS as information transfer would be predicted to occur not only in response to action potentials but also to sub-threshold membrane voltage oscillations. Thus, the SS synapse may serve as a homeostatic signalling mechanism between select neurons in the DRG and could play a role in abnormal sensation such as neuropathic pain.

  15. Dysfunctional Hyperpolarization-Activated Cyclic Nucleotide-gated Ion Channels in Cardiac Diseases

    Directory of Open Access Journals (Sweden)

    Xiaoqi Zhao

    Full Text Available Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN channels are reverse voltage-dependent, and their activation depends on the hyperpolarization of the membrane and may be directly or indirectly regulated by the cyclic adenosine monophosphate (cAMP or other signal-transduction cascades. The distribution, quantity and activation states of HCN channels differ in tissues throughout the body. Evidence exhibits that HCN channels play critical roles in the generation and conduction of the electrical impulse and the physiopathological process of some cardiac diseases. They may constitute promising drug targets in the treatment of these cardiac diseases. Pharmacological treatment targeting HCN channels is of benefit to these cardiac conditions.

  16. Nanosecond pulsed electric fields depolarize transmembrane potential via voltage-gated K+, Ca2+ and TRPM8 channels in U87 glioblastoma cells.

    Science.gov (United States)

    Burke, Ryan C; Bardet, Sylvia M; Carr, Lynn; Romanenko, Sergii; Arnaud-Cormos, Delia; Leveque, Philippe; O'Connor, Rodney P

    2017-10-01

    Nanosecond pulsed electric fields (nsPEFs) have a variety of applications in the biomedical and biotechnology industries. Cancer treatment has been at the forefront of investigations thus far as nsPEFs permeabilize cellular and intracellular membranes leading to apoptosis and necrosis. nsPEFs may also influence ion channel gating and have the potential to modulate cell physiology without poration of the membrane. This phenomenon was explored using live cell imaging and a sensitive fluorescent probe of transmembrane voltage in the human glioblastoma cell line, U87 MG, known to express a number of voltage-gated ion channels. The specific ion channels involved in the nsPEF response were screened using a membrane potential imaging approach and a combination of pharmacological antagonists and ion substitutions. It was found that a single 10ns pulsed electric field of 34kV/cm depolarizes the transmembrane potential of cells by acting on specific voltage-sensitive ion channels; namely the voltage and Ca2 + gated BK potassium channel, L- and T-type calcium channels, and the TRPM8 transient receptor potential channel. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Anti-addiction drug ibogaine inhibits voltage-gated ionic currents: A study to assess the drug's cardiac ion channel profile

    Energy Technology Data Exchange (ETDEWEB)

    Koenig, Xaver; Kovar, Michael; Rubi, Lena; Mike, Agnes K.; Lukacs, Peter; Gawali, Vaibhavkumar S.; Todt, Hannes [Center for Physiology and Pharmacology, Department of Neurophysiology and -pharmacology, Medical University of Vienna, 1090 Vienna (Austria); Hilber, Karlheinz, E-mail: karlheinz.hilber@meduniwien.ac.at [Center for Physiology and Pharmacology, Department of Neurophysiology and -pharmacology, Medical University of Vienna, 1090 Vienna (Austria); Sandtner, Walter [Center for Physiology and Pharmacology, Institute of Pharmacology, Medical University of Vienna, 1090 Vienna (Austria)

    2013-12-01

    The plant alkaloid ibogaine has promising anti-addictive properties. Albeit not licenced as a therapeutic drug, and despite hints that ibogaine may perturb the heart rhythm, this alkaloid is used to treat drug addicts. We have recently reported that ibogaine inhibits human ERG (hERG) potassium channels at concentrations similar to the drugs affinity for several of its known brain targets. Thereby the drug may disturb the heart's electrophysiology. Here, to assess the drug's cardiac ion channel profile in more detail, we studied the effects of ibogaine and its congener 18-Methoxycoronaridine (18-MC) on various cardiac voltage-gated ion channels. We confirmed that heterologously expressed hERG currents are reduced by ibogaine in low micromolar concentrations. Moreover, at higher concentrations, the drug also reduced human Na{sub v}1.5 sodium and Ca{sub v}1.2 calcium currents. Ion currents were as well reduced by 18-MC, yet with diminished potency. Unexpectedly, although blocking hERG channels, ibogaine did not prolong the action potential (AP) in guinea pig cardiomyocytes at low micromolar concentrations. Higher concentrations (≥ 10 μM) even shortened the AP. These findings can be explained by the drug's calcium channel inhibition, which counteracts the AP-prolonging effect generated by hERG blockade. Implementation of ibogaine's inhibitory effects on human ion channels in a computer model of a ventricular cardiomyocyte, on the other hand, suggested that ibogaine does prolong the AP in the human heart. We conclude that therapeutic concentrations of ibogaine have the propensity to prolong the QT interval of the electrocardiogram in humans. In some cases this may lead to cardiac arrhythmias. - Highlights: • We study effects of anti-addiction drug ibogaine on ionic currents in cardiomyocytes. • We assess the cardiac ion channel profile of ibogaine. • Ibogaine inhibits hERG potassium, sodium and calcium channels. • Ibogaine’s effects on

  18. RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum.

    Science.gov (United States)

    Abd El Halim, Hesham M; Alshukri, Baida M H; Ahmad, Munawar S; Nakasu, Erich Y T; Awwad, Mohammed H; Salama, Elham M; Gatehouse, Angharad M R; Edwards, Martin G

    2016-07-14

    The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30-60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control.

  19. Ion channeling

    International Nuclear Information System (INIS)

    Erramli, H.; Blondiaux, G.

    1994-01-01

    Channeling phenomenon was predicted, many years ago, by stark. The first channeling experiments were performed in 1963 by Davies and his coworkers. Parallely Robinson and Oen have investigated this process by simulating trajectories of ions in monocrystals. This technique has been combined with many methods like Rutherford Backscattering Spectrometry (R.B.S.), Particles Induced X-rays Emission (P.I.X.E) and online Nuclear Reaction (N.R.A.) to localize trace elements in the crystal or to determine crystalline quality. To use channeling for material characterization we need data about the stopping power of the incident particle in the channeled direction. The ratios of channeled to random stopping powers of silicon for irradiation in the direction have been investigated and compared to the available theoretical results. We describe few applications of ion channeling in the field of materials characterization. Special attention is given to ion channeling combined with Charged Particle Activation Analysis (C.P.A.A.) for studying the behaviour of oxygen atoms in Czochralski silicon lattices under the influence of internal gettering and in different gaseous atmospheres. Association between ion channeling and C.P.A.A was also utilised for studying the influence of the growing conditions on concentration and position of carbon atoms at trace levels in the MOVPE Ga sub (1-x) Al sub x lattice. 6 figs., 1 tab., 32 refs. (author)

  20. Structure of Voltage-gated Two-pore Channel TPC1 from Arabidopsis thaliana

    Science.gov (United States)

    Guo, Jiangtao; Zeng, Weizhong; Chen, Qingfeng; Lee, Changkeun; Chen, Liping; Yang, Yi; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

    2015-01-01

    Two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here, we present the first crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca2+. Ca2+ binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices (IS6 helices) from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain (VSD2) whose conformational changes are coupled to the pair of inner helices (IIS6 helices) from the second 6-TM domains. Luminal Ca2+ or Ba2+ can modulate voltage activation by stabilizing VSD2 in the resting state and shifts voltage activation towards more positive potentials. Our Ba2+ bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels. PMID:26689363

  1. New insights on the voltage dependence of the KCa3.1 channel block by internal TBA.

    Science.gov (United States)

    Banderali, Umberto; Klein, Hélène; Garneau, Line; Simoes, Manuel; Parent, Lucie; Sauvé, Rémy

    2004-10-01

    We present in this work a structural model of the open IKCa (KCa3.1) channel derived by homology modeling from the MthK channel structure, and used this model to compute the transmembrane potential profile along the channel pore. This analysis showed that the selectivity filter and the region extending from the channel inner cavity to the internal medium should respectively account for 81% and 16% of the transmembrane potential difference. We found however that the voltage dependence of the IKCa block by the quaternary ammonium ion TBA applied internally is compatible with an apparent electrical distance delta of 0.49 +/- 0.02 (n = 6) for negative potentials. To reconcile this observation with the electrostatic potential profile predicted for the channel pore, we modeled the IKCa block by TBA assuming that the voltage dependence of the block is governed by both the difference in potential between the channel cavity and the internal medium, and the potential profile along the selectivity filter region through an effect on the filter ion occupancy states. The resulting model predicts that delta should be voltage dependent, being larger at negative than positive potentials. The model also indicates that raising the internal K+ concentration should decrease the value of delta measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect delta for concentrations >50 mM. All these predictions are born out by our current experimental results. Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter. Globally, these results support a model whereby the voltage dependence of the TBA block in IKCa is mainly governed by the voltage dependence of the ion occupancy states of the selectivity filter.

  2. Voltage and pH sensing by the voltage-gated proton channel, HV1.

    Science.gov (United States)

    DeCoursey, Thomas E

    2018-04-01

    Voltage-gated proton channels are unique ion channels, membrane proteins that allow protons but no other ions to cross cell membranes. They are found in diverse species, from unicellular marine life to humans. In all cells, their function requires that they open and conduct current only under certain conditions, typically when the electrochemical gradient for protons is outwards. Consequently, these proteins behave like rectifiers, conducting protons out of cells. Their activity has electrical consequences and also changes the pH on both sides of the membrane. Here we summarize what is known about the way these proteins sense the membrane potential and the pH inside and outside the cell. Currently, it is hypothesized that membrane potential is sensed by permanently charged arginines (with very high p K a ) within the protein, which results in parts of the protein moving to produce a conduction pathway. The mechanism of pH sensing appears to involve titratable side chains of particular amino acids. For this purpose their p K a needs to be within the operational pH range. We propose a 'counter-charge' model for pH sensing in which electrostatic interactions within the protein are selectively disrupted by protonation of internally or externally accessible groups. © 2018 The Author.

  3. Voltage and pH sensing by the voltage-gated proton channel, HV1

    Science.gov (United States)

    2018-01-01

    Voltage-gated proton channels are unique ion channels, membrane proteins that allow protons but no other ions to cross cell membranes. They are found in diverse species, from unicellular marine life to humans. In all cells, their function requires that they open and conduct current only under certain conditions, typically when the electrochemical gradient for protons is outwards. Consequently, these proteins behave like rectifiers, conducting protons out of cells. Their activity has electrical consequences and also changes the pH on both sides of the membrane. Here we summarize what is known about the way these proteins sense the membrane potential and the pH inside and outside the cell. Currently, it is hypothesized that membrane potential is sensed by permanently charged arginines (with very high pKa) within the protein, which results in parts of the protein moving to produce a conduction pathway. The mechanism of pH sensing appears to involve titratable side chains of particular amino acids. For this purpose their pKa needs to be within the operational pH range. We propose a ‘counter-charge’ model for pH sensing in which electrostatic interactions within the protein are selectively disrupted by protonation of internally or externally accessible groups. PMID:29643227

  4. Activation of CRH receptor type 1 expressed on glutamatergic neurons increases excitability of CA1 pyramidal neurons by the modulation of voltage-gated ion channels

    Directory of Open Access Journals (Sweden)

    Stephan eKratzer

    2013-07-01

    Full Text Available Corticotropin-releasing hormone (CRH plays an important role in a substantial number of patients with stress-related mental disorders, such as anxiety disorders and depression. CRH has been shown to increase neuronal excitability in the hippocampus, but the underlying mechanisms are poorly understood. The effects of CRH on neuronal excitability were investigated in acute hippocampal brain slices. Population spikes (PS and field excitatory postsynaptic potentials (fEPSP were evoked by stimulating Schaffer-collaterals and recorded simultaneously from the somatic and dendritic region of CA1 pyramidal neurons. CRH was found to increase PS amplitudes (mean  Standard error of the mean; 231.8  31.2% of control; n=10 while neither affecting fEPSPs (104.3 ± 4.2%; n=10 nor long-term potentiation (LTP. However, when Schaffer-collaterals were excited via action potentials (APs generated by stimulation of CA3 pyramidal neurons, CRH increased fEPSP amplitudes (119.8 ± 3.6%; n=8 and the magnitude of LTP in the CA1 region. Experiments in slices from transgenic mice revealed that the effect on PS amplitude is mediated exclusively by CRH receptor 1 (CRHR1 expressed on glutamatergic neurons. The effects of CRH on PS were dependent on phosphatase-2B, L- and T-type calcium channels and voltage-gated potassium channels but independent on intracellular Ca2+-elevation. In patch-clamp experiments, CRH increased the frequency and decay times of APs and decreased currents through A-type and delayed-rectifier potassium channels. These results suggest that CRH does not affect synaptic transmission per se, but modulates voltage-gated ion currents important for the generation of APs and hence elevates by this route overall neuronal activity.

  5. Gating transitions in the selectivity filter region of a sodium channel are coupled to the domain IV voltage sensor.

    Science.gov (United States)

    Capes, Deborah L; Arcisio-Miranda, Manoel; Jarecki, Brian W; French, Robert J; Chanda, Baron

    2012-02-14

    Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltage-sensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels.

  6. Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel.

    Science.gov (United States)

    Heusser, Stephanie A; Yoluk, Özge; Klement, Göran; Riederer, Erika A; Lindahl, Erik; Howard, Rebecca J

    2016-07-01

    The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate

  7. C-terminus-mediated voltage gating of Arabidopsis guard cell anion channel QUAC1.

    Science.gov (United States)

    Mumm, Patrick; Imes, Dennis; Martinoia, Enrico; Al-Rasheid, Khaled A S; Geiger, Dietmar; Marten, Irene; Hedrich, Rainer

    2013-09-01

    Anion transporters in plants play a fundamental role in volume regulation and signaling. Currently, two plasma membrane-located anion channel families—SLAC/SLAH and ALMT—are known. Among the ALMT family, the root-expressed ALuminium-activated Malate Transporter 1 was identified by comparison of aluminum-tolerant and Al(3+)-sensitive wheat cultivars and was subsequently shown to mediate voltage-independent malate currents. In contrast, ALMT12/QUAC1 (QUickly activating Anion Channel1) is expressed in guard cells transporting malate in an Al(3+)-insensitive and highly voltage-dependent manner. So far, no information is available about the structure and mechanism of voltage-dependent gating with the QUAC1 channel protein. Here, we analyzed gating of QUAC1-type currents in the plasma membrane of guard cells and QUAC1-expressing oocytes revealing similar voltage dependencies and activation–deactivation kinetics. In the heterologous expression system, QUAC1 was electrophysiologically characterized at increasing extra- and intracellular malate concentrations. Thereby, malate additively stimulated the voltage-dependent QUAC1 activity. In search of structural determinants of the gating process, we could not identify transmembrane domains common for voltage-sensitive channels. However, site-directed mutations and deletions at the C-terminus of QUAC1 resulted in altered voltage-dependent channel activity. Interestingly, the replacement of a single glutamate residue, which is conserved in ALMT channels from different clades, by an alanine disrupted QUAC1 activity. Together with C- and N-terminal tagging, these results indicate that the cytosolic C-terminus is involved in the voltage-dependent gating mechanism of QUAC1.

  8. Disulfide mapping the voltage-sensing mechanism of a voltage-dependent potassium channel.

    Science.gov (United States)

    Nozaki, Tomohiro; Ozawa, Shin-Ichiro; Harada, Hitomi; Kimura, Tomomi; Osawa, Masanori; Shimada, Ichio

    2016-11-17

    Voltage-dependent potassium (Kv) channels allow for the selective permeability of potassium ions in a membrane potential dependent manner, playing crucial roles in neurotransmission and muscle contraction. Kv channel is a tetramer, in which each subunit possesses a voltage-sensing domain (VSD) and a pore domain (PD). Although several lines of evidence indicated that membrane depolarization is sensed as the movement of helix S4 of the VSD, the detailed voltage-sensing mechanism remained elusive, due to the difficulty of structural analyses at resting potential. In this study, we conducted a comprehensive disulfide locking analysis of the VSD using 36 double Cys mutants, in order to identify the proximal residue pairs of the VSD in the presence or absence of a membrane potential. An intramolecular SS-bond was formed between 6 Cys pairs under both polarized and depolarized environment, and one pair only under depolarized environment. The multiple conformations captured by the SS-bond can be divided by two states, up and down, where S4 lies on the extracellular and intracellular sides of the membrane, respectively, with axial rotation of 180°. The transition between these two states is caused by the S4 translocation of 12 Å, enabling allosteric regulation of the gating at the PD.

  9. Ion Channels of Pituitary Gonadotrophs and Their Roles in Signaling and Secretion

    Directory of Open Access Journals (Sweden)

    Stanko S. Stojilkovic

    2017-06-01

    Full Text Available Gonadotrophs are basophilic cells of the anterior pituitary gland specialized to secrete gonadotropins in response to elevation in intracellular calcium concentration. These cells fire action potentials (APs spontaneously, coupled with voltage-gated calcium influx of insufficient amplitude to trigger gonadotropin release. The spontaneous excitability of gonadotrophs reflects the expression of voltage-gated sodium, calcium, potassium, non-selective cation-conducting, and chloride channels at their plasma membrane (PM. These cells also express the hyperpolarization-activated and cyclic nucleotide-gated cation channels at the PM, as well as GABAA, nicotinic, and purinergic P2X channels gated by γ-aminobutyric acid (GABA, acetylcholine (ACh, and ATP, respectively. Activation of these channels leads to initiation or amplification of the pacemaking activity, facilitation of calcium influx, and activation of the exocytic pathway. Gonadotrophs also express calcium-conducting channels at the endoplasmic reticulum membranes gated by inositol trisphosphate and intracellular calcium. These channels are activated potently by hypothalamic gonadotropin-releasing hormone (GnRH and less potently by several paracrine calcium-mobilizing agonists, including pituitary adenylate cyclase-activating peptides, endothelins, ACh, vasopressin, and oxytocin. Activation of these channels causes oscillatory calcium release and a rapid gonadotropin release, accompanied with a shift from tonic firing of single APs to periodic bursting type of electrical activity, which accounts for a sustained calcium signaling and gonadotropin secretion. This review summarizes our current understanding of ion channels as signaling molecules in gonadotrophs, the role of GnRH and paracrine agonists in their gating, and the cross talk among channels.

  10. Voltage gated potassium channels expressed in Xenopus laevis(AMPHIBIA oocytes

    Directory of Open Access Journals (Sweden)

    Hedna Chaves

    2003-01-01

    Full Text Available Heterologous expression has been an important tool for structural and functionalcharacterization of proteins. The study of biophysical properties of ion channels,pumps and transporters has been possible thanks to their expression in Xenopuslaevisoocytes. Here we report the expression of two voltage gated channels, Kv1.1and Shaker, in X. laevisoocytes using a method for oocyte extraction, isolation, cul-ture, and microinjection adapted to the latitude and altitude conditions of Bogotá,Colombia.

  11. Contribution of S4 segments and S4-S5 linkers to the low-voltage activation properties of T-type CaV3.3 channels.

    Directory of Open Access Journals (Sweden)

    Ana Laura Sanchez-Sandoval

    Full Text Available Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA and low-voltage (LVA activated calcium channels is around 30-40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers.

  12. Contribution of S4 segments and S4-S5 linkers to the low-voltage activation properties of T-type CaV3.3 channels

    Science.gov (United States)

    Sanchez-Sandoval, Ana Laura; Herrera Carrillo, Zazil; Díaz Velásquez, Clara Estela; Delgadillo, Dulce María; Rivera, Heriberto Manuel

    2018-01-01

    Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA) and low-voltage (LVA) activated calcium channels is around 30–40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C) induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers. PMID:29474447

  13. Contribution of S4 segments and S4-S5 linkers to the low-voltage activation properties of T-type CaV3.3 channels.

    Science.gov (United States)

    Sanchez-Sandoval, Ana Laura; Herrera Carrillo, Zazil; Díaz Velásquez, Clara Estela; Delgadillo, Dulce María; Rivera, Heriberto Manuel; Gomora, Juan Carlos

    2018-01-01

    Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA) and low-voltage (LVA) activated calcium channels is around 30-40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C) induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers.

  14. KCNQ1 channel modulation by KCNE proteins via the voltage-sensing domain.

    Science.gov (United States)

    Nakajo, Koichi; Kubo, Yoshihiro

    2015-06-15

    The gating of the KCNQ1 potassium channel is drastically regulated by auxiliary subunit KCNE proteins. KCNE1, for example, slows the activation kinetics of KCNQ1 by two orders of magnitude. Like other voltage-gated ion channels, the opening of KCNQ1 is regulated by the voltage-sensing domain (VSD; S1-S4 segments). Although it has been known that KCNE proteins interact with KCNQ1 via the pore domain, some recent reports suggest that the VSD movement may be altered by KCNE. The altered VSD movement of KCNQ1 by KCNE proteins has been examined by site-directed mutagenesis, the scanning cysteine accessibility method (SCAM), voltage clamp fluorometry (VCF) and gating charge measurements. These accumulated data support the idea that KCNE proteins interact with the VSDs of KCNQ1 and modulate the gating of the KCNQ1 channel. In this review, we will summarize recent findings and current views of the KCNQ1 modulation by KCNE via the VSD. In this context, we discuss our recent findings that KCNE1 may alter physical interactions between the S4 segment (VSD) and the S5 segment (pore domain) of KCNQ1. Based on these findings from ourselves and others, we propose a hypothetical mechanism for how KCNE1 binding alters the VSD movement and the gating of the channel. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  15. Gabapentin Modulates HCN4 Channel Voltage-Dependence

    Directory of Open Access Journals (Sweden)

    Han-Shen Tae

    2017-08-01

    Full Text Available Gabapentin (GBP is widely used to treat epilepsy and neuropathic pain. There is evidence that GBP can act on hyperpolarization-activated cation (HCN channel-mediated Ih in brain slice experiments. However, evidence showing that GBP directly modulates HCN channels is lacking. The effect of GBP was tested using two-electrode voltage clamp recordings from human HCN1, HCN2, and HCN4 channels expressed in Xenopus oocytes. Whole-cell recordings were also made from mouse spinal cord slices targeting either parvalbumin positive (PV+ or calretinin positive (CR+ inhibitory neurons. The effect of GBP on Ih was measured in each inhibitory neuron population. HCN4 expression was assessed in the spinal cord using immunohistochemistry. When applied to HCN4 channels, GBP (100 μM caused a hyperpolarizing shift in the voltage of half activation (V1/2 thereby reducing the currents. Gabapentin had no impact on the V1/2 of HCN1 or HCN2 channels. There was a robust increase in the time to half activation for HCN4 channels with only a small increase noted for HCN1 channels. Gabapentin also caused a hyperpolarizing shift in the V1/2 of Ih measured from HCN4-expressing PV+ inhibitory neurons in the spinal dorsal horn. Gabapentin had minimal effect on Ih recorded from CR+ neurons. Consistent with this, immunohistochemical analysis revealed that the majority of CR+ inhibitory neurons do not express somatic HCN4 channels. In conclusion, GBP reduces HCN4 channel-mediated currents through a hyperpolarized shift in the V1/2. The HCN channel subtype selectivity of GBP provides a unique tool for investigating HCN4 channel function in the central nervous system. The HCN4 channel is a candidate molecular target for the acute analgesic and anticonvulsant actions of GBP.

  16. Differential distribution of voltage-gated ion channels in cortical neurons: implications for epilepsy.

    Science.gov (United States)

    Child, Nicholas D; Benarroch, Eduardo E

    2014-03-18

    Neurons contain different functional somatodendritic and axonal domains, each with a characteristic distribution of voltage-gated ion channels, synaptic inputs, and function. The dendritic tree of a cortical pyramidal neuron has 2 distinct domains, the basal and the apical dendrites, both containing dendritic spines; the different domains of the axon are the axonal initial segment (AIS), axon proper (which in myelinated axons includes the node of Ranvier, paranodes, juxtaparanodes, and internodes), and the axon terminals. In the cerebral cortex, the dendritic spines of the pyramidal neurons receive most of the excitatory synapses; distinct populations of γ-aminobutyric acid (GABA)ergic interneurons target specific cellular domains and thus exert different influences on pyramidal neurons. The multiple synaptic inputs reaching the somatodendritic region and generating excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) sum and elicit changes in membrane potential at the AIS, the site of initiation of the action potential.

  17. The Function of the Novel Mechanical Activated Ion Channel Piezo1 in the Human Osteosarcoma Cells

    OpenAIRE

    Jiang, Long; Zhao, Yi-ding; Chen, Wei-xiang

    2017-01-01

    Background The Piezo1 protein ion channel is a novel mechanical activated ion channel which is related to mechanical signal transduction. However, the function of the mechanically activated ion channel Piezo1 had not been explored. In this study, we explored the function of the Piezo1 ion channel in human osteosarcoma (OS) cells related to apoptosis, invasion, and the cell proliferation. Material/Methods Reverse transcription polymerase chain reaction (RT-PCR) and western-blotting were used t...

  18. A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel.

    Science.gov (United States)

    Li-Smerin, Y; Hackos, D H; Swartz, K J

    2000-02-01

    Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.

  19. Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History.

    Directory of Open Access Journals (Sweden)

    Janin Riedelsberger

    Full Text Available Voltage-gated potassium (K+ channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD, this K+ channel type segregates into at least two main functional categories-hyperpolarization-activated, inward-rectifying (Kin and depolarization-activated, outward-rectifying (Kout channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.

  20. Sigma-1 receptor agonist increases axon outgrowth of hippocampal neurons via voltage-gated calcium ions channels.

    Science.gov (United States)

    Li, Dong; Zhang, Shu-Zhuo; Yao, Yu-Hong; Xiang, Yun; Ma, Xiao-Yun; Wei, Xiao-Li; Yan, Hai-Tao; Liu, Xiao-Yan

    2017-12-01

    Sigma-1 receptors (Sig-1Rs) are unique endoplasmic reticulum proteins that have been implicated in both neurodegenerative and ischemic diseases, such as Alzheimer's disease and stroke. Accumulating evidence has suggested that Sig-1R plays a role in neuroprotection and axon outgrowth. The underlying mechanisms of Sig-1R-mediated neuroprotection have been well elucidated. However, the mechanisms underlying the effects of Sig-1R on axon outgrowth are not fully understood. To clarify this issue, we utilized immunofluorescence to compare the axon lengths of cultured naïve hippocampal neurons before and after the application of the Sig-1R agonist, SA4503. Then, electrophysiology and immunofluorescence were used to examine voltage-gated calcium ion channel (VGCCs) currents in the cell membranes and growth cones. We found that Sig-1R activation dramatically enhanced the axonal length of the naïve hippocampal neurons. Application of the Sig-1R antagonist NE100 and gene knockdown techniques both demonstrated the effects of Sig-1R. The growth-promoting effect of SA4503 was accompanied by the inhibition of voltage-gated Ca 2+ influx and was recapitulated by incubating the neurons with the L-type, N-type, and P/Q-type VGCC blockers, nimodipine, MVIIA and ω-agatoxin IVA, respectively. This effect was unrelated to glial cells. The application of SA4503 transformed the growth cone morphologies from complicated to simple, which favored axon outgrowth. Sig-1R activation can enhance axon outgrowth and may have a substantial influence on neurogenesis and neurodegenerative diseases. © 2017 John Wiley & Sons Ltd.

  1. Voltage-Gated Proton Channels: Molecular Biology, Physiology, and Pathophysiology of the HV Family

    Science.gov (United States)

    2013-01-01

    Voltage-gated proton channels (HV) are unique, in part because the ion they conduct is unique. HV channels are perfectly selective for protons and have a very small unitary conductance, both arguably manifestations of the extremely low H+ concentration in physiological solutions. They open with membrane depolarization, but their voltage dependence is strongly regulated by the pH gradient across the membrane (ΔpH), with the result that in most species they normally conduct only outward current. The HV channel protein is strikingly similar to the voltage-sensing domain (VSD, the first four membrane-spanning segments) of voltage-gated K+ and Na+ channels. In higher species, HV channels exist as dimers in which each protomer has its own conduction pathway, yet gating is cooperative. HV channels are phylogenetically diverse, distributed from humans to unicellular marine life, and perhaps even plants. Correspondingly, HV functions vary widely as well, from promoting calcification in coccolithophores and triggering bioluminescent flashes in dinoflagellates to facilitating killing bacteria, airway pH regulation, basophil histamine release, sperm maturation, and B lymphocyte responses in humans. Recent evidence that hHV1 may exacerbate breast cancer metastasis and cerebral damage from ischemic stroke highlights the rapidly expanding recognition of the clinical importance of hHV1. PMID:23589829

  2. Gating of the two-pore cation channel AtTPC1 in the plant vacuole is based on a single voltage-sensing domain.

    Science.gov (United States)

    Jaślan, D; Mueller, T D; Becker, D; Schultz, J; Cuin, T A; Marten, I; Dreyer, I; Schönknecht, G; Hedrich, R

    2016-09-01

    The two-pore cation channel TPC1 operates as a dimeric channel in animal and plant endomembranes. Each subunit consists of two homologous Shaker-like halves, with 12 transmembrane domains in total (S1-S6, S7-S12). In plants, TPC1 channels reside in the vacuolar membrane, and upon voltage stimulation, give rise to the well-known slow-activating SV currents. Here, we combined bioinformatics, structure modelling, site-directed mutagenesis, and in planta patch clamp studies to elucidate the molecular mechanisms of voltage-dependent channel gating in TPC1 in its native plant background. Structure-function analysis of the Arabidopsis TPC1 channel in planta confirmed that helix S10 operates as the major voltage-sensing site, with Glu450 and Glu478 identified as possible ion-pair partners for voltage-sensing Arg537. The contribution of helix S4 to voltage sensing was found to be negligible. Several conserved negative residues on the luminal site contribute to calcium binding, stabilizing the closed channel. During evolution of plant TPC1s from two separate Shaker-like domains, the voltage-sensing function in the N-terminal Shaker-unit (S1-S4) vanished. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

  3. Computing rates of Markov models of voltage-gated ion channels by inverting partial differential equations governing the probability density functions of the conducting and non-conducting states.

    Science.gov (United States)

    Tveito, Aslak; Lines, Glenn T; Edwards, Andrew G; McCulloch, Andrew

    2016-07-01

    Markov models are ubiquitously used to represent the function of single ion channels. However, solving the inverse problem to construct a Markov model of single channel dynamics from bilayer or patch-clamp recordings remains challenging, particularly for channels involving complex gating processes. Methods for solving the inverse problem are generally based on data from voltage clamp measurements. Here, we describe an alternative approach to this problem based on measurements of voltage traces. The voltage traces define probability density functions of the functional states of an ion channel. These probability density functions can also be computed by solving a deterministic system of partial differential equations. The inversion is based on tuning the rates of the Markov models used in the deterministic system of partial differential equations such that the solution mimics the properties of the probability density function gathered from (pseudo) experimental data as well as possible. The optimization is done by defining a cost function to measure the difference between the deterministic solution and the solution based on experimental data. By evoking the properties of this function, it is possible to infer whether the rates of the Markov model are identifiable by our method. We present applications to Markov model well-known from the literature. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Differential subcellular distribution of ion channels and the diversity of neuronal function.

    Science.gov (United States)

    Nusser, Zoltan

    2012-06-01

    Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Temperature and Voltage Coupling to Channel Opening in Transient Receptor Potential Melastatin 8 (TRPM8)*♦

    Science.gov (United States)

    Raddatz, Natalia; Castillo, Juan P.; Gonzalez, Carlos; Alvarez, Osvaldo; Latorre, Ramon

    2014-01-01

    Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca2+-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol−1. The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening. PMID:25352597

  6. Voltage-Gated Potassium Channels: A Structural Examination of Selectivity and Gating

    Science.gov (United States)

    Kim, Dorothy M.; Nimigean, Crina M.

    2016-01-01

    Voltage-gated potassium channels play a fundamental role in the generation and propagation of the action potential. The discovery of these channels began with predictions made by early pioneers, and has culminated in their extensive functional and structural characterization by electrophysiological, spectroscopic, and crystallographic studies. With the aid of a variety of crystal structures of these channels, a highly detailed picture emerges of how the voltage-sensing domain reports changes in the membrane electric field and couples this to conformational changes in the activation gate. In addition, high-resolution structural and functional studies of K+ channel pores, such as KcsA and MthK, offer a comprehensive picture on how selectivity is achieved in K+ channels. Here, we illustrate the remarkable features of voltage-gated potassium channels and explain the mechanisms used by these machines with experimental data. PMID:27141052

  7. LRRK2 regulates voltage-gated calcium channel function.

    Directory of Open Access Journals (Sweden)

    Cade eBedford

    2016-05-01

    Full Text Available Voltage-gated Ca2+ (CaV channels enable Ca2+ influx in response to membrane depolarization. CaV2.1 channels are localized to the presynaptic membrane of many types of neurons where they are involved in triggering neurotransmitter release. Several signaling proteins have been identified as important CaV2.1 regulators including protein kinases, G-proteins and Ca2+ binding proteins. Recently, we discovered that leucine rich repeat kinase 2 (LRRK2, a protein associated with inherited Parkinson’s disease, interacts with specific synaptic proteins and influences synaptic transmission. Since synaptic proteins functionally interact with CaV2.1 channels and synaptic transmission is triggered by Ca2+ entry via CaV2.1, we investigated whether LRRK2 could impact CaV2.1 channel function. CaV2.1 channel properties were measured using whole cell patch clamp electrophysiology in HEK293 cells transfected with CaV2.1 subunits and various LRRK2 constructs. Our results demonstrate that both wild type LRRK2 and the G2019S LRRK2 mutant caused a significant increase in whole cell Ca2+ current density compared to cells expressing only the CaV2.1 channel complex. In addition, LRRK2 expression caused a significant hyperpolarizing shift in voltage-dependent activation while having no significant effect on inactivation properties. These functional changes in CaV2.1 activity are likely due to a direct action of LRRK2 as we detected a physical interaction between LRRK2 and the β3 CaV channel subunit via coimmunoprecipitation. Furthermore, effects on CaV2.1 channel function are dependent on LRRK2 kinase activity as these could be reversed via treatment with a LRRK2 inhibitor. Interestingly, LRRK2 also augmented endogenous voltage-gated Ca2+ channel function in PC12 cells suggesting other CaV channels could also be regulated by LRRK2. Overall, our findings support a novel physiological role for LRRK2 in regulating CaV2.1 function that could have implications for how

  8. Niflumic acid alters gating of HCN2 pacemaker channels by interaction with the outer region of S4 voltage sensing domains.

    Science.gov (United States)

    Cheng, Lan; Sanguinetti, Michael C

    2009-05-01

    Niflumic acid, 2-[[3-(trifluoromethyl)phenyl]amino]pyridine-3-carboxylic acid (NFA), is a nonsteroidal anti-inflammatory drug that also blocks or modifies the gating of many ion channels. Here, we investigated the effects of NFA on hyperpolarization-activated cyclic nucleotide-gated cation (HCN) pacemaker channels expressed in X. laevis oocytes using site-directed mutagenesis and the two-electrode voltage-clamp technique. Extracellular NFA acted rapidly and caused a slowing of activation and deactivation and a hyperpolarizing shift in the voltage dependence of HCN2 channel activation (-24.5 +/- 1.2 mV at 1 mM). Slowed channel gating and reduction of current magnitude was marked in oocytes treated with NFA, while clamped at 0 mV but minimal in oocytes clamped at -100 mV, indicating the drug preferentially interacts with channels in the closed state. NFA at 0.1 to 3 mM shifted the half-point for channel activation in a concentration-dependent manner, with an EC(50) of 0.54 +/- 0.068 mM and a predicted maximum shift of -38 mV. NFA at 1 mM also reduced maximum HCN2 conductance by approximately 20%, presumably by direct block of the pore. The rapid onset and state-dependence of NFA-induced changes in channel gating suggests an interaction with the extracellular region of the S4 transmembrane helix, the primary voltage-sensing domain of HCN2. Neutralization (by mutation to Gln) of any three of the outer four basic charged residues in S4, but not single mutations, abrogated the NFA-induced shift in channel activation. We conclude that NFA alters HCN2 gating by interacting with the extracellular end of the S4 voltage sensor domains.

  9. Equilibrium fluctuation relations for voltage coupling in membrane proteins.

    Science.gov (United States)

    Kim, Ilsoo; Warshel, Arieh

    2015-11-01

    A general theoretical framework is developed to account for the effects of an external potential on the energetics of membrane proteins. The framework is based on the free energy relation between two (forward/backward) probability densities, which was recently generalized to non-equilibrium processes, culminating in the work-fluctuation theorem. Starting from the probability densities of the conformational states along the "voltage coupling" reaction coordinate, we investigate several interconnected free energy relations between these two conformational states, considering voltage activation of ion channels. The free energy difference between the two conformational states at zero (depolarization) membrane potential (i.e., known as the chemical component of free energy change in ion channels) is shown to be equivalent to the free energy difference between the two "equilibrium" (resting and activated) conformational states along the one-dimensional voltage couplin reaction coordinate. Furthermore, the requirement that the application of linear response approximation to the free energy functionals of voltage coupling should satisfy the general free energy relations, yields a novel closed-form expression for the gating charge in terms of other basic properties of ion channels. This connection is familiar in statistical mechanics, known as the equilibrium fluctuation-response relation. The theory is illustrated by considering the coupling of a unit charge to the external voltage in the two sites near the surface of membrane, representing the activated and resting states. This is done using a coarse-graining (CG) model of membrane proteins, which includes the membrane, the electrolytes and the electrodes. The CG model yields Marcus-type voltage dependent free energy parabolas for the response of the electrostatic environment (electrolytes etc.) to the transition from the initial to the final configuratinal states, leading to equilibrium free energy difference and free

  10. Simple Ion Channels: From Structure to Electrophysiology and Back

    Science.gov (United States)

    Pohorille, Andrzej

    2018-01-01

    A reliable way to establish whether our understanding of a channel is satisfactory is to reproduce its measured ionic conductance over a broad range of applied voltages in computer simulations. In molecular dynamics (MD), this can be done by way of applying an external electric field to the system and counting the number of ions that traverse the channel per unit time. Since this approach is computationally very expensive, we have developed a markedly more efficient alternative in which MD is combined with the electrodiffusion (ED) equation. In this approach, the assumptions of the ED equation can be rigorously tested, and the precision and consistency of the calculated conductance can be determined. We have demonstrated that the full current/voltage dependence and the underlying free energy profile for a simple channel can be reliably calculated from equilibrium or non-equilibrium MD simulations at a single voltage. To carry out MD simulations, a structural model of a channel has to be assumed, which is an important constraint, considering that high-resolution structures are available for only very few simple channels. If the comparison of calculated ionic conductance with electrophysiological data is satisfactory, it greatly increases our confidence that the structure and the function are described sufficiently accurately. We examined the validity of the ED for several channels embedded in phospholipid membranes - four naturally occurring channels: trichotoxin, alamethicin, p7 from hepatitis C virus (HCV) and Vpu from the HIV-1 virus, and a synthetic, hexameric channel, formed by a 21-residue peptide that contains only leucine and serine. All these channels mediate transport of potassium and chloride ions. It was found that the ED equation is satisfactory for these systems. In some of them experimental and calculated electrophysiological properties are in good agreement, whereas in others there are strong indications that the structural models are incorrect.

  11. Autoantibodies to neurotransmitter receptors and ion channels: from neuromuscular to neuropsychiatric disorders

    Directory of Open Access Journals (Sweden)

    Pilar eMartinez-Martinez

    2013-09-01

    Full Text Available Changes of voltage-gated ion channels and ligand-gated receptor channels caused by mutation or autoimmune attack are the cause of so-called channelopathies in the central and peripheral nervous system. We present the pathophysiology of channelopathies of the neuromuscular junction in terms of loss-of-function and gain-of-function principles. Autoantibodies generally have reduced access to the CNS, but in some cases this is enough to cause disease. A review is provided of recent findings implicating autoantibodies against ligand–activated receptor channels and potassium channels in psychiatric and neurological disorders, including schizophrenia and limbic encephalitis. The emergence of channelopathy-related neuropsychiatric disorders has implications for research and practice.

  12. Phosphorylation of purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal Kinase-3 modifies channel voltage-dependence

    Directory of Open Access Journals (Sweden)

    Rajeev Gupta

    2017-06-01

    Full Text Available Voltage-Dependent Anion Channel (VDAC phosphorylated by c-Jun N-terminal Kinase-3 (JNK3 was incorporated into the bilayer lipid membrane. Single-channel electrophysiological properties of the native and the phosphorylated VDAC were compared. The open probability versus voltage curve of the native VDAC displayed symmetry around the voltage axis, whereas that of the phosphorylated VDAC showed asymmetry. This result indicates that phosphorylation by JNK3 modifies voltage-dependence of VDAC.

  13. Phosphorylation of purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal Kinase-3 modifies channel voltage-dependence.

    Science.gov (United States)

    Gupta, Rajeev; Ghosh, Subhendu

    2017-06-01

    Voltage-Dependent Anion Channel (VDAC) phosphorylated by c-Jun N-terminal Kinase-3 (JNK3) was incorporated into the bilayer lipid membrane. Single-channel electrophysiological properties of the native and the phosphorylated VDAC were compared. The open probability versus voltage curve of the native VDAC displayed symmetry around the voltage axis, whereas that of the phosphorylated VDAC showed asymmetry. This result indicates that phosphorylation by JNK3 modifies voltage-dependence of VDAC.

  14. Conotoxins Targeting Neuronal Voltage-Gated Sodium Channel Subtypes: Potential Analgesics?

    Directory of Open Access Journals (Sweden)

    Jeffrey R. McArthur

    2012-11-01

    Full Text Available Voltage-gated sodium channels (VGSC are the primary mediators of electrical signal amplification and propagation in excitable cells. VGSC subtypes are diverse, with different biophysical and pharmacological properties, and varied tissue distribution. Altered VGSC expression and/or increased VGSC activity in sensory neurons is characteristic of inflammatory and neuropathic pain states. Therefore, VGSC modulators could be used in prospective analgesic compounds. VGSCs have specific binding sites for four conotoxin families: μ-, μO-, δ- and ί-conotoxins. Various studies have identified that the binding site of these peptide toxins is restricted to well-defined areas or domains. To date, only the μ- and μO-family exhibit analgesic properties in animal pain models. This review will focus on conotoxins from the μ- and μO-families that act on neuronal VGSCs. Examples of how these conotoxins target various pharmacologically important neuronal ion channels, as well as potential problems with the development of drugs from conotoxins, will be discussed.

  15. A common pathway for charge transport through voltage-sensing domains.

    Science.gov (United States)

    Chanda, Baron; Bezanilla, Francisco

    2008-02-07

    Voltage-gated ion channels derive their voltage sensitivity from the movement of specific charged residues in response to a change in transmembrane potential. Several studies on mechanisms of voltage sensing in ion channels support the idea that these gating charges move through a well-defined permeation pathway. This gating pathway in a voltage-gated ion channel can also be mutated to transport free cations, including protons. The recent discovery of proton channels with sequence homology to the voltage-sensing domains suggests that evolution has perhaps exploited the same gating pathway to generate a bona fide voltage-dependent proton transporter. Here we will discuss implications of these findings on the mechanisms underlying charge (and ion) transport by voltage-sensing domains.

  16. Bromodomain-containing Protein 4 Activates Voltage-gated Sodium Channel 1.7 Transcription in Dorsal Root Ganglia Neurons to Mediate Thermal Hyperalgesia in Rats.

    Science.gov (United States)

    Hsieh, Ming-Chun; Ho, Yu-Cheng; Lai, Cheng-Yuan; Wang, Hsueh-Hsiao; Lee, An-Sheng; Cheng, Jen-Kun; Chau, Yat-Pang; Peng, Hsien-Yu

    2017-11-01

    Bromodomain-containing protein 4 binds acetylated promoter histones and promotes transcription; however, the role of bromodomain-containing protein 4 in inflammatory hyperalgesia remains unclear. Male Sprague-Dawley rats received hind paw injections of complete Freund's adjuvant to induce hyperalgesia. The dorsal root ganglia were examined to detect changes in bromodomain-containing protein 4 expression and the activation of genes involved in the expression of voltage-gated sodium channel 1.7, which is a key pain-related ion channel. The intraplantar complete Freund's adjuvant injections resulted in thermal hyperalgesia (4.0 ± 1.5 s; n = 7). The immunohistochemistry and immunoblotting results demonstrated an increase in the bromodomain-containing protein 4-expressing dorsal root ganglia neurons (3.78 ± 0.38 fold; n = 7) and bromodomain-containing protein 4 protein levels (2.62 ± 0.39 fold; n = 6). After the complete Freund's adjuvant injection, histone H3 protein acetylation was enhanced in the voltage-gated sodium channel 1.7 promoter, and cyclin-dependent kinase 9 and phosphorylation of RNA polymerase II were recruited to this area. Furthermore, the voltage-gated sodium channel 1.7-mediated currents were enhanced in neurons of the complete Freund's adjuvant rats (55 ± 11 vs. 19 ± 9 pA/pF; n = 4 to 6 neurons). Using bromodomain-containing protein 4-targeted antisense small interfering RNA to the complete Freund's adjuvant-treated rats, the authors demonstrated a reduction in the expression of bromodomain-containing protein 4 (0.68 ± 0.16 fold; n = 7), a reduction in thermal hyperalgesia (7.5 ± 1.5 s; n = 7), and a reduction in the increased voltage-gated sodium channel 1.7 currents (21 ± 4 pA/pF; n = 4 to 6 neurons). Complete Freund's adjuvant triggers enhanced bromodomain-containing protein 4 expression, ultimately leading to the enhanced excitability of nociceptive neurons and thermal hyperalgesia. This effect is

  17. Biochemical and structural analysis of the hyperpolarization-activated K(+) channel MVP.

    Science.gov (United States)

    Randich, Amelia M; Cuello, Luis G; Wanderling, Sherry S; Perozo, Eduardo

    2014-03-18

    In contrast to the majority of voltage-gated ion channels, hyperpolarization-activated channels remain closed at depolarizing potentials and are activated at hyperpolarizing potentials. The basis for this reverse polarity is thought to be a result of differences in the way the voltage-sensing domain (VSD) couples to the pore domain. In the absence of structural data, the molecular mechanism of this reverse polarity coupling remains poorly characterized. Here we report the characterization of the structure and local dynamics of the closed activation gate (lower S6 region) of MVP, a hyperpolarization-activated potassium channel from Methanococcus jannaschii, by electron paramagnetic resonance (EPR) spectroscopy. We show that a codon-optimized version of MVP has high expression levels in Escherichia coli, is purified as a stable tetramer, and exhibits expected voltage-dependent activity when reconstituted in liposomes. EPR analysis of the mid to lower S6 region revealed positions exhibiting strong spin-spin coupling, indicating that the activation gate of MVP is closed at 0 mV. A comparison of local environmental parameters along the activation gate for MVP and KcsA indicates that MVP adopts a different closed conformation. These structural details set the stage for future evaluations of reverse electromechanical coupling in MVP.

  18. Biochemical and Structural Analysis of the Hyperpolarization-Activated K+ Channel MVP

    Science.gov (United States)

    2015-01-01

    In contrast to the majority of voltage-gated ion channels, hyperpolarization-activated channels remain closed at depolarizing potentials and are activated at hyperpolarizing potentials. The basis for this reverse polarity is thought to be a result of differences in the way the voltage-sensing domain (VSD) couples to the pore domain. In the absence of structural data, the molecular mechanism of this reverse polarity coupling remains poorly characterized. Here we report the characterization of the structure and local dynamics of the closed activation gate (lower S6 region) of MVP, a hyperpolarization-activated potassium channel from Methanococcus jannaschii, by electron paramagnetic resonance (EPR) spectroscopy. We show that a codon-optimized version of MVP has high expression levels in Escherichia coli, is purified as a stable tetramer, and exhibits expected voltage-dependent activity when reconstituted in liposomes. EPR analysis of the mid to lower S6 region revealed positions exhibiting strong spin–spin coupling, indicating that the activation gate of MVP is closed at 0 mV. A comparison of local environmental parameters along the activation gate for MVP and KcsA indicates that MVP adopts a different closed conformation. These structural details set the stage for future evaluations of reverse electromechanical coupling in MVP. PMID:24490868

  19. Dendritic calcium channels and their activation by synaptic signals in auditory coincidence detector neurons.

    Science.gov (United States)

    Blackmer, Trillium; Kuo, Sidney P; Bender, Kevin J; Apostolides, Pierre F; Trussell, Laurence O

    2009-08-01

    The avian nucleus laminaris (NL) encodes the azimuthal location of low-frequency sound sources by detecting the coincidence of binaural signals. Accurate coincidence detection requires precise developmental regulation of the lengths of the fine, bitufted dendrites that characterize neurons in NL. Such regulation has been suggested to be driven by local, synaptically mediated, dendritic signals such as Ca(2+). We examined Ca(2+) signaling through patch clamp and ion imaging experiments in slices containing nucleus laminaris from embryonic chicks. Voltage-clamp recordings of neurons located in the NL showed the presence of large Ca(2+) currents of two types, a low voltage-activated, fast inactivating Ni(2+) sensitive channel resembling mammalian T-type channels, and a high voltage-activated, slowly inactivating Cd(2+) sensitive channel. Two-photon Ca(2+) imaging showed that both channel types were concentrated on dendrites, even at their distal tips. Single action potentials triggered synaptically or by somatic current injection immediately elevated Ca(2+) throughout the entire cell. Ca(2+) signals triggered by subthreshold synaptic activity were highly localized. Thus when electrical activity is suprathreshold, Ca(2+) channels ensure that Ca(2+) rises in all dendrites, even those that are synaptically inactive.

  20. Voltage-Gated Calcium Channels

    Science.gov (United States)

    Zamponi, Gerald Werner

    Voltage Gated Calcium Channels is the first comprehensive book in the calcium channel field, encompassing over thirty years of progress towards our understanding of calcium channel structure, function, regulation, physiology, pharmacology, and genetics. This book balances contributions from many of the leading authorities in the calcium channel field with fresh perspectives from risings stars in the area, taking into account the most recent literature and concepts. This is the only all-encompassing calcium channel book currently available, and is an essential resource for academic researchers at all levels in the areas neuroscience, biophysics, and cardiovascular sciences, as well as to researchers in the drug discovery area.

  1. A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus.

    Science.gov (United States)

    Kajma, Anna; Szewczyk, Adam

    2012-10-01

    Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Dopamine negatively modulates the NCA ion channels in C. elegans.

    Science.gov (United States)

    Topalidou, Irini; Cooper, Kirsten; Pereira, Laura; Ailion, Michael

    2017-10-01

    The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.

  3. Recording ion channels across soy-extracted lecithin bilayer generated by water-soluble quantum dots

    Science.gov (United States)

    Sarma, Runjun; Mohanta, Dambarudhar

    2014-02-01

    We report on the quantum dot (QD)-induced ion channels across a soya-derived lecithin bilayer supported on a laser drilled of ~100 μm aperture of cellulose acetate substrate that separates two electrolytic chambers. Adequate current bursts were observed when the bilayer was subjected to a gating voltage. The voltage-dependent current fluctuation, across the bilayer, was attributed to the insertion of ~20 nm sized water-soluble CdSe QDs, forming nanopores due to their spontaneous aggregation. Apart from a closed state, the first observable conductance levels were found as 6.3 and 11 nS, as for the respective biasing voltages of -10 and -20 mV. The highest observable conductance states, at corresponding voltages were ~14.3 and 21.1 nS. Considering two simplified models, we predict that the non-spherical pores (dnspore) can be a better approximation over spherical nanopores (dspore) for exhibiting a definite conductance level. At times, even dnspore ≤ 4dspore and that the non-spherical nanopores were associated with a smaller No. of QDs than the case for spherical nanopores, for a definite conductance state. It seems like the current events are partly stochastic, possibly due to thermal effects on the aggregated QDs that would form nanopores. The dwell time of the states was predicted in the range of 384-411 μs. The ion channel mechanism in natural phospholipid bilayers over artificial ones will provide a closer account to understand ion transport mechanism in live cells and signaling activity including labelling with fluorescent QDs.

  4. Mapping the membrane-aqueous border for the voltage-sensing domain of a potassium channel.

    Science.gov (United States)

    Neale, Edward J; Rong, Honglin; Cockcroft, Christopher J; Sivaprasadarao, Asipu

    2007-12-28

    Voltage-sensing domains (VSDs) play diverse roles in biology. As integral components, they can detect changes in the membrane potential of a cell and couple these changes to activity of ion channels and enzymes. As independent proteins, homologues of the VSD can function as voltage-dependent proton channels. To sense voltage changes, the positively charged fourth transmembrane segment, S4, must move across the energetically unfavorable hydrophobic core of the bilayer, which presents a barrier to movement of both charged species and protons. To reduce the barrier to S4 movement, it has been suggested that aqueous crevices may penetrate the protein, reducing the extent of total movement. To investigate this hypothesis in a system containing fully functional channels in a native environment with an intact membrane potential, we have determined the contour of the membrane-aqueous border of the VSD of KvAP in Escherichia coli by examining the chemical accessibility of introduced cysteines. The results revealed the contour of the membrane-aqueous border of the VSD in its activated conformation. The water-inaccessible regions of S1 and S2 correspond to the standard width of the membrane bilayer (~28 A), but those of S3 and S4 are considerably shorter (> or = 40%), consistent with aqueous crevices pervading both the extracellular and intracellular ends. One face of S3b and the entire S3a were water-accessible, reducing the water-inaccessible region of S3 to just 10 residues, significantly shorter than for S4. The results suggest a key role for S3 in reducing the distance S4 needs to move to elicit gating.

  5. Soft Wall Ion Channel in Continuum Representation with Application to Modeling Ion Currents in α-Hemolysin

    Science.gov (United States)

    Simakov, Nikolay A.

    2010-01-01

    A soft repulsion (SR) model of short range interactions between mobile ions and protein atoms is introduced in the framework of continuum representation of the protein and solvent. The Poisson-Nernst-Plank (PNP) theory of ion transport through biological channels is modified to incorporate this soft wall protein model. Two sets of SR parameters are introduced: the first is parameterized for all essential amino acid residues using all atom molecular dynamic simulations; the second is a truncated Lennard – Jones potential. We have further designed an energy based algorithm for the determination of the ion accessible volume, which is appropriate for a particular system discretization. The effects of these models of short-range interaction were tested by computing current-voltage characteristics of the α-hemolysin channel. The introduced SR potentials significantly improve prediction of channel selectivity. In addition, we studied the effect of choice of some space-dependent diffusion coefficient distributions on the predicted current-voltage properties. We conclude that the diffusion coefficient distributions largely affect total currents and have little effect on rectifications, selectivity or reversal potential. The PNP-SR algorithm is implemented in a new efficient parallel Poisson, Poisson-Boltzman and PNP equation solver, also incorporated in a graphical molecular modeling package HARLEM. PMID:21028776

  6. Expression of voltage-activated calcium channels in the early zebrafish embryo.

    Science.gov (United States)

    Sanhueza, Dayán; Montoya, Andro; Sierralta, Jimena; Kukuljan, Manuel

    2009-05-01

    Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

  7. Critical voltage effects in electron channeling patterns

    International Nuclear Information System (INIS)

    Farrow, R.C.

    1984-01-01

    Electron channeling patterns were used to study critical voltage effects in the metals molybdenum and tungsten. The purpose was to characterize both theoretically and experimentally how a critical voltage will affect the channeling pattern line shapes. The study focused on the second order critical voltage that results from the degeneracy between the Bloch wave states of the (110) and (220) reflections. Theoretical (110) series electron channeling pattern line profiles were calculated using the dynamical theory of Hirsch and Humphreys (1970). A 10 beam dynamical electron diffraction calculation was performed (using complex Fourier lattice potentials) to generate Bloch wave coefficients, excitation amplitudes, and absorption coefficients needed for determining backscattering coefficients and subsequent backscattered electron intensities. The theoretical model is applicable to electron diffraction at all energies since no high energy approximation or perturbation method was used

  8. Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

    Science.gov (United States)

    Neely, Alan; Hidalgo, Patricia

    2014-01-01

    Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

  9. Asymmetric ion transport through ion-channel-mimetic solid-state nanopores.

    Science.gov (United States)

    Guo, Wei; Tian, Ye; Jiang, Lei

    2013-12-17

    Both scientists and engineers are interested in the design and fabrication of synthetic nanofluidic architectures that mimic the gating functions of biological ion channels. The effort to build such structures requires interdisciplinary efforts at the intersection of chemistry, materials science, and nanotechnology. Biological ion channels and synthetic nanofluidic devices have some structural and chemical similarities, and therefore, they share some common features in regulating the traverse ionic flow. In the past decade, researchers have identified two asymmetric ion transport phenomena in synthetic nanofluidic structures, the rectified ionic current and the net diffusion current. The rectified ionic current is a diode-like current-voltage response that occurs when switching the voltage bias. This phenomenon indicates a preferential direction of transport in the nanofluidic system. The net diffusion current occurs as a direct product of charge selectivity and is generated from the asymmetric diffusion through charged nanofluidic channels. These new ion transport phenomena and the elaborate structures that occur in biology have inspired us to build functional nanofluidic devices for both fundamental research and practical applications. In this Account, we review our recent progress in the design and fabrication of biomimetic solid-state nanofluidic devices with asymmetric ion transport behavior. We demonstrate the origin of the rectified ionic current and the net diffusion current. We also identify several influential factors and discuss how to build these asymmetric features into nanofluidic systems by controlling (1) nanopore geometry, (2) surface charge distribution, (3) chemical composition, (4) channel wall wettability, (5) environmental pH, (6) electrolyte concentration gradient, and (7) ion mobility. In the case of the first four features, we build these asymmetric features directly into the nanofluidic structures. With the final three, we construct

  10. Voltage-sensing domain mode shift is coupled to the activation gate by the N-terminal tail of hERG channels.

    Science.gov (United States)

    Tan, Peter S; Perry, Matthew D; Ng, Chai Ann; Vandenberg, Jamie I; Hill, Adam P

    2012-09-01

    Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique gating kinetics characterized by unusually slow activation and deactivation. The N terminus of the channel, which contains an amphipathic helix and an unstructured tail, has been shown to be involved in regulation of this slow deactivation. However, the mechanism of how this occurs and the connection between voltage-sensing domain (VSD) return and closing of the gate are unclear. To examine this relationship, we have used voltage-clamp fluorometry to simultaneously measure VSD motion and gate closure in N-terminally truncated constructs. We report that mode shifting of the hERG VSD results in a corresponding shift in the voltage-dependent equilibrium of channel closing and that at negative potentials, coupling of the mode-shifted VSD to the gate defines the rate of channel closure. Deletion of the first 25 aa from the N terminus of hERG does not alter mode shifting of the VSD but uncouples the shift from closure of the cytoplasmic gate. Based on these observations, we propose the N-terminal tail as an adaptor that couples voltage sensor return to gate closure to define slow deactivation gating in hERG channels. Furthermore, because the mode shift occurs on a time scale relevant to the cardiac action potential, we suggest a physiological role for this phenomenon in maximizing current flow through hERG channels during repolarization.

  11. The orientation and molecular movement of a k(+) channel voltage-sensing domain.

    Science.gov (United States)

    Gandhi, Chris S; Clark, Eliana; Loots, Eli; Pralle, Arnd; Isacoff, Ehud Y

    2003-10-30

    Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.

  12. Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation.

    Science.gov (United States)

    Lin, C S; Boltz, R C; Blake, J T; Nguyen, M; Talento, A; Fischer, P A; Springer, M S; Sigal, N H; Slaughter, R S; Garcia, M L

    1993-03-01

    The role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca(2+)-activated potassium channels (PK,Ca) and voltage-gated potassium channels (PK,V) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (IL-2 and interferon gamma) are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,Ca and PK,V, we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for PK,V. These studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca(2+)-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

  13. Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels.

    Science.gov (United States)

    Capes, Deborah L; Goldschen-Ohm, Marcel P; Arcisio-Miranda, Manoel; Bezanilla, Francisco; Chanda, Baron

    2013-08-01

    Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na(+) channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K(+) current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

  14. Evolutionary origins of mechanosensitive ion channels.

    Science.gov (United States)

    Martinac, Boris; Kloda, Anna

    2003-01-01

    According to the recent revision, the universal phylogenetic tree is composed of three domains: Eukarya (eukaryotes), Bacteria (eubacteria) and Archaea (archaebacteria). Mechanosensitive (MS) ion channels have been documented in cells belonging to all three domains suggesting their very early appearance during evolution of life on Earth. The channels show great diversity in conductance, selectivity and voltage dependence, while sharing the property of being gated by mechanical stimuli exerted on cell membranes. In prokaryotes, MS channels were first documented in Bacteria followed by their discovery in Archaea. The finding of MS channels in archaeal cells helped to recognize and establish the evolutionary relationship between bacterial and archaeal MS channels and to show that this relationship extends to eukaryotic Fungi (Schizosaccharomyces pombe) and Plants (Arabidopsis thaliana). Similar to their bacterial and archaeal homologues, MS channels in eukaryotic cell-walled Fungi and Plants may serve in protecting the cellular plasma membrane from excessive dilation and rupture that may occur during osmotic stress. This review summarizes briefly some of the recent developments in the MS channel research field that may ultimately lead to elucidation of the biophysical and evolutionary principles underlying the mechanosensory transduction in living cells.

  15. Integrative Approach with Electrophysiological and Theoretical Methods Reveals a New Role of S4 Positively Charged Residues in PKD2L1 Channel Voltage-Sensing.

    Science.gov (United States)

    Numata, Tomohiro; Tsumoto, Kunichika; Yamada, Kazunori; Kurokawa, Tatsuki; Hirose, Shinichi; Nomura, Hideki; Kawano, Mitsuhiro; Kurachi, Yoshihisa; Inoue, Ryuji; Mori, Yasuo

    2017-08-29

    Numerical model-based simulations provide important insights into ion channel gating when experimental limitations exist. Here, a novel strategy combining numerical simulations with patch clamp experiments was used to investigate the net positive charges in the putative transmembrane segment 4 (S4) of the atypical, positively-shifted voltage-dependence of polycystic kidney disease 2-like 1 (PKD2L1) channel. Charge-neutralising mutations (K452Q, K455Q and K461Q) in S4 reduced gating charges, positively shifted the Boltzmann-type activation curve [i.e., open probability (P open )-V curve] and altered the time-courses of activation/deactivation of PKD2L1, indicating that this region constitutes part of a voltage sensor. Numerical reconstruction of wild-type (WT) and mutant PKD2L1-mediated currents necessitated, besides their voltage-dependent gating parameters, a scaling factor that describes the voltage-dependence of maximal conductance, G max . Subsequent single-channel conductance (γ) measurements revealed that voltage-dependence of G max in WT can be explained by the inward-rectifying property of γ, which is greatly changed in PKD2L1 mutants. Homology modelling based on PKD2 and Na V Ab structures suggest that such voltage dependence of P open and γ in PKD2L1 could both reflect the charged state of the S4 domain. The present conjunctive experimental and theoretical approaches provide a framework to explore the undetermined mechanism(s) regulating TRP channels that possess non-classical voltage-dependent properties.

  16. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

    Science.gov (United States)

    Tang, Cheng; Zhou, Xi; Nguyen, Phuong Tran; Zhang, Yunxiao; Hu, Zhaotun; Zhang, Changxin; Yarov-Yarovoy, Vladimir; DeCaen, Paul G; Liang, Songping; Liu, Zhonghua

    2017-07-01

    Voltage-gated sodium channels (Na V s) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial Na V s have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial Na V s. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx-27 from the venom of tarantula Chilobrachys jingzhao as a high-affinity antagonist of the prokaryotic Na V s Ns V Ba (nonselective voltage-gated Bacillus alcalophilus ) and NaChBac (bacterial sodium channel from Bacillus halodurans ) (IC 50 = 112 nM and 30 nM, respectively). JZTx-27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of Ns V Ba, whereas the local anesthetic drug lidocaine was shown to antagonize Ns V Ba without affecting channel gating. Mutation analysis confirmed that JZTx-27 bound to S3-4 linker of Ns V Ba, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and in silico analysis suggested that JZTx-27 trapped VSM of Ns V Ba in one of the deactivated states. In mammalian Na V s, JZTx-27 preferably inhibited the inactivation of Na V 1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic Na V s. More important, we proposed that JZTx-27 stabilized the Ns V Ba VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of Na V s.-Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov-Yarovoy, V., DeCaen, P. G., Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel. © FASEB.

  17. The voltage-gated potassium channel subunit, Kv1.3, is expressed in epithelia

    DEFF Research Database (Denmark)

    Grunnet, Morten; Rasmussen, Hanne B; Hay-Schmidt, Anders

    2003-01-01

    The Shaker-type voltage-gated potassium channel, Kv1.3, is believed to be restricted in distribution to lymphocytes and neurons. In lymphocytes, this channel has gained intense attention since it has been proven that inhibition of Kv1.3 channels compromise T lymphocyte activation. To investigate...

  18. Voltage-gated sodium channel expression and action potential generation in differentiated NG108-15 cells.

    Science.gov (United States)

    Liu, Jinxu; Tu, Huiyin; Zhang, Dongze; Zheng, Hong; Li, Yu-Long

    2012-10-25

    The generation of action potential is required for stimulus-evoked neurotransmitter release in most neurons. Although various voltage-gated ion channels are involved in action potential production, the initiation of the action potential is mainly mediated by voltage-gated Na+ channels. In the present study, differentiation-induced changes of mRNA and protein expression of Na+ channels, Na+ currents, and cell membrane excitability were investigated in NG108-15 cells. Whole-cell patch-clamp results showed that differentiation (9 days) didn't change cell membrane excitability, compared to undifferentiated state. But differentiation (21 days) induced the action potential generation in 45.5% of NG108-15 cells (25/55 cells). In 9-day-differentiated cells, Na+ currents were mildly increased, which was also found in 21-day differentiated cells without action potential. In 21-day differentiated cells with action potential, Na+ currents were significantly enhanced. Western blot data showed that the expression of Na+ channels was increased with differentiated-time dependent manner. Single-cell real-time PCR data demonstrated that the expression of Na+ channel mRNA was increased by 21 days of differentiation in NG108-15 cells. More importantly, the mRNA level of Na+ channels in cells with action potential was higher than that in cells without action potential. Differentiation induces expression of voltage-gated Na+ channels and action potential generation in NG108-15 cells. A high level of the Na+ channel density is required for differentiation-triggered action potential generation.

  19. Modeling hysteresis observed in the human erythrocyte voltage-dependent cation channel

    DEFF Research Database (Denmark)

    Flyvbjerg, Henrik; Gudowska-Nowak, Ewa; Christophersen, Palle

    2012-01-01

    The non-selective voltage-activated cation channel from human red cells, which is activated at depolarizing potentials, has been shown to exhibit counter-clockwise gating hysteresis. Here, we analyze this phenomenon with the simplest possible phenomenological models. Specifically, the hysteresis ...

  20. New Role of P/Q-type Voltage-gated Calcium Channels

    DEFF Research Database (Denmark)

    Hansen, Pernille B L

    2015-01-01

    Voltage-gated calcium channels are important for the depolarization-evoked contraction of vascular smooth muscle cells (SMCs), with L-type channels being the classical channel involved in this mechanism. However, it has been demonstrated that the CaV2.1 subunit, which encodes a neuronal isoform...... of the voltage-gated calcium channels (P/Q-type), is also expressed and contributes functionally to contraction of renal blood vessels in both mice and humans. Furthermore, preglomerular vascular SMCs and aortic SMCs coexpress L-, P-, and Q-type calcium channels within the same cell. Calcium channel blockers...... are widely used as pharmacological treatments. However, calcium channel antagonists vary in their selectivity for the various calcium channel subtypes, and the functional contribution from P/Q-type channels as compared with L-type should be considered. Confirming the presence of P/Q-type voltage...

  1. Biological Membrane Ion Channels Dynamics, Structure, and Applications

    CERN Document Server

    Chung, Shin-Ho; Krishnamurthy, Vikram

    2007-01-01

    Ion channels are biological nanotubes that are formed by membrane proteins. Because ion channels regulate all electrical activities in living cells, understanding their mechanisms at a molecular level is a fundamental problem in biology. This book deals with recent breakthroughs in ion-channel research that have been brought about by the combined effort of experimental biophysicists and computational physicists, who together are beginning to unravel the story of these exquisitely designed biomolecules. With chapters by leading experts, the book is aimed at researchers in nanodevices and biosensors, as well as advanced undergraduate and graduate students in biology and the physical sciences. Key Features Presents the latest information on the molecular mechanisms of ion permeation through membrane ion channels Uses schematic diagrams to illustrate important concepts in biophysics Written by leading researchers in the area of ion channel investigations

  2. Proteoglycans, ion channels and cell-matrix adhesion

    DEFF Research Database (Denmark)

    Mitsou, Ioli; Multhaupt, Hinke A.B.; Couchman, John R.

    2017-01-01

    , maintenance, repair and disease.The cytoplasmic domains of syndecans, while having no intrinsic kinase activity, can nevertheless signal through binding proteins.All syndecans appear to be connected to the actin cytoskeleton and can therefore contribute to cell adhesion, notably to the ECM and migration.......Recent data now suggest that syndecans can regulate stretchactivated ion channels.The structure and function of the syndecans and the ion channels are reviewed here, along with an analysis of ion channel functions in cell-matrix adhesion.This area sheds new light on the syndecans, not least since evidence...

  3. Voltage-Gated Sodium Channels: Evolutionary History and Distinctive Sequence Features.

    Science.gov (United States)

    Kasimova, M A; Granata, D; Carnevale, V

    2016-01-01

    Voltage-gated sodium channels (Nav) are responsible for the rising phase of the action potential. Their role in electrical signal transmission is so relevant that their emergence is believed to be one of the crucial factors enabling development of nervous system. The presence of voltage-gated sodium-selective channels in bacteria (BacNav) has raised questions concerning the evolutionary history of the ones in animals. Here we review some of the milestones in the field of Nav phylogenetic analysis and discuss some of the most important sequence features that distinguish these channels from voltage-gated potassium channels and transient receptor potential channels. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Nonlinear drift-diffusion model of gating in K and nACh ion channels

    Energy Technology Data Exchange (ETDEWEB)

    Vaccaro, S.R. [Department of Physics, University of Adelaide, Adelaide, South Australia 5005 (Australia)], E-mail: svaccaro@physics.adelaide.edu.au

    2007-09-03

    The configuration of a sensor regulates the transition between the closed and open states of both voltage and ligand gated channels. The closed state dwell-time distribution f{sub c}(t) derived from a Fokker-Planck equation with a nonlinear diffusion coefficient is in good agreement with experimental data and can account for the power law approximation to f{sub c}(t) for a delayed rectifier K channel and a nicotinic acetylcholine (nACh) ion channel. The solution of a master equation which approximates the Fokker-Planck equation provides a better description of the small time behaviour of the dwell-time distribution and can account for the empirical rate-amplitude correlation for these ion channels.

  5. A unifying mechanism for cancer cell death through ion channel activation by HAMLET.

    Science.gov (United States)

    Storm, Petter; Klausen, Thomas Kjaer; Trulsson, Maria; Ho C S, James; Dosnon, Marion; Westergren, Tomas; Chao, Yinxia; Rydström, Anna; Yang, Henry; Pedersen, Stine Falsig; Svanborg, Catharina

    2013-01-01

    Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl2), preventing the changes in free cellular Na(+) and K(+) concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET's broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET's documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues.

  6. Ion Channel Trafficking: Control of Ion Channel Density as a Target for Arrhythmias?

    Directory of Open Access Journals (Sweden)

    Elise Balse

    2017-10-01

    Full Text Available The shape of the cardiac action potential (AP is determined by the contributions of numerous ion channels. Any dysfunction in the proper function or expression of these ion channels can result in a change in effective refractory period (ERP and lead to arrhythmia. The processes underlying the correct targeting of ion channels to the plasma membrane are complex, and have not been fully characterized in cardiac myocytes. Emerging evidence highlights ion channel trafficking as a potential causative factor in certain acquired and inherited arrhythmias, and therapies which target trafficking as opposed to pore block are starting to receive attention. In this review we present the current evidence for the mechanisms which underlie precise control of cardiac ion channel trafficking and targeting.

  7. Sensing voltage across lipid membranes

    Science.gov (United States)

    Swartz, Kenton J.

    2009-01-01

    The detection of electrical potentials across lipid bilayers by specialized membrane proteins is required for many fundamental cellular processes such as the generation and propagation of nerve impulses. These membrane proteins possess modular voltage-sensing domains, a notable example being the S1-S4 domains of voltage-activated ion channels. Ground-breaking structural studies on these domains explain how voltage sensors are designed and reveal important interactions with the surrounding lipid membrane. Although further structures are needed to fully understand the conformational changes that occur during voltage sensing, the available data help to frame several key concepts that are fundamental to the mechanism of voltage sensing. PMID:19092925

  8. Plant ion channels: gene families, physiology, and functional genomics analyses.

    Science.gov (United States)

    Ward, John M; Mäser, Pascal; Schroeder, Julian I

    2009-01-01

    Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.

  9. A single Markov-type kinetic model accounting for the macroscopic currents of all human voltage-gated sodium channel isoforms.

    Science.gov (United States)

    Balbi, Pietro; Massobrio, Paolo; Hellgren Kotaleski, Jeanette

    2017-09-01

    Modelling ionic channels represents a fundamental step towards developing biologically detailed neuron models. Until recently, the voltage-gated ion channels have been mainly modelled according to the formalism introduced by the seminal works of Hodgkin and Huxley (HH). However, following the continuing achievements in the biophysical and molecular comprehension of these pore-forming transmembrane proteins, the HH formalism turned out to carry limitations and inconsistencies in reproducing the ion-channels electrophysiological behaviour. At the same time, Markov-type kinetic models have been increasingly proven to successfully replicate both the electrophysiological and biophysical features of different ion channels. However, in order to model even the finest non-conducting molecular conformational change, they are often equipped with a considerable number of states and related transitions, which make them computationally heavy and less suitable for implementation in conductance-based neurons and large networks of those. In this purely modelling study we develop a Markov-type kinetic model for all human voltage-gated sodium channels (VGSCs). The model framework is detailed, unifying (i.e., it accounts for all ion-channel isoforms) and computationally efficient (i.e. with a minimal set of states and transitions). The electrophysiological data to be modelled are gathered from previously published studies on whole-cell patch-clamp experiments in mammalian cell lines heterologously expressing the human VGSC subtypes (from NaV1.1 to NaV1.9). By adopting a minimum sequence of states, and using the same state diagram for all the distinct isoforms, the model ensures the lightest computational load when used in neuron models and neural networks of increasing complexity. The transitions between the states are described by original ordinary differential equations, which represent the rate of the state transitions as a function of voltage (i.e., membrane potential). The

  10. Depression of voltage-activated Ca2+ release in skeletal muscle by activation of a voltage-sensing phosphatase.

    Science.gov (United States)

    Berthier, Christine; Kutchukian, Candice; Bouvard, Clément; Okamura, Yasushi; Jacquemond, Vincent

    2015-04-01

    Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) regulates the function of several types of plasma membrane ion channels. We investigated the potential role of PtdIns(4,5)P2 in Ca(2+) homeostasis and excitation-contraction (E-C) coupling of mouse muscle fibers using in vivo expression of the voltage-sensing phosphatases (VSPs) Ciona intestinalis VSP (Ci-VSP) or Danio rerio VSP (Dr-VSP). Confocal images of enhanced green fluorescent protein-tagged Dr-VSP revealed a banded pattern consistent with VSP localization within the transverse tubule membrane. Rhod-2 Ca(2+) transients generated by 0.5-s-long voltage-clamp depolarizing pulses sufficient to elicit Ca(2+) release from the sarcoplasmic reticulum (SR) but below the range at which VSPs are activated were unaffected by the presence of the VSPs. However, in Ci-VSP-expressing fibers challenged by 5-s-long depolarizing pulses, the Ca(2+) level late in the pulse (3 s after initiation) was significantly lower at 120 mV than at 20 mV. Furthermore, Ci-VSP-expressing fibers showed a reversible depression of Ca(2+) release during trains, with the peak Ca(2+) transient being reduced by ∼30% after the application of 10 200-ms-long pulses to 100 mV. A similar depression was observed in Dr-VSP-expressing fibers. Cav1.1 Ca(2+) channel-mediated current was unaffected by Ci-VSP activation. In fibers expressing Ci-VSP and a pleckstrin homology domain fused with monomeric red fluorescent protein (PLCδ1PH-mRFP), depolarizing pulses elicited transient changes in mRFP fluorescence consistent with release of transverse tubule-bound PLCδ1PH domain into the cytosol; the voltage sensitivity of these changes was consistent with that of Ci-VSP activation, and recovery occurred with a time constant in the 10-s range. Our results indicate that the PtdIns(4,5)P2 level is tightly maintained in the transverse tubule membrane of the muscle fibers

  11. Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid.

    Science.gov (United States)

    Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

    2016-07-05

    The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

  12. Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

    Science.gov (United States)

    Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

    2016-01-01

    The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane. PMID:27330112

  13. Single-channel kinetics of BK (Slo1 channels

    Directory of Open Access Journals (Sweden)

    Yanyan eGeng

    2015-01-01

    Full Text Available Single-channel kinetics has proven a powerful tool to reveal information about the gating mechanisms that control the opening and closing of ion channels. This introductory review focuses on the gating of large conductance Ca2+- and voltage-activated K+ (BK or Slo1 channels at the single-channel level. It starts with single-channel current records and progresses to presentation and analysis of single-channel data and the development of gating mechanisms in terms of discrete state Markov (DSM models. The DSM models are formulated in terms of the tetrameric modular structure of BK channels, consisting of a central transmembrane pore-gate domain (PGD attached to four surrounding transmembrane voltage sensing domains (VSD and a large intracellular cytosolic domain (CTD, also referred to as the gating ring. The modular structure and data analysis shows that the Ca2+ and voltage dependent gating considered separately can each be approximated by 10-state two-tiered models with 5 closed states on the upper tier and 5 open states on the lower tier. The modular structure and joint Ca2+ and voltage dependent gating are consistent with a 50 state two-tiered model with 25 closed states on the upper tier and 25 open states on the lower tier. Adding an additional tier of brief closed (flicker states to the 10-state or 50-state models improved the description of the gating. For fixed experimental conditions a channel would gate in only a subset of the potential number of states. The detected number of states and the correlations between adjacent interval durations are consistent with the tiered models. The examined models can account for the single-channel kinetics and the bursting behavior of gating. Ca2+ and voltage activate BK channels by predominantly increasing the effective opening rate of the channel with a smaller decrease in the effective closing rate. Ca2+ and depolarization thus activate by mainly destabilizing the closed states.

  14. Modulation of voltage-gated channel currents by harmaline and harmane.

    Science.gov (United States)

    Splettstoesser, Frank; Bonnet, Udo; Wiemann, Martin; Bingmann, Dieter; Büsselberg, Dietrich

    2005-01-01

    Harmala alkaloids are endogenous substances, which are involved in neurodegenerative disorders such as M. Parkinson, but some of them also have neuroprotective effects in the nervous system. While several sites of action at the cellular level (e.g. benzodiazepine receptors, 5-HT and GABA(A) receptors) have been identified, there is no report on how harmala alkaloids interact with voltage-gated membrane channels. The aim of this study was to investigate the effects of harmaline and harmane on voltage-activated calcium- (I(Ca(V))), sodium- (I(Na(V))) and potassium (I(K(V)))-channel currents, using the whole-cell patch-clamp method with cultured dorsal root ganglion neurones of 3-week-old rats. Currents were elicited by voltage steps from the holding potential to different command potentials. Harmaline and harmane reduced I(Ca(V)), I(Na(V)) and I(K(V)) concentration-dependent (10-500 microM) over the voltage range tested. I(Ca(V)) was reduced with an IC(50) of 100.6 microM for harmaline and by a significantly lower concentration of 75.8 microM (P<0.001, t-test) for harmane. The Hill coefficient was close to 1. Threshold concentration was around 10 microM for both substances. The steady state of inhibition of I(Ca(V)) by harmaline or harmane was reached within several minutes. The action was not use-dependent and at least partly reversible. It was mainly due to a reduction in the sustained calcium channel current (I(Ca(L+N))), while the transient voltage-gated calcium channel current (I(Ca(T))) was only partially affected. We conclude that harmaline and harmane are modulators of I(Ca(V)) in vitro. This might be related to their neuroprotective effects.

  15. Voltage-gated calcium channels of Paramecium cilia.

    Science.gov (United States)

    Lodh, Sukanya; Yano, Junji; Valentine, Megan S; Van Houten, Judith L

    2016-10-01

    Paramecium cells swim by beating their cilia, and make turns by transiently reversing their power stroke. Reversal is caused by Ca 2+ entering the cilium through voltage-gated Ca 2+ (Ca V ) channels that are found exclusively in the cilia. As ciliary Ca 2+ levels return to normal, the cell pivots and swims forward in a new direction. Thus, the activation of the Ca V channels causes cells to make a turn in their swimming paths. For 45 years, the physiological characteristics of the Paramecium ciliary Ca V channels have been known, but the proteins were not identified until recently, when the P. tetraurelia ciliary membrane proteome was determined. Three Ca V α1 subunits that were identified among the proteins were cloned and confirmed to be expressed in the cilia. We demonstrate using RNA interference that these channels function as the ciliary Ca V channels that are responsible for the reversal of ciliary beating. Furthermore, we show that Pawn (pw) mutants of Paramecium that cannot swim backward for lack of Ca V channel activity do not express any of the three Ca V 1 channels in their ciliary membrane, until they are rescued from the mutant phenotype by expression of the wild-type PW gene. These results reinforce the correlation of the three Ca V channels with backward swimming through ciliary reversal. The PwB protein, found in endoplasmic reticulum fractions, co-immunoprecipitates with the Ca V 1c channel and perhaps functions in trafficking. The PwA protein does not appear to have an interaction with the channel proteins but affects their appearance in the cilia. © 2016. Published by The Company of Biologists Ltd.

  16. Voltage-Gated Potassium Channels Kv1.3--Potentially New Molecular Target in Cancer Diagnostics and Therapy.

    Science.gov (United States)

    Teisseyre, Andrzej; Gąsiorowska, Justyna; Michalak, Krystyna

    2015-01-01

    Voltage-gated potassium channels, Kv1.3, which were discovered in 1984, are integral membrane proteins which are activated ("open") upon change of the cell membrane potential, enabling a passive flux of potassium ions across the cell membrane. The channels are expressed in many different tissues, both normal and cancer. Since 2005 it has been known that the channels are expressed not only in the plasma membrane, but also in the inner mitochondrial membrane. The activity of Kv1.3 channels plays an important role, among others, in setting the cell resting membrane potential, cell proliferation, apoptosis and volume regulation. For some years, these channels have been considered a potentially new molecular target in both the diagnostics and therapy of some cancer diseases. This review article focuses on: 1) changes of expression of the channels in cancer disorders with special regard to correlations between the channels' expression and stage of the disease, 2) influence of inhibitors of Kv1.3 channels on proliferation and apoptosis of cancer cells, 3) possible future applications of Kv1.3 channels' inhibitors in therapy of some cancer diseases. In the last section, the results of studies performed in our Laboratory of Bioelectricity on the influence of selected biologically active plant-derived compounds from the groups of flavonoids and stilbenes and their natural and synthetic derivatives on the activity of Kv1.3 channels in normal and cancer cells are reviewed. A possible application of some compounds from these groups to support therapy of cancer diseases, such as breast, colon and lymph node cancer, and melanoma or chronic lymphocytic leukemia (B-CLL), is announced.

  17. Cl- channels of the gastric parietal cell that are active at low pH.

    Science.gov (United States)

    Cuppoletti, J; Baker, A M; Malinowska, D H

    1993-06-01

    HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 m

  18. Intermediate state trapping of a voltage sensor

    DEFF Research Database (Denmark)

    Lacroix, Jérôme J; Pless, Stephan Alexander; Maragliano, Luca

    2012-01-01

    Voltage sensor domains (VSDs) regulate ion channels and enzymes by undergoing conformational changes depending on membrane electrical signals. The molecular mechanisms underlying the VSD transitions are not fully understood. Here, we show that some mutations of I241 in the S1 segment of the Shaker...... Kv channel positively shift the voltage dependence of the VSD movement and alter the functional coupling between VSD and pore domains. Among the I241 mutants, I241W immobilized the VSD movement during activation and deactivation, approximately halfway between the resting and active states......, and drastically shifted the voltage activation of the ionic conductance. This phenotype, which is consistent with a stabilization of an intermediate VSD conformation by the I241W mutation, was diminished by the charge-conserving R2K mutation but not by the charge-neutralizing R2Q mutation. Interestingly, most...

  19. ERG voltage-gated K+ channels regulate excitability and discharge dynamics of the medial vestibular nucleus neurones.

    Science.gov (United States)

    Pessia, Mauro; Servettini, Ilenio; Panichi, Roberto; Guasti, Leonardo; Grassi, Silvarosa; Arcangeli, Annarosa; Wanke, Enzo; Pettorossi, Vito Enrico

    2008-10-15

    The discharge properties of the medial vestibular nucleus neurones (MVNn) critically depend on the activity of several ion channel types. In this study we show, immunohistochemically, that the voltage-gated K(+) channels ERG1A, ERG1B, ERG2 and ERG3 are highly expressed within the vestibular nuclei of P10 and P60 mice. The role played by these channels in the spike-generating mechanisms of the MVNn and in temporal information processing was investigated electrophysiologically from mouse brain slices, in vitro, by analysing the spontaneous discharge and the response to square-, ramp- and sinusoid-like intracellular DC current injections in extracellular and whole-cell patch-clamp studies. We show that more than half of the recorded MVNn were responsive to ERG channel block (WAY-123,398, E4031), displaying an increase in spontaneous activity and discharge irregularity. The response to step and ramp current injection was also modified by ERG block showing a reduction of first spike latency, enhancement of discharge rate and reduction of the slow spike-frequency adaptation process. ERG channels influence the interspike slope without affecting the spike shape. Moreover, in response to sinusoid-like current, ERG channel block caused frequency-dependent gain enhancement and phase-lead shift. Taken together, the data demonstrate that ERG channels control the excitability of MVNn, their discharge regularity and probably their resonance properties.

  20. The Molecular Basis of Polyunsaturated Fatty Acid Interactions with the Shaker Voltage-Gated Potassium Channel.

    Directory of Open Access Journals (Sweden)

    Samira Yazdi

    2016-01-01

    Full Text Available Voltage-gated potassium (KV channels are membrane proteins that respond to changes in membrane potential by enabling K+ ion flux across the membrane. Polyunsaturated fatty acids (PUFAs induce channel opening by modulating the voltage-sensitivity, which can provide effective treatment against refractory epilepsy by means of a ketogenic diet. While PUFAs have been reported to influence the gating mechanism by electrostatic interactions to the voltage-sensor domain (VSD, the exact PUFA-protein interactions are still elusive. In this study, we report on the interactions between the Shaker KV channel in open and closed states and a PUFA-enriched lipid bilayer using microsecond molecular dynamics simulations. We determined a putative PUFA binding site in the open state of the channel located at the protein-lipid interface in the vicinity of the extracellular halves of the S3 and S4 helices of the VSD. In particular, the lipophilic PUFA tail covered a wide range of non-specific hydrophobic interactions in the hydrophobic central core of the protein-lipid interface, while the carboxylic head group displayed more specific interactions to polar/charged residues at the extracellular regions of the S3 and S4 helices, encompassing the S3-S4 linker. Moreover, by studying the interactions between saturated fatty acids (SFA and the Shaker KV channel, our study confirmed an increased conformational flexibility in the polyunsaturated carbon tails compared to saturated carbon chains, which may explain the specificity of PUFA action on channel proteins.

  1. Gambierol, a toxin produced by the dinoflagellate Gambierdiscus toxicus, is a potent blocker of voltage-gated potassium channels☆

    Science.gov (United States)

    Cuypers, Eva; Abdel-Mottaleb, Yousra; Kopljar, Ivan; Rainier, Jon D.; Raes, Adam L.; Snyders, Dirk J.; Tytgat, Jan

    2008-01-01

    In this study, we pharmacologically characterized gambierol, a marine polycyclic ether toxin which is produced by the dinoflagellate Gambierdiscus toxicus. Besides several other polycyclic ether toxins like ciguatoxins, this scarcely studied toxin is one of the compounds that may be responsible for ciguatera fish poisoning (CFP). Unfortunately, the biological target(s) that underlies CFP is still partly unknown. Today, ciguatoxins are described to specifically activate voltage-gated sodium channels by interacting with their receptor site 5. But some dispute about the role of gambierol in the CFP story shows up: some describe voltage-gated sodium channels as the target, while others pinpoint voltage-gated potassium channels as targets. Since gambierol was never tested on isolated ion channels before, it was subjected in this work to extensive screening on a panel of 17 ion channels: nine cloned voltage-gated ion channels (mammalian Nav1.1–Nav1.8 and insect Para) and eight cloned voltage-gated potassium channels (mammalian Kv1.1–Kv1.6, hERG and insect ShakerIR) expressed in Xenopus laevis oocytes using two-electrode voltage-clamp technique. All tested sodium channel subtypes are insensitive to gambierol concentrations up to 10 μM. In contrast, Kv1.2 is the most sensitive voltage-gated potassium channel subtype with almost full block (>97%) and an half maximal inhibitory concentration (IC50) of 34.5 nM. To the best of our knowledge, this is the first study where the selectivity of gambierol is tested on isolated voltage-gated ion channels. Therefore, these results lead to a better understanding of gambierol and its possible role in CFP and they may also be useful in the development of more effective treatments. PMID:18313714

  2. Effects of tetracaine on voltage-activated calcium sparks in frog intact skeletal muscle fibers.

    Science.gov (United States)

    Hollingworth, Stephen; Chandler, W Knox; Baylor, Stephen M

    2006-03-01

    The properties of Ca(2+) sparks in frog intact skeletal muscle fibers depolarized with 13 mM [K(+)] Ringer's are well described by a computational model with a Ca(2+) source flux of amplitude 2.5 pA (units of current) and duration 4.6 ms (18 degrees C; Model 2 of Baylor et al., 2002). This result, in combination with the values of single-channel Ca(2+) current reported for ryanodine receptors (RyRs) in bilayers under physiological ion conditions, 0.5 pA (Kettlun et al., 2003) to 2 pA (Tinker et al., 1993), suggests that 1-5 RyR Ca(2+) release channels open during a voltage-activated Ca(2+) spark in an intact fiber. To distinguish between one and greater than one channel per spark, sparks were measured in 8 mM [K(+)] Ringer's in the absence and presence of tetracaine, an inhibitor of RyR channel openings in bilayers. The most prominent effect of 75-100 microM tetracaine was an approximately sixfold reduction in spark frequency. The remaining sparks showed significant reductions in the mean values of peak amplitude, decay time constant, full duration at half maximum (FDHM), full width at half maximum (FWHM), and mass, but not in the mean value of rise time. Spark properties in tetracaine were simulated with an updated spark model that differed in minor ways from our previous model. The simulations show that (a) the properties of sparks in tetracaine are those expected if tetracaine reduces the number of active RyR Ca(2+) channels per spark, and (b) the single-channel Ca(2+) current of an RyR channel is normal voltage-activated sparks (i.e., in the absence of tetracaine) are produced by two or more active RyR Ca(2+) channels. The question of how the activation of multiple RyRs is coordinated is discussed.

  3. Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH.

    Science.gov (United States)

    Pinelli, Laurent; Nissant, Antoine; Edwards, Aurélie; Lourdel, Stéphane; Teulon, Jacques; Paulais, Marc

    2016-09-01

    ClC-K2, a member of the ClC family of Cl(-) channels and transporters, forms the major basolateral Cl(-) conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl(-) absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl(-), and Ca(2+) on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca(2+) strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl(-) has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl(-)/HCO3 (-) exchange in type B intercalated cells. © 2016 Pinelli et al.

  4. Glioblastoma cancer stem cell lines express functional acid sensing ion channels ASIC1a and ASIC3

    DEFF Research Database (Denmark)

    Tian, Yuemin; Bresenitz, Pia; Reska, Anna

    2017-01-01

    Acidic microenvironment is commonly observed in tumour tissues, including glioblastoma (GBM), the most aggressive and lethal brain tumour in adults. Acid sensing ion channels (ASICs) are neuronal voltage-insensitive sodium channels, which are sensors of extracellular protons. Here we studied...

  5. External pH modulates EAG superfamily K+ channels through EAG-specific acidic residues in the voltage sensor

    Science.gov (United States)

    Kazmierczak, Marcin; Zhang, Xiaofei; Chen, Bihan; Mulkey, Daniel K.; Shi, Yingtang; Wagner, Paul G.; Pivaroff-Ward, Kendra; Sassic, Jessica K.; Bayliss, Douglas A.

    2013-01-01

    The Ether-a-go-go (EAG) superfamily of voltage-gated K+ channels consists of three functionally distinct gene families (Eag, Elk, and Erg) encoding a diverse set of low-threshold K+ currents that regulate excitability in neurons and muscle. Previous studies indicate that external acidification inhibits activation of three EAG superfamily K+ channels, Kv10.1 (Eag1), Kv11.1 (Erg1), and Kv12.1 (Elk1). We show here that Kv10.2, Kv12.2, and Kv12.3 are similarly inhibited by external protons, suggesting that high sensitivity to physiological pH changes is a general property of EAG superfamily channels. External acidification depolarizes the conductance–voltage (GV) curves of these channels, reducing low threshold activation. We explored the mechanism of this high pH sensitivity in Kv12.1, Kv10.2, and Kv11.1. We first examined the role of acidic voltage sensor residues that mediate divalent cation block of voltage activation in EAG superfamily channels because protons reduce the sensitivity of Kv12.1 to Zn2+. Low pH similarly reduces Mg2+ sensitivity of Kv10.1, and we found that the pH sensitivity of Kv11.1 was greatly attenuated at 1 mM Ca2+. Individual neutralizations of a pair of EAG-specific acidic residues that have previously been implicated in divalent block of diverse EAG superfamily channels greatly reduced the pH response in Kv12.1, Kv10.2, and Kv11.1. Our results therefore suggest a common mechanism for pH-sensitive voltage activation in EAG superfamily channels. The EAG-specific acidic residues may form the proton-binding site or alternatively are required to hold the voltage sensor in a pH-sensitive conformation. The high pH sensitivity of EAG superfamily channels suggests that they could contribute to pH-sensitive K+ currents observed in vivo. PMID:23712551

  6. External pH modulates EAG superfamily K+ channels through EAG-specific acidic residues in the voltage sensor.

    Science.gov (United States)

    Kazmierczak, Marcin; Zhang, Xiaofei; Chen, Bihan; Mulkey, Daniel K; Shi, Yingtang; Wagner, Paul G; Pivaroff-Ward, Kendra; Sassic, Jessica K; Bayliss, Douglas A; Jegla, Timothy

    2013-06-01

    The Ether-a-go-go (EAG) superfamily of voltage-gated K(+) channels consists of three functionally distinct gene families (Eag, Elk, and Erg) encoding a diverse set of low-threshold K(+) currents that regulate excitability in neurons and muscle. Previous studies indicate that external acidification inhibits activation of three EAG superfamily K(+) channels, Kv10.1 (Eag1), Kv11.1 (Erg1), and Kv12.1 (Elk1). We show here that Kv10.2, Kv12.2, and Kv12.3 are similarly inhibited by external protons, suggesting that high sensitivity to physiological pH changes is a general property of EAG superfamily channels. External acidification depolarizes the conductance-voltage (GV) curves of these channels, reducing low threshold activation. We explored the mechanism of this high pH sensitivity in Kv12.1, Kv10.2, and Kv11.1. We first examined the role of acidic voltage sensor residues that mediate divalent cation block of voltage activation in EAG superfamily channels because protons reduce the sensitivity of Kv12.1 to Zn(2+). Low pH similarly reduces Mg(2+) sensitivity of Kv10.1, and we found that the pH sensitivity of Kv11.1 was greatly attenuated at 1 mM Ca(2+). Individual neutralizations of a pair of EAG-specific acidic residues that have previously been implicated in divalent block of diverse EAG superfamily channels greatly reduced the pH response in Kv12.1, Kv10.2, and Kv11.1. Our results therefore suggest a common mechanism for pH-sensitive voltage activation in EAG superfamily channels. The EAG-specific acidic residues may form the proton-binding site or alternatively are required to hold the voltage sensor in a pH-sensitive conformation. The high pH sensitivity of EAG superfamily channels suggests that they could contribute to pH-sensitive K(+) currents observed in vivo.

  7. Functional interactions at the interface between voltage-sensing and pore domains in the Shaker K(v) channel.

    Science.gov (United States)

    Soler-Llavina, Gilberto J; Chang, Tsg-Hui; Swartz, Kenton J

    2006-11-22

    Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.

  8. The secret life of ion channels: Kv1.3 potassium channels and proliferation.

    Science.gov (United States)

    Pérez-García, M Teresa; Cidad, Pilar; López-López, José R

    2018-01-01

    Kv1.3 channels are involved in the switch to proliferation of normally quiescent cells, being implicated in the control of cell cycle in many different cell types and in many different ways. They modulate membrane potential controlling K + fluxes, sense changes in potential, and interact with many signaling molecules through their intracellular domains. From a mechanistic point of view, we can describe the role of Kv1.3 channels in proliferation with at least three different models. In the "membrane potential model," membrane hyperpolarization resulting from Kv1.3 activation provides the driving force for Ca 2+ influx required to activate Ca 2+ -dependent transcription. This model explains most of the data obtained from several cells from the immune system. In the "voltage sensor model," Kv1.3 channels serve mainly as sensors that transduce electrical signals into biochemical cascades, independently of their effect on membrane potential. Kv1.3-dependent proliferation of vascular smooth muscle cells (VSMCs) could fit this model. Finally, in the "channelosome balance model," the master switch determining proliferation may be related to the control of the Kv1.3 to Kv1.5 ratio, as described in glial cells and also in VSMCs. Since the three mechanisms cannot function independently, these models are obviously not exclusive. Nevertheless, they could be exploited differentially in different cells and tissues. This large functional flexibility of Kv1.3 channels surely gives a new perspective on their functions beyond their elementary role as ion channels, although a conclusive picture of the mechanisms involved in Kv1.3 signaling to proliferation is yet to be reached.

  9. Hyperpolarization moves S4 sensors inward to open MVP, a methanococcal voltage-gated potassium channel.

    Science.gov (United States)

    Sesti, Federico; Rajan, Sindhu; Gonzalez-Colaso, Rosana; Nikolaeva, Natalia; Goldstein, Steve A N

    2003-04-01

    MVP, a Methanococcus jannaschii voltage-gated potassium channel, was cloned and shown to operate in eukaryotic and prokaryotic cells. Like pacemaker channels, MVP opens on hyperpolarization using S4 voltage sensors like those in classical channels activated by depolarization. The MVP S4 span resembles classical sensors in sequence, charge, topology and movement, traveling inward on hyperpolarization and outward on depolarization (via canaliculi in the protein that bring the extracellular and internal solutions into proximity across a short barrier). Thus, MVP opens with sensors inward indicating a reversal of S4 position and pore state compared to classical channels. Homologous channels in mammals and plants are expected to function similarly.

  10. Arginine side chain interactions and the role of arginine as a gating charge carrier in voltage sensitive ion channels

    Science.gov (United States)

    Armstrong, Craig T.; Mason, Philip E.; Anderson, J. L. Ross; Dempsey, Christopher E.

    2016-02-01

    Gating charges in voltage-sensing domains (VSD) of voltage-sensitive ion channels and enzymes are carried on arginine side chains rather than lysine. This arginine preference may result from the unique hydration properties of the side chain guanidinium group which facilitates its movement through a hydrophobic plug that seals the center of the VSD, as suggested by molecular dynamics simulations. To test for side chain interactions implicit in this model we inspected interactions of the side chains of arginine and lysine with each of the 19 non-glycine amino acids in proteins in the protein data bank. The arginine guanidinium interacts with non-polar aromatic and aliphatic side chains above and below the guanidinium plane while hydrogen bonding with polar side chains is restricted to in-plane positions. In contrast, non-polar side chains interact largely with the aliphatic part of the lysine side chain. The hydration properties of arginine and lysine are strongly reflected in their respective interactions with non-polar and polar side chains as observed in protein structures and in molecular dynamics simulations, and likely underlie the preference for arginine as a mobile charge carrier in VSD.

  11. Ion channel electrophysiology via integrated planar patch-clamp chip with on-demand drug exchange.

    Science.gov (United States)

    Chen, Chang-Yu; Tu, Ting-Yuan; Jong, De-Shien; Wo, Andrew M

    2011-06-01

    Planar patch clamp has revolutionized characterization of ion channel behavior in drug discovery primarily via advancement in high throughput. Lab use of planar technology, however, addresses different requirements and suffers from inflexibility to enable wide range of interrogation via a single cell. This work presents integration of planar patch clamp with microfluidics, achieving multiple solution exchanges for tailor-specific measurement and allowing rapid replacement of the cell-contacting aperture. Studies via endogenously expressed ion channels in HEK 293T cells were commenced to characterize the device. Results reveal the microfluidic concentration generator produces distinct solution/drug combination/concentrations on-demand. Volume-regulated chloride channel and voltage-gated potassium channels in HEK 293T cells immersed in generated solutions under various osmolarities or drug concentrations show unique channel signature under specific condition. Excitation and blockage of ion channels in a single cell was demonstrated via serial solution exchange. Robustness of the reversible bonding and ease of glass substrate replacement were proven via repeated usage of the integrated device. The present approach reveals the capability and flexibility of integrated microfluidic planar patch-clamp system for ion channel assays. Copyright © 2011 Wiley Periodicals, Inc.

  12. "Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA.

    Directory of Open Access Journals (Sweden)

    Lei Zhu

    Full Text Available CaV2.2 (N-type voltage-gated calcium channels (Ca2+ channels play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

  13. Ion channels in the central regulation of energy and glucose homeostasis

    Directory of Open Access Journals (Sweden)

    Jong-Woo eSohn

    2013-05-01

    Full Text Available Ion channels are critical regulators of neuronal excitability and synaptic function in the brain. Recent evidence suggests that ion channels expressed by neurons within the brain are responsible for regulating energy and glucose homeostasis. In addition, the central effects of neurotransmitters and hormones are at least in part achieved by modifications of ion channel activity. This review focuses on ion channels and their neuronal functions followed by a discussion of the identified roles for specific ion channels in the central pathways regulating food intake, energy expenditure, and glucose balance.

  14. Down-regulation of voltage-dependent sodium channels initiated by sodium influx in developing neurons

    International Nuclear Information System (INIS)

    Dargent, B.; Couraud, F.

    1990-01-01

    To address the issue of whether regulatory feedback exists between the electrical activity of a neuron and ion-channel density, the authors investigated the effect of Na + -channel activators (scorpion α toxin, batrachotoxin, and veratridine) on the density of Na + channels in fetal rat brain neurons in vitro. A partial but rapid (t 1/2 , 15 min) disappearance of surface Na + channels was observed as measured by a decrease in the specific binding of [ 3 H]saxitoxin and 125 I-labeled scorpion β toxin and a decrease in specific 22 Na + uptake. Moreover, the increase in the number of Na + channels that normally occurs during neuronal maturation in vitro was inhibited by chronic channel activator treatment. The induced disappearance of Na + channels was abolished by tetrodotoxin, was found to be dependent on the external Na + concentration, and was prevented when either choline (a nonpermeant ion) or Li + (a permeant ion) was substituted for Na + . Amphotericin B, a Na + ionophore, and monensin were able to mimick the effect of Na + -channel activators, while a KCl depolarization failed to do this. This feedback regulation seems to be a neuronal property since Na + -channel density in cultured astrocytes was not affected by channel activator treatment or by amphotericin B. The present evidence suggests that an increase in intracellular Na + concentration, whether elicited by Na + -channel activators or mediated by a Na + ionophore, can induce a decrease in surface Na + channels and therefore is involved in down-regulation of Na + -channel density in fetal rat brain neurons in vitro

  15. A Low-Noise Transimpedance Amplifier for BLM-Based Ion Channel Recording

    Directory of Open Access Journals (Sweden)

    Marco Crescentini

    2016-05-01

    Full Text Available High-throughput screening (HTS using ion channel recording is a powerful drug discovery technique in pharmacology. Ion channel recording with planar bilayer lipid membranes (BLM is scalable and has very high sensitivity. A HTS system based on BLM ion channel recording faces three main challenges: (i design of scalable microfluidic devices; (ii design of compact ultra-low-noise transimpedance amplifiers able to detect currents in the pA range with bandwidth >10 kHz; (iii design of compact, robust and scalable systems that integrate these two elements. This paper presents a low-noise transimpedance amplifier with integrated A/D conversion realized in CMOS 0.35 μm technology. The CMOS amplifier acquires currents in the range ±200 pA and ±20 nA, with 100 kHz bandwidth while dissipating 41 mW. An integrated digital offset compensation loop balances any voltage offsets from Ag/AgCl electrodes. The measured open-input input-referred noise current is as low as 4 fA/√Hz at ±200 pA range. The current amplifier is embedded in an integrated platform, together with a microfluidic device, for current recording from ion channels. Gramicidin-A, α-haemolysin and KcsA potassium channels have been used to prove both the platform and the current-to-digital converter.

  16. A Low-Noise Transimpedance Amplifier for BLM-Based Ion Channel Recording.

    Science.gov (United States)

    Crescentini, Marco; Bennati, Marco; Saha, Shimul Chandra; Ivica, Josip; de Planque, Maurits; Morgan, Hywel; Tartagni, Marco

    2016-05-19

    High-throughput screening (HTS) using ion channel recording is a powerful drug discovery technique in pharmacology. Ion channel recording with planar bilayer lipid membranes (BLM) is scalable and has very high sensitivity. A HTS system based on BLM ion channel recording faces three main challenges: (i) design of scalable microfluidic devices; (ii) design of compact ultra-low-noise transimpedance amplifiers able to detect currents in the pA range with bandwidth >10 kHz; (iii) design of compact, robust and scalable systems that integrate these two elements. This paper presents a low-noise transimpedance amplifier with integrated A/D conversion realized in CMOS 0.35 μm technology. The CMOS amplifier acquires currents in the range ±200 pA and ±20 nA, with 100 kHz bandwidth while dissipating 41 mW. An integrated digital offset compensation loop balances any voltage offsets from Ag/AgCl electrodes. The measured open-input input-referred noise current is as low as 4 fA/√Hz at ±200 pA range. The current amplifier is embedded in an integrated platform, together with a microfluidic device, for current recording from ion channels. Gramicidin-A, α-haemolysin and KcsA potassium channels have been used to prove both the platform and the current-to-digital converter.

  17. Evaluation of stochastic differential equation approximation of ion channel gating models.

    Science.gov (United States)

    Bruce, Ian C

    2009-04-01

    Fox and Lu derived an algorithm based on stochastic differential equations for approximating the kinetics of ion channel gating that is simpler and faster than "exact" algorithms for simulating Markov process models of channel gating. However, the approximation may not be sufficiently accurate to predict statistics of action potential generation in some cases. The objective of this study was to develop a framework for analyzing the inaccuracies and determining their origin. Simulations of a patch of membrane with voltage-gated sodium and potassium channels were performed using an exact algorithm for the kinetics of channel gating and the approximate algorithm of Fox & Lu. The Fox & Lu algorithm assumes that channel gating particle dynamics have a stochastic term that is uncorrelated, zero-mean Gaussian noise, whereas the results of this study demonstrate that in many cases the stochastic term in the Fox & Lu algorithm should be correlated and non-Gaussian noise with a non-zero mean. The results indicate that: (i) the source of the inaccuracy is that the Fox & Lu algorithm does not adequately describe the combined behavior of the multiple activation particles in each sodium and potassium channel, and (ii) the accuracy does not improve with increasing numbers of channels.

  18. Fatty acids and related Kv2 channel blockers: electrophysiology and toxicity on mosquitoes

    Science.gov (United States)

    Ligand-gated ion channels form an important superfamily of proteins involved in many biological processes. Among them, the potassium channels constitute a very diverse group involved in neural signaling, neuronal activity and action potential. Among the different types of channel activation, voltage...

  19. Sensing with Ion Channels

    CERN Document Server

    Martinac, Boris

    2008-01-01

    All living cells are able to detect and translate environmental stimuli into biologically meaningful signals. Sensations of touch, hearing, sight, taste, smell or pain are essential to the survival of all living organisms. The importance of sensory input for the existence of life thus justifies the effort made to understand its molecular origins. Sensing with Ion Channels focuses on ion channels as key molecules enabling biological systems to sense and process the physical and chemical stimuli that act upon cells in their living environment. Its aim is to serve as a reference to ion channel specialists and as a source of new information to non specialists who want to learn about the structural and functional diversity of ion channels and their role in sensory physiology.

  20. Microscopic origin of gating current fluctuations in a potassium channel voltage sensor.

    Science.gov (United States)

    Freites, J Alfredo; Schow, Eric V; White, Stephen H; Tobias, Douglas J

    2012-06-06

    Voltage-dependent ion channels open and close in response to changes in membrane electrical potential due to the motion of their voltage-sensing domains (VSDs). VSD charge displacements within the membrane electric field are observed in electrophysiology experiments as gating currents preceding ionic conduction. The elementary charge motions that give rise to the gating current cannot be observed directly, but appear as discrete current pulses that generate fluctuations in gating current measurements. Here we report direct observation of gating-charge displacements in an atomistic molecular dynamics simulation of the isolated VSD from the KvAP channel in a hydrated lipid bilayer on the timescale (10-μs) expected for elementary gating charge transitions. The results reveal that gating-charge displacements are associated with the water-catalyzed rearrangement of salt bridges between the S4 arginines and a set of conserved acidic side chains on the S1-S3 transmembrane segments in the hydrated interior of the VSD. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Ladder-Shaped Ion Channel Ligands: Current State of Knowledge

    Science.gov (United States)

    Shmukler, Yuri B.; Nikishin, Denis A.

    2017-01-01

    Ciguatoxins (CTX) and brevetoxins (BTX) are polycyclic ethereal compounds biosynthesized by the worldwide distributed planktonic and epibenthic dinoflagellates of Gambierdiscus and Karenia genera, correspondingly. Ciguatera, evoked by CTXs, is a type of ichthyosarcotoxism, which involves a variety of gastrointestinal and neurological symptoms, while BTXs cause so-called neurotoxic shellfish poisoning. Both types of toxins are reviewed together because of similar mechanisms of their action. These are the only molecules known to activate voltage-sensitive Na+-channels in mammals through a specific interaction with site 5 of its α-subunit and may compete for it, which results in an increase in neuronal excitability, neurotransmitter release and impairment of synaptic vesicle recycling. Most marine ciguatoxins potentiate Nav channels, but a considerable number of them, such as gambierol and maitotoxin, have been shown to affect another ion channel. Although the extrinsic function of these toxins is probably associated with the function of a feeding deterrent, it was suggested that their intrinsic function is coupled with the regulation of photosynthesis via light-harvesting complex II and thioredoxin. Antagonistic effects of BTXs and brevenal may provide evidence of their participation as positive and negative regulators of this mechanism. PMID:28726749

  2. Ladder-Shaped Ion Channel Ligands: Current State of Knowledge

    Directory of Open Access Journals (Sweden)

    Yuri B. Shmukler

    2017-07-01

    Full Text Available Ciguatoxins (CTX and brevetoxins (BTX are polycyclic ethereal compounds biosynthesized by the worldwide distributed planktonic and epibenthic dinoflagellates of Gambierdiscus and Karenia genera, correspondingly. Ciguatera, evoked by CTXs, is a type of ichthyosarcotoxism, which involves a variety of gastrointestinal and neurological symptoms, while BTXs cause so-called neurotoxic shellfish poisoning. Both types of toxins are reviewed together because of similar mechanisms of their action. These are the only molecules known to activate voltage-sensitive Na+-channels in mammals through a specific interaction with site 5 of its α-subunit and may compete for it, which results in an increase in neuronal excitability, neurotransmitter release and impairment of synaptic vesicle recycling. Most marine ciguatoxins potentiate Nav channels, but a considerable number of them, such as gambierol and maitotoxin, have been shown to affect another ion channel. Although the extrinsic function of these toxins is probably associated with the function of a feeding deterrent, it was suggested that their intrinsic function is coupled with the regulation of photosynthesis via light-harvesting complex II and thioredoxin. Antagonistic effects of BTXs and brevenal may provide evidence of their participation as positive and negative regulators of this mechanism.

  3. Ion channels in the central regulation of energy and glucose homeostasis

    OpenAIRE

    Sohn, Jong-Woo

    2013-01-01

    Ion channels are critical regulators of neuronal excitability and synaptic function in the brain. Recent evidence suggests that ion channels expressed by neurons within the brain are responsible for regulating energy and glucose homeostasis. In addition, the central effects of neurotransmitters and hormones are at least in part achieved by modifications of ion channel activity. This review focuses on ion channels and their neuronal functions followed by a discussion of the identified roles fo...

  4. Direct Interaction between the Voltage Sensors Produces Cooperative Sustained Deactivation in Voltage-gated H+ Channel Dimers*

    OpenAIRE

    Okuda, Hiroko; Yonezawa, Yasushige; Takano, Yu; Okamura, Yasushi; Fujiwara, Yuichiro

    2016-01-01

    The voltage-gated H+ channel (Hv) is a voltage sensor domain-like protein consisting of four transmembrane segments (S1?S4). The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively. Here we show that the two voltage sensor S4 helices within the dimer directly cooperate via a ?-stacking interaction between Trp residues at the middle of each segment. Scanning mutagenesis showed that Trp situated around the original position provides the slow gating kineti...

  5. The cooperative voltage sensor motion that gates a potassium channel.

    Science.gov (United States)

    Pathak, Medha; Kurtz, Lisa; Tombola, Francesco; Isacoff, Ehud

    2005-01-01

    The four arginine-rich S4 helices of a voltage-gated channel move outward through the membrane in response to depolarization, opening and closing gates to generate a transient ionic current. Coupling of voltage sensing to gating was originally thought to operate with the S4s moving independently from an inward/resting to an outward/activated conformation, so that when all four S4s are activated, the gates are driven to open or closed. However, S4 has also been found to influence the cooperative opening step (Smith-Maxwell et al., 1998a), suggesting a more complex mechanism of coupling. Using fluorescence to monitor structural rearrangements in a Shaker channel mutant, the ILT channel (Ledwell and Aldrich, 1999), that energetically isolates the steps of activation from the cooperative opening step, we find that opening is accompanied by a previously unknown and cooperative movement of S4. This gating motion of S4 appears to be coupled to the internal S6 gate and to two forms of slow inactivation. Our results suggest that S4 plays a direct role in gating. While large transmembrane rearrangements of S4 may be required to unlock the gating machinery, as proposed before, it appears to be the gating motion of S4 that drives the gates to open and close.

  6. Characterization of Eag1 channel lateral mobility in rat hippocampal cultures by single-particle-tracking with quantum dots.

    Directory of Open Access Journals (Sweden)

    David Gómez-Varela

    2010-01-01

    Full Text Available Voltage-gated ion channels are main players involved in fast synaptic events. However, only slow intracellular mechanisms have so far been described for controlling their localization as real-time visualization of endogenous voltage-gated channels at high temporal and spatial resolution has not been achieved yet. Using a specific extracellular antibody and quantum dots we reveal and characterize lateral mobility as a faster mechanism to dynamically control the number of endogenous ether-a-go-go (Eag1 ion channels inside synapses. We visualize Eag1 entering and leaving synapses by lateral diffusion in the plasma membrane of rat hippocampal neurons. Mathematical analysis of their trajectories revealed how the motion of Eag1 gets restricted when the channels diffuse into the synapse, suggesting molecular interactions between Eag1 and synaptic components. In contrast, Eag1 channels switch to Brownian movement when they exit synapses and diffuse into extrasynaptic membranes. Furthermore, we demonstrate that the mobility of Eag1 channels is specifically regulated inside synapses by actin filaments, microtubules and electrical activity. In summary, using single-particle-tracking techniques with quantum dots nanocrystals, our study shows for the first time the lateral diffusion of an endogenous voltage-gated ion channel in neurons. The location-dependent constraints imposed by cytoskeletal elements together with the regulatory role of electrical activity strongly suggest a pivotal role for the mobility of voltage-gated ion channels in synaptic activity.

  7. Structure of the TRPV1 ion channel determined by electron cryo-microscopy.

    Science.gov (United States)

    Liao, Maofu; Cao, Erhu; Julius, David; Cheng, Yifan

    2013-12-05

    Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5-6 (S5-S6) and the intervening pore loop, which is flanked by S1-S4 voltage-sensor-like domains. TRPV1 has a wide extracellular 'mouth' with a short selectivity filter. The conserved 'TRP domain' interacts with the S4-S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.

  8. Marine Toxins That Target Voltage-gated Sodium Channels

    Directory of Open Access Journals (Sweden)

    Robert J. French

    2006-04-01

    Full Text Available Abstract: Eukaryotic, voltage-gated sodium (NaV channels are large membrane proteins which underlie generation and propagation of rapid electrical signals in nerve, muscle and heart. Nine different NaV receptor sites, for natural ligands and/or drugs, have been identified, based on functional analyses and site-directed mutagenesis. In the marine ecosystem, numerous toxins have evolved to disrupt NaV channel function, either by inhibition of current flow through the channels, or by modifying the activation and inactivation gating processes by which the channels open and close. These toxins function in their native environment as offensive or defensive weapons in prey capture or deterrence of predators. In composition, they range from organic molecules of varying size and complexity to peptides consisting of ~10-70 amino acids. We review the variety of known NaV-targeted marine toxins, outlining, where known, their sites of interaction with the channel protein and their functional effects. In a number of cases, these natural ligands have the potential applications as drugs in clinical settings, or as models for drug development.

  9. Dual patch voltage clamp study of low membrane resistance astrocytes in situ.

    Science.gov (United States)

    Ma, Baofeng; Xu, Guangjin; Wang, Wei; Enyeart, John J; Zhou, Min

    2014-03-17

    Whole-cell patch clamp recording has been successfully used in identifying the voltage-dependent gating and conductance properties of ion channels in a variety of cells. However, this powerful technique is of limited value in studying low membrane resistance cells, such as astrocytes in situ, because of the inability to control or accurately measure the real amplitude of command voltages. To facilitate the study of ionic conductances of astrocytes, we have developed a dual patch recording method which permits membrane current and membrane potential to be simultaneously recorded from astrocytes in spite of their extraordinarily low membrane resistance. The utility of this technique is demonstrated by measuring the voltage-dependent activation of the inwardly rectifying K+ current abundantly expressed in astrocytes and multiple ionic events associated with astrocytic GABAA receptor activation. This protocol can be performed routinely in the study of astrocytes. This method will be valuable for identifying and characterizing the individual ion channels that orchestrate the electrical activity of low membrane resistance cells.

  10. Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides

    Science.gov (United States)

    Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

    2015-01-01

    Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor PI(4)P from the plasma membrane through Ca2+-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 or PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin. PMID:25670203

  11. Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities.

    Science.gov (United States)

    Hsieh, Ying-Hsin; Huang, Ying-Ju; Jin, Jin-Shan; Yu, Liyan; Yang, Hsiuchin; Jiang, Chun; Wang, Binghe; Tai, Phang C

    2014-11-14

    SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Chronic Ca2+ influx through voltage-dependent Ca2+ channels enhance delayed rectifier K+ currents via activating Src family tyrosine kinase in rat hippocampal neurons.

    Science.gov (United States)

    Yang, Yoon-Sil; Jeon, Sang-Chan; Kim, Dong-Kwan; Eun, Su-Yong; Jung, Sung-Cherl

    2017-03-01

    Excessive influx and the subsequent rapid cytosolic elevation of Ca 2+ in neurons is the major cause to induce hyperexcitability and irreversible cell damage although it is an essential ion for cellular signalings. Therefore, most neurons exhibit several cellular mechanisms to homeostatically regulate cytosolic Ca 2+ level in normal as well as pathological conditions. Delayed rectifier K + channels (I DR channels) play a role to suppress membrane excitability by inducing K + outflow in various conditions, indicating their potential role in preventing pathogenic conditions and cell damage under Ca 2+ -mediated excitotoxic conditions. In the present study, we electrophysiologically evaluated the response of I DR channels to hyperexcitable conditions induced by high Ca 2+ pretreatment (3.6 mM, for 24 hours) in cultured hippocampal neurons. In results, high Ca 2+ -treatment significantly increased the amplitude of I DR without changes of gating kinetics. Nimodipine but not APV blocked Ca 2+ -induced I DR enhancement, confirming that the change of I DR might be targeted by Ca 2+ influx through voltage-dependent Ca 2+ channels (VDCCs) rather than NMDA receptors (NMDARs). The VDCC-mediated I DR enhancement was not affected by either Ca 2+ -induced Ca 2+ release (CICR) or small conductance Ca 2+ -activated K + channels (SK channels). Furthermore, PP2 but not H89 completely abolished I DR enhancement under high Ca 2+ condition, indicating that the activation of Src family tyrosine kinases (SFKs) is required for Ca 2+ -mediated I DR enhancement. Thus, SFKs may be sensitive to excessive Ca 2+ influx through VDCCs and enhance I DR to activate a neuroprotective mechanism against Ca 2+ -mediated hyperexcitability in neurons.

  13. Cloning and functional expression of a plant voltage-dependent chloride channel.

    Science.gov (United States)

    Lurin, C; Geelen, D; Barbier-Brygoo, H; Guern, J; Maurel, C

    1996-01-01

    Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants. PMID:8624442

  14. Substitutions in the domain III voltage-sensing module enhance the sensitivity of an insect sodium channel to a scorpion beta-toxin.

    Science.gov (United States)

    Song, Weizhong; Du, Yuzhe; Liu, Zhiqi; Luo, Ningguang; Turkov, Michael; Gordon, Dalia; Gurevitz, Michael; Goldin, Alan L; Dong, Ke

    2011-05-06

    Scorpion β-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant β-toxin, Lqh-dprIT(3), from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNa(v)). Like other scorpion β-toxins, Lqh-dprIT(3) shifts the voltage dependence of activation of BgNa(v) channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNa(v) splice variants tested for their sensitivity to the toxin, only BgNa(v)1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT(3). Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion β-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation.

  15. Ion collection from laser-induced plasma by applying radio-frequency voltage

    International Nuclear Information System (INIS)

    Shibata, Takemasa; Ogura, Koichi

    1995-01-01

    Ions were collected on the electrodes from a laser resonance photoionized plasma by applying 1.8MHz radio-frequency voltage to the electrode. It was demonstrated that the ions are collected in a shorter time at the same kinetic energy of the collected ions compared with ion collection by applying DC voltage to the electrode. A simple one-dimensional model was extended for prediction of ion collection times in the cases of applications of not only the DC voltage but also the radio-frequency voltage. The ion collection times estimated using the simple one-dimensional model agreed with experimental values in both cases of DC and radio-frequency voltages. (author)

  16. High throughput electrophysiology: new perspectives for ion channel drug discovery

    DEFF Research Database (Denmark)

    Willumsen, Niels J; Bech, Morten; Olesen, Søren-Peter

    2003-01-01

    Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels....... A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct...... characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion...

  17. Ion channel density regulates switches between regular and fast spiking in soma but not in axons.

    Directory of Open Access Journals (Sweden)

    Hugo Zeberg

    2010-04-01

    Full Text Available The threshold firing frequency of a neuron is a characterizing feature of its dynamical behaviour, in turn determining its role in the oscillatory activity of the brain. Two main types of dynamics have been identified in brain neurons. Type 1 dynamics (regular spiking shows a continuous relationship between frequency and stimulation current (f-I(stim and, thus, an arbitrarily low frequency at threshold current; Type 2 (fast spiking shows a discontinuous f-I(stim relationship and a minimum threshold frequency. In a previous study of a hippocampal neuron model, we demonstrated that its dynamics could be of both Type 1 and Type 2, depending on ion channel density. In the present study we analyse the effect of varying channel density on threshold firing frequency on two well-studied axon membranes, namely the frog myelinated axon and the squid giant axon. Moreover, we analyse the hippocampal neuron model in more detail. The models are all based on voltage-clamp studies, thus comprising experimentally measurable parameters. The choice of analysing effects of channel density modifications is due to their physiological and pharmacological relevance. We show, using bifurcation analysis, that both axon models display exclusively Type 2 dynamics, independently of ion channel density. Nevertheless, both models have a region in the channel-density plane characterized by an N-shaped steady-state current-voltage relationship (a prerequisite for Type 1 dynamics and associated with this type of dynamics in the hippocampal model. In summary, our results suggest that the hippocampal soma and the two axon membranes represent two distinct kinds of membranes; membranes with a channel-density dependent switching between Type 1 and 2 dynamics, and membranes with a channel-density independent dynamics. The difference between the two membrane types suggests functional differences, compatible with a more flexible role of the soma membrane than that of the axon membrane.

  18. Effects of Voltage-Gated K+ Channel on Cell Proliferation in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Wei Wang

    2014-01-01

    Full Text Available Objective. To study the effects and underlying mechanisms of voltage-gated K+ channels on the proliferation of multiple myeloma cells. Methods. RPMI-8226 MM cell line was used for the experiments. Voltage-gated K+ currents and the resting potential were recorded by whole-cell patch-clamp technique. RT-PCR detected Kv channel mRNA expression. Cell viability was analyzed with MTT assay. Cell counting system was employed to monitor cell proliferation. DNA contents and cell volume were analyzed by flow cytometry. Results. Currents recorded in RPMI-8226 cells were confirmed to be voltage-gated K+ channels. A high level of Kv1.3 mRNA was detected but no Kv3.1 mRNA was detected in RPMI-8226 cells. Voltage-gated K+ channel blocker 4-aminopyridine (4-AP (2 mM depolarized the resting potential from −42 ± 1.7 mV to −31.8 ± 2.8 mV (P0.05. Conclusions. In RPMI-8226, voltage-gated K+ channels are involved in proliferation and cell cycle progression its influence on the resting potential and cell volume may be responsible for this process; the inhibitory effect of the voltage-gated K+ channel blocker on RPMI-8226 cell proliferation is a phase-specific event.

  19. Voltage dependence of a stochastic model of activation of an alpha helical S4 sensor in a K channel membrane

    Science.gov (United States)

    Vaccaro, S. R.

    2011-09-01

    The voltage dependence of the ionic and gating currents of a K channel is dependent on the activation barriers of a voltage sensor with a potential function which may be derived from the principal electrostatic forces on an S4 segment in an inhomogeneous dielectric medium. By variation of the parameters of a voltage-sensing domain model, consistent with x-ray structures and biophysical data, the lowest frequency of the survival probability of each stationary state derived from a solution of the Smoluchowski equation provides a good fit to the voltage dependence of the slowest time constant of the ionic current in a depolarized membrane, and the gating current exhibits a rising phase that precedes an exponential relaxation. For each depolarizing potential, the calculated time dependence of the survival probabilities of the closed states of an alpha helical S4 sensor are in accord with an empirical model of the ionic and gating currents recorded during the activation process.

  20. The CaV2.3 R-type voltage-gated Ca2+ channel in mouse sleep architecture.

    Science.gov (United States)

    Siwek, Magdalena Elisabeth; Müller, Ralf; Henseler, Christina; Broich, Karl; Papazoglou, Anna; Weiergräber, Marco

    2014-05-01

    Voltage-gated Ca(2+) channels (VGCCs) are key elements in mediating thalamocortical rhythmicity. Low-voltage activated (LVA) CaV 3 T-type Ca(2+) channels have been related to thalamic rebound burst firing and to generation of non-rapid eye movement (NREM) sleep. High-voltage activated (HVA) CaV 1 L-type Ca(2+) channels, on the opposite, favor the tonic mode of action associated with higher levels of vigilance. However, the role of the HVA Non-L-type CaV2.3 Ca(2+) channels, which are predominantly expressed in the reticular thalamic nucleus (RTN), still remains unclear. Recently, CaV2.3(-/-) mice were reported to exhibit altered spike-wave discharge (SWD)/absence seizure susceptibility supported by the observation that CaV2.3 mediated Ca(2+) influx into RTN neurons can trigger small-conductance Ca(2+)-activated K(+)-channel type 2 (SK2) currents capable of maintaining thalamic burst activity. Based on these studies we investigated the role of CaV2.3 R-type Ca(2+) channels in rodent sleep. The role of CaV2.3 Ca(2+) channels was analyzed in CaV2.3(-/-) mice and controls in both spontaneous and artificial urethane-induced sleep, using implantable video-EEG radiotelemetry. Data were analyzed for alterations in sleep architecture using sleep staging software and time-frequency analysis. CaV2.3 deficient mice exhibited reduced wake duration and increased slow-wave sleep (SWS). Whereas mean sleep stage durations remained unchanged, the total number of SWS epochs was increased in CaV2.3(-/-) mice. Additional changes were observed for sleep stage transitions and EEG amplitudes. Furthermore, urethane-induced SWS mimicked spontaneous sleep results obtained from CaV2.3 deficient mice. Quantitative Real-time PCR did not reveal changes in thalamic CaV3 T-type Ca(2+) channel expression. The detailed mechanisms of SWS increase in CaV2.3(-/-) mice remain to be determined. Low-voltage activated CaV2.3 R-type Ca(2+) channels in the thalamocortical loop and extra

  1. Ion channel regulation by phosphoinositides analyzed with VSPs – PI(4,5P2 affinity, phosphoinositide selectivity, and PI(4,5P2 pool accessibility

    Directory of Open Access Journals (Sweden)

    Alexandra eRjasanow

    2015-06-01

    Full Text Available The activity of many proteins depends on the phosphoinositide (PI content of the membrane. E.g., dynamic changes of the concentration of PI(4,5P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids.Voltage-sensitive phosphatases (VSPs turn over PI(4,5P2 to PI(4P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5P2. Because cellular PI(4,5P2 is resynthesized rapidly, steady state PI(4,5P2 changes with the degree of VSP activation and thus depends on membrane potential.Here we show that titration of endogenous PI(4,5P2 with Ci-VSP allows for the quantification of relative PI(4,5P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K+ channels to Ci-VSP allowed for comparison of PI(4,5P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5P2 and PI(4P was insensitive to VSP.Surprisingly, despite comparable PI(4,5P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5P2 that differ in their accessibility to PLC and VSPs.

  2. Two separate interfaces between the voltage sensor and pore are required for the function of voltage-dependent K(+ channels.

    Directory of Open Access Journals (Sweden)

    Seok-Yong Lee

    2009-03-01

    Full Text Available Voltage-dependent K(+ (Kv channels gate open in response to the membrane voltage. To further our understanding of how cell membrane voltage regulates the opening of a Kv channel, we have studied the protein interfaces that attach the voltage-sensor domains to the pore. In the crystal structure, three physical interfaces exist. Only two of these consist of amino acids that are co-evolved across the interface between voltage sensor and pore according to statistical coupling analysis of 360 Kv channel sequences. A first co-evolved interface is formed by the S4-S5 linkers (one from each of four voltage sensors, which form a cuff surrounding the S6-lined pore opening at the intracellular surface. The crystal structure and published mutational studies support the hypothesis that the S4-S5 linkers convert voltage-sensor motions directly into gate opening and closing. A second co-evolved interface forms a small contact surface between S1 of the voltage sensor and the pore helix near the extracellular surface. We demonstrate through mutagenesis that this interface is necessary for the function and/or structure of two different Kv channels. This second interface is well positioned to act as a second anchor point between the voltage sensor and the pore, thus allowing efficient transmission of conformational changes to the pore's gate.

  3. A voltage-gated H+ channel underlying pH homeostasis in calcifying coccolithophores.

    Directory of Open Access Journals (Sweden)

    Alison R Taylor

    2011-06-01

    Full Text Available Marine coccolithophorid phytoplankton are major producers of biogenic calcite, playing a significant role in the global carbon cycle. Predicting the impacts of ocean acidification on coccolithophore calcification has received much recent attention and requires improved knowledge of cellular calcification mechanisms. Uniquely amongst calcifying organisms, coccolithophores produce calcified scales (coccoliths in an intracellular compartment and secrete them to the cell surface, requiring large transcellular ionic fluxes to support calcification. In particular, intracellular calcite precipitation using HCO₃⁻ as the substrate generates equimolar quantities of H+ that must be rapidly removed to prevent cytoplasmic acidification. We have used electrophysiological approaches to identify a plasma membrane voltage-gated H+ conductance in Coccolithus pelagicus ssp braarudii with remarkably similar biophysical and functional properties to those found in metazoans. We show that both C. pelagicus and Emiliania huxleyi possess homologues of metazoan H(v1 H+ channels, which function as voltage-gated H+ channels when expressed in heterologous systems. Homologues of the coccolithophore H+ channels were also identified in a diversity of eukaryotes, suggesting a wide range of cellular roles for the H(v1 class of proteins. Using single cell imaging, we demonstrate that the coccolithophore H+ conductance mediates rapid H+ efflux and plays an important role in pH homeostasis in calcifying cells. The results demonstrate a novel cellular role for voltage gated H+ channels and provide mechanistic insight into biomineralisation by establishing a direct link between pH homeostasis and calcification. As the coccolithophore H+ conductance is dependent on the trans-membrane H+ electrochemical gradient, this mechanism will be directly impacted by, and may underlie adaptation to, ocean acidification. The presence of this H+ efflux pathway suggests that there is no obligate

  4. Differential association of GABAB receptors with their effector ion channels in Purkinje cells.

    Science.gov (United States)

    Luján, Rafael; Aguado, Carolina; Ciruela, Francisco; Cózar, Javier; Kleindienst, David; de la Ossa, Luis; Bettler, Bernhard; Wickman, Kevin; Watanabe, Masahiko; Shigemoto, Ryuichi; Fukazawa, Yugo

    2018-04-01

    Metabotropic GABA B receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABA B receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABA B1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABA B receptors with two key effector ion channels, the G protein-gated inwardly rectifying K + (GIRK/Kir3) channel and the voltage-dependent Ca 2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABA B receptors co-assembled with GIRK and Ca V 2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABA B1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABA B1 and Ca V 2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABA B1 and GIRK2 or Ca V 2.1 channels was detected, inter-cluster distance for GABA B1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABA B1 and Ca V 2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABA B receptors are associated with GIRK and Ca V 2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABA B receptors and their effector ion channels in the cerebellar network.

  5. Cells exposed to a huntingtin fragment containing an expanded polyglutamine tract show no sign of ion channel formation: results arguing against the ion channel hypothesis

    DEFF Research Database (Denmark)

    Nørremølle, Anne; Grunnet, Morten; Hasholt, Lis

    2003-01-01

    Ion channels formed by expanded polyglutamine tracts have been proposed to play an important role in the pathological processes leading to neurodegeneration in Huntington's disease and other CAG repeat diseases. We tested the capacity of a huntingtin fragment containing an expanded polyglutamine...... in the currents recorded in any of the two expression systems, indicating no changes in ion channel activity. The results therefore argue against the proposed hypothesis of expanded polyglutamines forming ion channels....

  6. Suppression of ion conductance by electro-osmotic flow in nano-channels with weakly overlapping electrical double layers

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2016-08-01

    Full Text Available This theoretical study investigates the nonlinear ionic current-voltage characteristics of nano-channels that have weakly overlapping electrical double layers. Numerical simulations as well as a 1-D mathematical model are developed to reveal that the electro-osmotic flow (EOF interplays with the concentration-polarization process and depletes the ion concentration inside the channels, thus significantly suppressing the channel conductance. The conductance may be restored at high electrical biases in the presence of recirculating vortices within the channels. As a result of the EOF-driven ion depletion, a limiting-conductance behavior is identified, which is intrinsically different from the classical limiting-current behavior.

  7. Extracts and compounds active on TRP ion channels from Waldheimia glabra, a ritual medicinal plant from Himalaya.

    Science.gov (United States)

    Giorgi, Annamaria; Bassoli, Angela; Borgonovo, Gigliola; Panseri, Sara; Manzo, Alessandra; Pentimalli, Daniela; Schiano Moriello, Aniello; De Petrocellis, Luciano

    2017-08-15

    Waldheimia glabra (Decne.) Regel is a wild plant from the Himalayan Mountains, commonly known as Smooth Ground Daisy. This plant is traditionally used by local populations in religious rituals (incense) or in traditional herbal medicine to treat skin diseases, headache, joint pain and fever. In literature few data are available on the investigation of this aromatic plant. The present work aims at deepening knowledge about the chemical composition of W. glabra extracts and incense, as well as its activity on TRP ion channels. Extracts and incense of W. glabra were analyzed by using HS-SPME GC/MS, GC/MS and NMR analysis. Tests on the activity of W. glabra extracts and isolated compounds (+)-ludartin 1 and B-ring-homo-tonghaosu 2 on TRP channels were also performed. Some extracts and pure compounds from W. glabra showed an interesting activity in terms of efficacy and potency on rat TRPA1, an ion channel involved in several sensory mechanisms, including pungency, environmental irritation and pain perception. Activity is discussed and compared with that of other known TRPA1 natural agonists with different chemical structures. All compounds showed only a negligible inhibition activity on rat TRPM8 ion channel. Our findings demonstrate that W. glabra is involved in the receptor activation mechanism and therefore represents a new natural product potentially useful in pharmaceutical and agrifood research. Copyright © 2017 Elsevier GmbH. All rights reserved.

  8. Ciguatoxins Evoke Potent CGRP Release by Activation of Voltage-Gated Sodium Channel Subtypes NaV1.9, NaV1.7 and NaV1.1

    Directory of Open Access Journals (Sweden)

    Filip Touska

    2017-08-01

    Full Text Available Ciguatoxins (CTXs are marine toxins that cause ciguatera fish poisoning, a debilitating disease dominated by sensory and neurological disturbances that include cold allodynia and various painful symptoms as well as long-lasting pruritus. Although CTXs are known as the most potent mammalian sodium channel activator toxins, the etiology of many of its neurosensory symptoms remains unresolved. We recently described that local application of 1 nM Pacific Ciguatoxin-1 (P-CTX-1 into the skin of human subjects induces a long-lasting, painful axon reflex flare and that CTXs are particularly effective in releasing calcitonin-gene related peptide (CGRP from nerve terminals. In this study, we used mouse and rat skin preparations and enzyme-linked immunosorbent assays (ELISA to study the molecular mechanism by which P-CTX-1 induces CGRP release. We show that P-CTX-1 induces CGRP release more effectively in mouse as compared to rat skin, exhibiting EC50 concentrations in the low nanomolar range. P-CTX-1-induced CGRP release from skin is dependent on extracellular calcium and sodium, but independent from the activation of various thermosensory transient receptor potential (TRP ion channels. In contrast, lidocaine and tetrodotoxin (TTX reduce CGRP release by 53–75%, with the remaining fraction involving L-type and T-type voltage-gated calcium channels (VGCC. Using transgenic mice, we revealed that the TTX-resistant voltage-gated sodium channel (VGSC NaV1.9, but not NaV1.8 or NaV1.7 alone and the combined activation of the TTX-sensitive VGSC subtypes NaV1.7 and NaV1.1 carry the largest part of the P-CTX-1-caused CGRP release of 42% and 34%, respectively. Given the contribution of CGRP to nociceptive and itch sensing pathways, our findings contribute to a better understanding of sensory symptoms of acute and chronic ciguatera that may help in the identification of potential therapeutics.

  9. Ciguatoxins Evoke Potent CGRP Release by Activation of Voltage-Gated Sodium Channel Subtypes NaV1.9, NaV1.7 and NaV1.1

    Science.gov (United States)

    Touska, Filip; Sattler, Simon; Malsch, Philipp; Lewis, Richard J.; Zimmermann, Katharina

    2017-01-01

    Ciguatoxins (CTXs) are marine toxins that cause ciguatera fish poisoning, a debilitating disease dominated by sensory and neurological disturbances that include cold allodynia and various painful symptoms as well as long-lasting pruritus. Although CTXs are known as the most potent mammalian sodium channel activator toxins, the etiology of many of its neurosensory symptoms remains unresolved. We recently described that local application of 1 nM Pacific Ciguatoxin-1 (P-CTX-1) into the skin of human subjects induces a long-lasting, painful axon reflex flare and that CTXs are particularly effective in releasing calcitonin-gene related peptide (CGRP) from nerve terminals. In this study, we used mouse and rat skin preparations and enzyme-linked immunosorbent assays (ELISA) to study the molecular mechanism by which P-CTX-1 induces CGRP release. We show that P-CTX-1 induces CGRP release more effectively in mouse as compared to rat skin, exhibiting EC50 concentrations in the low nanomolar range. P-CTX-1-induced CGRP release from skin is dependent on extracellular calcium and sodium, but independent from the activation of various thermosensory transient receptor potential (TRP) ion channels. In contrast, lidocaine and tetrodotoxin (TTX) reduce CGRP release by 53–75%, with the remaining fraction involving L-type and T-type voltage-gated calcium channels (VGCC). Using transgenic mice, we revealed that the TTX-resistant voltage-gated sodium channel (VGSC) NaV1.9, but not NaV1.8 or NaV1.7 alone and the combined activation of the TTX-sensitive VGSC subtypes NaV1.7 and NaV1.1 carry the largest part of the P-CTX-1-caused CGRP release of 42% and 34%, respectively. Given the contribution of CGRP to nociceptive and itch sensing pathways, our findings contribute to a better understanding of sensory symptoms of acute and chronic ciguatera that may help in the identification of potential therapeutics. PMID:28867800

  10. Constraints on voltage sensor movement in the shaker K+ channel.

    Science.gov (United States)

    Darman, Rachel B; Ivy, Allison A; Ketty, Vina; Blaustein, Robert O

    2006-12-01

    In nerve and muscle cells, the voltage-gated opening and closing of cation-selective ion channels is accompanied by the translocation of 12-14 elementary charges across the membrane's electric field. Although most of these charges are carried by residues in the S4 helix of the gating module of these channels, the precise nature of their physical movement is currently the topic of spirited debate. Broadly speaking, two classes of models have emerged: those that suggest that small-scale motions can account for the extensive charge displacement, and those that invoke a much larger physical movement. In the most recent incarnation of the latter type of model, which is based on structural and functional data from the archaebacterial K(+) channel KvAP, a "voltage-sensor paddle" comprising a helix-turn-helix of S3-S4 translocates approximately 20 A through the bilayer during the gating cycle (Jiang, Y., A. Lee, J. Chen, V. Ruta, M. Cadene, B.T. Chait, and R. MacKinnon. 2003. Nature. 423:33-41; Jiang, Y., V. Ruta, J. Chen, A. Lee, and R. MacKinnon. 2003. Nature. 423:42-48.; Ruta, V., J. Chen, and R. MacKinnon. 2005. Cell. 123:463-475). We used two methods to test for analogous motions in the Shaker K(+) channel, each examining the aqueous exposure of residues near S3. In the first, we employed a pore-blocking maleimide reagent (Blaustein, R.O., P.A. Cole, C. Williams, and C. Miller. 2000. Nat. Struct. Biol. 7:309-311) to probe for state-dependent changes in the chemical reactivity of substituted cysteines; in the second, we tested the state-dependent accessibility of a tethered biotin to external streptavidin (Qiu, X.Q., K.S. Jakes, A. Finkelstein, and S.L. Slatin. 1994. J. Biol. Chem. 269:7483-7488; Slatin, S.L., X.Q. Qiu, K.S. Jakes, and A. Finkelstein. 1994. Nature. 371:158-161). In both types of experiments, residues predicted to lie near the top of S3 did not exhibit any change in aqueous exposure during the gating cycle. This lack of state dependence argues against

  11. T-type voltage-gated calcium channels regulate the tone of mouse efferent arterioles

    DEFF Research Database (Denmark)

    Poulsen, Christian B; Al-Mashhadi, Rozh H; Cribbs, Leanne L

    2011-01-01

    Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation-contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar...... tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium...... by immunocytochemistry to be located in mouse efferent arterioles, human pre- and postglomerular vasculature, and Ca(v)3.2 in rat glomerular arterioles. Inhibition of endothelial nitric oxide synthase by L-NAME or its deletion by gene knockout changed the potassium-elicited transient constriction to a sustained response...

  12. Scorpion β-toxin interference with NaV channel voltage sensor gives rise to excitatory and depressant modes

    Science.gov (United States)

    Leipold, Enrico; Borges, Adolfo

    2012-01-01

    Scorpion β toxins, peptides of ∼70 residues, specifically target voltage-gated sodium (NaV) channels to cause use-dependent subthreshold channel openings via a voltage–sensor trapping mechanism. This excitatory action is often overlaid by a not yet understood depressant mode in which NaV channel activity is inhibited. Here, we analyzed these two modes of gating modification by β-toxin Tz1 from Tityus zulianus on heterologously expressed NaV1.4 and NaV1.5 channels using the whole cell patch-clamp method. Tz1 facilitated the opening of NaV1.4 in a use-dependent manner and inhibited channel opening with a reversed use dependence. In contrast, the opening of NaV1.5 was exclusively inhibited without noticeable use dependence. Using chimeras of NaV1.4 and NaV1.5 channels, we demonstrated that gating modification by Tz1 depends on the specific structure of the voltage sensor in domain 2. Although residue G658 in NaV1.4 promotes the use-dependent transitions between Tz1 modification phenotypes, the equivalent residue in NaV1.5, N803, abolishes them. Gating charge neutralizations in the NaV1.4 domain 2 voltage sensor identified arginine residues at positions 663 and 669 as crucial for the outward and inward movement of this sensor, respectively. Our data support a model in which Tz1 can stabilize two conformations of the domain 2 voltage sensor: a preactivated outward position leading to NaV channels that open at subthreshold potentials, and a deactivated inward position preventing channels from opening. The results are best explained by a two-state voltage–sensor trapping model in that bound scorpion β toxin slows the activation as well as the deactivation kinetics of the voltage sensor in domain 2. PMID:22450487

  13. Active voltage contrast imaging of cross-sectional surface of multilayer ceramic capacitor using helium ion microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Sakai, C., E-mail: SAKAI.Chikako@nims.go.jp; Ishida, N.; Masuda, H.; Nagano, S.; Kitahara, M.; Fujita, D. [National Institute for Materials Science, Tsukuba, Ibaraki 305-0047 (Japan); Ogata, Y. [TAIYO YUDEN CO., LTD., Takasaki-shi, Gunma 370-3347 (Japan)

    2016-08-01

    We studied active voltage contrast (AVC) imaging using helium ion microscopy (HIM). We observed secondary electron (SE) images of the cross-sectional surface of multilayer ceramic capacitors (MLCCs) with and without a voltage applied to the internal electrodes. When no voltage was applied, we obtained an image reflecting the material contrast between the Ni internal electrode region and the BaTiO{sub 3} dielectric region of the cross-sectional surface of the MLCC. When a voltage was applied, the electrical potential difference between the grounded and the positively biased internal electrodes affected the contrast (voltage contrast). Moreover, attenuation of the SE intensity from the grounded to the positively biased internal electrodes was observed in the dielectric region. Kelvin probe force microscopy (KPFM) measurements of the contact potential difference (CPD) were performed on the same sample. By using the AVC image from the HIM observation and the CPD image from the KPFM measurement, we could quantitatively evaluate the electrical potential. We think that the results of this study will lead to an expansion in the number of applications of HIM.

  14. Active Dendrites and Differential Distribution of Calcium Channels Enable Functional Compartmentalization of Golgi Cells.

    Science.gov (United States)

    Rudolph, Stephanie; Hull, Court; Regehr, Wade G

    2015-11-25

    Interneurons are essential to controlling excitability, timing, and synaptic integration in neuronal networks. Golgi cells (GoCs) serve these roles at the input layer of the cerebellar cortex by releasing GABA to inhibit granule cells (grcs). GoCs are excited by mossy fibers (MFs) and grcs and provide feedforward and feedback inhibition to grcs. Here we investigate two important aspects of GoC physiology: the properties of GoC dendrites and the role of calcium signaling in regulating GoC spontaneous activity. Although GoC dendrites are extensive, previous studies concluded they are devoid of voltage-gated ion channels. Hence, the current view holds that somatic voltage signals decay passively within GoC dendrites, and grc synapses onto distal dendrites are not amplified and are therefore ineffective at firing GoCs because of strong passive attenuation. Using whole-cell recording and calcium imaging in rat slices, we find that dendritic voltage-gated sodium channels allow somatic action potentials to activate voltage-gated calcium channels (VGCCs) along the entire dendritic length, with R-type and T-type VGCCs preferentially located distally. We show that R- and T-type VGCCs located in the dendrites can boost distal synaptic inputs and promote burst firing. Active dendrites are thus critical to the regulation of GoC activity, and consequently, to the processing of input to the cerebellar cortex. In contrast, we find that N-type channels are preferentially located near the soma, and control the frequency and pattern of spontaneous firing through their close association with calcium-activated potassium (KCa) channels. Thus, VGCC types are differentially distributed and serve specialized functions within GoCs. Interneurons are essential to neural processing because they modulate excitability, timing, and synaptic integration within circuits. At the input layer of the cerebellar cortex, a single type of interneuron, the Golgi cell (GoC), carries these functions. The

  15. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    Science.gov (United States)

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  16. Analytical threshold voltage modeling of ion-implanted strained-Si double-material double-gate (DMDG) MOSFETs

    Science.gov (United States)

    Goel, Ekta; Singh, Balraj; Kumar, Sanjay; Singh, Kunal; Jit, Satyabrata

    2017-04-01

    Two dimensional threshold voltage model of ion-implanted strained-Si double-material double-gate MOSFETs has been done based on the solution of two dimensional Poisson's equation in the channel region using the parabolic approximation method. Novelty of the proposed device structure lies in the amalgamation of the advantages of both the strained-Si channel and double-material double-gate structure with a vertical Gaussian-like doping profile. The effects of different device parameters (such as device channel length, gate length ratios, germanium mole fraction) and doping parameters (such as projected range, straggle parameter) on threshold voltage of the proposed structure have been investigated. It is observed that the subthreshold performance of the device can be improved by simply controlling the doping parameters while maintaining other device parameters constant. The modeling results show a good agreement with the numerical simulation data obtained by using ATLAS™, a 2D device simulator from SILVACO.

  17. Curcumin ((E,E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) activates and desensitizes the nociceptor ion channel TRPA1.

    Science.gov (United States)

    Leamy, Andrew W; Shukla, Praveen; McAlexander, Michael A; Carr, Michael J; Ghatta, Srinivas

    2011-10-10

    The ion channel TRPA1 is activated by a wide variety of noxious stimuli, such as pollutants, products of oxidative tissue damage, and pungent natural products. Many TRPA1 activators are reactive electrophiles that form Michael adducts with cysteine and lysine residues of TRPA1's intracellular N-terminus. Curcumin, the active principle of turmeric root (Curcuma longa), can also form Michael adducts. In order to test the hypothesis that the electrophilic curcumin activates TRPA1, we have performed whole-cell, voltage-clamp analysis on both HEK293 cells expressing human TRPA1 (hTRPA1-HEK) and native mouse vagal neurons. In nominally calcium-free extracellular and intracellular solutions which minimized the chances of calcium-dependent activation of TRPA1, curcumin increased TRPA1 currents in hTRPA1-HEK cells in a concentration-dependent manner (1-30μM) but did not cause block or activation of recombinant TRPM8 and TRPV1. In addition, 7 out of 11 vagal sensory neurons from wild type mice responded to curcumin (30μM) with inward currents (11.6±5.4pA/pF) that were largely reversed by TRPA1 blockers. In marked contrast, neurons from TRPA1-deficient mice did not respond to curcumin (30μM). With physiological levels of calcium added to the external solution to facilitate channel desensitization, curcumin-dependent currents in hTRPA1-HEK cells were completely desensitized and exhibited marked tachyphylaxis upon subsequent application of curcumin. Taken together, these results demonstrate that curcumin causes activation and subsequent desensitization of native and recombinant TRPA1 ion channels of multiple mammalian species. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP

    DEFF Research Database (Denmark)

    Lundby, Alicia; Akemann, Walther; Knöpfel, Thomas

    2010-01-01

    A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane...... as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy....... Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing...

  19. Identification of an HV 1 voltage-gated proton channel in insects.

    Science.gov (United States)

    Chaves, Gustavo; Derst, Christian; Franzen, Arne; Mashimo, Yuta; Machida, Ryuichiro; Musset, Boris

    2016-04-01

    The voltage-gated proton channel 1 (HV 1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV 1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV 1 channel. In the present study, we report the existence of an HV 1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative HV 1 orthologues in various polyneopteran insects. To confirm that these putative HV 1 orthologues were functional channels, we studied the HV 1 channel of Nicoletia phytophila (NpHV 1), an insect of the Zygentoma order, in more detail. NpHV 1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, NpHV 1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels. Nucleotide sequence data have been deposited in the GenBank database under accession number KT780722. © 2016 Federation of European Biochemical Societies.

  20. Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

    Energy Technology Data Exchange (ETDEWEB)

    Gaudioso, Christelle; Carlier, Edmond; Youssouf, Fahamoe [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France); Clare, Jeffrey J. [Eaton Pharma Consulting, Eaton Socon, Cambridgeshire PE19 8EF (United Kingdom); Debanne, Dominique [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France); Alcaraz, Gisele, E-mail: gisele.alcaraz@univmed.fr [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France)

    2011-07-29

    Highlights: {yields} Both Ca{sup ++}-Calmodulin (CaM) and Ca{sup ++}-free CaM bind to the C-terminal region of Nav1.1. {yields} Ca{sup ++} and CaM have both opposite and convergent effects on I{sub Nav1.1}. {yields} Ca{sup ++}-CaM modulates I{sub Nav1.1} amplitude. {yields} CaM hyperpolarizes the voltage-dependence of activation, and increases the inactivation rate. {yields} Ca{sup ++} alone antagonizes CaM for both effects, and depolarizes the voltage-dependence of inactivation. -- Abstract: Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca{sup ++} depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca{sup ++} could bind the Nav1.1 C-terminal region with micromolar affinity.

  1. Current-voltage characteristics of quantum-point contacts in the closed-channel regime: Transforming the bias voltage into an energy scale

    DEFF Research Database (Denmark)

    Gloos, K.; Utko, P.; Aagesen, M.

    2006-01-01

    We investigate the I(V) characteristics (current versus bias voltage) of side-gated quantum-point contacts, defined in GaAs/AlxGa1-xAs heterostructures. These point contacts are operated in the closed-channel regime, that is, at fixed gate voltages below zero-bias pinch-off for conductance. Our....... Such a built-in energy-voltage calibration allows us to distinguish between the different contributions to the electron transport across the pinched-off contact due to thermal activation or quantum tunneling. The first involves the height of the barrier, and the latter also its length. In the model that we...

  2. The Challenge of Interpreting Glutamate-Receptor Ion-Channel Structures.

    Science.gov (United States)

    Mayer, Mark L

    2017-11-21

    Ion channels activated by glutamate mediate excitatory synaptic transmission in the central nervous system. Similar to other ligand-gated ion channels, their gating cycle begins with transitions from a ligand-free closed state to glutamate-bound active and desensitized states. In an attempt to reveal the molecular mechanisms underlying gating, numerous structures for glutamate receptors have been solved in complexes with agonists, antagonists, allosteric modulators, and auxiliary proteins. The embarrassingly rich library of structures emerging from this work reveals very dynamic molecules with a more complex conformational spectrum than anticipated from functional studies. Unanticipated conformations solved for complexes with competitive antagonists and a lack of understanding of the structural basis for ion channel subconductance states further highlight challenges that have yet to be addressed. Published by Elsevier Inc.

  3. Ion channel gene expressions in infertile men: A case-control study

    Directory of Open Access Journals (Sweden)

    Serkan Carkci

    2017-12-01

    Full Text Available Background: Infertility is described as not receiving pregnancy despite unprotected and regular sexual intercourse in a 1 yr period. It is detected by 15% of the couples. Male and female factor in the etiology may be detected in similar rates. Objective: The present study aims to investigate ion channel gene expression in semen samples of infertile male compared with fertile men. Materials and Methods: A total of 150 men who applied to the urology clinic due to infertility were divided into five equal groups: asthenozoospermia, oligozoospermia, oligoasthenoteratozoospermia, teratozoospermia, and normozoospermia (control. All paticipants were evaluated with Cation Channel Spermia (CatSper 1, 2, 3, 4, Proton Voltage Gated Ion Channel1 (Hv1, Potassium Channel Subfamily U1 (KCNU1, and transmembrane protein (TMEM16A gene expression in semen samples. Results: “CatSper1, 4, HV1, KCNU1, and TMEM16A gene expression were detected higher in the oligozoospermia group compared to the controls. CatSper1, 2, 3, 4, KCNU1, and TMEM16A gene expression in the asthenozoospermia group and CatSper1, 2, 3, 4, KCNU1, and TMEM16A gene expression in the teratozoospermia group were detected lower compared to the controls. CatSper1, 4, HV1, and TMEM16A gen expression were higher in the oligoasthenoteratozoospermia men than the controls while CatSper3 gen expression was detected as lower.” Conclusion: It was detected that these ion channels have an effect on sperm progressive motility and morphology. It may be considered that mutations in these ion channels may result in infertility

  4. IBiSA_Tools: A Computational Toolkit for Ion-Binding State Analysis in Molecular Dynamics Trajectories of Ion Channels.

    Directory of Open Access Journals (Sweden)

    Kota Kasahara

    Full Text Available Ion conduction mechanisms of ion channels are a long-standing conundrum. Although the molecular dynamics (MD method has been extensively used to simulate ion conduction dynamics at the atomic level, analysis and interpretation of MD results are not straightforward due to complexity of the dynamics. In our previous reports, we proposed an analytical method called ion-binding state analysis to scrutinize and summarize ion conduction mechanisms by taking advantage of a variety of analytical protocols, e.g., the complex network analysis, sequence alignment, and hierarchical clustering. This approach effectively revealed the ion conduction mechanisms and their dependence on the conditions, i.e., ion concentration and membrane voltage. Here, we present an easy-to-use computational toolkit for ion-binding state analysis, called IBiSA_tools. This toolkit consists of a C++ program and a series of Python and R scripts. From the trajectory file of MD simulations and a structure file, users can generate several images and statistics of ion conduction processes. A complex network named ion-binding state graph is generated in a standard graph format (graph modeling language; GML, which can be visualized by standard network analyzers such as Cytoscape. As a tutorial, a trajectory of a 50 ns MD simulation of the Kv1.2 channel is also distributed with the toolkit. Users can trace the entire process of ion-binding state analysis step by step. The novel method for analysis of ion conduction mechanisms of ion channels can be easily used by means of IBiSA_tools. This software is distributed under an open source license at the following URL: http://www.ritsumei.ac.jp/~ktkshr/ibisa_tools/.

  5. Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains.

    Science.gov (United States)

    Hsu, Eric J; Zhu, Wandi; Schubert, Angela R; Voelker, Taylor; Varga, Zoltan; Silva, Jonathan R

    2017-03-06

    Functional eukaryotic voltage-gated Na + (Na V ) channels comprise four domains (DI-DIV), each containing six membrane-spanning segments (S1-S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1-S4), which together form a voltage-sensing domain (VSD). A critical Na V channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair Na V channel inactivation. These locations include the DIII-DIV linker, the DIII S4-S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIV-VSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII-DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery. © 2017 Hsu et al.

  6. Developmental regulation of voltage-sensitive sodium channels in rat skeletal muscle

    International Nuclear Information System (INIS)

    Sherman, S.J.

    1985-01-01

    The developmental regulation of the voltage-sensitive Na + channel in rat skeletal muscle was studied in vivo and in vitro. In triceps surae muscle developing in vivo the development of TTX-sensitive Na + channel occurred primarily during the first three postnatal weeks as determined by the specific binding of [ 3 H]saxitoxin. This development proceeded in two separate phases. The first phase occurs independently of continuing motor neuron innervation and accounts for 60% of the adult density of TTX-sensitive Na + channels. The second phase, which begins about day 11, requires innervation. Muscle cells in primary culture were found to have both TTX-sensitive and insensitive Na + channels. The development of the TTX-sensitive channel, in vitro, paralleled the initial innervation-independent phase of development observed in vivo. The density of TTX-sensitive Na + channels in cultured muscle cells was regulated by electrical activity and cytosolic Ca ++ levels. Pharmacological blockade of the spontaneous electrical activity present in these cells lead to a nearly 2-fold increase in the surface density of TTX-sensitive channels. The turnover time of the TTX-sensitive Na + channel was measured by blocking the incorporation of newly synthesized channels with tunicamycin, an inhibitor of N-linked protein glycosylation. The regulation of channel density by electrical activity, cytosolic Ca ++ levels, and agents affecting cyclic neucleotide levels had no effect on the turnover time of the TTX-sensitive Na + channel, indicating that these regulatory agents instead affect the synthesis of the channel

  7. Cation gating and selectivity in a purified, reconstituted, voltage-dependent sodium channel

    International Nuclear Information System (INIS)

    Barchi, R.L.; Tanaka, J.C.

    1984-01-01

    In excitable membranes, the voltage-dependent sodium channel controls the primary membrane conductance change necessary for the generation of an action potential. Over the past four decades, the time- and voltage-dependent sodium currents gated by this channel have been thoroughly documented with increasingly sophisticated voltage-clamp techniques. Recent advances in the biochemistry of membrane proteins have led to the solubilization and purification of this channel protein from nerve (6) and from muscle (4) or muscle-derived (1) membranes, and have provided an approach to the correlation of the channel's molecular structure with its functional properties. Each of these sodium channel preparations appears to contain a large glycoprotein either as its sole component (2) or in association with several small subunits (6, 3). Evidence that these purified proteins represent the excitable membrane sodium channel is presented. 8 refs., 1 fig., 1 tab

  8. A Statistical Thermodynamic Model for Ligands Interacting With Ion Channels: Theoretical Model and Experimental Validation of the KCNQ2 Channel

    Directory of Open Access Journals (Sweden)

    Fang Bai

    2018-03-01

    Full Text Available Ion channels are important therapeutic targets, and their pharmacology is becoming increasingly important. However, knowledge of the mechanism of interaction of the activators and ion channels is still limited due to the complexity of the mechanisms. A statistical thermodynamic model has been developed in this study to characterize the cooperative binding of activators to ion channels. By fitting experimental concentration-response data, the model gives eight parameters for revealing the mechanism of an activator potentiating an ion channel, i.e., the binding affinity (KA, the binding cooperative coefficients for two to four activator molecules interacting with one channel (γ, μ, and ν, and the channel conductance coefficients for four activator binding configurations of the channel (a, b, c, and d. Values for the model parameters and the mechanism underlying the interaction of ztz240, a proven KCNQ2 activator, with the wild-type channel have been obtained and revealed by fitting the concentration-response data of this activator potentiating the outward current amplitudes of KCNQ2. With these parameters, our model predicted an unexpected bi-sigmoid concentration-response curve of ztz240 activation of the WT-F137A mutant heteromeric channel that was in good agreement with the experimental data determined in parallel in this study, lending credence to the assumptions on which the model is based and to the model itself. Our model can provide a better fit to the measured data than the Hill equation and estimates the binding affinity, as well as the cooperative coefficients for the binding of activators and conductance coefficients for binding states, which validates its use in studying ligand-channel interaction mechanisms.

  9. Channel sialic acids limit hERG channel activity during the ventricular action potential.

    Science.gov (United States)

    Norring, Sarah A; Ednie, Andrew R; Schwetz, Tara A; Du, Dongping; Yang, Hui; Bennett, Eric S

    2013-02-01

    Activity of human ether-a-go-go-related gene (hERG) 1 voltage-gated K(+) channels is responsible for portions of phase 2 and phase 3 repolarization of the human ventricular action potential. Here, we questioned whether and how physiologically and pathophysiologically relevant changes in surface N-glycosylation modified hERG channel function. Voltage-dependent hERG channel gating and activity were evaluated as expressed in a set of Chinese hamster ovary (CHO) cell lines under conditions of full glycosylation, no sialylation, no complex N-glycans, and following enzymatic deglycosylation of surface N-glycans. For each condition of reduced glycosylation, hERG channel steady-state activation and inactivation relationships were shifted linearly by significant depolarizing ∼9 and ∼18 mV, respectively. The hERG window current increased significantly by 50-150%, and the peak shifted by a depolarizing ∼10 mV. There was no significant change in maximum hERG current density. Deglycosylated channels were significantly more active (20-80%) than glycosylated controls during phases 2 and 3 of action potential clamp protocols. Simulations of hERG current and ventricular action potentials corroborated experimental data and predicted reduced sialylation leads to a 50-70-ms decrease in action potential duration. The data describe a novel mechanism by which hERG channel gating is modulated through physiologically and pathophysiologically relevant changes in N-glycosylation; reduced channel sialylation increases hERG channel activity during the action potential, thereby increasing the rate of action potential repolarization.

  10. ‘Sleepy’ inward rectifier channels in guinea-pig cardiomyocytes are activated only during strong hyperpolarization

    Science.gov (United States)

    Liu, Gong Xin; Daut, Jürgen

    2002-01-01

    K+ channels of isolated guinea-pig cardiomyocytes were studied using the patch-clamp technique. At transmembrane potentials between −120 and −220 mV we observed inward currents through an apparently novel channel. The novel channel was strongly rectifying, no outward currents could be recorded. Between −200 and −160 mV it had a slope conductance of 42.8 ± 3.0 pS (s.d.; n = 96). The open probability (Po) showed a sigmoid voltage dependence and reached a maximum of 0.93 at −200 mV, half-maximal activation was approximately −150 mV. The voltage dependence of Po was not affected by application of 50 μm isoproterenol. The open-time distribution could be described by a single exponential function, the mean open time ranged between 73.5 ms at −220 mV and 1.4 ms at −160 mV. At least two exponential components were required to fit the closed time distribution. Experiments with different external Na+, K+ and Cl− concentrations suggested that the novel channel is K+ selective. Extracellular Ba2+ ions gave rise to a voltage-dependent reduction in Po by inducing long closed states; Cs+ markedly reduced mean open time at −200 mV. In cell-attached recordings the novel channel frequently converted to a classical inward rectifier channel, and vice versa. This conversion was not voltage dependent. After excision of the patch, the novel channel always converted to a classical inward rectifier channel within 0–3 min. This conversion was not affected by intracellular Mg2+, phosphatidylinositol (4,5)-bisphosphate or spermine. Taken together, our findings suggest that the novel K+ channel represents a different ‘mode’ of the classical inward rectifier channel in which opening occurs only at very negative potentials. PMID:11897847

  11. Induction of divalent cation permeability by heterologous expression of a voltage sensor domain.

    Science.gov (United States)

    Arima, Hiroki; Tsutsui, Hidekazu; Sakamoto, Ayako; Yoshida, Manabu; Okamura, Yasushi

    2018-01-06

    The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K + channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Ion peak narrowing by applying additional AC voltage (ripple voltage) to FAIMS extractor electrode.

    Science.gov (United States)

    Pervukhin, Viktor V; Sheven, Dmitriy G

    2010-01-01

    The use of a non-uniform electric field in a high-field asymmetric waveform ion mobility spectrometry (FAIMS) analyzer increases sensitivity but decreases resolution. The application of an additional AC voltage to the extractor electrode ("ripple" voltage, U(ripple)) can overcome this effect, which decreases the FAIMS peak width. In this approach, the diffusion ion loss remains minimal in the non-uniform electric field in the cylindrical part of the device, and all ion losses under U(ripple) occur in a short portion of their path. Application of the ripple voltage to the extractor electrode is twice as efficient as the applying of U(ripple) along the total length of the device. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

  13. Ca2+ and voltage dependence of cardiac ryanodine receptor channel block by sphingosylphosphorylcholine.

    Science.gov (United States)

    Yasukochi, Midori; Uehara, Akira; Kobayashi, Sei; Berlin, Joshua R

    2003-03-01

    The effect of sphingosylphosphorylcholine (SPC) on the cytoplasmic Ca(2+) and voltage dependence of channel gating by cardiac ryanodine receptors (RyR) was examined in lipid bilayer experiments. Micromolar concentrations of the lysosphingolipid SPC added to cis solutions rapidly and reversibly decreased the single-channel open probability (P(o)) of reconstituted RyR channels. The SPC-induced decrease in P(o) was marked by an increase in mean closed time and burst-like channel gating. Gating kinetics during intraburst periods were unchanged from those observed in the absence of the sphingolipid, although SPC induced a long-lived closed state that appeared to explain the observed decrease in channel P(o). SPC effects were observed over a broad range of cis [Ca(2+)] but were not competitive with Ca(2+). Interestingly, the sphingolipid-induced, long-lived closed state displayed voltage-dependent kinetics, even though other channel gating kinetics were not sensitive to voltage. Assuming SPC effects represent channel blockade, these results suggest that the blocking rate is independent of voltage whereas the unblocking rate is voltage dependent. Together, these results suggest that SPC binds directly to the cytoplasmic side of the RyR protein in a location in or near the membrane dielectric, but distinct from cytoplasmic Ca(2+) binding sites on the protein.

  14. Localization and Molecular Determinants of the Hanatoxin Receptors on the Voltage-Sensing Domains of a K+ Channel

    Science.gov (United States)

    Li-Smerin, Yingying; Swartz, Kenton J.

    2000-01-01

    Hanatoxin inhibits voltage-gated K+ channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K+ channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1–S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K+ channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K+ channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20–25 Å away from the central pore axis. PMID:10828242

  15. Ion channels versus ion pumps: the principal difference, in principle.

    Science.gov (United States)

    Gadsby, David C

    2009-05-01

    The incessant traffic of ions across cell membranes is controlled by two kinds of border guards: ion channels and ion pumps. Open channels let selected ions diffuse rapidly down electrical and concentration gradients, whereas ion pumps labour tirelessly to maintain the gradients by consuming energy to slowly move ions thermodynamically uphill. Because of the diametrically opposed tasks and the divergent speeds of channels and pumps, they have traditionally been viewed as completely different entities, as alike as chalk and cheese. But new structural and mechanistic information about both of these classes of molecular machines challenges this comfortable separation and forces its re-evaluation.

  16. New Trends in Cancer Therapy: Targeting Ion Channels and Transporters

    Directory of Open Access Journals (Sweden)

    Annarosa Arcangeli

    2010-04-01

    Full Text Available The expression and activity of different channel types mark and regulate specific stages of cancer establishment and progression. Blocking channel activity impairs the growth of some tumors, both in vitro and in vivo, which opens a new field for pharmaceutical research. However, ion channel blockers may produce serious side effects, such as cardiac arrhythmias. For instance, Kv11.1 (hERG1 channels are aberrantly expressed in several human cancers, in which they control different aspects of the neoplastic cell behaviour. hERG1 blockers tend to inhibit cancer growth. However they also retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, which can lead to life-threatening ventricular arrhythmias. Several possibilities exist to produce less harmful compounds, such as developing specific drugs that bind hERG1 channels in the open state or disassemble the ion channel/integrin complex which appears to be crucial in certain stages of neoplastic progression. The potential approaches to improve the efficacy and safety of ion channel targeting in oncology include: (1 targeting specific conformational channel states; (2 finding ever more specific inhibitors, including peptide toxins, for channel subtypes mainly expressed in well-identified tumors; (3 using specific ligands to convey traceable or cytotoxic compounds; (4 developing channel blocking antibodies; (5 designing new molecular tools to decrease channel expression in selected cancer types. Similar concepts apply to ion transporters such as the Na+/K+ pump and the Na+/H+ exchanger. Pharmacological targeting of these transporters is also currently being considered in anti-neoplastic therapy.

  17. Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants.

    Directory of Open Access Journals (Sweden)

    Toru Kobayashi

    Full Text Available Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+ (GIRK, Kir3 channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.

  18. Dysfunctional HCN ion channels in neurological diseases

    Directory of Open Access Journals (Sweden)

    Jacopo C. DiFrancesco

    2015-03-01

    Full Text Available Hyperpolarization-activated cyclic nucleotide-gated (HCN channels are expressed as four different isoforms (HCN1-4 in the heart and in the central and peripheral nervous systems. HCN channels are activated by membrane hyperpolarization at voltages close to resting membrane potentials and carry the hyperpolarization-activated current, dubbed If (funny current in heart and Ih in neurons. HCN channels contribute in several ways to neuronal activity and are responsible for many important cellular functions, including cellular excitability, generation and modulation of rhythmic activity, dendritic integration, transmission of synaptic potentials and plasticity phenomena. Because of their role, defective HCN channels are natural candidates in the search for potential causes of neurological disorders in humans. Several data, including growing evidence that some forms of epilepsy are associated with HCN mutations, support the notion of an involvement of dysfunctional HCN channels in different experimental models of the disease. Additionally, some anti-epileptic drugs are known to modify the activity of the Ih current. HCN channels are widely expressed in the peripheral nervous system and recent evidence has highlighted the importance of the HCN2 isoform in the transmission of pain. HCN channels are also present in the midbrain system, where they finely regulate the activity of dopaminergic neurons, and a potential role of these channels in the pathogenesis of Parkinson’s disease has recently emerged. The function of HCN channels is regulated by specific accessory proteins, which control the correct expression and modulation of the neuronal Ih current. Alteration of these proteins can severely interfere with the physiological channel function, potentially predisposing to pathological conditions. In this review we address the present knowledge of the association between HCN dysfunctions and neurological diseases, including clinical, genetic and

  19. The ion-channel laser

    International Nuclear Information System (INIS)

    Whittum, D.H.; Sessler, A.M.; Dawson, J.M.

    1990-01-01

    A relativistic electron beam propagating through a plasma in the ion-focused regime exhibits an electromagnetic instability at a resonant frequency ω ∼ 2γ 2 ω β . Growth is enhanced by optical guiding in the ion channel, which acts as dielectric waveguide, with fiber parameter V ∼ 2 (I/I A ) 1/2 . A 1-D theory for such an ''ion-channel laser'' is formulated, scaling laws are derived and numerical examples are given. Possible experimental evidence is noted. 23 refs., 1 fig., 1 tab

  20. Voltage Gated Calcium Channel Activation by Backpropagating Action Potentials Downregulates NMDAR Function

    Directory of Open Access Journals (Sweden)

    Anne-Kathrin Theis

    2018-04-01

    Full Text Available The majority of excitatory synapses are located on dendritic spines of cortical glutamatergic neurons. In spines, compartmentalized Ca2+ signals transduce electrical activity into specific long-term biochemical and structural changes. Action potentials (APs propagate back into the dendritic tree and activate voltage gated Ca2+ channels (VGCCs. For spines, this global mode of spine Ca2+ signaling is a direct biochemical feedback of suprathreshold neuronal activity. We previously demonstrated that backpropagating action potentials (bAPs result in long-term enhancement of spine VGCCs. This activity-dependent VGCC plasticity results in a large interspine variability of VGCC Ca2+ influx. Here, we investigate how spine VGCCs affect glutamatergic synaptic transmission. We combined electrophysiology, two-photon Ca2+ imaging and two-photon glutamate uncaging in acute brain slices from rats. T- and R-type VGCCs were the dominant depolarization-associated Ca2+conductances in dendritic spines of excitatory layer 2 neurons and do not affect synaptic excitatory postsynaptic potentials (EPSPs measured at the soma. Using two-photon glutamate uncaging, we compared the properties of glutamatergic synapses of single spines that express different levels of VGCCs. While VGCCs contributed to EPSP mediated Ca2+ influx, the amount of EPSP mediated Ca2+ influx is not determined by spine VGCC expression. On a longer timescale, the activation of VGCCs by bAP bursts results in downregulation of spine NMDAR function.

  1. Mining the Virgin Land of Neurotoxicology: A Novel Paradigm of Neurotoxic Peptides Action on Glycosylated Voltage-Gated Sodium Channels

    Directory of Open Access Journals (Sweden)

    Zhirui Liu

    2012-01-01

    Full Text Available Voltage-gated sodium channels (VGSCs are important membrane protein carrying on the molecular basis for action potentials (AP in neuronal firings. Even though the structure-function studies were the most pursued spots, the posttranslation modification processes, such as glycosylation, phosphorylation, and alternative splicing associating with channel functions captured less eyesights. The accumulative research suggested an interaction between the sialic acids chains and ion-permeable pores, giving rise to subtle but significant impacts on channel gating. Sodium channel-specific neurotoxic toxins, a family of long-chain polypeptides originated from venomous animals, are found to potentially share the binding sites adjacent to glycosylated region on VGSCs. Thus, an interaction between toxin and glycosylated VGSC might hopefully join the campaign to approach the role of glycosylation in modulating VGSCs-involved neuronal network activity. This paper will cover the state-of-the-art advances of researches on glycosylation-mediated VGSCs function and the possible underlying mechanisms of interactions between toxin and glycosylated VGSCs, which may therefore, fulfill the knowledge in identifying the pharmacological targets and therapeutic values of VGSCs.

  2. Direct evidence that scorpion α-toxins (site-3 modulate sodium channel inactivation by hindrance of voltage-sensor movements.

    Directory of Open Access Journals (Sweden)

    Zhongming Ma

    Full Text Available The position of the voltage-sensing transmembrane segment, S4, in voltage-gated ion channels as a function of voltage remains incompletely elucidated. Site-3 toxins bind primarily to the extracellular loops connecting transmembrane helical segments S1-S2 and S3-S4 in Domain 4 (D4 and S5-S6 in Domain 1 (D1 and slow fast-inactivation of voltage-gated sodium channels. As S4 of the human skeletal muscle voltage-gated sodium channel, hNav1.4, moves in response to depolarization from the resting to the inactivated state, two D4S4 reporters (R2C and R3C, Arg1451Cys and Arg1454Cys, respectively move from internal to external positions as deduced by reactivity to internally or externally applied sulfhydryl group reagents, methane thiosulfonates (MTS. The changes in reporter reactivity, when cycling rapidly between hyperpolarized and depolarized voltages, enabled determination of the positions of the D4 voltage-sensor and of its rate of movement. Scorpion α-toxin binding impedes D4S4 segment movement during inactivation since the modification rates of R3C in hNav1.4 with methanethiosulfonate (CH3SO2SCH2CH2R, where R = -N(CH33 (+ trimethylammonium, MTSET and benzophenone-4-carboxamidocysteine methanethiosulfonate (BPMTS were slowed ~10-fold in toxin-modified channels. Based upon the different size, hydrophobicity and charge of the two reagents it is unlikely that the change in reactivity is due to direct or indirect blockage of access of this site to reagent in the presence of toxin (Tx, but rather is the result of inability of this segment to move outward to the normal extent and at the normal rate in the toxin-modified channel. Measurements of availability of R3C to internally applied reagent show decreased access (slower rates of thiol reaction providing further evidence for encumbered D4S4 movement in the presence of toxins consistent with the assignment of at least part of the toxin binding site to the region of D4S4 region of the voltage

  3. Domain-domain interactions determine the gating, permeation, pharmacology, and subunit modulation of the IKs ion channel.

    Science.gov (United States)

    Zaydman, Mark A; Kasimova, Marina A; McFarland, Kelli; Beller, Zachary; Hou, Panpan; Kinser, Holly E; Liang, Hongwu; Zhang, Guohui; Shi, Jingyi; Tarek, Mounir; Cui, Jianmin

    2014-12-23

    Voltage-gated ion channels generate electrical currents that control muscle contraction, encode neuronal information, and trigger hormonal release. Tissue-specific expression of accessory (β) subunits causes these channels to generate currents with distinct properties. In the heart, KCNQ1 voltage-gated potassium channels coassemble with KCNE1 β-subunits to generate the IKs current (Barhanin et al., 1996; Sanguinetti et al., 1996), an important current for maintenance of stable heart rhythms. KCNE1 significantly modulates the gating, permeation, and pharmacology of KCNQ1 (Wrobel et al., 2012; Sun et al., 2012; Abbott, 2014). These changes are essential for the physiological role of IKs (Silva and Rudy, 2005); however, after 18 years of study, no coherent mechanism explaining how KCNE1 affects KCNQ1 has emerged. Here we provide evidence of such a mechanism, whereby, KCNE1 alters the state-dependent interactions that functionally couple the voltage-sensing domains (VSDs) to the pore.

  4. Domain–domain interactions determine the gating, permeation, pharmacology, and subunit modulation of the IKs ion channel

    Science.gov (United States)

    Zaydman, Mark A; Kasimova, Marina A; McFarland, Kelli; Beller, Zachary; Hou, Panpan; Kinser, Holly E; Liang, Hongwu; Zhang, Guohui; Shi, Jingyi; Tarek, Mounir; Cui, Jianmin

    2014-01-01

    Voltage-gated ion channels generate electrical currents that control muscle contraction, encode neuronal information, and trigger hormonal release. Tissue-specific expression of accessory (β) subunits causes these channels to generate currents with distinct properties. In the heart, KCNQ1 voltage-gated potassium channels coassemble with KCNE1 β-subunits to generate the IKs current (Barhanin et al., 1996; Sanguinetti et al., 1996), an important current for maintenance of stable heart rhythms. KCNE1 significantly modulates the gating, permeation, and pharmacology of KCNQ1 (Wrobel et al., 2012; Sun et al., 2012; Abbott, 2014). These changes are essential for the physiological role of IKs (Silva and Rudy, 2005); however, after 18 years of study, no coherent mechanism explaining how KCNE1 affects KCNQ1 has emerged. Here we provide evidence of such a mechanism, whereby, KCNE1 alters the state-dependent interactions that functionally couple the voltage-sensing domains (VSDs) to the pore. DOI: http://dx.doi.org/10.7554/eLife.03606.001 PMID:25535795

  5. Lysine and the Na+/K+ Selectivity in Mammalian Voltage-Gated Sodium Channels.

    Directory of Open Access Journals (Sweden)

    Yang Li

    Full Text Available Voltage-gated sodium (Nav channels are critical in the generation and transmission of neuronal signals in mammals. The crystal structures of several prokaryotic Nav channels determined in recent years inspire the mechanistic studies on their selection upon the permeable cations (especially between Na+ and K+ ions, a property that is proposed to be mainly determined by residues in the selectivity filter. However, the mechanism of cation selection in mammalian Nav channels lacks direct explanation at atomic level due to the difference in amino acid sequences between mammalian and prokaryotic Nav homologues, especially at the constriction site where the DEKA motif has been identified to determine the Na+/K+ selectivity in mammalian Nav channels but is completely absent in the prokaryotic counterparts. Among the DEKA residues, Lys is of the most importance since its mutation to Arg abolishes the Na+/K+ selectivity. In this work, we modeled the pore domain of mammalian Nav channels by mutating the four residues at the constriction site of a prokaryotic Nav channel (NavRh to DEKA, and then mechanistically investigated the contribution of Lys in cation selection using molecular dynamics simulations. The DERA mutant was generated as a comparison to understand the loss of ion selectivity caused by the K-to-R mutation. Simulations and free energy calculations on the mutants indicate that Lys facilitates Na+/K+ selection by electrostatically repelling the cation to a highly Na+-selective location sandwiched by the carboxylate groups of Asp and Glu at the constriction site. In contrast, the electrostatic repulsion is substantially weakened when Lys is mutated to Arg, because of two intrinsic properties of the Arg side chain: the planar geometric design and the sparse charge distribution of the guanidine group.

  6. Lysine and the Na+/K+ Selectivity in Mammalian Voltage-Gated Sodium Channels.

    Science.gov (United States)

    Li, Yang; Liu, Huihui; Xia, Mengdie; Gong, Haipeng

    2016-01-01

    Voltage-gated sodium (Nav) channels are critical in the generation and transmission of neuronal signals in mammals. The crystal structures of several prokaryotic Nav channels determined in recent years inspire the mechanistic studies on their selection upon the permeable cations (especially between Na+ and K+ ions), a property that is proposed to be mainly determined by residues in the selectivity filter. However, the mechanism of cation selection in mammalian Nav channels lacks direct explanation at atomic level due to the difference in amino acid sequences between mammalian and prokaryotic Nav homologues, especially at the constriction site where the DEKA motif has been identified to determine the Na+/K+ selectivity in mammalian Nav channels but is completely absent in the prokaryotic counterparts. Among the DEKA residues, Lys is of the most importance since its mutation to Arg abolishes the Na+/K+ selectivity. In this work, we modeled the pore domain of mammalian Nav channels by mutating the four residues at the constriction site of a prokaryotic Nav channel (NavRh) to DEKA, and then mechanistically investigated the contribution of Lys in cation selection using molecular dynamics simulations. The DERA mutant was generated as a comparison to understand the loss of ion selectivity caused by the K-to-R mutation. Simulations and free energy calculations on the mutants indicate that Lys facilitates Na+/K+ selection by electrostatically repelling the cation to a highly Na+-selective location sandwiched by the carboxylate groups of Asp and Glu at the constriction site. In contrast, the electrostatic repulsion is substantially weakened when Lys is mutated to Arg, because of two intrinsic properties of the Arg side chain: the planar geometric design and the sparse charge distribution of the guanidine group.

  7. Voltage-gated sodium channels: action players with many faces

    NARCIS (Netherlands)

    Koopmann, Tamara T.; Bezzina, Connie R.; Wilde, Arthur A. M.

    2006-01-01

    Voltage-gated sodium channels are responsible for the upstroke of the action potential and thereby play an important role in propagation of the electrical impulse in excitable tissues like muscle, nerve and the heart. Duplication of the sodium channels encoding genes during evolution generated the

  8. Allosteric substrate switching in a voltage-sensing lipid phosphatase.

    Science.gov (United States)

    Grimm, Sasha S; Isacoff, Ehud Y

    2016-04-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We found that the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), has not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage-sensing domain (VSD). Using fast fluorescence resonance energy transfer (FRET) reporters of PIPs to monitor enzyme activity and voltage-clamp fluorometry to monitor conformational changes in the VSD, we found that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage-sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This two-step allosteric control over a dual-specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility, endocytosis and exocytosis.

  9. Allosteric substrate switching in a voltage sensing lipid phosphatase

    Science.gov (United States)

    Grimm, Sasha S.; Isacoff, Ehud Y.

    2016-01-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We find the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), to have not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage sensing domain (VSD). Using fast FRET reporters of PIPs to monitor enzyme activity and voltage clamp fluorometry to monitor conformational changes in the VSD, we find that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This novel 2-step allosteric control over a dual specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility and endo/exocytosis. PMID:26878552

  10. Strong activation of bile acid-sensitive ion channel (BASIC) by ursodeoxycholic acid

    Science.gov (United States)

    Wiemuth, Dominik; Sahin, Hacer; Lefèvre, Cathérine M.T.; Wasmuth, Hermann E.; Gründer, Stefan

    2013-01-01

    Bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC gene family of unknown function. Rat BASIC (rBASIC) is inactive at rest. We have recently shown that cholangiocytes, the epithelial cells lining the bile ducts, are the main site of BASIC expression in the liver and identified bile acids, in particular hyo- and chenodeoxycholic acid, as agonists of rBASIC. Moreover, it seems that extracellular divalent cations stabilize the resting state of rBASIC, because removal of extracellular divalent cations opens the channel. In this addendum, we demonstrate that removal of extracellular divalent cations potentiates the activation of rBASIC by bile acids, suggesting an allosteric mechanism. Furthermore, we show that rBASIC is strongly activated by the anticholestatic bile acid ursodeoxycholic acid (UDCA), suggesting that BASIC might mediate part of the therapeutic effects of UDCA. PMID:23064163

  11. A permeation theory for single-file ion channels: one- and two-step models.

    Science.gov (United States)

    Nelson, Peter Hugo

    2011-04-28

    How many steps are required to model permeation through ion channels? This question is investigated by comparing one- and two-step models of permeation with experiment and MD simulation for the first time. In recent MD simulations, the observed permeation mechanism was identified as resembling a Hodgkin and Keynes knock-on mechanism with one voltage-dependent rate-determining step [Jensen et al., PNAS 107, 5833 (2010)]. These previously published simulation data are fitted to a one-step knock-on model that successfully explains the highly non-Ohmic current-voltage curve observed in the simulation. However, these predictions (and the simulations upon which they are based) are not representative of real channel behavior, which is typically Ohmic at low voltages. A two-step association/dissociation (A/D) model is then compared with experiment for the first time. This two-parameter model is shown to be remarkably consistent with previously published permeation experiments through the MaxiK potassium channel over a wide range of concentrations and positive voltages. The A/D model also provides a first-order explanation of permeation through the Shaker potassium channel, but it does not explain the asymmetry observed experimentally. To address this, a new asymmetric variant of the A/D model is developed using the present theoretical framework. It includes a third parameter that represents the value of the "permeation coordinate" (fractional electric potential energy) corresponding to the triply occupied state n of the channel. This asymmetric A/D model is fitted to published permeation data through the Shaker potassium channel at physiological concentrations, and it successfully predicts qualitative changes in the negative current-voltage data (including a transition to super-Ohmic behavior) based solely on a fit to positive-voltage data (that appear linear). The A/D model appears to be qualitatively consistent with a large group of published MD simulations, but no

  12. High voltage holding in the negative ion sources with cesium deposition

    Energy Technology Data Exchange (ETDEWEB)

    Belchenko, Yu.; Abdrashitov, G.; Ivanov, A.; Sanin, A.; Sotnikov, O., E-mail: O.Z.Sotnikov@inp.nsk.su [Budker Institute of Nuclear Physics, Siberian Branch of Russian Academy of Sciences, Novosibirsk (Russian Federation)

    2016-02-15

    High voltage holding of the large surface-plasma negative ion source with cesium deposition was studied. It was found that heating of ion-optical system electrodes to temperature >100 °C facilitates the source conditioning by high voltage pulses in vacuum and by beam shots. The procedure of electrode conditioning and the data on high-voltage holding in the negative ion source with small cesium seed are described. The mechanism of high voltage holding improvement by depletion of cesium coverage is discussed.

  13. Independent and cooperative motions of the Kv1.2 channel: voltage sensing and gating.

    Science.gov (United States)

    Yeheskel, Adva; Haliloglu, Turkan; Ben-Tal, Nir

    2010-05-19

    Voltage-gated potassium (Kv) channels, such as Kv1.2, are involved in the generation and propagation of action potentials. The Kv channel is a homotetramer, and each monomer is composed of a voltage-sensing domain (VSD) and a pore domain (PD). We analyzed the fluctuations of a model structure of Kv1.2 using elastic network models. The analysis suggested a network of coupled fluctuations of eight rigid structural units and seven hinges that may control the transition between the active and inactive states of the channel. For the most part, the network is composed of amino acids that are known to affect channel activity. The results suggested allosteric interactions and cooperativity between the subunits in the coupling between the motion of the VSD and the selectivity filter of the PD, in accordance with recent empirical data. There are no direct contacts between the VSDs of the four subunits, and the contacts between these and the PDs are loose, suggesting that the VSDs are capable of functioning independently. Indeed, they manifest many inherent fluctuations that are decoupled from the rest of the structure. In general, the analysis suggests that the two domains contribute to the channel function both individually and cooperatively. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  14. Atom-by-atom engineering of voltage-gated ion channels: Magnified insights into function and pharmacology

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Kim, Robin Y; Ahern, Christopher A

    2015-01-01

    Unnatural amino acid incorporation into ion channels has proven to be a valuable approach to interrogate detailed hypotheses arising from atomic resolution structures. In this short review, we provide a brief overview of some of the basic principles and methods for incorporation of unnatural amino...

  15. Antibodies to voltage-gated potassium and calcium channels in epilepsy.

    Science.gov (United States)

    Majoie, H J Marian; de Baets, Mark; Renier, Willy; Lang, Bethan; Vincent, Angela

    2006-10-01

    To determine the prevalence of antibodies to ion channels in patients with long standing epilepsy. Although the CNS is thought to be protected from circulating antibodies by the blood brain barrier, glutamate receptor antibodies have been reported in Rasmussen's encephalitis, glutamic acid decarboxylase (GAD) antibodies have been found in a few patients with epilepsy, and antibodies to voltage-gated potassium channels (VGKC) have been found in a non-paraneoplastic form of limbic encephalitis (with amnesia and seizures) that responds to immunosuppressive therapy. We retrospectively screened sera from female epilepsy patients (n=106) for autoantibodies to VGKC (Kv 1.1, 1.2 or 1.6), voltage-gated calcium channels (VGCC) (P/Q-type), and GAD. All positive results, based on the values of control data [McKnight, K., Jiang, Y., et al. (2005). Serum antibodies in epilepsy and seizure-associated disorders. Neurology 65, 1730-1735], were retested at lower serum concentrations, and results compared with previously published control data. Demographics, medical history, and epilepsy related information was gathered. The studied group consisted predominantly of patients with long standing drug resistant epilepsy. VGKC antibodies were raised (>100 pM) in six patients. VGCC antibodies (>45 pM) were slightly raised in only one patient. GAD antibodies were VGKC antibodies differed from previously described patients with limbic encephalitis-like syndrome, and were not different with respect to seizure type, age at first seizure, duration of epilepsy, or use of anti-epileptic drugs from the VGKC antibody negative patients. The results demonstrate that antibodies to VGKC are present in 6% of patients with typical long-standing epilepsy, but whether these antibodies are pathogenic or secondary to the primary disease process needs to be determined.

  16. Ion channeling revisited

    Energy Technology Data Exchange (ETDEWEB)

    Doyle, Barney Lee [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Corona, Aldo [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Nguyen, Anh [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2014-09-01

    A MS Excel program has been written that calculates accidental, or unintentional, ion channeling in cubic bcc, fcc and diamond lattice crystals or polycrystalline materials. This becomes an important issue when simulating the creation by energetic neutrons of point displacement damage and extended defects using beams of ions. All of the tables and graphs in the three Ion Beam Analysis Handbooks that previously had to be manually looked up and read from were programed into Excel in handy lookup tables, or parameterized, for the case of the graphs, using rather simple exponential functions with different powers of the argument. The program then offers an extremely convenient way to calculate axial and planar half-angles and minimum yield or dechanneling probabilities, effects on half-angles of amorphous overlayers, accidental channeling probabilities for randomly oriented crystals or crystallites, and finally a way to automatically generate stereographic projections of axial and planar channeling half-angles. The program can generate these projections and calculate these probabilities for axes and [hkl] planes up to (555).

  17. Electrophysiological characterisation of KCNQ channel modulators

    DEFF Research Database (Denmark)

    Schrøder, R.L

    Potassium (K+) ion channels are ubiquitously expressed in mammalian cells, and each channel serves a precise physiological role due to its specific biophysical characteristics and expression pattern. A few K+ channels are targets for certain drugs, and in this thesis it is suggested that the KCNQ K......+ channels may be targets for neuroprotective, anti-epileptic and anti-nociceptive compounds. The importance of these channels is underscored by the fact that four out of five KCNQ channel subtypes are involved in severe human diseases. However, the pharmacology of the KCNQ channels is yet poorly understood...... as these channels were identified only recently. Therefore, there is a need for understanding the biophysical behavior and pharmacology of these ion channels. KCNQ channels belong to the group of voltage-activated K+ channels. The subfamily consists of KCNQ1-5, which is primarily expressed in the CNS, heart, ear...

  18. Quantum Interference and Selectivity through Biological Ion Channels.

    Science.gov (United States)

    Salari, Vahid; Naeij, Hamidreza; Shafiee, Afshin

    2017-01-30

    The mechanism of selectivity in ion channels is still an open question in biology for more than half a century. Here, we suggest that quantum interference can be a solution to explain the selectivity mechanism in ion channels since interference happens between similar ions through the same size of ion channels. In this paper, we simulate two neighboring ion channels on a cell membrane with the famous double-slit experiment in physics to investigate whether there is any possibility of matter-wave interference of ions via movement through ion channels. Our obtained decoherence timescales indicate that the quantum states of ions can only survive for short times, i.e. ≈100 picoseconds in each channel and ≈17-53 picoseconds outside the channels, giving the result that the quantum interference of ions seems unlikely due to environmental decoherence. However, we discuss our results and raise few points, which increase the possibility of interference.

  19. Channeling ion implantation through palladium films

    International Nuclear Information System (INIS)

    Ishiwara, H.; Furukawa, S.

    1975-01-01

    The possibility of channeling ion implantation into semiconductors through polycrystalline metallic layers is studied. Minimum values and standard deviations of channeling angular yield in polycrystalline Pd 2 Si layers formed on Si have been measured by protons and 4 He, and 14 N ion backscattering and channeling measurements. Depth distributions of the spread of crystallite orientations and scattering centers such as lattice defects have been separately derived by using the above two quantities. It has been concluded that the channeling-ion-implantation technique will become a practical one by using the parallel scanning system

  20. Common molecular determinants of tarantula huwentoxin-IV inhibition of Na+ channel voltage sensors in domains II and IV.

    Science.gov (United States)

    Xiao, Yucheng; Jackson, James O; Liang, Songping; Cummins, Theodore R

    2011-08-05

    The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxin-binding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.

  1. Neuronal trafficking of voltage-gated potassium channels

    DEFF Research Database (Denmark)

    Jensen, Camilla S; Rasmussen, Hanne Borger; Misonou, Hiroaki

    2011-01-01

    The computational ability of CNS neurons depends critically on the specific localization of ion channels in the somatodendritic and axonal membranes. Neuronal dendrites receive synaptic inputs at numerous spines and integrate them in time and space. The integration of synaptic potentials is regul......The computational ability of CNS neurons depends critically on the specific localization of ion channels in the somatodendritic and axonal membranes. Neuronal dendrites receive synaptic inputs at numerous spines and integrate them in time and space. The integration of synaptic potentials...

  2. Amphotericin B channels in phospholipid membrane-coated nanoporous silicon surfaces: implications for photovoltaic driving of ions across membranes.

    Science.gov (United States)

    Yilma, Solomon; Liu, Nangou; Samoylov, Alexander; Lo, Ting; Brinker, C Jeffrey; Vodyanoy, Vitaly

    2007-03-15

    The antimycotic agent amphotericin B (AmB) functions by forming complexes with sterols to form ion channels that cause membrane leakage. When AmB and cholesterol mixed at 2:1 ratio were incorporated into phospholipid bilayer membranes formed on the tip of patch pipettes, ion channel current fluctuations with characteristic open and closed states were observed. These channels were also functional in phospholipid membranes formed on nanoporous silicon surfaces. Electrophysiological studies of AmB-cholesterol mixtures that were incorporated into phospholipid membranes formed on the surface of nanoporous (6.5 nm pore diameter) silicon plates revealed large conductance ion channels ( approximately 300 pS) with distinct open and closed states. Currents through the AmB-cholesterol channels on nanoporous silicon surfaces can be driven by voltage applied via conventional electrical circuits or by photovoltaic electrical potential entirely generated when the nanoporous silicon surface is illuminated with a narrow laser beam. Electrical recordings made during laser illumination of AmB-cholesterol containing membrane-coated nanoporous silicon surfaces revealed very large conductance ion channels with distinct open and closed states. Our findings indicate that nanoporous silicon surfaces can serve as mediums for ion-channel-based biosensors. The photovoltaic properties of nanoporous silicon surfaces show great promise for making such biosensors addressable via optical technologies.

  3. Acid-sensing ion channels and migraine

    Directory of Open Access Journals (Sweden)

    Yu-qi KANG

    2015-09-01

    Full Text Available Acid-sensing ion channels (ASICs are ligand-gated ion channels that are activated by extracellular protons (H+, which belong to epithelial sodium channels/degenerin (ENaC/DEG superfamily. ASICs are widely distributed in central nervous system, peripheral nervous system, digestive system and some tumor tissues. Different ASIC subunits play important roles in various pathophysiological processes such as touch, sour taste, learning and memory, including inflammation, ischemic stroke, pain, learning and memory decline, epilepsy, multiple sclerosis (MS, migraine, irritable bowel syndrome and tumor. Research over the last 2 decades has achieved substantial advances in migraine pathophysiology. It is now largely accepted that inflammatory pathways play a key role and three main events seem to take place: cortical spreading depression (CSD, activation of the trigeminovascular system (i.e. dural nociceptors, peripheral and central sensitization of this pain pathway. However, the exact mechanisms that link these three events to each other and to inflammation have so far remained to be studied. This article takes an overview of newly research advances in structure, distribution and the relationship with migraine of ASICs.  DOI: 10.3969/j.issn.1672-6731.2015.09.013

  4. The temperature dependence of the BK channel activity - kinetics, thermodynamics, and long-range correlations.

    Science.gov (United States)

    Wawrzkiewicz-Jałowiecka, Agata; Dworakowska, Beata; Grzywna, Zbigniew J

    2017-10-01

    Large-conductance, voltage dependent, Ca 2+ -activated potassium channels (BK) are transmembrane proteins that regulate many biological processes by controlling potassium flow across cell membranes. Here, we investigate to what extent temperature (in the range of 17-37°C with ΔT=5°C step) is a regulating parameter of kinetic properties of the channel gating and memory effect in the series of dwell-time series of subsequent channel's states, at membrane depolarization and hyperpolarization. The obtained results indicate that temperature affects strongly the BK channels' gating, but, counterintuitively, it exerts no effect on the long-range correlations, as measured by the Hurst coefficient. Quantitative differences between dependencies of appropriate channel's characteristics on temperature are evident for different regimes of voltage. Examining the characteristics of BK channel activity as a function of temperature allows to estimate the net activation energy (E act ) and changes of thermodynamic parameters (ΔH, ΔS, ΔG) by channel opening. Larger E act corresponds to the channel activity at membrane hyperpolarization. The analysis of entropy and enthalpy changes of closed to open channel's transition suggest the entropy-driven nature of the increase of open state probability during voltage activation and supports the hypothesis about the voltage-dependent geometry of the channel vestibule. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. TAURINE REGULATION OF VOLTAGE-GATED CHANNELS IN RETINAL NEURONS

    Science.gov (United States)

    Rowan, Matthew JM; Bulley, Simon; Purpura, Lauren; Ripps, Harris; Shen, Wen

    2017-01-01

    Taurine activates not only Cl−-permeable ionotropic receptors, but also receptors that mediate metabotropic responses. The metabotropic property of taurine was revealed in electrophysiological recordings obtained after fully blocking Cl−-permeable receptors with an inhibitory “cocktail” consisting of picrotoxin, SR95531, and strychnine. We found that taurine’s metabotropic effects regulate voltage-gated channels in retinal neurons. After applying the inhibitory cocktail, taurine enhanced delayed outward rectifier K+ channels preferentially in Off-bipolar cells, and the effect was completely blocked by the specific PKC inhibitor, GF109203X. Additionally, taurine also acted through a metabotropic pathway to suppress both L- and N-type Ca2+ channels in retinal neurons, which were insensitive to the potent GABAB receptor inhibitor, CGP55845. This study reinforces our previous finding that taurine in physiological concentrations produces a multiplicity of metabotropic effects that precisely govern the integration of signals being transmitted from the retina to the brain. PMID:23392926

  6. Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening.

    Directory of Open Access Journals (Sweden)

    Zeineb Es-Salah-Lamoureux

    2010-05-01

    Full Text Available hERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening.Tetramethylrhodamine-5-maleimide (TMRM fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449 in the S1-S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the V((1/2 of activation to -27.5+/-2.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1-S2 linker cysteines with valines allowed unobstructed recording of S3-S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-V(ON, with V((1/2 (,1 = -37.8+/-1.7 mV, and V((1/2 (,2 = 43.5+/-7.9 mV. The first phase, V((1/2 (,1, was approximately 20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-V((1/2 = -18.3+/-1.2 mV, and relatively unchanged in a non-inactivating E519C:S620T mutant (V((1/2 = -34.4+/-1.5 mV, suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarization (V((1/2 = -20.6+/-1.2, k = 11.4 mV and L520C quenching during depolarization (V((1/2 = -26.8+/-1.0, k = 13.3 mV matched the respective voltage dependencies of hERG ionic tails, and deactivation time constants from -40 to -110 mV, suggesting they detected pore-S4 rearrangements related to ionic current flow during pore opening and closing.THE DATA INDICATE: 1 that rapid environmental changes occur at the

  7. K+ channel openers restore verapamil-inhibited lung fluid resolution and transepithelial ion transport

    Directory of Open Access Journals (Sweden)

    Su Xue-Feng

    2010-05-01

    Full Text Available Abstract Background Lung epithelial Na+ channels (ENaC are regulated by cell Ca2+ signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K+ channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K+ channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC by up-regulating both apical and basolateral ion transport. Methods Verapamil-induced depression of heterologously expressed human αβγ ENaC in Xenopus oocytes, apical and basolateral ion transport in monolayers of human lung epithelial cells (H441, and in vivo alveolar fluid clearance were measured, respectively, using the two-electrode voltage clamp, Ussing chamber, and BSA protein assays. Ca2+ signal in H441 cells was analyzed using Fluo 4AM. Results The rate of in vivo AFC was reduced significantly (40.6 ± 6.3% of control, P Ca3.1 (1-EBIO and KATP (minoxidil channel openers significantly recovered AFC. In addition to short-circuit current (Isc in intact H441 monolayers, both apical and basolateral Isc levels were reduced by verapamil in permeabilized monolayers. Moreover, verapamil significantly altered Ca2+ signal evoked by ionomycin in H441 cells. Depletion of cytosolic Ca2+ in αβγ ENaC-expressing oocytes completely abolished verapamil-induced inhibition. Intriguingly, KV (pyrithione-Na, K Ca3.1 (1-EBIO, and KATP (minoxidil channel openers almost completely restored the verapamil-induced decrease in Isc levels by diversely up-regulating apical and basolateral Na+ and K+ transport pathways. Conclusions Our observations demonstrate that K+ channel openers are capable of rescuing reduced vectorial Na+ transport across lung epithelial cells with impaired Ca2+ signal.

  8. Differential Activity of Voltage- and Ca2+-Dependent Potassium Channels in Leukemic T Cell Lines: Jurkat Cells Represent an Exceptional Case

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    Salvador Valle-Reyes

    2018-05-01

    Full Text Available Activation of resting T cells relies on sustained Ca2+ influx across the plasma membrane, which in turn depends on the functional expression of potassium channels, whose activity repolarizes the membrane potential. Depending on the T-cells subset, upon activation the expression of Ca2+- or voltage-activated K+ channels, KCa or Kv, is up-regulated. In this study, by means of patch-clamp technique in the whole cell mode, we have studied in detail the characteristics of Kv and KCa currents in resting and activated human T cells, the only well explored human T-leukemic cell line Jurkat, and two additional human leukemic T cell lines, CEM and MOLT-3. Voltage dependence of activation and inactivation of Kv1.3 current were shifted up to by 15 mV to more negative potentials upon a prolonged incubation in the whole cell mode and displayed little difference at a stable state in all cell lines but CEM, where the activation curve was biphasic, with a high and low potential components. In Jurkat, KCa currents were dominated by apamine-sensitive KCa2.2 channels, whereas only KCa3.1 current was detected in healthy T and leukemic CEM and MOLT-3 cells. Despite a high proliferation potential of Jurkat cells, Kv and KCa currents were unexpectedly small, more than 10-fold lesser as compared to activated healthy human T cells, CEM and MOLT-3, which displayed characteristic Kv1.3high:KCa3.1high phenotype. Our results suggest that Jurkat cells represent perhaps a singular case and call for more extensive studies on primary leukemic T cell lines as well as a verification of the therapeutic potential of specific KCa3.1 blockers to combat acute lymphoblastic T leukemias.

  9. Electric organ discharge diversification in mormyrid weakly electric fish is associated with differential expression of voltage-gated ion channel genes.

    Science.gov (United States)

    Nagel, Rebecca; Kirschbaum, Frank; Tiedemann, Ralph

    2017-03-01

    In mormyrid weakly electric fish, the electric organ discharge (EOD) is used for species recognition, orientation and prey localization. Produced in the muscle-derived adult electric organ, the EOD exhibits a wide diversity across species in both waveform and duration. While certain defining EOD characteristics can be linked to anatomical features of the electric organ, many factors underlying EOD differentiation are yet unknown. Here, we report the differential expression of 13 Kv1 voltage-gated potassium channel genes, two inwardly rectifying potassium channel genes, two previously studied sodium channel genes and an ATPase pump in two sympatric species of the genus Campylomormyrus in both the adult electric organ and skeletal muscle. Campylomormyrus compressirostris displays a basal EOD, largely unchanged during development, while C. tshokwe has an elongated, putatively derived discharge. We report an upregulation in all Kv1 genes in the electric organ of Campylomormyrus tshokwe when compared to both skeletal muscle and C. compressirostris electric organ. This pattern of upregulation in a species with a derived EOD form suggests that voltage-gated potassium channels are potentially involved in the diversification of the EOD signal among mormyrid weakly electric fish.

  10. Aluminium and hydrogen ions inhibit a mechanosensory calcium-selective cation channel

    Science.gov (United States)

    Ding, J. P.; Pickard, B. G.

    1993-01-01

    The tension-dependent activity of mechanosensory calcium-selective cation channels in excised plasmalemmal patches from onion bulb scale epidermis is modulated by pH in the physiologically meaningful range between 4.5 and 7.2. It is rapidly lowered by lowering pH and rapidly raised by raising pH. Channel activity is effectively inhibited by low levels of aluminium ions and activity can be partially restored by washing for a few minutes. We suggest that under normal conditions the sensitivity of the mechanosensory channels to pH of the wall free space plays important roles in regulation of plant activities such as growth. We further suggest that, when levels of acid and aluminium ions in the soil solution are high, they might inhibit similar sensory channels in cells of the root tip, thus contributing critically to the acid soil syndrome.

  11. High throughput electrophysiology: new perspectives for ion channel drug discovery

    DEFF Research Database (Denmark)

    Willumsen, Niels J; Bech, Morten; Olesen, Søren-Peter

    2003-01-01

    . A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct....... The introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery....

  12. Additional ion bombardment in PVD processes generated by a superimposed pulse bias voltage

    International Nuclear Information System (INIS)

    Olbrich, W.; Kampschulte, G.

    1993-01-01

    The superimposed pulse bias voltage is a tool to apply an additional ion bombardment during deposition in physical vapour deposition (PVD) processes. It is generated by the combination of a d.c. ground voltage and a higher d.c. pulse voltage. Using a superimposed pulse bias voltage in ion-assisted PVD processes effects an additional all-around ion bombardment on the surface with ions of higher energy. Both metal and reactive or inert-gas ions are accelerated to the surface. The basic principles and important characteristics of this newly developed process such as ion fluxes or deposition rates are shown. Because of pulsing the high voltage, the deposition temperature does not increase much. The adhesion, structure, morphology and internal stresses are influenced by these additional ion impacts. The columnar growth of the deposited films could be suppressed by using the superimposed pulse bias voltage without increasing the deposition temperature. Different metallizations (Cr and Cu) produced by arc and sputter ion plating are investigated. Carbon-fibre-reinforced epoxy are coated with PVD copper films for further treatment in electrochemical processes. (orig.)

  13. Mechanically Gated Ion Channels in Mammalian Hair Cells

    Directory of Open Access Journals (Sweden)

    Xufeng Qiu

    2018-04-01

    Full Text Available Hair cells in the inner ear convert mechanical stimuli provided by sound waves and head movements into electrical signal. Several mechanically evoked ionic currents with different properties have been recorded in hair cells. The search for the proteins that form the underlying ion channels is still in progress. The mechanoelectrical transduction (MET channel near the tips of stereociliary in hair cells, which is responsible for sensory transduction, has been studied most extensively. Several components of the sensory mechanotransduction machinery in stereocilia have been identified, including the multi-transmembrane proteins tetraspan membrane protein in hair cell stereocilia (TMHS/LHFPL5, transmembrane inner ear (TMIE and transmembrane channel-like proteins 1 and 2 (TMC1/2. However, there remains considerable uncertainty regarding the molecules that form the channel pore. In addition to the sensory MET channel, hair cells express the mechanically gated ion channel PIEZO2, which is localized near the base of stereocilia and not essential for sensory transduction. The function of PIEZO2 in hair cells is not entirely clear but it might have a role in damage sensing and repair processes. Additional stretch-activated channels of unknown molecular identity and function have been found to localize at the basolateral membrane of hair cells. Here, we review current knowledge regarding the different mechanically gated ion channels in hair cells and discuss open questions concerning their molecular composition and function.

  14. Mitigating Voltage Decay of Li-Rich Cathode Material via Increasing Ni Content for Lithium-Ion Batteries.

    Science.gov (United States)

    Shi, Ji-Lei; Zhang, Jie-Nan; He, Min; Zhang, Xu-Dong; Yin, Ya-Xia; Li, Hong; Guo, Yu-Guo; Gu, Lin; Wan, Li-Jun

    2016-08-10

    Li-rich layered materials have been considered as the most promising cathode materials for future high-energy-density lithium-ion batteries. However, they suffer from severe voltage decay upon cycling, which hinders their further commercialization. Here, we report a Li-rich layered material 0.5Li2MnO3·0.5LiNi0.8Co0.1Mn0.1O2 with high nickel content, which exhibits much slower voltage decay during long-term cycling compared to conventional Li-rich materials. The voltage decay after 200 cycles is 201 mV. Combining in situ X-ray diffraction (XRD), ex situ XRD, ex situ X-ray photoelectron spectroscopy, and scanning transmission electron microscopy, we demonstrate that nickel ions act as stabilizing ions to inhibit the Jahn-Teller effect of active Mn(3+) ions, improving d-p hybridization and supporting the layered structure as a pillar. In addition, nickel ions can migrate between the transition-metal layer and the interlayer, thus avoiding the formation of spinel-like structures and consequently mitigating the voltage decay. Our results provide a simple and effective avenue for developing Li-rich layered materials with mitigated voltage decay and a long lifespan, thereby promoting their further application in lithium-ion batteries with high energy density.

  15. Properties of Single K+ and Cl− Channels in Asclepias tuberosa Protoplasts 1

    Science.gov (United States)

    Schauf, Charles L.; Wilson, Kathryn J.

    1987-01-01

    Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K+ and Cl−, respectively. Whole cell K+ currents activated exponentially during step depolarizations, while voltage-dependent Cl− channels were activated by hyperpolarizations. Single K+ channel conductance was 40 ± 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs+ and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 ± 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn2+ and ethacrynic acid. Whole-cell Cl− currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development. Images Fig. 5 PMID:16665712

  16. Microvillar ion channels: cytoskeletal modulation of ion fluxes.

    Science.gov (United States)

    Lange, K

    2000-10-21

    movement of the system (electro-mechanical coupling). Because ionic transmission through linear polyelectrolytes is very slow compared with electronic conduction, only low-frequency electromagnetic fields can interact with the condensed counterion systems of linear polyelectrolytes. The delineated characteristics of microvillar ion conduction are strongly supported by the phenomenon of electro-mechanical coupling (reverse transduction) in microvilli of the audioreceptor (hair) cells and the recently reported dynamics of Ca(2+)signaling in microvilli of audio- and photoreceptor cells. Due to the cell-specific expression of different types and combinations of ion channels and transporters in the microvillar tip membrane of differentiated cells, the functional properties of this cell surface organelle are highly variable serving a multitude of different cellular functions including receptor-mediated effects such as Ca(2+)signaling, regulation of glucose and amino acid transport, as well as modulation of membrane potential. Even mechanical channel activation involved in cell volume regulation can be deduced from the systematic properties of the microvillar channel concept. In addition, the specific ion conduction properties of microfilaments combined with their proposed role in Ca(2+)signaling make microvilli the most likely cellular site for the interaction with external electric and magnetic fields. Copyright 2000 Academic Press.

  17. Lithium-Ion Electrolytes with Improved Safety Tolerance to High Voltage Systems

    Science.gov (United States)

    Smart, Marshall C. (Inventor); Bugga, Ratnakumar V. (Inventor); Prakash, Surya G. (Inventor); Krause, Frederick C. (Inventor)

    2015-01-01

    The invention discloses various embodiments of electrolytes for use in lithium-ion batteries, the electrolytes having improved safety and the ability to operate with high capacity anodes and high voltage cathodes. In one embodiment there is provided an electrolyte for use in a lithium-ion battery comprising an anode and a high voltage cathode. The electrolyte has a mixture of a cyclic carbonate of ethylene carbonate (EC) or mono-fluoroethylene carbonate (FEC) co-solvent, ethyl methyl carbonate (EMC), a flame retardant additive, a lithium salt, and an electrolyte additive that improves compatibility and performance of the lithium-ion battery with a high voltage cathode. The lithium-ion battery is charged to a voltage in a range of from about 2.0 V (Volts) to about 5.0 V (Volts).

  18. Phosphatidylinositol (4,5)bisphosphate inhibits K+-efflux channel activity in NT1 tobacco cultured cells.

    Science.gov (United States)

    Ma, Xiaohong; Shor, Oded; Diminshtein, Sofia; Yu, Ling; Im, Yang Ju; Perera, Imara; Lomax, Aaron; Boss, Wendy F; Moran, Nava

    2009-02-01

    In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed "cytosolic" Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: "Low PIs" had depressed levels of these PIs, and "High PIs" had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decreasing PtdInsP2 levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 microM) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (i.e. NtORK activity). Moreover, increasing PtdInsP2 levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5-4 microM), decreased NtORK activity. In all cases, IK decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP2 in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells.

  19. Channeled-ion implantation of group-III and group-V ions into silicon

    International Nuclear Information System (INIS)

    Furuya, T.; Nishi, H.; Inada, T.; Sakurai, T.

    1978-01-01

    Implantation of group-III and group-V ions along [111] and [110] axes of silicon have been performed using a backscattering technique, and the depth profiles of implanted ions have been measured by the C-V method. The range of channeled Ga ions is the largest among the present data, and a p-type layer of about 6 μm is obtained by implantation at only 150 keV. The carrier profiles of channeled Al and Ga ions with deep ranges do not show any distinguishable channeled peak contrasting with the B, P, and As channeling which gives a well-defined peak. The electronic stopping cross section (S/sub e/) of channeled P ions agree well with the results of Eisen and Reddi, but in B channeling, the discrepancies of 10--20% are observed among S/sub e/ values obtained experimentally by three different groups

  20. Interactions between charged residues in the transmembrane segments of the voltage-sensing domain in the hERG channel.

    Science.gov (United States)

    Zhang, M; Liu, J; Jiang, M; Wu, D-M; Sonawane, K; Guy, H R; Tseng, G-N

    2005-10-01

    Studies on voltage-gated K channels such as Shaker have shown that positive charges in the voltage-sensor (S4) can form salt bridges with negative charges in the surrounding transmembrane segments in a state-dependent manner, and different charge pairings can stabilize the channels in closed or open states. The goal of this study is to identify such charge interactions in the hERG channel. This knowledge can provide constraints on the spatial relationship among transmembrane segments in the channel's voltage-sensing domain, which are necessary for modeling its structure. We first study the effects of reversing S4's positive charges on channel activation. Reversing positive charges at the outer (K525D) and inner (K538D) ends of S4 markedly accelerates hERG activation, whereas reversing the 4 positive charges in between either has no effect or slows activation. We then use the 'mutant cycle analysis' to test whether D456 (outer end of S2) and D411 (inner end of S1) can pair with K525 and K538, respectively. Other positive charges predicted to be able, or unable, to interact with D456 or D411 are also included in the analysis. The results are consistent with predictions based on the distribution of these charged residues, and confirm that there is functional coupling between D456 and K525 and between D411 and K538.

  1. Progress in Development of Improved Ion-Channel Biosensors

    Science.gov (United States)

    Nadeau, Jay L.; White, Victor E.; Maurer, Joshua A.; Dougherty, Dennis A.

    2008-01-01

    Further improvements have recently been made in the development of the devices described in Improved Ion-Channel Biosensors (NPO-30710), NASA Tech Briefs, Vol. 28, No. 10 (October 2004), page 30. As discussed in more detail in that article, these sensors offer advantages of greater stability, greater lifetime, and individual electrical addressability, relative to prior ion-channel biosensors. In order to give meaning to a brief description of the recent improvements, it is necessary to recapitulate a substantial portion of the text of the cited previous article. The figure depicts one sensor that incorporates the recent improvements, and can be helpful in understanding the recapitulated text, which follows: These sensors are microfabricated from silicon and other materials compatible with silicon. Typically, the sensors are fabricated in arrays in silicon wafers on glass plates. Each sensor in the array can be individually electrically addressed, without interference with its neighbors. Each sensor includes a well covered by a thin layer of silicon nitride, in which is made a pinhole for the formation of a lipid bilayer membrane. In one stage of fabrication, the lower half of the well is filled with agarose, which is allowed to harden. Then the upper half of the well is filled with a liquid electrolyte (which thereafter remains liquid) and a lipid bilayer is painted over the pinhole. The liquid contains a protein that forms an ion channel on top of the hardened agarose. The combination of enclosure in the well and support by the hardened agarose provides the stability needed to keep the membrane functional for times as long as days or even weeks. An electrode above the well, another electrode below the well, and all the materials between the electrodes together constitute a capacitor. What is measured is the capacitive transient current in response to an applied voltage pulse. One notable feature of this sensor, in comparison with prior such sensors, is a

  2. X-ray generation in an ion channel

    International Nuclear Information System (INIS)

    Kostyukov, I.; Kiselev, S.; Pukhov, A.

    2003-01-01

    X-ray generation by relativistic electrons in an ion channel is studied. The emission process is analyzed in the regime of high harmonic generation when the plasma wiggler strength is large. Like for the conventional free electron laser, the synchrotron-like broadband spectrum is generated in this regime. An asymptotic expression for the radiation spectrum of the spontaneous emission is derived. The radiation spectrum emitted from an axisymmetric monoenergetic electron beam is analyzed. The stimulated emission in the ion channel is studied and the gain of the ion-channel synchrotron-radiation laser is calculated. It is shown that the use of laser-produced ion channels leads to a much higher power of x-ray radiation than the one in a self-generated channel. In addition, the mean photon energy, the number of emitted photons and the brilliance of the photon beam increase dramatically. Three-dimensional particle-in-cell simulations of a 25-GeV electron bunch propagating in a laser-produced ion channel are made. Several GeV γ-quants are produced in a good agreement with the analytical results

  3. Simulation of channelled ion ranges in crystalline silicon

    International Nuclear Information System (INIS)

    Kabadayi, Oender; Guemues, Hasan

    2004-01-01

    We present results from a channelled ion range simulation model based on separation of ion trajectories into three different categories known as random, channelled, and well-channelled. We present this for boron projectiles incident along the Si direction. Stopping powers for channelled particles, well-channelled, and random particles are determined using experimental ratios of random and channelled stopping powers for a boron/silicon system. We have found the particle range distributions and the mean range of particles in crystalline channels. A new program code has been developed for the implementation of the presented model. The results are compared with experimental data as well as Crystal-transport and range of ions in matter, stopping and ranges of ions in matter, and projected range algorithm programs. We have found good agreement between our calculations and experiment, with an average discrepancy of 7%. Our model is also able to simulate the observed shift towards larger depths for the main ion distribution under channelling conditions

  4. Acid-sensing ion channel (ASIC) 4 predominantly localizes to an early endosome-related organelle upon heterologous expression.

    Science.gov (United States)

    Schwartz, Verena; Friedrich, Katharina; Polleichtner, Georg; Gründer, Stefan

    2015-12-15

    Acid-sensing ion channels (ASICs) are voltage-independent proton-gated amiloride sensitive sodium channels, belonging to the DEG/ENaC gene family. Six different ASICs have been identified (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) that are activated by a drop in extracellular pH, either as homo- or heteromers. An exception is ASIC4, which is not activated by protons as a homomer and which does not contribute to functional heteromeric ASICs. Insensitivity of ASIC4 to protons and its comparatively low sequence identity to other ASICs (45%) raises the question whether ASIC4 may have different functions than other ASICs. In this study, we therefore investigated the subcellular localization of ASIC4 in heterologous cell lines, which revealed a surprising accumulation of the channel in early endosome-related vacuoles. Moreover, we identified an unique amino-terminal motif as important for forward-trafficking from the ER/Golgi to the early endosome-related compartment. Collectively, our results show that heterologously expressed ASIC4 predominantly resides in an intracellular endosomal compartment.

  5. [Mechanism of action of neurotoxins acting on the inactivation of voltage-gated sodium channels].

    Science.gov (United States)

    Benoit, E

    1998-01-01

    This review focuses on the mechanism(s) of action of neurotoxins acting on the inactivation of voltage-gated Na channels. Na channels are transmembrane proteins which are fundamental for cellular communication. These proteins form pores in the plasma membrane allowing passive ionic movements to occur. Their opening and closing are controlled by gating systems which depend on both membrane potential and time. Na channels have three functional properties, mainly studied using electrophysiological and biochemical techniques, to ensure their role in the generation and propagation of action potentials: 1) a highly selectivity for Na ions, 2) a rapid opening ("activation"), responsible for the depolarizing phase of the action potential, and 3) a late closing ("inactivation") involved in the repolarizing phase of the action potential. As an essential protein for membrane excitability, the Na channel is the specific target of a number of vegetal and animal toxins which, by binding to the channel, alter its activity by affecting one or more of its properties. At least six toxin receptor sites have been identified on the neuronal Na channel on the basis of binding studies. However, only toxins interacting with four of these sites (sites 2, 3, 5 et 6) produce alterations of channel inactivation. The maximal percentage of Na channels modified by the binding of neurotoxins to sites 2 (batrachotoxin and some alkaloids), 3 (alpha-scorpion and sea anemone toxins), 5 (brevetoxins and ciguatoxins) et 6 (delta-conotoxins) is different according to the site considered. However, in all cases, these channels do not inactivate. Moreover, Na channels modified by toxins which bind to sites 2, 5 and 6 activate at membrane potentials more negative than do unmodified channels. The physiological consequences of Na channel modifications, induced by the binding of neurotoxins to sites 2, 3, 5 and 6, are (i) an inhibition of cellular excitability due to an important membrane depolarization (site

  6. C-terminal modulatory domain controls coupling of voltage-sensing to pore opening in Cav1.3 L-type Ca(2+) channels.

    Science.gov (United States)

    Lieb, Andreas; Ortner, Nadine; Striessnig, Jörg

    2014-04-01

    Activity of voltage-gated Cav1.3 L-type Ca(2+) channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Cav1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (QON-V) and of current activation (ICa-V) of the long (Cav1.3L) and a short Cav1.3 splice variant (Cav1.342A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (QON) of Cav1.3L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Cav1.2 and Cav3.1. However, a significantly higher fraction of the total charge had to move for activation of Cav1.3 half-maximal conductance (Cav1.3: 68%; Cav1.2: 52%; Cav3.1: 22%). This indicated a weaker coupling of Cav1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Cav1.342A, thereby shifting ICa-V by 7.2 mV to potentials that were more negative without changing QON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of QON-V and a more negative activation of ICa-V of all three channels. These findings illustrate that the voltage sensors of Cav1.3 channels respond more sensitively to depolarization than those of Cav1.2 or Cav3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Cav1.342A splice variants to activate at lower voltages

  7. Major Channels Involved In Neuropsychiatric Disorders And Therapeutic Perspectives

    Directory of Open Access Journals (Sweden)

    Paola eImbrici

    2013-05-01

    Full Text Available Voltage-gated ion channels are important mediators of physiological functions in the central nervous system. The cyclic activation of these channels influences neurotransmitter release, neuron excitability, gene transcription and plasticity, providing distinct brain areas with unique physiological and pharmacological response. A growing body of data has implicated ion channels in the susceptibility or pathogenesis of psychiatric diseases. Indeed, population studies support the association of polymorphisms in calcium and potassium channels with the genetic risk for bipolar disorders or schizophrenia. Moreover, point mutations in calcium, sodium and potassium channel genes have been identified in some childhood developmental disorders. Finally, antibodies against potassium channel complexes occur in a series of autoimmune psychiatric diseases. Here we report recent studies assessing the role of calcium, sodium and potassium channels in bipolar disorder, schizophrenia and autism spectrum disorders, and briefly summarize promising pharmacological strategies targeted on ion channels for the therapy of mental illness and related genetic tests.

  8. A two-dimensional analytical model for channel potential and threshold voltage of short channel dual material gate lightly doped drain MOSFET

    International Nuclear Information System (INIS)

    Tripathi Shweta

    2014-01-01

    An analytical model for the channel potential and the threshold voltage of the short channel dual-material-gate lightly doped drain (DMG-LDD) metal—oxide—semiconductor field-effect transistor (MOSFET) is presented using the parabolic approximation method. The proposed model takes into account the effects of the LDD region length, the LDD region doping, the lengths of the gate materials and their respective work functions, along with all the major geometrical parameters of the MOSFET. The impact of the LDD region length, the LDD region doping, and the channel length on the channel potential is studied in detail. Furthermore, the threshold voltage of the device is calculated using the minimum middle channel potential, and the result obtained is compared with the DMG MOSFET threshold voltage to show the improvement in the threshold voltage roll-off. It is shown that the DMG-LDD MOSFET structure alleviates the problem of short channel effects (SCEs) and the drain induced barrier lowering (DIBL) more efficiently. The proposed model is verified by comparing the theoretical results with the simulated data obtained by using the commercially available ATLAS™ 2D device simulator. (interdisciplinary physics and related areas of science and technology)

  9. A two-dimensional analytical model for channel potential and threshold voltage of short channel dual material gate lightly doped drain MOSFET

    Science.gov (United States)

    Shweta, Tripathi

    2014-11-01

    An analytical model for the channel potential and the threshold voltage of the short channel dual-material-gate lightly doped drain (DMG-LDD) metal—oxide—semiconductor field-effect transistor (MOSFET) is presented using the parabolic approximation method. The proposed model takes into account the effects of the LDD region length, the LDD region doping, the lengths of the gate materials and their respective work functions, along with all the major geometrical parameters of the MOSFET. The impact of the LDD region length, the LDD region doping, and the channel length on the channel potential is studied in detail. Furthermore, the threshold voltage of the device is calculated using the minimum middle channel potential, and the result obtained is compared with the DMG MOSFET threshold voltage to show the improvement in the threshold voltage roll-off. It is shown that the DMG-LDD MOSFET structure alleviates the problem of short channel effects (SCEs) and the drain induced barrier lowering (DIBL) more efficiently. The proposed model is verified by comparing the theoretical results with the simulated data obtained by using the commercially available ATLAS™ 2D device simulator.

  10. Channeling of molecular ions with relativistic energy

    International Nuclear Information System (INIS)

    Azuma, Toshiyuki; Muranaka, Tomoko; Kondo, Chikara; Hatakeyama, Atsushi; Komaki, Kenichiro; Yamazaki, Yasunori; Takabayashi, Yuichi; Murakami, Takeshi; Takada, Eiichi

    2003-01-01

    When energetic ions are injected into a single crystal parallel to a crystal axis or plane, they proceed in an open space guided by the crystal potential without colliding with atoms in the atomic plane or string, which is called channeling. We aimed to study dynamics of molecular ions, H 2 + , of 160 MeV/u and their fragment ions, H + ions in a Si crystal under the channeling condition. The molecular ions, H 2 + , are soon ionized, i.e. electron-stripped in the crystal, and a pair of bare nuclei, H + ions, travels in the crystal potential with mutual Coulomb repulsion. We developed a 2D position sensitive detector for the angular-distribution measurement of the H + ions transmitted through the crystal, and observed the detailed angular distribution. In addition we measured the case of H + on incidence for comparison. As a result, the channeled component and non-channeling were clearly separated. The incident angular divergence is critical to discuss the effect of Coulomb explosion of molecular H 2 + ions. (author)

  11. On the mechanism of TBA block of the TRPV1 channel.

    Science.gov (United States)

    Oseguera, Andrés Jara; Islas, León D; García-Villegas, Refugio; Rosenbaum, Tamara

    2007-06-01

    The transient receptor potential vanilloid 1 (TRPV1) channel is a nonselective cation channel activated by capsaicin and responsible for thermosensation. To date, little is known about the gating characteristics of these channels. Here we used tetrabutylammonium (TBA) to determine whether this molecule behaves as an ion conduction blocker in TRPV1 channels and to gain insight into the nature of the activation gate of this protein. TBA belongs to a family of classic potassium channel blockers that have been widely used as tools for determining the localization of the activation gate and the properties of the pore of several ion channels. We found TBA to be a voltage-dependent pore blocker and that the properties of block are consistent with an open-state blocker, with the TBA molecule binding to multiple open states, each with different blocker affinities. Kinetics of channel closure and burst-length analysis in the presence of blocker are consistent with a state-dependent blocking mechanism, with TBA interfering with closing of an activation gate. This activation gate may be located cytoplasmically with respect to the binding site of TBA ions, similar to what has been observed in potassium channels. We propose an allosteric model for TRPV1 activation and block by TBA, which explains our experimental data.

  12. The voltage-sensing domain of a phosphatase gates the pore of a potassium channel.

    Science.gov (United States)

    Arrigoni, Cristina; Schroeder, Indra; Romani, Giulia; Van Etten, James L; Thiel, Gerhard; Moroni, Anna

    2013-03-01

    The modular architecture of voltage-gated K(+) (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates Kv(Synth1), a functional voltage-gated, outwardly rectifying K(+) channel. Kv(Synth1) displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V(1/2) = +56 mV; z of ~1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.

  13. Piezo proteins are pore-forming subunits of mechanically activated channels.

    Science.gov (United States)

    Coste, Bertrand; Xiao, Bailong; Santos, Jose S; Syeda, Ruhma; Grandl, Jörg; Spencer, Kathryn S; Kim, Sung Eun; Schmidt, Manuela; Mathur, Jayanti; Dubin, Adrienne E; Montal, Mauricio; Patapoutian, Ardem

    2012-02-19

    Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

  14. CHANNELING OF B-IONS IN SILICON

    NARCIS (Netherlands)

    VOS, M; MITCHELL, [No Value; SMULDERS, PJM

    We present new results on the channeling of B ions in Si crystals. Standard surface barrier detectors have been used to record energy spectra for B ions backscattered from the near surface (approximately 1500 angstrom) of a silicon crystal, under perfect, and near axial and planar channeling

  15. New Conotoxin SO-3 Targeting N-type Voltage-Sensitive Calcium Channels

    Directory of Open Access Journals (Sweden)

    Lei Wen

    2006-04-01

    Full Text Available Selective blockers of the N-type voltage-sensitive calcium (CaV channels are useful in the management of severe chronic pain. Here, the structure and function characteristics of a novel N-type CaV channel blocker, SO-3, are reviewed. SO-3 is a 25-amino acid conopeptide originally derived from the venom of Conus striatus, and contains the same 4-loop, 6-cysteine framework (C-C-CC-C-C as O-superfamily conotoxins. The synthetic SO-3 has high analgesic activity similar to ω-conotoxin MVIIA (MVIIA, a selective N-type CaV channel blocker approved in the USA and Europe for the alleviation of persistent pain states. In electrophysiological studies, SO-3 shows more selectivity towards the N-type CaV channels than MVIIA. The dissimilarity between SO-3 and MVIIA in the primary and tertiary structures is further discussed in an attempt to illustrate the difference in selectivity of SO-3 and MVIIA towards N-type CaV channels.

  16. The human red cell voltage-dependent cation channel. Part III: Distribution homogeneity and pH dependence

    DEFF Research Database (Denmark)

    Bennekou, P.; Barksmann, T. L.; Christophersen, P.

    2006-01-01

    The homogeneity of the distribution of the non-selective voltage-dependent cation channel (the NSVDC channel) in the human erythrocyte, and the pH dependence was investigated. Activation of this channel caused a uniform cellular dehydration, which was characterized by the changes in the erythrocyte...... osmotic resistance profiles: After 1/2 h of activation, the osmolarity at 50% hemolysis changed from 73 mM (control) to 34 mM NaCl, corresponding to 0.48% and 0.21% NaCl respectively. Unchanging standard deviations show participation of the entire erythrocyte population, which implies an even distribution...... of the NSVDC channel among the cells. Inactivation of the NSVDC channel with N-ethyl-maleimide (NEM) or blocking of the Cl- conductance with NS1652 retarded the migration of the resistance profiles towards lower osmolarities. The NSVDC channel activation was blocked by a decrease of the intracellular...

  17. Investigation of channel width-dependent threshold voltage variation in a-InGaZnO thin-film transistors

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Kuan-Hsien; Chou, Wu-Ching [Department of Electrophysics, National Chiao Tung University, Hsinchu, Taiwan (China); Chang, Ting-Chang, E-mail: tcchang@mail.phys.nsysu.edu.tw [Department of Physics, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Advanced Optoelectronics Technology Center, National Cheng Kung University, Taiwan (China); Wu, Ming-Siou; Hung, Yi-Syuan; Sze, Simon M. [Department of Electronics Engineering, National Chiao Tung University, Hsinchu, Taiwan (China); Hung, Pei-Hua; Chu, Ann-Kuo [Department of Photonics, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Hsieh, Tien-Yu [Department of Physics, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Yeh, Bo-Liang [Advanced Display Technology Research Center, AU Optronics, No. 1, Li-Hsin Rd. 2, Hsinchu Science Park, Hsinchu 30078, Taiwan (China)

    2014-03-31

    This Letter investigates abnormal channel width-dependent threshold voltage variation in amorphous indium-gallium-zinc-oxide (a-IGZO) thin-film transistors. Unlike drain-induced source barrier lowering effect, threshold voltage increases with increasing drain voltage. Furthermore, the wider the channel, the larger the threshold voltage observed. Because of the surrounding oxide and other thermal insulating material and the low thermal conductivity of the IGZO layer, the self-heating effect will be pronounced in wider channel devices and those with a larger operating drain bias. To further clarify the physical mechanism, fast IV measurement is utilized to demonstrate the self-heating induced anomalous channel width-dependent threshold voltage variation.

  18. Comparison of Langevin and Markov channel noise models for neuronal signal generation.

    Science.gov (United States)

    Sengupta, B; Laughlin, S B; Niven, J E

    2010-01-01

    The stochastic opening and closing of voltage-gated ion channels produce noise in neurons. The effect of this noise on the neuronal performance has been modeled using either an approximate or Langevin model based on stochastic differential equations or an exact model based on a Markov process model of channel gating. Yet whether the Langevin model accurately reproduces the channel noise produced by the Markov model remains unclear. Here we present a comparison between Langevin and Markov models of channel noise in neurons using single compartment Hodgkin-Huxley models containing either Na+ and K+, or only K+ voltage-gated ion channels. The performance of the Langevin and Markov models was quantified over a range of stimulus statistics, membrane areas, and channel numbers. We find that in comparison to the Markov model, the Langevin model underestimates the noise contributed by voltage-gated ion channels, overestimating information rates for both spiking and nonspiking membranes. Even with increasing numbers of channels, the difference between the two models persists. This suggests that the Langevin model may not be suitable for accurately simulating channel noise in neurons, even in simulations with large numbers of ion channels.

  19. Functional diversity of voltage-sensing phosphatases in two urodele amphibians.

    Science.gov (United States)

    Mutua, Joshua; Jinno, Yuka; Sakata, Souhei; Okochi, Yoshifumi; Ueno, Shuichi; Tsutsui, Hidekazu; Kawai, Takafumi; Iwao, Yasuhiro; Okamura, Yasushi

    2014-07-16

    Voltage-sensing phosphatases (VSPs) share the molecular architecture of the voltage sensor domain (VSD) with voltage-gated ion channels and the phosphoinositide phosphatase region with the phosphatase and tensin homolog (PTEN), respectively. VSPs enzymatic activities are regulated by the motions of VSD upon depolarization. The physiological role of these proteins has remained elusive, and insights may be gained by investigating biological variations in different animal species. Urodele amphibians are vertebrates with potent activities of regeneration and also show diverse mechanisms of polyspermy prevention. We cloned cDNAs of VSPs from the testes of two urodeles; Hynobius nebulosus and Cynops pyrrhogaster, and compared their expression and voltage-dependent activation. Their molecular architecture is highly conserved in both Hynobius VSP (Hn-VSP) and Cynops VSP (Cp-VSP), including the positively-charged arginine residues in the S4 segment of the VSD and the enzymatic active site for substrate binding, yet the C-terminal C2 domain of Hn-VSP is significantly shorter than that of Cp-VSP and other VSP orthologs. RT-PCR analysis showed that gene expression pattern was distinct between two VSPs. The voltage sensor motions and voltage-dependent phosphatase activities were investigated electrophysiologically by expression in Xenopus oocytes. Both VSPs showed "sensing" currents, indicating that their voltage sensor domains are functional. The phosphatase activity of Cp-VSP was found to be voltage dependent, as shown by its ability to regulate the conductance of coexpressed GIRK2 channels, but Hn-VSP lacked such phosphatase activity due to the truncation of its C2 domain. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  20. Acid-sensing ion channels and transient-receptor potential ion channels in zebrafish taste buds.

    Science.gov (United States)

    Levanti, M; Randazzo, B; Viña, E; Montalbano, G; Garcia-Suarez, O; Germanà, A; Vega, J A; Abbate, F

    2016-09-01

    Sensory information from the environment is required for life and survival, and it is detected by specialized cells which together make up the sensory system. The fish sensory system includes specialized organs that are able to detect mechanical and chemical stimuli. In particular, taste buds are small organs located on the tongue in terrestrial vertebrates that function in the perception of taste. In fish, taste buds occur on the lips, the flanks, and the caudal (tail) fins of some species and on the barbels of others. In fish taste receptor cells, different classes of ion channels have been detected which, like in mammals, presumably participate in the detection and/or transduction of chemical gustatory signals. However, since some of these ion channels are involved in the detection of additional sensory modalities, it can be hypothesized that taste cells sense stimuli other than those specific for taste. This mini-review summarizes current knowledge on the presence of transient-receptor potential (TRP) and acid-sensing (ASIC) ion channels in the taste buds of teleosts, especially adult zebrafish. Up to now ASIC4, TRPC2, TRPA1, TRPV1 and TRPV4 ion channels have been found in the sensory cells, while ASIC2 was detected in the nerves supplying the taste buds. Copyright © 2016 Elsevier GmbH. All rights reserved.

  1. Action of insecticidal N-alkylamides at site 2 of the voltage-sensitive sodium channel

    International Nuclear Information System (INIS)

    Ottea, J.A.; Payne, G.T.; Soderlund, D.M.

    1990-01-01

    Nine synthetic N-alkylamides were examined as inhibitors of the specific binding of [ 3 H]batrachotoxinin A 20α-benzoate ([ 3 H]BTX-B) to sodium channels and as activators of sodium uptake in mouse brain synaptoneurosomes. In the presence of scorpion (Leiurus quinquestriatus) venom, the six insecticidal analogues were active as both inhibitors of [ 3 H]BTX-B binding and stimulators of sodium uptake. These findings are consistent with an action of these compounds at the alkaloid activator recognition site (site 2) of the voltage-sensitive sodium channel. The three noninsecticidal N-alkylamides also inhibited [ 3 H]BTX-B binding but were ineffective as activators of sodium uptake. Concentration-response studies revealed that some of the insecticidal amides also enhanced sodium uptake through a second, high-affinity interaction that does not involve site 2, but this secondary effect does not appear to be correlated with insecticidal activity. The activities of N-alkylamides as sodium channel activators were influenced by the length of the alkenyl chain and the location of unsaturation within the molecule. These results further define the actions of N-alkylamides on sodium channels and illustrate the significance of the multiple binding domains of the sodium channel as target sites for insect control agents

  2. Sodium Channel (Dys)Function and Cardiac Arrhythmias

    NARCIS (Netherlands)

    Remme, Carol Ann; Bezzina, Connie R.

    2010-01-01

    P>Cardiac voltage-gated sodium channels are transmembrane proteins located in the cell membrane of cardiomyocytes. Influx of sodium ions through these ion channels is responsible for the initial fast upstroke of the cardiac action potential. This inward sodium current thus triggers the initiation

  3. Local anesthetics disrupt energetic coupling between the voltage-sensing segments of a sodium channel.

    Science.gov (United States)

    Muroi, Yukiko; Chanda, Baron

    2009-01-01

    Local anesthetics block sodium channels in a state-dependent fashion, binding with higher affinity to open and/or inactivated states. Gating current measurements show that local anesthetics immobilize a fraction of the gating charge, suggesting that the movement of voltage sensors is modified when a local anesthetic binds to the pore of the sodium channel. Here, using voltage clamp fluorescence measurements, we provide a quantitative description of the effect of local anesthetics on the steady-state behavior of the voltage-sensing segments of a sodium channel. Lidocaine and QX-314 shifted the midpoints of the fluorescence-voltage (F-V) curves of S4 domain III in the hyperpolarizing direction by 57 and 65 mV, respectively. A single mutation in the S6 of domain IV (F1579A), a site critical for local anesthetic block, abolished the effect of QX-314 on the voltage sensor of domain III. Both local anesthetics modestly shifted the F-V relationships of S4 domain IV toward hyperpolarized potentials. In contrast, the F-V curve of the S4 domain I was shifted by 11 mV in the depolarizing direction upon QX-314 binding. These antagonistic effects of the local anesthetic indicate that the drug modifies the coupling between the voltage-sensing domains of the sodium channel. Our findings suggest a novel role of local anesthetics in modulating the gating apparatus of the sodium channel.

  4. Structural mechanism of voltage-dependent gating in an isolated voltage-sensing domain.

    Science.gov (United States)

    Li, Qufei; Wanderling, Sherry; Paduch, Marcin; Medovoy, David; Singharoy, Abhishek; McGreevy, Ryan; Villalba-Galea, Carlos A; Hulse, Raymond E; Roux, Benoît; Schulten, Klaus; Kossiakoff, Anthony; Perozo, Eduardo

    2014-03-01

    The transduction of transmembrane electric fields into protein motion has an essential role in the generation and propagation of cellular signals. Voltage-sensing domains (VSDs) carry out these functions through reorientations of positive charges in the S4 helix. Here, we determined crystal structures of the Ciona intestinalis VSD (Ci-VSD) in putatively active and resting conformations. S4 undergoes an ~5-Å displacement along its main axis, accompanied by an ~60° rotation. This movement is stabilized by an exchange in countercharge partners in helices S1 and S3 that generates an estimated net charge transfer of ~1 eo. Gating charges move relative to a ''hydrophobic gasket' that electrically divides intra- and extracellular compartments. EPR spectroscopy confirms the limited nature of S4 movement in a membrane environment. These results provide an explicit mechanism for voltage sensing and set the basis for electromechanical coupling in voltage-dependent enzymes and ion channels.

  5. Search after new agents for hyperpolarization-activated and cyclic nucleotide-gated ion channels; Suche nach neuen Wirkstoffen fuer Hyperpolarisationsaktivierte und zyklisch Nukleotid-gesteuerte Ionenkanaele

    Energy Technology Data Exchange (ETDEWEB)

    Struenker, T.

    2005-12-01

    Rhythmic activity of single cells or cellular networks is a common feature of most organisms. Cellular rhythms govern the beating of the heart, cycles of sleep and wakefulness, breathing, and the release of hormones. The endogenous rhythmic activity of many neurons and cardiac relies on a complex interplay between several distinct ion channels. In particular, one type of ion channel plays a prominent role in the control of rhythmic electrical activity because it determines the frequency of the oscillations. The activity of the channels is thus setting the ''pace'' of the activity; therefore, these channels are often referred to as ''pacemaker'' channels. Despite their obvious physiological importance it hasn't been until a few years ago that the genes encoding pacemaker channels have been identified. Because both hyperpolarization and cyclic nucleotides are key elements that control their activity, pacemaker channels have now been designated hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels. From a scientific as well as medical point of view, HCN channels are interesting drug targets. Only a few substances are known that specifically affect HCN channels. In the present study, a microtiter plate-based high throughput screening assay for HCN1 and HCN4 channels was developed. With this assay, known drugs for HCN channels were characterized. Subsequently, venoms of snails, spiders, scorpions, and snakes were screened for toxins affecting HCN channel activity. A few venoms were identified that possibly contain drugs that act on HCN channels. (orig.)

  6. Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

    Directory of Open Access Journals (Sweden)

    Tzilhav Shem-Ad

    Full Text Available The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

  7. Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

    Science.gov (United States)

    Shem-Ad, Tzilhav; Irit, Orr; Yifrach, Ofer

    2013-01-01

    The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

  8. The tarantula toxins ProTx-II and huwentoxin-IV differentially interact with human Nav1.7 voltage sensors to inhibit channel activation and inactivation.

    Science.gov (United States)

    Xiao, Yucheng; Blumenthal, Kenneth; Jackson, James O; Liang, Songping; Cummins, Theodore R

    2010-12-01

    The voltage-gated sodium channel Na(v)1.7 plays a crucial role in pain, and drugs that inhibit hNa(v)1.7 may have tremendous therapeutic potential. ProTx-II and huwentoxin-IV (HWTX-IV), cystine knot peptides from tarantula venoms, preferentially block hNa(v)1.7. Understanding the interactions of these toxins with sodium channels could aid the development of novel pain therapeutics. Whereas both ProTx-II and HWTX-IV have been proposed to preferentially block hNa(v)1.7 activation by trapping the domain II voltage-sensor in the resting configuration, we show that specific residues in the voltage-sensor paddle of domain II play substantially different roles in determining the affinities of these toxins to hNa(v)1.7. The mutation E818C increases ProTx-II's and HWTX-IV's IC(50) for block of hNa(v)1.7 currents by 4- and 400-fold, respectively. In contrast, the mutation F813G decreases ProTx-II affinity by 9-fold but has no effect on HWTX-IV affinity. It is noteworthy that we also show that ProTx-II, but not HWTX-IV, preferentially interacts with hNa(v)1.7 to impede fast inactivation by trapping the domain IV voltage-sensor in the resting configuration. Mutations E1589Q and T1590K in domain IV each decreased ProTx-II's IC(50) for impairment of fast inactivation by ~6-fold. In contrast mutations D1586A and F1592A in domain-IV increased ProTx-II's IC(50) for impairment of fast inactivation by ~4-fold. Our results show that whereas ProTx-II and HWTX-IV binding determinants on domain-II may overlap, domain II plays a much more crucial role for HWTX-IV, and contrary to what has been proposed to be a guiding principle of sodium channel pharmacology, molecules do not have to exclusively target the domain IV voltage-sensor to influence sodium channel inactivation.

  9. Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator

    International Nuclear Information System (INIS)

    Fitzpatrick, L.A.; Yasumoto, T.; Aurbach, G.D.

    1989-01-01

    Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release

  10. L-Type Voltage-Gated Ca2+ Channels Regulate Synaptic-Activity-Triggered Recycling Endosome Fusion in Neuronal Dendrites

    Directory of Open Access Journals (Sweden)

    Brian G. Hiester

    2017-11-01

    Full Text Available The repertoire and abundance of proteins displayed on the surface of neuronal dendrites are tuned by regulated fusion of recycling endosomes (REs with the dendritic plasma membrane. While this process is critical for neuronal function and plasticity, how synaptic activity drives RE fusion remains unexplored. We demonstrate a multistep fusion mechanism that requires Ca2+ from distinct sources. NMDA receptor Ca2+ initiates RE fusion with the plasma membrane, while L-type voltage-gated Ca2+ channels (L-VGCCs regulate whether fused REs collapse into the membrane or reform without transferring their cargo to the cell surface. Accordingly, NMDA receptor activation triggered AMPA-type glutamate receptor trafficking to the dendritic surface in an L-VGCC-dependent manner. Conversely, potentiating L-VGCCs enhanced AMPA receptor surface expression only when NMDA receptors were also active. Thus L-VGCCs play a role in tuning activity-triggered surface expression of key synaptic proteins by gating the mode of RE fusion.

  11. [Compared Markov with fractal models by using single-channel experimental and simulation data].

    Science.gov (United States)

    Lan, Tonghan; Wu, Hongxiu; Lin, Jiarui

    2006-10-01

    The gating mechanical kinetical of ion channels has been modeled as a Markov process. In these models it is assumed that the channel protein has a small number of discrete conformational states and kinetic rate constants connecting these states are constant, the transition rate constants among the states is independent both of time and of the previous channel activity. It is assumed in Liebovitch's fractal model that the channel exists in an infinite number of energy states, consequently, transitions from one conductance state to another would be governed by a continuum of rate constants. In this paper, a statistical comparison is presented of Markov and fractal models of ion channel gating, the analysis is based on single-channel data from ion channel voltage-dependence K+ single channel of neuron cell and simulation data from three-states Markov model.

  12. Skin secretion of Siphonops paulensis (Gymnophiona, Amphibia forms voltage-dependent ionic channels in lipid membranes

    Directory of Open Access Journals (Sweden)

    E.F. Schwartz

    2003-09-01

    Full Text Available The effect of the skin secretion of the amphibian Siphonops paulensis was investigated by monitoring the changes in conductance of an artificial planar lipid bilayer. Skin secretion was obtained by exposure of the animals to ether-saturated air, and then rinsing the animals with distilled water. Artificial lipid bilayers were obtained by spreading a solution of azolectin over an aperture of a Delrin cup inserted into a cut-away polyvinyl chloride block. In 9 of 12 experiments, the addition of the skin secretion to lipid bilayers displayed voltage-dependent channels with average unitary conductance of 258 ± 41.67 pS, rather than nonspecific changes in bilayer conductance. These channels were not sensitive to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid or tetraethylammonium ion, but the experimental protocol used does not permit us to specify their characteristics.

  13. From Brownian Dynamics to Markov Chain: An Ion Channel Example

    KAUST Repository

    Chen, Wan

    2014-02-27

    A discrete rate theory for multi-ion channels is presented, in which the continuous dynamics of ion diffusion is reduced to transitions between Markovian discrete states. In an open channel, the ion permeation process involves three types of events: an ion entering the channel, an ion escaping from the channel, or an ion hopping between different energy minima in the channel. The continuous dynamics leads to a hierarchy of Fokker-Planck equations, indexed by channel occupancy. From these the mean escape times and splitting probabilities (denoting from which side an ion has escaped) can be calculated. By equating these with the corresponding expressions from the Markov model, one can determine the Markovian transition rates. The theory is illustrated with a two-ion one-well channel. The stationary probability of states is compared with that from both Brownian dynamics simulation and the hierarchical Fokker-Planck equations. The conductivity of the channel is also studied, and the optimal geometry maximizing ion flux is computed. © 2014 Society for Industrial and Applied Mathematics.

  14. Incorporating Born solvation energy into the three-dimensional Poisson-Nernst-Planck model to study ion selectivity in KcsA K+ channels

    Science.gov (United States)

    Liu, Xuejiao; Lu, Benzhuo

    2017-12-01

    Potassium channels are much more permeable to potassium than sodium ions, although potassium ions are larger and both carry the same positive charge. This puzzle cannot be solved based on the traditional Poisson-Nernst-Planck (PNP) theory of electrodiffusion because the PNP model treats all ions as point charges, does not incorporate ion size information, and therefore cannot discriminate potassium from sodium ions. The PNP model can qualitatively capture some macroscopic properties of certain channel systems such as current-voltage characteristics, conductance rectification, and inverse membrane potential. However, the traditional PNP model is a continuum mean-field model and has no or underestimates the discrete ion effects, in particular the ion solvation or self-energy (which can be described by Born model). It is known that the dehydration effect (closely related to ion size) is crucial to selective permeation in potassium channels. Therefore, we incorporated Born solvation energy into the PNP model to account for ion hydration and dehydration effects when passing through inhomogeneous dielectric channel environments. A variational approach was adopted to derive a Born-energy-modified PNP (BPNP) model. The model was applied to study a cylindrical nanopore and a realistic KcsA channel, and three-dimensional finite element simulations were performed. The BPNP model can distinguish different ion species by ion radius and predict selectivity for K+ over Na+ in KcsA channels. Furthermore, ion current rectification in the KcsA channel was observed by both the PNP and BPNP models. The I -V curve of the BPNP model for the KcsA channel indicated an inward rectifier effect for K+ (rectification ratio of ˜3 /2 ) but indicated an outward rectifier effect for Na+ (rectification ratio of ˜1 /6 ) .

  15. Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution.

    Science.gov (United States)

    Shen, Huaizong; Zhou, Qiang; Pan, Xiaojing; Li, Zhangqiang; Wu, Jianping; Yan, Nieng

    2017-03-03

    Voltage-gated sodium (Na v ) channels are responsible for the initiation and propagation of action potentials. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. Here, we report the cryogenic electron microscopy structure of a putative Na v channel from American cockroach (designated Na v PaS) at 3.8 angstrom resolution. The voltage-sensing domains (VSDs) of the four repeats exhibit distinct conformations. The entrance to the asymmetric selectivity filter vestibule is guarded by heavily glycosylated and disulfide bond-stabilized extracellular loops. On the cytoplasmic side, a conserved amino-terminal domain is placed below VSD I , and a carboxy-terminal domain binds to the III-IV linker. The structure of Na v PaS establishes an important foundation for understanding function and disease mechanism of Na v and related voltage-gated calcium channels. Copyright © 2017, American Association for the Advancement of Science.

  16. Effect of 50 and 80 MeV phosphorous ions on the contribution of interface and oxide state density in n-channel MOSFETs

    Energy Technology Data Exchange (ETDEWEB)

    Shinde, N.S.; Dhole, S.D.; Kanjilal, D.; Bhoraskar, V.N. E-mail: vnb@physics.unipune.ernet.in

    1999-07-02

    n-channel depletion MOS devices were irradiated with 50 and 80 MeV phosphorous ions, with different fluences varying in the range from 10{sup 11} to 10{sup 13} ions/cm{sup 2}. The pre and post irradiation I-V characteristics were measured and the corresponding threshold shift {delta}V{sub TH} was estimated. In both the cases, the drain current I{sub D} and the threshold voltage V{sub TH} were found to decrease with the ion fluence. The increase in the threshold voltage shift {delta}V{sub TH} with the ion fluence, was greater for the devices irradiated with 80 MeV ions than those irradiated with 50 MeV ions. The interface and oxide state densities were determined through the subthreshold voltage measurements. To separate the contributions of oxide and interface states towards the threshold voltage shift, the ion irradiated MOS devices were annealed at 150 deg. C. The threshold shift during annealing initially decreased and later increased with increasing annealing period. The rate of change of the interface states during annealing was higher than that of the oxide states. It was also found that depletion mode (normally ON) MOSFETs switched operation to enhancement mode (normally OFF)

  17. Imaging the PCP site of the NMDA ion channel

    Energy Technology Data Exchange (ETDEWEB)

    Waterhouse, Rikki N. E-mail: rnw7@columbia.edu

    2003-11-01

    The N-methyl-D-aspartate (NMDA) ion channel plays a role in neuroprotection, neurodegeneration, long-term potentiation, memory, and cognition. It is implicated in the pathophysiology of several neurological and neuropsychiatric disorders including Parkinson's Disease, Huntington's Chorea, schizophrenia, alcoholism and stroke. The development of effective radiotracers for the study of NMDA receptors is critical for our understanding of their function, and their modulation by endogenousr substances or therapeutic drugs. Since the NMDA/PCP receptor lies within the channel, it is a unique target and is theoretically accessible only when the channel is in the active and 'open' state, but not when it is in the inactive or 'closed' state. The physical location of the NMDA/PCP receptor not only makes it an important imaging target but also complicates the development of suitable PET and SPECT radiotracers for this site. An intimate understanding of the biochemical, pharmacological, physiological and behavioral processes associated with the NMDA ion channel is essential to develop improved imaging agents. This review outlines progress made towards the development of radiolabeled agents for PCP sites of the NMDA ion channel. In addition, the animal and pharmacological models used for in vitro and in vivo assessment of NMDA receptor targeted agents are discussed.

  18. Imaging the PCP site of the NMDA ion channel

    International Nuclear Information System (INIS)

    Waterhouse, Rikki N.

    2003-01-01

    The N-methyl-D-aspartate (NMDA) ion channel plays a role in neuroprotection, neurodegeneration, long-term potentiation, memory, and cognition. It is implicated in the pathophysiology of several neurological and neuropsychiatric disorders including Parkinson's Disease, Huntington's Chorea, schizophrenia, alcoholism and stroke. The development of effective radiotracers for the study of NMDA receptors is critical for our understanding of their function, and their modulation by endogenousr substances or therapeutic drugs. Since the NMDA/PCP receptor lies within the channel, it is a unique target and is theoretically accessible only when the channel is in the active and 'open' state, but not when it is in the inactive or 'closed' state. The physical location of the NMDA/PCP receptor not only makes it an important imaging target but also complicates the development of suitable PET and SPECT radiotracers for this site. An intimate understanding of the biochemical, pharmacological, physiological and behavioral processes associated with the NMDA ion channel is essential to develop improved imaging agents. This review outlines progress made towards the development of radiolabeled agents for PCP sites of the NMDA ion channel. In addition, the animal and pharmacological models used for in vitro and in vivo assessment of NMDA receptor targeted agents are discussed

  19. Artificial modulation of the gating behavior of a K+ channel in a KvAP-DNA chimera.

    Directory of Open Access Journals (Sweden)

    Andrew Wang

    Full Text Available We present experiments where the gating behavior of a voltage-gated ion channel is modulated by artificial ligand binding. We construct a channel-DNA chimera with the KvAP potassium channel reconstituted in an artificial membrane. The channel is functional and the single channel ion conductivity unperturbed by the presence of the DNA. However, the channel opening probability vs. bias voltage, i.e., the gating, can be shifted considerably by the electrostatic force between the charges on the DNA and the voltage sensing domain of the protein. Different hybridization states of the chimera DNA thus lead to different response curves of the channel.

  20. Glycosylation of voltage-gated calcium channels in health and disease

    Czech Academy of Sciences Publication Activity Database

    Lazniewska, Joanna; Weiss, Norbert

    2017-01-01

    Roč. 1859, č. 5 (2017), s. 662-668 ISSN 0005-2736 R&D Projects: GA ČR GA15-13556S; GA MŠk 7AMB15FR015 Institutional support: RVO:61388963 Keywords : calcium channels * voltage-gated calcium channels * N-glycosylation * ancillary subunit * trafficking * stability Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.498, year: 2016

  1. The Activation Effect of Hainantoxin-I, a Peptide Toxin from the Chinese Spider, Ornithoctonus hainana, on Intermediate-Conductance Ca2+-Activated K+ Channels

    Directory of Open Access Journals (Sweden)

    Pengfei Huang

    2014-08-01

    Full Text Available Intermediate-conductance Ca2+-activated K+ (IK channels are calcium/calmodulin-regulated voltage-independent K+ channels. Activation of IK currents is important in vessel and respiratory tissues, rendering the channels potential drug targets. A variety of small organic molecules have been synthesized and found to be potent activators of IK channels. However, the poor selectivity of these molecules limits their therapeutic value. Venom-derived peptides usually block their targets with high specificity. Therefore, we searched for novel peptide activators of IK channels by testing a series of toxins from spiders. Using electrophysiological experiments, we identified hainantoxin-I (HNTX-I as an IK-channel activator. HNTX-I has little effect on voltage-gated Na+ and Ca2+ channels from rat dorsal root ganglion neurons and on the heterologous expression of voltage-gated rapidly activating delayed rectifier K+ channels (human ether-à-go-go-related gene; human ERG in HEK293T cells. Only 35.2% ± 0.4% of the currents were activated in SK channels, and there was no effect on BK channels. We demonstrated that HNTX-I was not a phrenic nerve conduction blocker or acutely toxic. This is believed to be the first report of a peptide activator effect on IK channels. Our study suggests that the activity and selectivity of HNTX-I on IK channels make HNTX-I a promising template for designing new drugs for cardiovascular diseases.

  2. Ion Selectivity Mechanism in a Bacterial Pentameric Ligand-Gated Ion Channel

    International Nuclear Information System (INIS)

    Wang, Hailong; Cheng, Xiaolin

    2011-01-01

    The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor (nAChR) that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential of mean force (PMF) profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel is open for a sodium ion to transport, but presents a ∼10 kcal/mol free energy barrier for a chloride ion, which arises primarily from the unfavorable interactions with a ring of negatively charged glutamate residues (E-2) at the intracellular end and a ring of hydrophobic residues (I9) in the middle of the transmembrane domain. Our collective findings further suggest that the charge selection mechanism can, to a large extent, be attributed to the narrow intracellular end and a ring of glutamate residues in this position their strong negative electrostatics and ability to bind cations. By contrast, E19 at the extracellular entrance only plays a minor role in ion selectivity of GLIC. In addition to electrostatics, both ion hydration and protein dynamics are found to be crucial for ion conduction as well, which explains why a chloride ion experiences a much greater barrier than a sodium ion in the hydrophobic region of the pore.

  3. AlGaN/GaN metal-insulator-semiconductor high electron mobility transistors with reduced leakage current and enhanced breakdown voltage using aluminum ion implantation

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Shichuang [Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074 (China); Key Laboratory of Nanodevices and Applications, Suzhou Institute of Nano-Tech and Nano-Bionics, CAS, Suzhou 215123 (China); Fu, Kai, E-mail: kfu2009@sinano.ac.cn, E-mail: cqchen@mail.hust.edu.cn; Yu, Guohao; Zhang, Zhili; Song, Liang; Deng, Xuguang; Li, Shuiming; Sun, Qian; Cai, Yong; Zhang, Baoshun [Key Laboratory of Nanodevices and Applications, Suzhou Institute of Nano-Tech and Nano-Bionics, CAS, Suzhou 215123 (China); Qi, Zhiqiang; Dai, Jiangnan; Chen, Changqing, E-mail: kfu2009@sinano.ac.cn, E-mail: cqchen@mail.hust.edu.cn [Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074 (China)

    2016-01-04

    This letter has studied the performance of AlGaN/GaN metal-insulator-semiconductor high electron mobility transistors on silicon substrate with GaN buffer treated by aluminum ion implantation for insulating followed by a channel regrown by metal–organic chemical vapor deposition. For samples with Al ion implantation of multiple energies of 140 keV (dose: 1.4 × 10{sup 14} cm{sup −2}) and 90 keV (dose: 1 × 10{sup 14} cm{sup −2}), the OFF-state leakage current is decreased by more than 3 orders and the breakdown voltage is enhanced by nearly 6 times compared to the samples without Al ion implantation. Besides, little degradation of electrical properties of the 2D electron gas channel is observed where the maximum drain current I{sub DSmax} at a gate voltage of 3 V was 701 mA/mm and the maximum transconductance g{sub mmax} was 83 mS/mm.

  4. Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

    DEFF Research Database (Denmark)

    Timmermann, D B; Lund, Trine Meldgaard; Belhage, B

    2001-01-01

    The physiological significance and subcellular distribution of voltage dependent calcium channels was defined using calcium channel blockers to inhibit potassium induced rises in cytosolic calcium concentration in cultured mouse neocortical neurons. The cytosolic calcium concentration was measured...... channels were differentially distributed in somata, neurites and nerve terminals. omega-conotoxin MVIIC (omega-CgTx MVIIC) inhibited approximately 40% of the Ca(2+)-rise in both somata and neurites and 60% of the potassium induced [3H]GABA release, indicating that the Q-type channel is the quantitatively...... most important voltage dependent calcium channel in all parts of the neuron. After treatment with thapsigargin the increase in cytosolic calcium was halved, indicating that calcium release from thapsigargin sensitive intracellular calcium stores is an important component of the potassium induced rise...

  5. Mechanosensitive Piezo Channels in the Gastrointestinal Tract.

    Science.gov (United States)

    Alcaino, C; Farrugia, G; Beyder, A

    2017-01-01

    Sensation of mechanical forces is critical for normal function of the gastrointestinal (GI) tract and abnormalities in mechanosensation are linked to GI pathologies. In the GI tract there are several mechanosensitive cell types-epithelial enterochromaffin cells, intrinsic and extrinsic enteric neurons, smooth muscle cells and interstitial cells of Cajal. These cells use mechanosensitive ion channels that respond to mechanical forces by altering transmembrane ionic currents in a process called mechanoelectrical coupling. Several mechanosensitive ionic conductances have been identified in the mechanosensory GI cells, ranging from mechanosensitive voltage-gated sodium and calcium channels to the mechanogated ion channels, such as the two-pore domain potassium channels K2P (TREK-1) and nonselective cation channels from the transient receptor potential family. The recently discovered Piezo channels are increasingly recognized as significant contributors to cellular mechanosensitivity. Piezo1 and Piezo2 are nonselective cationic ion channels that are directly activated by mechanical forces and have well-defined biophysical and pharmacologic properties. The role of Piezo channels in the GI epithelium is currently under investigation and their role in the smooth muscle syncytium and enteric neurons is still not known. In this review, we outline the current state of knowledge on mechanosensitive ion channels in the GI tract, with a focus on the known and potential functions of the Piezo channels. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein-Protein Interaction Inhibitors of CaV Function.

    Science.gov (United States)

    Findeisen, Felix; Campiglio, Marta; Jo, Hyunil; Abderemane-Ali, Fayal; Rumpf, Christine H; Pope, Lianne; Rossen, Nathan D; Flucher, Bernhard E; DeGrado, William F; Minor, Daniel L

    2017-06-21

    For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (Ca V ) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (Ca V β). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Ca V β interactions and reduce the entropic penalty associated with AID binding to Ca V β. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Ca V α 1 :Ca V β interaction that modulate Ca V function in an Ca V β isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based Ca V modulator design.

  7. Dynamics of the resistive state of a narrow superconducting channel in the ac voltage-driven regime

    International Nuclear Information System (INIS)

    Yerin, Yu.S.; Fenchenko, V.N.

    2013-01-01

    Within the time-dependent Ginzburg-Landau equations the dynamics of the order parameter in superconducting narrow channels of different lengths is investigated in the ac voltage-driven regime. The resistive state of the system at low frequencies of the applied voltage is characterized by the formation of periodic-in-time groups of oscillating phase-slip centers (PSC). An increase in frequency reduces the duration of the existence of these periodic groups. Depending on the length of the channel the ac voltage either tends to revert the channel to the state with one central PSC in periodic groups or minimizes the number of forming PSCs and orders their pattern in the system. A further increase in frequency for rather short channels leads to suppression of the order parameter without any creation of PSCs. For systems, whose length exceeds the specified limit, the formation of PSC occurs after a certain time which increases rapidly with frequency. The current-voltage characteristics of rather short channels at different applied voltage frequencies are calculated too. It is found that the current-voltage characteristics have a step-like structure, and the height of the first step is determined by the quadruple value of the Josephson frequency.

  8. Effects of the small molecule HERG activator NS1643 on Kv11.3 channels.

    Directory of Open Access Journals (Sweden)

    Arne Bilet

    Full Text Available NS1643 is one of the small molecule HERG (Kv11.1 channel activators and has also been found to increase erg2 (Kv11.2 currents. We now investigated whether NS1643 is also able to act as an activator of Kv11.3 (erg3 channels expressed in CHO cells. Activation of rat Kv11.3 current occurred in a dose-dependent manner and maximal current increasing effects were obtained with 10 µM NS1643. At this concentration, steady-state outward current increased by about 80% and the current increase was associated with a significant shift in the voltage dependence of activation to more negative potentials by about 15 mV. In addition, activation kinetics were accelerated, whereas deactivation was slowed. There was no significant effect on the kinetics of inactivation and recovery from inactivation. The strong current-activating agonistic effect of NS1643 did not result from a shift in the voltage dependence of Kv11.3 channel inactivation and was independent from external Na(+ or Ca(2+. At the higher concentration of 20 µM, NS1643 induced clearly less current increase. The left shift in the voltage dependence of activation reversed and the voltage sensitivity of activation dramatically decreased along with a slowing of Kv11.3 channel activation. These data show that, in comparison to other Kv11 family members, NS1643 exerts distinct effects on Kv11.3 channels with especially pronounced partial antagonistic effects at higher concentration.

  9. A molecular switch between the outer and the inner vestibule of the voltage-gated Na+ channel

    International Nuclear Information System (INIS)

    Zarrabi, T.

    2010-01-01

    Na+ channels permit rapid transmission of depolarizing impulses throughout cells and cell networks, and are essential to the proper function of skeletal muscle, the heart and the nervous system. The selectivity filter of the channel is considered to be formed by the amino acids D400, E755, K1237, and A1529 ('DEKA' motif) which are located at the innermost turn of the P-loops connecting S5 and S6 segments of each domain. The inner vestibule is believed to be lined by four S6 helices, one from each domain. Comparison of crystal structures of K+ channels in open and closed states as well as electron paramagnetic resonance spectroscopic studies suggest that the activation gate of voltage-gated ion channels is located at the inner part of the S6 segments. This may also hold true for voltage-gated Na+ channels because mutations in S6 segments alter activation gating. The gate for fast inactivation of the channel has been mapped to the intracellular linker between domains III and IV. This intracellular loop is currently considered to produce channel inactivation by transiently occluding the intracellular vestibule of the channel. The time constants of entry into and recovery from fast inactivation are on the order of milliseconds. Apart from 'fast inactivation' a number of slower inactivated states have been described. During very long depolarizations, on the order of several minutes, rNaV1.4 channels enter a very stable inactivated state which we refer to as 'ultra-slow' inactivation (IUS). In these channels the likelihood of entry into IUS is substantially increased by a mutation in the selectivity filter, K1237E. IUS can be modulated by molecules binding to the outer vestibule, suggesting that a conformational change of the outer vestibule gives rise to this kinetic state. On the other hand, the local anesthetic drug lidocaine, which binds to the internal part of the channel pore, inhibits entry into IUS by a 'foot-in-the-door' mechanism indicating that a

  10. Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP.

    Science.gov (United States)

    Lundby, Alicia; Akemann, Walther; Knöpfel, Thomas

    2010-11-01

    A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy transfer) signal. Here we report sensing current measurements from VSFP2.3, and show that VSFP2.3 carries 1.2 e sensing charges, which are displaced within 1.5 ms. The sensing currents become faster at higher temperatures, and the voltage dependence of the decay time constants is temperature dependent. Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing mechanism of Ci-VSP, which will allow us to further improve the sensitivity and kinetics of the family of VSFP proteins.

  11. Effect of ciguatoxin 3C on voltage-gated Na+ and K+ currents in mouse taste cells.

    Science.gov (United States)

    Ghiaroni, Valeria; Fuwa, Haruhiko; Inoue, Masayuki; Sasaki, Makoto; Miyazaki, Keisuke; Hirama, Masahiro; Yasumoto, Takeshi; Rossini, Gian Paolo; Scalera, Giuseppe; Bigiani, Albertino

    2006-09-01

    The marine dinoflagellate Gambierdiscus toxicus produces highly lipophilic, polycyclic ether toxins that cause a seafood poisoning called ciguatera. Ciguatoxins (CTXs) and gambierol represent the two major causative agents of ciguatera intoxication, which include taste alterations (dysgeusiae). However, information on the mode of action of ciguatera toxins in taste cells is scarce. Here, we have studied the effect of synthetic CTX3C (a CTX congener) on mouse taste cells. By using the patch-clamp technique to monitor membrane ion currents, we found that CTX3C markedly affected the operation of voltage-gated Na(+) channels but was ineffective on voltage-gated K(+) channels. This result was the exact opposite of what we obtained earlier with gambierol, which inhibits K(+) channels but not Na(+) channels. Thus, CTXs and gambierol affect with high potency the operation of separate classes of voltage-gated ion channels in taste cells. Our data suggest that taste disturbances reported in ciguatera poisoning might be due to the ability of ciguatera toxins to interfere with ion channels in taste buds.

  12. Toxic β-Amyloid (Aβ) Alzheimer's Ion Channels: From Structure to Function and Design

    Science.gov (United States)

    Nussinov, Ruth

    2012-02-01

    Full-length amyloid beta peptides (Aβ1-40/42) form neuritic amyloid plaques in Alzheimer's disease (AD) patients and are implicated in AD pathology. Recent biophysical and cell biological studies suggest a direct mechanism of amyloid beta toxicity -- ion channel mediated loss of calcium homeostasis. Truncated amyloid beta fragments (Aβ11-42 and Aβ17-42), commonly termed as non-amyloidogenic are also found in amyloid plaques of Alzheimer's disease (AD) and in the preamyloid lesions of Down's syndrome (DS), a model system for early onset AD study. Very little is known about the structure and activity of these smaller peptides although they could be key AD and DS pathological agents. Using complementary techniques of explicit solvent molecular dynamics (MD) simulations, atomic force microscopy (AFM), channel conductance measurements, cell calcium uptake assays, neurite degeneration and cell death assays, we have shown that non-amyloidogenic Aβ9-42 and Aβ17-42 peptides form ion channels with loosely attached subunits and elicit single channel conductances. The subunits appear mobile suggesting insertion of small oligomers, followed by dynamic channel assembly and dissociation. These channels allow calcium uptake in APP-deficient cells and cause neurite degeneration in human cortical neurons. Channel conductance, calcium uptake and neurite degeneration are selectively inhibited by zinc, a blocker of amyloid ion channel activity. Thus truncated Aβ fragments could account for undefined roles played by full length Aβs and provide a novel mechanism of AD and DS pathology. The emerging picture from our large-scale simulations is that toxic ion channels formed by β-sheets are highly polymorphic, and spontaneously break into loosely interacting dynamic units (though still maintaining ion channel structures as imaged with AFM), that associate and dissociate leading to toxic ion flux. This sharply contrasts intact conventional gated ion channels that consist of tightly

  13. Acid-sensing ion channels: trafficking and synaptic function

    Directory of Open Access Journals (Sweden)

    Zha Xiang-ming

    2013-01-01

    Full Text Available Abstract Extracellular acidification occurs in the brain with elevated neural activity, increased metabolism, and neuronal injury. This reduction in pH can have profound effects on brain function because pH regulates essentially every single biochemical reaction. Therefore, it is not surprising to see that Nature evolves a family of proteins, the acid-sensing ion channels (ASICs, to sense extracellular pH reduction. ASICs are proton-gated cation channels that are mainly expressed in the nervous system. In recent years, a growing body of literature has shown that acidosis, through activating ASICs, contributes to multiple diseases, including ischemia, multiple sclerosis, and seizures. In addition, ASICs play a key role in fear and anxiety related psychiatric disorders. Several recent reviews have summarized the importance and therapeutic potential of ASICs in neurological diseases, as well as the structure-function relationship of ASICs. However, there is little focused coverage on either the basic biology of ASICs or their contribution to neural plasticity. This review will center on these topics, with an emphasis on the synaptic role of ASICs and molecular mechanisms regulating the spatial distribution and function of these ion channels.

  14. Acid-sensing ion channels: trafficking and synaptic function.

    Science.gov (United States)

    Zha, Xiang-ming

    2013-01-02

    Extracellular acidification occurs in the brain with elevated neural activity, increased metabolism, and neuronal injury. This reduction in pH can have profound effects on brain function because pH regulates essentially every single biochemical reaction. Therefore, it is not surprising to see that Nature evolves a family of proteins, the acid-sensing ion channels (ASICs), to sense extracellular pH reduction. ASICs are proton-gated cation channels that are mainly expressed in the nervous system. In recent years, a growing body of literature has shown that acidosis, through activating ASICs, contributes to multiple diseases, including ischemia, multiple sclerosis, and seizures. In addition, ASICs play a key role in fear and anxiety related psychiatric disorders. Several recent reviews have summarized the importance and therapeutic potential of ASICs in neurological diseases, as well as the structure-function relationship of ASICs. However, there is little focused coverage on either the basic biology of ASICs or their contribution to neural plasticity. This review will center on these topics, with an emphasis on the synaptic role of ASICs and molecular mechanisms regulating the spatial distribution and function of these ion channels.

  15. Novel Insights into Acid-Sensing Ion Channels: Implications for Degenerative Diseases.

    Science.gov (United States)

    Zhou, Ren-Peng; Wu, Xiao-Shan; Wang, Zhi-Sen; Xie, Ya-Ya; Ge, Jin-Fang; Chen, Fei-Hu

    2016-08-01

    Degenerative diseases often strike older adults and are characterized by progressive deterioration of cells, eventually leading to tissue and organ degeneration for which limited effective treatment options are currently available. Acid-sensing ion channels (ASICs), a family of extracellular H(+)-activated ligand-gated ion channels, play critical roles in physiological and pathological conditions. Aberrant activation of ASICs is reported to regulate cell apoptosis, differentiation and autophagy. Accumulating evidence has highlighted a dramatic increase and activation of ASICs in degenerative disorders, including multiple sclerosis, Parkinson's disease, Huntington's disease, intervertebral disc degeneration and arthritis. In this review, we have comprehensively discussed the critical roles of ASICs and their potential utility as therapeutic targets in degenerative diseases.

  16. Functional Importance of L- and P/Q-Type Voltage-Gated Calcium Channels in Human Renal Vasculature

    DEFF Research Database (Denmark)

    Hansen, Pernille B; Poulsen, Christian B; Walter, Steen

    2011-01-01

    Calcium channel blockers are widely used for treatment of hypertension, because they decrease peripheral vascular resistance through inhibition of voltage-gated calcium channels. Animal studies of renal vasculature have shown expression of several types of calcium channels that are involved......-type subtype (Ca(v) 3.1 and Ca(v) 3.2) voltage-gated calcium channels (Ca(v)s), and quantitative PCR showed highest expression of L-type channels in renal arteries and variable expression between patients of subtypes of calcium channels in intrarenal vessels. Immunohistochemical labeling of kidney sections...

  17. Downregulation of Kv7.4 channel activity in primary and secondary hypertension

    DEFF Research Database (Denmark)

    Jepps, Thomas Andrew; Chadha, Preet S; Davis, Alison J

    2011-01-01

    Voltage-gated potassium (K(+)) channels encoded by KCNQ genes (Kv7 channels) have been identified in various rodent and human blood vessels as key regulators of vascular tone; however, nothing is known about the functional impact of these channels in vascular disease. We ascertained the effect of...... structurally different activators of Kv7.2 through Kv7.5 channels (BMS-204352, S-1, and retigabine) on blood vessels from normotensive and hypertensive animals.......Voltage-gated potassium (K(+)) channels encoded by KCNQ genes (Kv7 channels) have been identified in various rodent and human blood vessels as key regulators of vascular tone; however, nothing is known about the functional impact of these channels in vascular disease. We ascertained the effect of 3...

  18. Ion channel profile of TRPM8 cold receptors reveals a novel role of TASK-3 potassium channels in thermosensation

    Science.gov (United States)

    Morenilla-Palao, Cruz; Luis, Enoch; Fernández-Peña, Carlos; Quintero, Eva; Weaver, Janelle L.; Bayliss, Douglas A.; Viana, Félix

    2017-01-01

    Summary Animals sense cold ambient temperatures through the activation of peripheral thermoreceptors that express TRPM8, a cold- and menthol-activated ion channel. These receptors can discriminate a very wide range of temperatures from innocuous to noxious. The molecular mechanism responsible for the variable sensitivity of individual cold receptors to temperature is unclear. To address this question, we performed a detailed ion channel expression analysis of cold sensitive neurons, combining BAC transgenesis with a molecular profiling approach in FACS purified TRPM8 neurons. We found that TASK-3 leak potassium channels are highly enriched in a subpopulation of these sensory neurons. The thermal threshold of TRPM8 cold neurons is decreased during TASK-3 blockade and in mice lacking TASK-3 and, most importantly, these mice display hypersensitivity to cold. Our results demonstrate a novel role of TASK-3 channels in thermosensation, showing that a channel-based combinatorial strategy in TRPM8 cold thermoreceptors leads to molecular specialization and functional diversity. PMID:25199828

  19. H2O2 augments cytosolic calcium in nucleus tractus solitarii neurons via multiple voltage-gated calcium channels.

    Science.gov (United States)

    Ostrowski, Tim D; Dantzler, Heather A; Polo-Parada, Luis; Kline, David D

    2017-05-01

    Reactive oxygen species (ROS) play a profound role in cardiorespiratory function under normal physiological conditions and disease states. ROS can influence neuronal activity by altering various ion channels and transporters. Within the nucleus tractus solitarii (nTS), a vital brainstem area for cardiorespiratory control, hydrogen peroxide (H 2 O 2 ) induces sustained hyperexcitability following an initial depression of neuronal activity. The mechanism(s) associated with the delayed hyperexcitability are unknown. Here we evaluate the effect(s) of H 2 O 2 on cytosolic Ca 2+ (via fura-2 imaging) and voltage-dependent calcium currents in dissociated rat nTS neurons. H 2 O 2 perfusion (200 µM; 1 min) induced a delayed, slow, and moderate increase (~27%) in intracellular Ca 2+ concentration ([Ca 2+ ] i ). The H 2 O 2 -mediated increase in [Ca 2+ ] i prevailed during thapsigargin, excluding the endoplasmic reticulum as a Ca 2+ source. The effect, however, was abolished by removal of extracellular Ca 2+ or the addition of cadmium to the bath solution, suggesting voltage-gated Ca 2+ channels (VGCCs) as targets for H 2 O 2 modulation. Recording of the total voltage-dependent Ca 2+ current confirmed H 2 O 2 enhanced Ca 2+ entry. Blocking VGCC L, N, and P/Q subtypes decreased the number of cells and their calcium currents that respond to H 2 O 2 The number of responder cells to H 2 O 2 also decreased in the presence of dithiothreitol, suggesting the actions of H 2 O 2 were dependent on sulfhydryl oxidation. In summary, here, we have shown that H 2 O 2 increases [Ca 2+ ] i and its Ca 2+ currents, which is dependent on multiple VGCCs likely by oxidation of sulfhydryl groups. These processes presumably contribute to the previously observed delayed hyperexcitability of nTS neurons in in vitro brainstem slices. Copyright © 2017 the American Physiological Society.

  20. Trajectory separation of channeled ions in crystalline materials

    International Nuclear Information System (INIS)

    Temkin, Misha; Chakarov, Ivan; Webb, Roger

    2000-01-01

    Spatial distributions of ions implanted into crystals can be of a very complex shape with 'lobes' due to ions penetrating through open channels in several directions. This paper suggests an analytical model which represents such a distribution as a linear combination of 'random' distribution and one or more 'channeled' distributions. This study is focused on the algorithm of the separation of ion trajectories into several distributions. The first distribution includes those ions which have undergone predominantly random collisions. The other distributions include those ions which have undergone mainly 'weak' collisions and traveled mostly along the main channeling directions. Our binary collision approximation (BCA) simulator is used for generating and analyzing ion trajectories. The spatial moments can be extracted from each separated distribution. It is shown that 2D analytical distributions obtained as a linear combination of distributions derived from these moments and aligned along corresponding channeling direction are in a very good agreement with direct BCA calculations

  1. Unique battery with an active membrane separator having uniform physico-chemically functionalized ion channels and a method making the same

    Science.gov (United States)

    Gerald, II, Rex E.; Ruscic, Katarina J [Chicago, IL; Sears, Devin N [Spruce Grove, CA; Smith, Luis J [Natick, MA; Klingler, Robert J [Glenview, IL; Rathke, Jerome W [Homer Glen, IL

    2012-02-21

    The invention relates to a unique battery having an active, porous membrane and method of making the same. More specifically the invention relates to a sealed battery system having a porous, metal oxide membrane with uniform, physicochemically functionalized ion channels capable of adjustable ionic interaction. The physicochemically-active porous membrane purports dual functions: an electronic insulator (separator) and a unidirectional ion-transporter (electrolyte). The electrochemical cell membrane is activated for the transport of ions by contiguous ion coordination sites on the interior two-dimensional surfaces of the trans-membrane unidirectional pores. The membrane material is designed to have physicochemical interaction with ions. Control of the extent of the interactions between the ions and the interior pore walls of the membrane and other materials, chemicals, or structures contained within the pores provides adjustability of the ionic conductivity of the membrane.

  2. Channeling effect for low energy ion implantation in Si

    International Nuclear Information System (INIS)

    Cho, K.; Allen, W.R.; Finstad, T.G.; Chu, W.K.; Liu, J.; Wortman, J.J.

    1985-01-01

    Ion implantation is one of the most important processes in semiconductor device fabrication. Due to the crystalline nature of Si, channeling of implanted ions occurs during this process. Modern devices become smaller and shallower and therefore require ion implantation at lower energies. The effect of channeling on ion implantation becomes a significant problem for low energy ion implantation. The critical angle for axial and planar channeling increases with decreasing energy. This corresponds to an increased probability for channeling with lowering of ion energy. The industry approach to avoid the channeling problem is to employ a tilt angle of 7 0 between the ion implantation direction and the surface normal. We approach the problem by mapping major crystalline axes and planes near the [100] surface normal. Our analysis indicates that a 7 0 tilt is not an optimum selection in channeling reduction. Tilt angles in the range 5 0 to 6 0 combined with 7 0 +- 0.5 0 rotation from the (100) plane are better selections for the reduction of the channeling effect. The range of suitable angles is a function of the implantation energy. Implantations of boron along well specified crystallographic directions have been carried out by careful alignment and the resulting boron profiles measured by SIMS. (orig.)

  3. Twenty-channel high-voltage pulse generators

    International Nuclear Information System (INIS)

    Anan'in, P.S.; Kashirin, A.P.

    1980-01-01

    A 20-channel high-voltage pulse generator operating with a mismatched load is described. The generator contains shaping lines 20 m long made of coaxial cable, a trigatron-type discharged, and isolating plates. The channel characteristic impedance is 50 Ohm. The maximum pulse amplitude is up to 15 kV on a high-resistance load and 7.5 kV on a matched one. The pulse duration is 100 ns at a pulse rise time of 12 ns, the delay introduced by the generator is 200 +-2.5 ns. Provision is made in the control circuit for compensation of the shaped pulse and separation of a pulse reflected from the load. The reflected pulse shape and amplitude characterize load parameters. Generator tests proved its high operational reliability (after 10 5 operations no significant changes in generator performances have been observed). The generator is intended for filmless data output from spark chambers

  4. Ion Channels of Pituitary Gonadotrophs and Their Roles in Signaling and Secretion

    Czech Academy of Sciences Publication Activity Database

    Stojilkovic, S. S.; Bjelobaba, I.; Zemková, Hana

    2017-01-01

    Roč. 8, Jun 9 (2017), č. článku 126. ISSN 1664-2392 R&D Projects: GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GA16-12695S Institutional support: RVO:67985823 Keywords : gonadotrophs * voltage-gated channels * ligand-gated channels * electrical activity Subject RIV: FH - Neurology OBOR OECD: Physiology (including cytology) Impact factor: 3.675, year: 2016

  5. Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca2+ channels

    DEFF Research Database (Denmark)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin

    2015-01-01

    Our previous studies revealed that L-type voltage-dependent Ca2+ channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular......M), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin–agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1.......2 channels revealed that enzymatic digestion of highly sulfated heparan sulfates with heparinase 1 affects neither voltage-dependence of channel activation nor the level of steady state inactivation, but did speed up channel inactivation. Treatment of hippocampal cultures with heparinase 1 reduced the firing...

  6. Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium

    Directory of Open Access Journals (Sweden)

    Adela Banciu

    2018-05-01

    Full Text Available Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1, estrogen receptors (ESR1, ESR2, GPR30, and nuclear receptor coactivator 3 (NCOA3. Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM antagonized the effect of BAY K8644 (2.5 or 5 µM in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant

  7. Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium.

    Science.gov (United States)

    Banciu, Adela; Banciu, Daniel Dumitru; Mustaciosu, Cosmin Catalin; Radu, Mihai; Cretoiu, Dragos; Xiao, Junjie; Cretoiu, Sanda Maria; Suciu, Nicolae; Radu, Beatrice Mihaela

    2018-05-09

    Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human uterine

  8. Reconstruction of Cell Surface Densities of Ion Pumps, Exchangers, and Channels from mRNA Expression, Conductance Kinetics, Whole-Cell Calcium, and Current-Clamp Voltage Recordings, with an Application to Human Uterine Smooth Muscle Cells.

    Directory of Open Access Journals (Sweden)

    Jolene Atia

    2016-04-01

    Full Text Available Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the 'conductance repertoire' being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations.

  9. Molecular basis of inhibition of acid sensing ion channel 1A by diminazene.

    Directory of Open Access Journals (Sweden)

    Aram J Krauson

    Full Text Available Acid-sensing ion channels (ASICs are trimeric proton-gated cation permeable ion channels expressed primarily in neurons. Here we employed site-directed mutagenesis and electrophysiology to investigate the mechanism of inhibition of ASIC1a by diminazene. This compound inhibits mouse ASIC1a with a half-maximal inhibitory concentration (IC50 of 2.4 μM. At first, we examined whether neutralizing mutations of Glu79 and Glu416 alter diminazene block. These residues form a hexagonal array in the lower palm domain that was previously shown to contribute to pore opening in response to extracellular acidification. Significantly, single Gln substitutions at positions 79 and 416 in ASIC1a reduced diminazene apparent affinity by 6-7 fold. This result suggests that diminazene inhibits ASIC1a in part by limiting conformational rearrangement in the lower palm domain. Because diminazene is charged at physiological pHs, we assessed whether it inhibits ASIC1a by blocking the ion channel pore. Consistent with the notion that diminazene binds to a site within the membrane electric field, diminazene block showed a strong dependence with the membrane potential. Moreover, a Gly to Ala mutation at position 438, in the ion conduction pathway of ASIC1a, increased diminazene IC50 by one order of magnitude and eliminated the voltage dependence of block. Taken together, our results indicate that the inhibition of ASIC1a by diminazene involves both allosteric modulation and blocking of ion flow through the conduction pathway. Our findings provide a foundation for the development of more selective and potent ASIC pore blockers.

  10. Expression and activity of acid-sensing ion channels in the mouse anterior pituitary.

    Directory of Open Access Journals (Sweden)

    Jianyang Du

    Full Text Available Acid sensing ion channels (ASICs are proton-gated cation channels that are expressed in the nervous system and play an important role in fear learning and memory. The function of ASICs in the pituitary, an endocrine gland that contributes to emotions, is unknown. We sought to investigate which ASIC subunits were present in the pituitary and found mRNA expression for all ASIC isoforms, including ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4. We also observed acid-evoked ASIC-like currents in isolated anterior pituitary cells that were absent in mice lacking ASIC1a. The biophysical properties and the responses to PcTx1, amiloride, Ca2+ and Zn2+ suggested that ASIC currents were mediated predominantly by heteromultimeric channels that contained ASIC1a and ASIC2a or ASIC2b. ASIC currents were also sensitive to FMRFamide (Phe-Met-Arg-Phe amide, suggesting that FMRFamide-like compounds might endogenously regulate pituitary ASICs. To determine whether ASICs might regulate pituitary cell function, we applied low pH and found that it increased the intracellular Ca2+ concentration. These data suggest that ASIC channels are present and functionally active in anterior pituitary cells and may therefore influence their function.

  11. Tuning the ion selectivity of two-pore channels

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jiangtao; Zeng, Weizhong; Jiang, Youxing (UTSMC)

    2017-01-17

    Organellar two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in plants and animals. Interestingly, plant and animal TPCs share high sequence similarity in the filter region, yet exhibit drastically different ion selectivity. Plant TPC1 functions as a nonselective cation channel on the vacuole membrane, whereas mammalian TPC channels have been shown to be endo/lysosomal Na+-selective or Ca2+-release channels. In this study, we performed systematic characterization of the ion selectivity of TPC1 from Arabidopsis thaliana (AtTPC1) and compared its selectivity with the selectivity of human TPC2 (HsTPC2). We demonstrate that AtTPC1 is selective for Ca2+ over Na+, but nonselective among monovalent cations (Li+, Na+, and K+). Our results also confirm that HsTPC2 is a Na+-selective channel activated by phosphatidylinositol 3,5-bisphosphate. Guided by our recent structure of AtTPC1, we converted AtTPC1 to a Na+-selective channel by mimicking the selectivity filter of HsTPC2 and identified key residues in the TPC filters that differentiate the selectivity between AtTPC1 and HsTPC2. Furthermore, the structure of the Na+-selective AtTPC1 mutant elucidates the structural basis for Na+ selectivity in mammalian TPCs.

  12. Structure-based assessment of disease-related mutations in human voltage-gated sodium channels

    Directory of Open Access Journals (Sweden)

    Weiyun Huang

    2017-02-01

    Full Text Available ABSTRACT Voltage-gated sodium (Nav channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Nav channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Nav channels, with Nav1.1 and Nav1.5 each harboring more than 400 mutations. Nav channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Nav channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Cav channel Cav1.1 provides a template for homology-based structural modeling of the evolutionarily related Nav channels. In this Resource article, we summarized all the reported disease-related mutations in human Nav channels, generated a homologous model of human Nav1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Nav channels, the analysis presented here serves as the base framework for mechanistic investigation of Nav channelopathies and for potential structure-based drug discovery.

  13. Structure-based assessment of disease-related mutations in human voltage-gated sodium channels.

    Science.gov (United States)

    Huang, Weiyun; Liu, Minhao; Yan, S Frank; Yan, Nieng

    2017-06-01

    Voltage-gated sodium (Na v ) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na v channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na v channels, with Na v 1.1 and Na v 1.5 each harboring more than 400 mutations. Na v channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na v channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca v ) channel Ca v 1.1 provides a template for homology-based structural modeling of the evolutionarily related Na v channels. In this Resource article, we summarized all the reported disease-related mutations in human Na v channels, generated a homologous model of human Na v 1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na v channels, the analysis presented here serves as the base framework for mechanistic investigation of Na v channelopathies and for potential structure-based drug discovery.

  14. Voltage-gated sodium channels as targets for pyrethroid insecticides.

    Science.gov (United States)

    Field, Linda M; Emyr Davies, T G; O'Reilly, Andrias O; Williamson, Martin S; Wallace, B A

    2017-10-01

    The pyrethroid insecticides are a very successful group of compounds that have been used extensively for the control of arthropod pests of agricultural crops and vectors of animal and human disease. Unfortunately, this has led to the development of resistance to the compounds in many species. The mode of action of pyrethroids is known to be via interactions with the voltage-gated sodium channel. Understanding how binding to the channel is affected by amino acid substitutions that give rise to resistance has helped to elucidate the mode of action of the compounds and the molecular basis of their selectivity for insects vs mammals and between insects and other arthropods. Modelling of the channel/pyrethroid interactions, coupled with the ability to express mutant channels in oocytes and study function, has led to knowledge of both how the channels function and potentially how to design novel insecticides with greater species selectivity.

  15. Pulsed voltage electrospray ion source and method for preventing analyte electrolysis

    Science.gov (United States)

    Kertesz, Vilmos [Knoxville, TN; Van Berkel, Gary [Clinton, TN

    2011-12-27

    An electrospray ion source and method of operation includes the application of pulsed voltage to prevent electrolysis of analytes with a low electrochemical potential. The electrospray ion source can include an emitter, a counter electrode, and a power supply. The emitter can include a liquid conduit, a primary working electrode having a liquid contacting surface, and a spray tip, where the liquid conduit and the working electrode are in liquid communication. The counter electrode can be proximate to, but separated from, the spray tip. The power system can supply voltage to the working electrode in the form of a pulse wave, where the pulse wave oscillates between at least an energized voltage and a relaxation voltage. The relaxation duration of the relaxation voltage can range from 1 millisecond to 35 milliseconds. The pulse duration of the energized voltage can be less than 1 millisecond and the frequency of the pulse wave can range from 30 to 800 Hz.

  16. Demystifying Mechanosensitive Piezo Ion Channels.

    Science.gov (United States)

    Xu, X Z Shawn

    2016-06-01

    Mechanosensitive channels mediate touch, hearing, proprioception, and blood pressure regulation. Piezo proteins, including Piezo1 and Piezo2, represent a new class of mechanosensitive channels that have been reported to play key roles in most, if not all, of these modalities. The structural architecture and molecular mechanisms by which Piezos act as mechanosensitive channels, however, remain mysterious. Two new studies have now provided critical insights into the atomic structure and molecular basis of the ion permeation and mechano-gating properties of the Piezo1 channel.

  17. Dielectrophoretic analysis of changes in cytoplasmic ion levels due to ion channel blocker action reveals underlying differences between drug-sensitive and multidrug-resistant leukaemic cells

    International Nuclear Information System (INIS)

    Duncan, L; Shelmerdine, H; Hughes, M P; Coley, H M; Huebner, Y; Labeed, F H

    2008-01-01

    Dielectrophoresis (DEP)-the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K + and Ca 2+ levels were extremely similar between sensitive and resistant lines, levels of Cl - were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function. (note)

  18. High-current and low acceleration voltage arsenic ion implanted polysilicon-gate and source-drain electrode Si mos transistor

    International Nuclear Information System (INIS)

    Saito, Yasuyuki; Sugimura, Yoshiro; Sugihara, Michiyuki

    1993-01-01

    The fabrication process of high current arsenic (As) ion implanted polysilicon (Si) gate and source drain (SD) electrode Si n-channel metal oxide-semiconductor field effect transistor (MOSFET) was examined. Poly Si film n-type doping was performed by using high current (typical current: 2mA) and relatively low acceleration voltage (40keV) As ion implantation technique (Lintott series 3). It was observed that high dose As implanted poly Si films as is show refractoriness against radical fluorine excited by microwave. Using GCA MANN4800 (m/c ID No.2, resist: OFPR) mask pattern printing technique, the high current As ion implantation technique and radical fluorine gas phase etching (Chemical dry etching: CDE) technique, the n-channel Poly Si gate (ρs = ≅100Ω/□) enhancement MQSFETs(ρs source drain = ≅50Ω/□, SiO 2 gate=380 angstrom) with off-leak-less were obtained on 3 inch Czochralski grown 2Ωcm boron doped p type wafers (Osaka titanium). By the same process, a 8 bit single chip μ-processor with 26MHz full operation was performed

  19. The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel.

    Science.gov (United States)

    Mishima, Eriko; Sato, Yoko; Nanatani, Kei; Hoshi, Naomi; Lee, Jong-Kook; Schiller, Nina; von Heijne, Gunnar; Sakaguchi, Masao; Uozumi, Nobuyuki

    2016-12-01

    Voltage-dependent K + (K V ) channels control K + permeability in response to shifts in the membrane potential. Voltage sensing in K V channels is mediated by the positively charged transmembrane domain S4. The best-characterized K V channel, KvAP, lacks the distinct hydrophilic region corresponding to the S3-S4 extracellular loop that is found in other K + channels. In the present study, we evaluated the topogenic properties of the transmembrane regions within the voltage-sensing domain in KvAP. S3 had low membrane insertion activity, whereas S4 possessed a unique type-I signal anchor (SA-I) function, which enabled it to insert into the membrane by itself. S4 was also found to function as a stop-transfer signal for retention in the membrane. The length and structural nature of the extracellular S3-S4 loop affected the membrane insertion of S3 and S4, suggesting that S3 membrane insertion was dependent on S4. Replacement of charged residues within the transmembrane regions with residues of opposite charge revealed that Asp 72 in S2 and Glu 93 in S3 contributed to membrane insertion of S3 and S4, and increased the stability of S4 in the membrane. These results indicate that the SA-I function of S4, unique among K + channels studied to date, promotes the insertion of S3 into the membrane, and that the charged residues essential for voltage sensing contribute to the membrane-insertion of the voltage sensor domain in KvAP. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  20. Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by proteoliposomes and cultured rat sertoli cells: Evidence for involvement of voltage-activated and voltage-independent calcium channels

    International Nuclear Information System (INIS)

    Grasso, P.; Reichert, L.E. Jr.

    1989-01-01

    We have previously reported incorporation into liposomes of Triton X-100-solubilized FSH receptor-G-protein complexes derived from purified bovine calf testis membranes. In the present study we have used this model system to show that FSH induces flux of 45Ca2+ into such proteoliposomes in a hormone-specific concentration-dependent manner. FSH, inactivated by boiling, had no stimulatory effect on 45Ca2+ flux, nor did isolated alpha- or beta-subunits of FSH. Addition of GTP (or its analogs 5'-guanylylimidodiphosphate and guanosine-5'-O-[3-thiotriphosphate]) or sodium fluoride (in the presence or absence of GTP or its analogs) failed to induce 45Ca2+ flux into proteoliposomes, suggesting that the uptake of 45Ca2+ was receptor, and not G-protein, related. Voltage-independent (ruthenium red and gadolinium chloride) and voltage-activated (methyoxyverapamil and nifedipine) calcium channel-blocking agents reduced FSH-stimulated 45Ca2+ flux into proteoliposomes to control levels. FSH also induced uptake of 45Ca2+ by cultured rat Sertoli cells. Ruthenium red and gadolinium chloride had no effect on basal levels of 45Ca2+ uptake or estradiol secretion by cultured rat Sertoli cells, nor did methoxyverapamil or nifedipine. All four calcium channel blockers, however, were able to reduce FSH-induced 45Ca2+ uptake to basal levels and FSH-stimulated conversion of androstenedione to estradiol by up to 50%, indicating an involvement of Ca2+ in FSH-stimulated steroidogenesis. Our results suggest that the well documented changes in intracellular calcium levels consequent to FSH binding may be due, at least in part, to an influx of calcium through FSH receptor-regulated calcium channels

  1. Neuroprotective effect of interleukin-6 regulation of voltage-gated Na+ channels of cortical neurons is time- and dose-dependent

    Directory of Open Access Journals (Sweden)

    Wei Xia

    2015-01-01

    Full Text Available Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour exposure of interleukin-6 on cortical neurons at various concentrations (0.1, 1, 5 and 10 ng/mL and the effects of 10 ng/mL interleukin-6 exposure to cortical neurons for various durations (2, 4, 8, 24 and 48 hours by studying voltage-gated Na + channels using a patch-clamp technique. Voltage-clamp recording results demonstrated that interleukin-6 suppressed Na + currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of cortical neurons, but did not change the action potential threshold. The regulation of voltage-gated Na + channels in rat cortical neurons by interleukin-6 is time- and dose-dependent.

  2. Monitoring voltage-sensitive membrane impedance change using radio frequency interrogation.

    Science.gov (United States)

    Dharia, Sameera; Rabbitt, Richard D

    2010-01-01

    Here we present a new technique to monitor dynamic conformational changes in voltage-sensitive membrane-bound proteins using radio frequency (RF) impedance measurements. Xenopus oocytes were transfected to express ShakerB-IR K(+) ion channels, and step changes in membrane potential were applied using two-electrode voltage clamp (TEVC). Simultaneously, bipolar extracellular electrodes were used to measure the RF electrical impedance across the cell (300 kHz - 1 MHz). RF current will either pass through the media, around the cell, or displace charge across the cell membrane. The change in displacement current in the cell membrane during voltage clamp resulted in measurable RF impedance change. RF impedance change during DC membrane depolarization was significantly greater in ShakerB-IR expressing oocytes than in endogenous controls at 300 kHz, 500 kHz and, to a lesser extent, 1 MHz. Since the RF were too high to modulate ShakerB-IR protein conformational state (e.g. open channel probability), impedance changes are interpreted as reflections of voltage-dependent protein conformation and associated biophysics such as ion-channel dipole interactions, fluctuations in bound water, or charged lipid head-group rotations.

  3. Computational optogenetics: empirically-derived voltage- and light-sensitive channelrhodopsin-2 model.

    Directory of Open Access Journals (Sweden)

    John C Williams

    Full Text Available Channelrhodospin-2 (ChR2, a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1 accurate inward rectification in the current-voltage response across irradiances; 2 empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation; and 3 accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10 were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and

  4. Molecular identity of dendritic voltage-gated sodium channels.

    Science.gov (United States)

    Lorincz, Andrea; Nusser, Zoltan

    2010-05-14

    Active invasion of the dendritic tree by action potentials (APs) generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In cortical pyramidal cells (PCs), this AP back-propagation is supported by dendritic voltage-gated Na+ (Nav) channels, whose molecular identity is unknown. Using a highly sensitive electron microscopic immunogold technique, we revealed the presence of the Nav1.6 subunit in hippocampal CA1 PC proximal and distal dendrites. Here, the subunit density is lower by a factor of 35 to 80 than that found in axon initial segments. A gradual decrease in Nav1.6 density along the proximodistal axis of the dendritic tree was also detected without any labeling in dendritic spines. Our results reveal the characteristic subcellular distribution of the Nav1.6 subunit, identifying this molecule as a key substrate enabling dendritic excitability.

  5. High voltage and high specific capacity dual intercalating electrode Li-ion batteries

    Science.gov (United States)

    West, William C. (Inventor); Blanco, Mario (Inventor)

    2010-01-01

    The present invention provides high capacity and high voltage Li-ion batteries that have a carbonaceous cathode and a nonaqueous electrolyte solution comprising LiF salt and an anion receptor that binds the fluoride ion. The batteries can comprise dual intercalating electrode Li ion batteries. Methods of the present invention use a cathode and electrode pair, wherein each of the electrodes reversibly intercalate ions provided by a LiF salt to make a high voltage and high specific capacity dual intercalating electrode Li-ion battery. The present methods and systems provide high-capacity batteries particularly useful in powering devices where minimizing battery mass is important.

  6. Modeling the concentration-dependent permeation modes of the KcsA potassium ion channel.

    Science.gov (United States)

    Nelson, Peter Hugo

    2003-12-01

    The potassium channel from Streptomyces lividans (KcsA) is an integral membrane protein with sequence similarity to all known potassium channels, particularly in the selectivity filter region. A recently proposed model for ion channels containing either n or (n-1) single-file ions in their selectivity filters [P. H. Nelson, J. Chem. Phys. 177, 11396 (2002)] is applied to published KcsA channel K+ permeation data that exhibit a high-affinity process at low concentrations and a low-affinity process at high concentrations [M. LeMasurier et al., J. Gen. Physiol. 118, 303 (2001)]. The kinetic model is shown to provide a reasonable first-order explanation for both the high- and low-concentration permeation modes observed experimentally. The low-concentration mode ([K+]200 mM) has a 200-mV dissociation constant of 1100 mM and a conductance of 500 pS. Based on the permeation model, and x-ray analysis [J. H. Morais-Cabral et al., Nature (London) 414, 37 (2001)], it is suggested that the experimentally observed K+ permeation modes correspond to an n=3 mechanism at high concentrations and an n=2 mechanism at low concentrations. The ratio of the electrical dissociation distances for the high- and low-concentration modes is 3:2, also consistent with the proposed n=3 and n=2 modes. Model predictions for K+ channels that exhibit asymmetric current-voltage (I-V) curves are presented, and further validation of the kinetic model via molecular simulation and experiment is discussed. The qualitatively distinct I-V characteristics exhibited experimentally by Tl+, NH+4, and Rb+ ions at 100 mM concentration can also be explained using the model, but more extensive experimental tests are required for quantitative validation of the model predictions.

  7. A novel ZVS high voltage power supply for micro-channel plate photomultiplier tubes

    International Nuclear Information System (INIS)

    Pei, Chengquan; Tian, Jinshou; Liu, Zhen; Qin, Hong; Wu, Shengli

    2017-01-01

    A novel resonant high voltage power supply (HVPS) with zero voltage switching (ZVS), to reduce the voltage stress on switching devices and improve conversion efficiency, is proposed. The proposed HVPS includes a drive circuit, a transformer, several voltage multiplying circuits, and a regulator circuit. The HVPS contains several secondary windings that can be precisely regulated. The proposed HVPS performed better than the traditional resistor voltage divider, which requires replacing matching resistors resulting in resistor dispersibility in the Micro-Channel Plate (MCP). The equivalent circuit of the proposed HVPS was established and the operational principle analyzed. The entire switching element can achieve ZVS, which was validated by a simulation and experiments. The properties of this HVPS were tested including minimum power loss (240 mW), maximum power loss (1 W) and conversion efficiency (85%). The results of this research are that the proposed HVPS was suitable for driving the micro-channel plate photomultiplier tube (MCP-PMT). It was therefore adopted to test the MCP-PMT, which will be used in Daya Bay reactor neutrino experiment II in China.

  8. A novel ZVS high voltage power supply for micro-channel plate photomultiplier tubes

    Energy Technology Data Exchange (ETDEWEB)

    Pei, Chengquan [Key Laboratory for Physical Electronics and Devices of the Ministry of Education, Xi' an Jiaotong University, Xi’an 710049 (China); Tian, Jinshou [Xi’an Institute of Optics and Precision Mechanics, Chinese Academy of Sciences, Xi' an 710119 (China); Liu, Zhen [Key Laboratory for Physical Electronics and Devices of the Ministry of Education, Xi' an Jiaotong University, Xi’an 710049 (China); Qin, Hong [School of Computer Science and Technology, Xi' an University of Science and Technology, Xi' an 710054 (China); Wu, Shengli, E-mail: slwu@mail.xjtu.edu.cn [Key Laboratory for Physical Electronics and Devices of the Ministry of Education, Xi' an Jiaotong University, Xi’an 710049 (China)

    2017-04-11

    A novel resonant high voltage power supply (HVPS) with zero voltage switching (ZVS), to reduce the voltage stress on switching devices and improve conversion efficiency, is proposed. The proposed HVPS includes a drive circuit, a transformer, several voltage multiplying circuits, and a regulator circuit. The HVPS contains several secondary windings that can be precisely regulated. The proposed HVPS performed better than the traditional resistor voltage divider, which requires replacing matching resistors resulting in resistor dispersibility in the Micro-Channel Plate (MCP). The equivalent circuit of the proposed HVPS was established and the operational principle analyzed. The entire switching element can achieve ZVS, which was validated by a simulation and experiments. The properties of this HVPS were tested including minimum power loss (240 mW), maximum power loss (1 W) and conversion efficiency (85%). The results of this research are that the proposed HVPS was suitable for driving the micro-channel plate photomultiplier tube (MCP-PMT). It was therefore adopted to test the MCP-PMT, which will be used in Daya Bay reactor neutrino experiment II in China.

  9. A quantized mechanism for activation of pannexin channels

    Science.gov (United States)

    Chiu, Yu-Hsin; Jin, Xueyao; Medina, Christopher B.; Leonhardt, Susan A.; Kiessling, Volker; Bennett, Brad C.; Shu, Shaofang; Tamm, Lukas K.; Yeager, Mark; Ravichandran, Kodi S.; Bayliss, Douglas A.

    2017-01-01

    Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels. PMID:28134257

  10. C-terminal β9-strand of the cyclic nucleotide-binding homology domain stabilizes activated states of Kv11.1 channels.

    Directory of Open Access Journals (Sweden)

    Chai Ann Ng

    Full Text Available Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K(+ channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels.

  11. Activation of voltage-gated KCNQ/Kv7 channels by anticonvulsant retigabine attenuates mechanical allodynia of inflammatory temporomandibular joint in rats

    Directory of Open Access Journals (Sweden)

    Xu Wen

    2010-08-01

    Full Text Available Abstract Background Temporomandibular disorders (TMDs are characterized by persistent orofacial pain and have diverse etiologic factors that are not well understood. It is thought that central sensitization leads to neuronal hyperexcitability and contributes to hyperalgesia and spontaneous pain. Nonsteroidal anti-inflammatory drugs (NSAIDs are currently the first choice of drug to relieve TMD pain. NSAIDS were shown to exhibit anticonvulsant properties and suppress cortical neuron activities by enhancing neuronal voltage-gated potassium KCNQ/Kv7 channels (M-current, suggesting that specific activation of M-current might be beneficial for TMD pain. Results In this study, we selected a new anticonvulsant drug retigabine that specifically activates M-current, and investigated the effect of retigabine on inflammation of the temporomandibular joint (TMJ induced by complete Freund's adjuvant (CFA in rats. The results show that the head withdrawal threshold for escape from mechanical stimulation applied to facial skin over the TMJ in inflamed rats was significantly lower than that in control rats. Administration of centrally acting M-channel opener retigabine (2.5 and 7.5 mg/kg can dose-dependently raise the head withdrawal threshold of mechanical allodynia, and this analgesic effect can be reversed by the specific KCNQ channel blocker XE991 (3 mg/kg. Food intake is known to be negatively associated with TMJ inflammation. Food intake was increased significantly by the administration of retigabine (2.5 and 7.5 mg/kg, and this effect was reversed by XE991 (3 mg/kg. Furthermore, intracerebralventricular injection of retigabine further confirmed the analgesic effect of central retigabine on inflammatory TMJ. Conclusions Our findings indicate that central sensitization is involved in inflammatory TMJ pain and pharmacological intervention for controlling central hyperexcitability by activation of neuronal KCNQ/M-channels may have therapeutic potential for

  12. 4-Aminopyridine: a pan voltage-gated potassium channel inhibitor that enhances K7.4 currents and inhibits noradrenaline-mediated contraction of rat mesenteric small arteries

    DEFF Research Database (Denmark)

    Khammy, Makhala M; Kim, Sukhan; Bentzen, Bo H

    2018-01-01

    has not been systematically studied. The aim of this study was to investigate the pharmacological activity of 4-AP on Kv7.4 and Kv7.5 channels and characterize the effect of 4-AP on rat resistance arteries. EXPERIMENTAL APPROACH: Voltage clamp experiments were performed on Xenopus laevis oocytes......BACKGROUND AND PURPOSE: Kv7.4 and Kv7.5 channels are regulators of vascular tone. 4-Aminopyridine (4-AP) is considered a broad inhibitor of voltage-gated potassium (KV) channels, with little inhibitory effect on Kv7 family members at mmol concentrations. However, the effect of 4-AP on Kv7 channels...

  13. Characterization of a Cyclic Nucleotide-Activated K+ Channel and its Lipid Environment by Using Solid-State NMR Spectroscopy

    NARCIS (Netherlands)

    Cukkemane, A.A.; Baldus, M.

    2013-01-01

    Voltage-gated ion channels are large tetrameric multidomain membrane proteins that play crucial roles in various cellular transduction pathways. Because of their large size and domain-related mobility, structural characterization has proved challenging. We analyzed high-resolution solid-state NMR

  14. Conformational changes and slow dynamics through microsecond polarized atomistic molecular simulation of an integral Kv1.2 ion channel

    DEFF Research Database (Denmark)

    Bjelkmar, Pär; Niemelä, Perttu S; Vattulainen, Ilpo

    2009-01-01

    transitions occur in membrane proteins-not to mention numerous applications in drug design. Here, we present a full 1 micros atomic-detail molecular dynamics simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements......Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how...... and significant thinning of the membrane also observed in experiments, this provides additional support for the predictive power of microsecond-scale membrane protein simulations....

  15. Functional characterization of Kv11.1 (hERG) potassium channels split in the voltage-sensing domain.

    Science.gov (United States)

    de la Peña, Pilar; Domínguez, Pedro; Barros, Francisco

    2018-03-23

    Voltage-dependent KCNH family potassium channel functionality can be reconstructed using non-covalently linked voltage-sensing domain (VSD) and pore modules (split channels). However, the necessity of a covalent continuity for channel function has not been evaluated at other points within the two functionally independent channel modules. We find here that by cutting Kv11.1 (hERG, KCNH2) channels at the different loops linking the transmembrane spans of the channel core, not only channels split at the S4-S5 linker level, but also those split at the intracellular S2-S3 and the extracellular S3-S4 loops, yield fully functional channel proteins. Our data indicate that albeit less markedly, channels split after residue 482 in the S2-S3 linker resemble the uncoupled gating phenotype of those split at the C-terminal end of the VSD S4 transmembrane segment. Channels split after residues 514 and 518 in the S3-S4 linker show gating characteristics similar to those of the continuous wild-type channel. However, breaking the covalent link at this level strongly accelerates the voltage-dependent accessibility of a membrane impermeable methanethiosulfonate reagent to an engineered cysteine at the N-terminal region of the S4 transmembrane helix. Thus, besides that of the S4-S5 linker, structural integrity of the intracellular S2-S3 linker seems to constitute an important factor for proper transduction of VSD rearrangements to opening and closing the cytoplasmic gate. Furthermore, our data suggest that the short and probably rigid characteristics of the extracellular S3-S4 linker are not an essential component of the Kv11.1 voltage sensing machinery.

  16. Well-Defined Microapertures for Ion Channel Biosensors

    NARCIS (Netherlands)

    Halza, Erik; Bro, Tobias Hedegaard; Bilenberg, Brian; Kocer, Armagan

    2013-01-01

    Gated ion channels are excitable nanopores in biological membranes. They sense and respond to different triggers in nature. The sensory characteristics of these channels can be modified by protein engineering tools and the channels can be functionally reconstituted into synthetic lipid bilayer

  17. The shaping of two distinct dendritic spikes by A-type voltage-gated K+ channels

    Directory of Open Access Journals (Sweden)

    Sungchil eYang

    2015-12-01

    Full Text Available Dendritic ion channels have been a subject of intense research in neuroscience because active ion channels in dendrites shape input signals. Ca2+-permeable channels including NMDA receptors (NMDARs have been implicated in supralinear dendritic integration, and the IA conductance in sublinear integration. Despite their essential roles in dendritic integration, it has remained uncertain whether these conductances coordinate with, or counteract, each other in the process of dendritic integration. To address this question, experiments were designed in hippocampal CA1 neurons with a recent 3D digital holography system that has shown excellent performance for spatial photoactivation. The results demonstrated a role of IA as a key contributor to two distinct dendritic spikes, low- and high-threshold Ca2+ spikes, through a preferential action of IA on Ca2+-permeable channel-mediated currents, over fast AMPAR-mediated currents. It is likely that the rapid kinetics of IA provides feed-forward inhibition to counteract the delayed Ca2+ channel-mediated dendritic excitability. This research reveals one dynamic ionic mechanism of dendritic integration, and may contribute to a new understanding of neuronal hyperexcitability embedded in several neural diseases such as epilepsy, fragile X syndrome and Alzheimer's disease.

  18. The Eag domain regulates the voltage-dependent inactivation of rat Eag1 K+ channels.

    Directory of Open Access Journals (Sweden)

    Ting-Feng Lin

    Full Text Available Eag (Kv10 and Erg (Kv11 belong to two distinct subfamilies of the ether-à-go-go K+ channel family (KCNH. While Erg channels are characterized by an inward-rectifying current-voltage relationship that results from a C-type inactivation, mammalian Eag channels display little or no voltage-dependent inactivation. Although the amino (N-terminal region such as the eag domain is not required for the C-type inactivation of Erg channels, an N-terminal deletion in mouse Eag1 has been shown to produce a voltage-dependent inactivation. To further discern the role of the eag domain in the inactivation of Eag1 channels, we generated N-terminal chimeras between rat Eag (rEag1 and human Erg (hERG1 channels that involved swapping the eag domain alone or the complete cytoplasmic N-terminal region. Functional analyses indicated that introduction of the homologous hERG1 eag domain led to both a fast phase and a slow phase of channel inactivation in the rEag1 chimeras. By contrast, the inactivation features were retained in the reverse hERG1 chimeras. Furthermore, an eag domain-lacking rEag1 deletion mutant also showed the fast phase of inactivation that was notably attenuated upon co-expression with the rEag1 eag domain fragment, but not with the hERG1 eag domain fragment. Additionally, we have identified a point mutation in the S4-S5 linker region of rEag1 that resulted in a similar inactivation phenotype. Biophysical analyses of these mutant constructs suggested that the inactivation gating of rEag1 was distinctly different from that of hERG1. Overall, our findings are consistent with the notion that the eag domain plays a critical role in regulating the inactivation gating of rEag1. We propose that the eag domain may destabilize or mask an inherent voltage-dependent inactivation of rEag1 K+ channels.

  19. Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons.

    Science.gov (United States)

    Cantrell, A R; Scheuer, T; Catterall, W A

    1999-07-01

    Activation of D1-like dopamine (DA) receptors reduces peak Na+ current in acutely isolated hippocampal neurons through phosphorylation of the alpha subunit of the Na+ channel by cAMP-dependent protein kinase (PKA). Here we report that neuromodulation of Na+ currents by DA receptors via PKA is voltage-dependent in the range of -110 to -70 mV and is also sensitive to concurrent activation of protein kinase C (PKC). Depolarization enhanced the ability of D1-like DA receptors to reduce peak Na+ currents via the PKA pathway. Similar voltage-dependent modulation was observed when PKA was activated directly with the membrane-permeant PKA activator DCl-cBIMPS (cBIMPS; 20 microM), indicating that the membrane potential dependence occurs downstream of PKA. PKA activation caused only a small (-2.9 mV) shift in the voltage dependence of steady-state inactivation and had no effect on slow inactivation or on the rates of entry into the fast or slow inactivated states, suggesting that another mechanism is responsible for coupling of membrane potential changes to PKA modulation. Activation of PKC with a low concentration of the membrane-permeant diacylglycerol analog oleylacetyl glycerol also potentiated modulation by SKF 81297 or cBIMPS, and these effects were most striking at hyperpolarized membrane potentials where PKA modulation was not stimulated by membrane depolarization. Thus, activation of D1-like DA receptors causes a strong reduction in Na+ current via the PKA pathway, but it is effective primarily when it is combined with depolarization or activation of PKC. The convergence of these three distinct signaling modalities on the Na+ channel provides an intriguing mechanism for integration of information from multiple signaling pathways in the hippocampus and CNS.

  20. Interface Engineering for Precise Threshold Voltage Control in Multilayer-Channel Thin Film Transistors

    KAUST Repository

    Park, Jihoon

    2016-11-29

    Multilayer channel structure is used to effectively manipulate the threshold voltage of zinc oxide transistors without degrading its field-effect mobility. Transistors operating in enhancement mode with good mobility are fabricated by optimizing the structure of the multilayer channel. The optimization is attributed to the formation of additional channel and suppression of the diffusion of absorbed water molecules and oxygen vacancies.

  1. Interface Engineering for Precise Threshold Voltage Control in Multilayer-Channel Thin Film Transistors

    KAUST Repository

    Park, Jihoon; Alshammari, Fwzah Hamud; Wang, Zhenwei; Alshareef, Husam N.

    2016-01-01

    Multilayer channel structure is used to effectively manipulate the threshold voltage of zinc oxide transistors without degrading its field-effect mobility. Transistors operating in enhancement mode with good mobility are fabricated by optimizing the structure of the multilayer channel. The optimization is attributed to the formation of additional channel and suppression of the diffusion of absorbed water molecules and oxygen vacancies.

  2. Bimodal voltage dependence of TRPA1: mutations of a key pore helix residue reveal strong intrinsic voltage-dependent inactivation.

    Science.gov (United States)

    Wan, Xia; Lu, Yungang; Chen, Xueqin; Xiong, Jian; Zhou, Yuanda; Li, Ping; Xia, Bingqing; Li, Min; Zhu, Michael X; Gao, Zhaobing

    2014-07-01

    Transient receptor potential A1 (TRPA1) is implicated in somatosensory processing and pathological pain sensation. Although not strictly voltage-gated, ionic currents of TRPA1 typically rectify outwardly, indicating channel activation at depolarized membrane potentials. However, some reports also showed TRPA1 inactivation at high positive potentials, implicating voltage-dependent inactivation. Here we report a conserved leucine residue, L906, in the putative pore helix, which strongly impacts the voltage dependency of TRPA1. Mutation of the leucine to cysteine (L906C) converted the channel from outward to inward rectification independent of divalent cations and irrespective to stimulation by allyl isothiocyanate. The mutant, but not the wild-type channel, displayed exclusively voltage-dependent inactivation at positive potentials. The L906C mutation also exhibited reduced sensitivity to inhibition by TRPA1 blockers, HC030031 and ruthenium red. Further mutagenesis of the leucine to all natural amino acids individually revealed that most substitutions at L906 (15/19) resulted in inward rectification, with exceptions of three amino acids that dramatically reduced channel activity and one, methionine, which mimicked the wild-type channel. Our data are plausibly explained by a bimodal gating model involving both voltage-dependent activation and inactivation of TRPA1. We propose that the key pore helix residue, L906, plays an essential role in responding to the voltage-dependent gating.

  3. An evolutionarily conserved gene family encodes proton-selective ion channels.

    Science.gov (United States)

    Tu, Yu-Hsiang; Cooper, Alexander J; Teng, Bochuan; Chang, Rui B; Artiga, Daniel J; Turner, Heather N; Mulhall, Eric M; Ye, Wenlei; Smith, Andrew D; Liman, Emily R

    2018-03-02

    Ion channels form the basis for cellular electrical signaling. Despite the scores of genetically identified ion channels selective for other monatomic ions, only one type of proton-selective ion channel has been found in eukaryotic cells. By comparative transcriptome analysis of mouse taste receptor cells, we identified Otopetrin1 (OTOP1), a protein required for development of gravity-sensing otoconia in the vestibular system, as forming a proton-selective ion channel. We found that murine OTOP1 is enriched in acid-detecting taste receptor cells and is required for their zinc-sensitive proton conductance. Two related murine genes, Otop2 and Otop3 , and a Drosophila ortholog also encode proton channels. Evolutionary conservation of the gene family and its widespread tissue distribution suggest a broad role for proton channels in physiology and pathophysiology. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  4. ModFossa: A library for modeling ion channels using Python.

    Science.gov (United States)

    Ferneyhough, Gareth B; Thibealut, Corey M; Dascalu, Sergiu M; Harris, Frederick C

    2016-06-01

    The creation and simulation of ion channel models using continuous-time Markov processes is a powerful and well-used tool in the field of electrophysiology and ion channel research. While several software packages exist for the purpose of ion channel modeling, most are GUI based, and none are available as a Python library. In an attempt to provide an easy-to-use, yet powerful Markov model-based ion channel simulator, we have developed ModFossa, a Python library supporting easy model creation and stimulus definition, complete with a fast numerical solver, and attractive vector graphics plotting.

  5. Dopamine Induces LTP Differentially in Apical and Basal Dendrites through BDNF and Voltage-Dependent Calcium Channels

    Science.gov (United States)

    Navakkode, Sheeja; Sajikumar, Sreedharan; Korte, Martin; Soong, Tuck Wah

    2012-01-01

    The dopaminergic modulation of long-term potentiation (LTP) has been studied well, but the mechanism by which dopamine induces LTP (DA-LTP) in CA1 pyramidal neurons is unknown. Here, we report that DA-LTP in basal dendrites is dependent while in apical dendrites it is independent of activation of L-type voltage-gated calcium channels (VDCC).…

  6. Molecular mechanisms of Cys-loop ion channel receptor modulation by ivermectin

    DEFF Research Database (Denmark)

    Lynagh, Timothy; Lynch, Joseph W.

    2012-01-01

    Ivermectin is an anthelmintic drug that works by inhibiting neuronal activity and muscular contractility in arthropods and nematodes. It works by activating glutamate-gated chloride channels (GluClRs) at nanomolar concentrations. These receptors, found exclusively in invertebrates, belong to the ...... to the neurotransmitter binding site, thus suggesting a mechanism by which ivermectin potentiates neurotransmitter-gated currents. Together, this information provides new insights into the mechanisms of action of this important drug.......) to its site. Several lines of evidence suggest that ivermectin opens the channel pore via a structural change distinct from that induced by the neurotransmitter agonist. Conformational changes occurring at locations distant from the pore can be probed using voltage-clamp fluorometry (VCF), a technique...

  7. Toxins That Affect Voltage-Gated Sodium Channels.

    Science.gov (United States)

    Ji, Yonghua

    2017-10-26

    Voltage-gated sodium channels (VGSCs) are critical in generation and conduction of electrical signals in multiple excitable tissues. Natural toxins, produced by animal, plant, and microorganisms, target VGSCs through diverse strategies developed over millions of years of evolutions. Studying of the diverse interaction between VGSC and VGSC-targeting toxins has been contributing to the increasing understanding of molecular structure and function, pharmacology, and drug development potential of VGSCs. This chapter aims to summarize some of the current views on the VGSC-toxin interaction based on the established receptor sites of VGSC for natural toxins.

  8. Ion channels in glioblastoma.

    Science.gov (United States)

    Molenaar, Remco J

    2011-01-01

    Glioblastoma is the most common primary brain tumor with the most dismal prognosis. It is characterized by extensive invasion, migration, and angiogenesis. Median survival is only 15 months due to this behavior, rendering focal surgical resection ineffective and adequate radiotherapy impossible. At this moment, several ion channels have been implicated in glioblastoma proliferation, migration, and invasion. This paper summarizes studies on potassium, sodium, chloride, and calcium channels of glioblastoma. It provides an up-to-date overview of the literature that could ultimately lead to new therapeutic targets.

  9. Phosphatidylinositol (4,5)Bisphosphate Inhibits K+-Efflux Channel Activity in NT1 Tobacco Cultured Cells1[W][OA

    Science.gov (United States)

    Ma, Xiaohong; Shor, Oded; Diminshtein, Sofia; Yu, Ling; Im, Yang Ju; Perera, Imara; Lomax, Aaron; Boss, Wendy F.; Moran, Nava

    2009-01-01

    In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed “cytosolic” Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: “Low PIs” had depressed levels of these PIs, and “High PIs” had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decreasing PtdInsP2 levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 μm) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (i.e. NtORK activity). Moreover, increasing PtdInsP2 levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5–4 μm), decreased NtORK activity. In all cases, IK decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP2 in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells. PMID:19052153

  10. Ion Permeation and Mechanotransduction Mechanisms of Mechanosensitive Piezo Channels.

    Science.gov (United States)

    Zhao, Qiancheng; Wu, Kun; Geng, Jie; Chi, Shaopeng; Wang, Yanfeng; Zhi, Peng; Zhang, Mingmin; Xiao, Bailong

    2016-03-16

    Piezo proteins have been proposed as the long-sought-after mechanosensitive cation channels in mammals that play critical roles in various mechanotransduction processes. However, the molecular bases that underlie their ion permeation and mechanotransduction have remained functionally undefined. Here we report our finding of the miniature pore-forming module of Piezo1 that resembles the pore architecture of other trimeric channels and encodes the essential pore properties. We further identified specific residues within the pore module that determine unitary conductance, pore blockage and ion selectivity for divalent and monovalent cations and anions. The non-pore-containing region of Piezo1 confers mechanosensitivity to mechano-insensitive trimeric acid-sensing ion channels, demonstrating that Piezo1 channels possess intrinsic mechanotransduction modules separate from their pore modules. In conclusion, this is the first report on the bona fide pore module and mechanotransduction components of Piezo channels, which define their ion-conducting properties and gating by mechanical stimuli, respectively. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Molecular motions that shape the cardiac action potential: Insights from voltage clamp fluorometry.

    Science.gov (United States)

    Zhu, Wandi; Varga, Zoltan; Silva, Jonathan R

    2016-01-01

    Very recently, voltage-clamp fluorometry (VCF) protocols have been developed to observe the membrane proteins responsible for carrying the ventricular ionic currents that form the action potential (AP), including those carried by the cardiac Na(+) channel, NaV1.5, the L-type Ca(2+) channel, CaV1.2, the Na(+)/K(+) ATPase, and the rapid and slow components of the delayed rectifier, KV11.1 and KV7.1. This development is significant, because VCF enables simultaneous observation of ionic current kinetics with conformational changes occurring within specific channel domains. The ability gained from VCF, to connect nanoscale molecular movement to ion channel function has revealed how the voltage-sensing domains (VSDs) control ion flux through channel pores, mechanisms of post-translational regulation and the molecular pathology of inherited mutations. In the future, we expect that this data will be of great use for the creation of multi-scale computational AP models that explicitly represent ion channel conformations, connecting molecular, cell and tissue electrophysiology. Here, we review the VCF protocol, recent results, and discuss potential future developments, including potential use of these experimental findings to create novel computational models. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Nuclear fusion ion beam source composed of optimum channel wall

    International Nuclear Information System (INIS)

    Furukaw, T.

    2007-01-01

    Full text of publication follows: Numerical and experimental researches of the hall-type beam accelerator was conducted by highlighting both neutral species and material of acceleration channel wall. The hall-type beam accelerator is expected as ion beam source for nuclear fusion since it could product ion beam density over 10 3 times as high as that of electrostatic accelerator, which is used regularly as beam heating device, because it is proven that the beam heating method could accelerate ion to high energy beam by electric field and heat plasma to ultra high temperature of 100 million degrees or more. At high-voltage mode of DC regime that is normal operational condition, however, the various plasma MHD (magneto-hydrodynamic) instabilities are generated. In particular, the large-amplitude and low-frequency plasma MHD instability in the tens of kHz among them has been a serious problem that should be solved to improve the operational stability and the system durability. So, we propose a hall-type beam accelerator with new design concepts; both acquisition of simultaneous solution for reducing the plasma MHD instability and the accelerator core overheating and optimum combination of the acceleration channel wall material. The technologies for this concept are as follows: 1) To increase neutral species velocity-inlet in acceleration channel by preheating propellant through circularly propellant conduit line inside accelerator system could bring about the lower amplitude of the instability. 2) Through this method, the accelerator system is cooled, and the higher thrust and specific-impulse is produced with hardly changing thrust efficiency at the same time. 3) To select BN (Boron- Nitride) and Al 2 O 3 as wall material of ionization- and acceleration-zone in acceleration channel respectively having different secondary-electron emission-coefficient could achieve the higher-efficiency and -durability. The hall-type beam accelerator designed using these technologies

  13. Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Elstone, Fisal D; Niciforovic, Ana P

    2014-01-01

    largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (NaV1......Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain.......4). The results show that the highly conserved aromatic side chain constituting the S2 HC makes distinct functional contributions in each of the four NaV domains. No obvious cation-pi interaction exists with nearby S4 charges in any domain, and natural and unnatural mutations at these aromatic sites produce...

  14. A low-voltage flash memory cell utilizing the gate-injection program/erase method with a recessed channel structure

    International Nuclear Information System (INIS)

    Wu Dake; Huang Ru; Wang Pengfei; Tang Poren; Wang Yangyuan

    2008-01-01

    In this paper, a low-voltage recessed channel SONOS flash memory using the gate-injection program/erase method is proposed and investigated for NAND application. It is shown that the proposed flash memory can achieve 8 V lower programming voltage compared with planar flash memory, due to the effective capacitance coupling and the electric-field enhancement by combining the recessed channel structure and the gate-injection program/erase method. In addition, more than 30% larger threshold voltage window and improved short channel effects can be obtained in the proposed flash memory

  15. Improved Ion-Channel Biosensors

    Science.gov (United States)

    Nadeau, Jay; White, Victor; Dougherty, Dennis; Maurer, Joshua

    2004-01-01

    An effort is underway to develop improved biosensors of a type based on ion channels in biomimetic membranes. These sensors are microfabricated from silicon and other materials compatible with silicon. As described, these sensors offer a number of advantages over prior sensors of this type.

  16. Micro- and nanofabrication methods for ion channel reconstitution in bilayer lipid membranes

    Science.gov (United States)

    Tadaki, Daisuke; Yamaura, Daichi; Arata, Kohei; Ohori, Takeshi; Ma, Teng; Yamamoto, Hideaki; Niwano, Michio; Hirano-Iwata, Ayumi

    2018-03-01

    The self-assembled bilayer lipid membrane (BLM) forms the basic structure of the cell membrane and serves as a major barrier against ion movement. Ion channel proteins function as gated pores that permit ion permeation across the BLM. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for investigating channel functions and screening drug effects on ion channels. In this review, we will discuss our recent microfabrication approaches to the formation of stable BLMs containing ion channel proteins as a potential platform for next-generation drug screening systems. BLMs formed in a microaperture having a tapered edge exhibited highly stable properties, such as a lifetime of ∼65 h and tolerance to solution changes even after the incorporation of the human ether-a-go-go-related gene (hERG) channel. We also explore a new method of efficiently incorporating human ion channels into BLMs by centrifugation. Our approaches to the formation of stable BLMs and efficient channel incorporation markedly improve the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based high-throughput platform for functional assays of various ion channels.

  17. KCNE1 constrains the voltage sensor of Kv7.1 K+ channels.

    Directory of Open Access Journals (Sweden)

    Liora Shamgar

    Full Text Available Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K(+ channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K(+ channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions.

  18. Supercooling Agent Icilin Blocks a Warmth-Sensing Ion Channel TRPV3

    Directory of Open Access Journals (Sweden)

    Muhammad Azhar Sherkheli

    2012-01-01

    Full Text Available Transient receptor potential vanilloid subtype 3 (TRPV3 is a thermosensitive ion channel expressed in a variety of neural cells and in keratinocytes. It is activated by warmth (33–39°C, and its responsiveness is dramatically increased at nociceptive temperatures greater than 40°C. Monoterpenoids and 2-APB are chemical activators of TRPV3 channels. We found that Icilin, a known cooling substance and putative ligand of TRPM8, reversibly inhibits TRPV3 activity at nanomolar concentrations in expression systems like Xenopus laeves oocytes, HEK-293 cells, and in cultured human keratinocytes. Our data show that icilin's antagonistic effects for the warm-sensitive TRPV3 ion channel occurs at very low concentrations. Therefore, the cooling effect evoked by icilin may at least in part be due to TRPV3 inhibition in addition to TRPM8 potentiation. Blockade of TRPV3 activity by icilin at such low concentrations might have important implications for overall cooling sensations detected by keratinocytes and free nerve endings in skin. We hypothesize that blockage of TRPV3 might be a signal for cool-sensing systems (like TRPM8 to beat up the basal activity resulting in increased cold perception when warmth sensors (like TRPV3 are shut off.

  19. Ion-Channeling Studies of Interfaces and Defect Properties in Silicon Carbide

    International Nuclear Information System (INIS)

    Jiang, Weilin; Weber, William J.; C.H. Carter, Jr., R.P. Devaty, and G.S. Rohrer

    2000-01-01

    Helium ion channeling has been used in a detailed study of 3C-SiC films on a Si/SiO2/Si (SIMOX) substrate. The strain-induced angular shift was determined to be 0.16?? 0.05?, indicating a kink between the SiC and Si layers along the axis. Single crystals of 6H-SiC have been irradiated with a variety of ions over a range of fluences. The relative disorder on Si sublattice shows a sigmoidal dependence on dose for all ions. In isochronal and isothermal annealing studies, two distinct recovery stages are identified with activation energies of 0.25? 0.1 eV and 1.5? 0.3 eV, respectively. Deuterium ion channeling is also applied to simultaneously study accumulated disorder on Si and C sublattices in 6H-SiC crystals irradiated at 100 and 300 K

  20. Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch

    Science.gov (United States)

    Poole, Kate; Herget, Regina; Lapatsina, Liudmila; Ngo, Ha-Duong; Lewin, Gary R.

    2014-01-01

    In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure–function experiments localize the Piezo modulatory activity of STOML3 to the stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch. PMID:24662763

  1. Symposia for a Meeting on Ion Channels and Gap Junctions

    CERN Document Server

    Sáez, Juan

    1997-01-01

    Ion channels allow us to see nature in all its magnificence, to hear a Bach suite, to smell the aroma of grandmother's cooking, and, in this regard, they put us in contact with the external world. These ion channels are protein molecules located in the cell membrane. In complex organisms, cells need to communicate in order to know about their metabolic status and to act in a coordinate manner. The latter is also accomplished by a class of ion channels able to pierce the lipid bilayer membranes of two adjacent cells. These intercellular channels are the functional subunits of gap junctions. Accordingly, the book is divided in two parts: the first part is dedicated to ion channels that look to the external world, and the second part is dedicated to gap junctions found at cell interfaces. This book is based on a series of symposia for a meeting on ion channels and gap junctions held in Santiago, Chile, on November 28-30, 1995. The book should be useful to graduate students taking the first steps in this field as...

  2. Structure and hydration of membranes embedded with voltage-sensing domains.

    Science.gov (United States)

    Krepkiy, Dmitriy; Mihailescu, Mihaela; Freites, J Alfredo; Schow, Eric V; Worcester, David L; Gawrisch, Klaus; Tobias, Douglas J; White, Stephen H; Swartz, Kenton J

    2009-11-26

    Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly charged S1-S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated ion channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1-S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations and cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings indicate that voltage sensors have evolved to interact with the lipid membrane while keeping energetic and structural perturbations to a minimum, and that water penetrates the membrane, to hydrate charged residues and shape the transmembrane electric field.

  3. Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions.

    Science.gov (United States)

    Kang, Bok Eum; Baker, Bradley J

    2016-04-04

    An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response.

  4. Heteromeric Kv7.2/7.3 channels differentially regulate action potential initiation and conduction in neocortical myelinated axons

    NARCIS (Netherlands)

    Battefeld, A.; Tran, B.T.; Gavrilis, J.; Cooper, E.C.; Kole, Maarten|info:eu-repo/dai/nl/256257574

    2014-01-01

    Rapid energy-efficient signaling along vertebrate axons is achieved through intricate subcellular arrangements of voltage-gated ion channels and myelination. One recently appreciated example is the tight colocalization of Kv7 potassium channels and voltage-gated sodium (Nav ) channels in the axonal

  5. Heteromeric Kv7.2/7.3 channels differentially regulate action potential initiation and conduction in neocortical myelinated axons

    NARCIS (Netherlands)

    Battefeld, A.; Tran, Baouyen T; Gavrilis, Jason; Cooper, Edward C; Kole, Maarten H P

    2014-01-01

    Rapid energy-efficient signaling along vertebrate axons is achieved through intricate subcellular arrangements of voltage-gated ion channels and myelination. One recently appreciated example is the tight colocalization of K(v)7 potassium channels and voltage-gated sodium (Na(v)) channels in the

  6. The peculiarities of spectral manifestations of high-voltage electric discharge in different phase states of ion systems.

    Science.gov (United States)

    Gafurov, M M; Aliev, A R; Ataev, M B; Rabadanov, K Sh

    2013-10-01

    The effects of high-voltage pulsed discharge (HVPD activation) on vibrational spectra of ion salt systems have been studied. The peculiarities of spectral display of HVPD in ion melts and aqueous solutions of electrolytes, in ion-conducting phases of crystalline and glassy salt systems have been investigated. After HVPD a salt system is in non-equilibrium activated state. In the activated state of a salt system, the relaxation time of the vibrational excited states of molecular ions is shorter than in the equilibrium state if the vibrational relaxation rate increases with temperature in the system. For those systems for which the relaxation rate decreases at elevated temperatures, the relaxation time of the vibrational excited states of molecular ions is longer than in the equilibrium state. HVPD activation of a salt system can change the configuration of the electron shell of molecular ions. Therefore, the lifetime values of activated state of salt systems are abnormally large. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Eslicarbazepine and the enhancement of slow inactivation of voltage-gated sodium channels: a comparison with carbamazepine, oxcarbazepine and lacosamide.

    Science.gov (United States)

    Hebeisen, Simon; Pires, Nuno; Loureiro, Ana I; Bonifácio, Maria João; Palma, Nuno; Whyment, Andrew; Spanswick, David; Soares-da-Silva, Patrício

    2015-02-01

    This study aimed at evaluating the effects of eslicarbazepine, carbamazepine (CBZ), oxcarbazepine (OXC) and lacosamide (LCM) on the fast and slow inactivated states of voltage-gated sodium channels (VGSC). The anti-epileptiform activity was evaluated in mouse isolated hippocampal slices. The anticonvulsant effects were evaluated in MES and the 6-Hz psychomotor tests. The whole-cell patch-clamp technique was used to investigate the effects of eslicarbazepine, CBZ, OXC and LCM on sodium channels endogenously expressed in N1E-115 mouse neuroblastoma cells. CBZ and eslicarbazepine exhibit similar concentration dependent suppression of epileptiform activity in hippocampal slices. In N1E-115 mouse neuroblastoma cells, at a concentration of 250 μM, the voltage dependence of the fast inactivation was not influenced by eslicarbazepine, whereas LCM, CBZ and OXC shifted the V0.5 value (mV) by -4.8, -12.0 and -16.6, respectively. Eslicarbazepine- and LCM-treated fast-inactivated channels recovered similarly to control conditions, whereas CBZ- and OXC-treated channels required longer pulses to recover. CBZ, eslicarbazepine and LCM shifted the voltage dependence of the slow inactivation (V0.5, mV) by -4.6, -31.2 and -53.3, respectively. For eslicarbazepine, LCM, CBZ and OXC, the affinity to the slow inactivated state was 5.9, 10.4, 1.7 and 1.8 times higher than to the channels in the resting state, respectively. In conclusion, eslicarbazepine did not share with CBZ and OXC the ability to alter fast inactivation of VGSC. Both eslicarbazepine and LCM reduce VGSC availability through enhancement of slow inactivation, but LCM demonstrated higher interaction with VGSC in the resting state and with fast inactivation gating. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Use of Ion-Channel Modulating Agents to Study Cyanobacterial Na+ - K+ Fluxes

    Directory of Open Access Journals (Sweden)

    Pomati Francesco

    2004-01-01

    Full Text Available Here we describe an experimental design aimed to investigate changes in total cellular levels of Na+ and K+ ions in cultures of freshwater filamentous cyanobacteria. Ion concentrations were measured in whole cells by flame photometry. Cellular Na+ levels increased exponentially with rising alkalinity, with K+ levels being maximal for optimal growth pH (~8. At standardized pH conditions, the increase in cellular Na+, as induced by NaCl at 10 mM, was coupled by the two sodium channel-modulating agents lidocaine hydrochloride at 1 &mgr;M and veratridine at 100 &mgr;M. Both the channel-blockers amiloride (1 mM and saxitoxin (1 &mgr;M, decreased cell-bound Na+ and K+ levels. Results presented demonstrate the robustness of well-defined channel blockers and channel-activators in the study of cyanobacterial Na+- K+ fluxes.

  9. A simulation study of antimatter-helium ion planar channeling in silicon

    International Nuclear Information System (INIS)

    Wijesundera, Dharshana; Jayarathna, Sandun; Bellwied, Rene; Chu, Wei-Kan

    2012-01-01

    With the physical significance arising with the reports on experimental observation of antimatter-He nuclei, we have investigated a case of 2 MeV antimatter-He ion planar channeling in Si (1 0 0) in comparison with He channeling, by simulation. For a negatively charged antimatter-He nucleus, the planar potential well is centered at the atomic plane itself as opposed to the center-channel minimum for He ions; the antimatter-He ion distribution therefore tends to concentrate toward the atomic lattice planes. The antimatter-He ion flux distribution and the resulting close encounter probability are crucial in determining the probability of close encounter events including annihilation at channeling incidence. We have therefore analyzed the variation of antimatter-He ion flux distribution within the channels with respect to the angle of incidence and have thereby derived the orientation dependence of probability of close encounter events, or an antimatter-He channeling angular scan. The angular scan is inverted with a maximum yield at the perfect beam-planar alignment. The half-angle is narrower compared to He channeling, as a consequence of the narrower planar channeling potential centered at the lattice planes. The high de-channeling rate associated with the higher antimatter-He ion concentration in the proximity of lattice planes causes the maximum yield to be less prominent and to decrease rapidly with depth. The shoulder region shows strong depth dependent reduction that can be associated to near surface depth dependent ion flux variation.

  10. Imaging large cohorts of single ion channels and their activity

    Directory of Open Access Journals (Sweden)

    Katia eHiersemenzel

    2013-09-01

    Full Text Available As calcium is the most important signaling molecule in neurons and secretory cells, amongst many other cell types, it follows that an understanding of calcium channels and their regulation of exocytosis is of vital importance. Calcium imaging using calcium dyes such as Fluo3, or FRET-based dyes that have been used widely has provided invaluable information, which combined with modeling has estimated the sub-types of channels responsible for triggering the exocytotic machinery as well as inferences about the relative distances away from vesicle fusion sites these molecules adopt. Importantly, new super-resolution microscopy techniques, combined with novel Ca2+ indicators and imaginative imaging approaches can now define directly the nanoscale locations of very large cohorts of single channel molecules in relation to single vesicles. With combinations of these techniques the activity of individual channels can be visualized and quantified using novel Ca2+ indicators. Fluorescently labeled specific channel toxins can also be used to localize endogenous assembled channel tetramers. Fluorescence lifetime imaging microscopy and other single-photon-resolution spectroscopic approaches offer the possibility to quantify protein-protein interactions between populations of channels and the SNARE protein machinery for the first time. Together with simultaneous electrophysiology, this battery of quantitative imaging techniques has the potential to provide unprecedented detail describing the locations, dynamic behaviours, interactions and conductance activities of many thousands of channel molecules and vesicles in living cells.

  11. The molecular mechanism of multi-ion conduction in K{sup +} channels

    Energy Technology Data Exchange (ETDEWEB)

    Gwan, J.F.

    2007-01-19

    Steered molecular dynamics (SMD) simulation method is applied to a fully solvated membrane-channel model for studying the ion permeation process in potassium channels. The channel model is based on the crystallographic structure of a prokaryotic K{sup +} channel- the KcsA channel, which is a representative of most known eukaryotic K{sup +} channels. It has long been proposed that the ion transportation in a conventional K{sup +}-channel follows a multi-ion fashion: permeating ions line in a queue in the channel pore and move in a single file through the channel. The conventional view of multi-ion transportation is that the electrostatic repulsion between ions helps to overcome the attraction between ions and the channel pore. In this study, we proposed two SMD simulation schemes, referred to 'the single-ion SMD' simulations and 'the multi-ion SMD' simulations. Concerted movements of a K-W-K sequence in the selectivity filter were observed in the single-ion SMD simulations. The analysis of the concerted movement reveals the molecular mechanism of the multi-ion transportation. It shows that, rather than the long range electrostatic interaction, the short range polar interaction is a more dominant factor in the multi-ion transportation. The polar groups which play a role in the concerted transportation are the water molecules and the backbone carbonyl groups of the selectivity filter. The polar interaction is sensitive to the relative orientation of the polar groups. By changing the orientation of a polar group, the interaction may switch from attractive to repulsive or vice versa. By this means, the energy barrier between binding sites in the selectivity filter can be switched on and off, and therefore the K{sup +} may be able to move to the neighboring binding site without an external driving force. The concerted transportation in the selectivity filter requires a delicate cooperation between K{sup +}, waters, and the backbone carbonyl groups. To

  12. High-voltage scanning ion microscope: Beam optics and design

    Energy Technology Data Exchange (ETDEWEB)

    Magilin, D., E-mail: dmitrymagilin@gmail.com; Ponomarev, A.; Rebrov, V.; Ponomarov, A.

    2015-05-01

    This article is devoted to the conceptual design of a compact high-voltage scanning ion microscope (HVSIM). In an HVSIM design, the ion optical system is based on a high-brightness ion source. Specifically, the ion optical system is divided into two components: an ion injector and a probe-forming system (PFS) that consists of an accelerating tube and a multiplet of quadrupole lenses. The crossover is formed and controlled by the injector, which acts as an object collimator, and is focused on the image plane by the PFS. The ion microprobe has a size of 0.1 μm and an energy of 2 MeV. When the influence of the chromatic and third-order aberrations is theoretically taken into account, the HVSIM forms an ion microprobe.

  13. Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation.

    Science.gov (United States)

    Gregorio-Teruel, Lucia; Valente, Pierluigi; González-Ros, José Manuel; Fernández-Ballester, Gregorio; Ferrer-Montiel, Antonio

    2014-03-01

    The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues-I696 and W697-with Ala markedly affects TRPV1's response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.

  14. Free energy dissipation of the spontaneous gating of a single voltage-gated potassium channel.

    Science.gov (United States)

    Wang, Jia-Zeng; Wang, Rui-Zhen

    2018-02-01

    Potassium channels mainly contribute to the resting potential and re-polarizations, with the potassium electrochemical gradient being maintained by the pump Na + /K + -ATPase. In this paper, we construct a stochastic model mimicking the kinetics of a potassium channel, which integrates temporal evolving of the membrane voltage and the spontaneous gating of the channel. Its stationary probability density functions (PDFs) are found to be singular at the boundaries, which result from the fact that the evolving rates of voltage are greater than the gating rates of the channel. We apply PDFs to calculate the power dissipations of the potassium current, the leakage, and the gating currents. On a physical perspective, the essential role of the system is the K + -battery charging the leakage (L-)battery. A part of power will inevitably be dissipated among the process. So, the efficiency of energy transference is calculated.

  15. Free energy dissipation of the spontaneous gating of a single voltage-gated potassium channel

    Science.gov (United States)

    Wang, Jia-Zeng; Wang, Rui-Zhen

    2018-02-01

    Potassium channels mainly contribute to the resting potential and re-polarizations, with the potassium electrochemical gradient being maintained by the pump Na+/K+-ATPase. In this paper, we construct a stochastic model mimicking the kinetics of a potassium channel, which integrates temporal evolving of the membrane voltage and the spontaneous gating of the channel. Its stationary probability density functions (PDFs) are found to be singular at the boundaries, which result from the fact that the evolving rates of voltage are greater than the gating rates of the channel. We apply PDFs to calculate the power dissipations of the potassium current, the leakage, and the gating currents. On a physical perspective, the essential role of the system is the K+-battery charging the leakage (L-)battery. A part of power will inevitably be dissipated among the process. So, the efficiency of energy transference is calculated.

  16. Ion-channeling analysis of As relocation in heavily doped Si:As irradiated with high-energy ions

    International Nuclear Information System (INIS)

    Lulli, G.; Albertazzi, E.; Bianconi, M.; Ferri, M.

    2003-01-01

    Silicon on insulator layers doped with 8x10 20 As cm -3 and thermally equilibrated at 1100 deg. C, have been irradiated with 2 MeV Si + ions. Rutherford backscattering-channeling analysis shows an increase in As disorder upon irradiation significantly larger than the increase in Si disorder, while electrical measurements show a large decrease in electrical activation. Monte Carlo simulation of channeling angular scans suggests that the enhanced As disorder effect is due to the preferential relocation of dopant atoms slightly displaced from lattice sites, which appear the main reason responsible for the electrical deactivation in the unirradiated sample and are believed to be in the form of As-vacancy clusters. Upon 600 deg. C 15 s annealing, the As atoms randomly relocated by ion irradiation almost completely recover their original configuration, probably capturing vacancies and forming, again, the complexes dissociated by ion irradiation

  17. Heteromeric K(v)7.2/7.3 Channels Differentially Regulate Action Potential Initiation and Conduction in Neocortical Myelinated Axons

    NARCIS (Netherlands)

    Battefeld, Arne; Tran, Baouyen T.; Gavrilis, Jason; Cooper, Edward C.; Kole, Maarten H. P.

    2014-01-01

    Rapid energy-efficient signaling along vertebrate axons is achieved through intricate subcellular arrangements of voltage-gated ion channels and myelination. One recently appreciated example is the tight colocalization of K(v)7 potassium channels and voltage-gated sodium (Na-v) channels in the

  18. Intron retention in mRNA encoding ancillary subunit of insect voltage-gated sodium channel modulates channel expression, gating regulation and drug sensitivity.

    Directory of Open Access Journals (Sweden)

    Céline M Bourdin

    Full Text Available Insect voltage-gated sodium (Nav channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation "temperature-induced-paralysis locus E." The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na(+ currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1 strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3'UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1 co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280 in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280. PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be

  19. Defect imaging and channeling studies using channeling scanning transmission ion microscopy

    NARCIS (Netherlands)

    King, PJC; Breese, MBH; Smulders, PJM; Wilshaw, PR; Grime, GW

    The technique of channeling scanning transmission ion microscopy (CSTIM) can be used to produce images of individual crystal defects (such as dislocations and stacking faults) using the scanned, focused ion beam from a nuclear microprobe. As well as offering a new method for studies of crystal

  20. A structural view of ligand-dependent activation in thermoTRP channels

    Directory of Open Access Journals (Sweden)

    Ximena eSteinberg

    2014-05-01

    Full Text Available Transient Receptor Potential (TRP proteins are a large family of ion channels, grouped intoseven sub-families. Although great advances have been made regarding the activation andmodulation of TRP channel activity, detailed molecular mechanisms governing TRPchannel gating are still needed. Sensitive to electric, chemical, mechanical, and thermalcues, TRP channels are tightly associated with the detection and integration of sensoryinput, emerging as a model to study the polymodal activation of ion channel proteins.Among TRP channels, the temperature-activated kind constitute a subgroup by itself,formed by Vanilloid receptors 1-4, Melastatin receptors 2, 4, 5 and 8, TRPC5, and TRPA1.Some of the so-called thermoTRP channels participate in the detection of noxious stimulimaking them an interesting pharmacological target for the treatment of pain. However, thepoor specificity of the compounds available in the market represents an important obstacleto overcome. Understanding the molecular mechanics underlying ligand-dependentmodulation of TRP channels may help with the rational design of novel syntheticanalgesics. The present review focuses on the structural basis of ligand-dependentactivation of TRPV1 and TRPM8 channels. Special attention is drawn to the dissection ofligand-binding sites within TRPV1, PIP 2 -dependent modulation of TRP channels, and thestructure of natural and synthetic ligands.

  1. Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D

    2001-01-01

    The distribution of voltage-dependent calcium channels in kidney pre- and postglomerular resistance vessels was determined at the molecular and functional levels. Reverse transcription-polymerase chain reaction analysis of microdissected rat preglomerular vessels and cultured smooth muscle cells...... on vascular diameter in the afferent arteriole. We conclude that voltage-dependent L- and T-type calcium channels are expressed and of functional significance in renal cortical preglomerular vessels, in juxtamedullary efferent arterioles, and in outer medullary vasa recta, but not in cortical efferent...

  2. A family of fluoride-specific ion channels with dual-topology architecture.

    Science.gov (United States)

    Stockbridge, Randy B; Robertson, Janice L; Kolmakova-Partensky, Ludmila; Miller, Christopher

    2013-08-27

    Fluoride ion, ubiquitous in soil, water, and marine environments, is a chronic threat to microorganisms. Many prokaryotes, archea, unicellular eukaryotes, and plants use a recently discovered family of F(-) exporter proteins to lower cytoplasmic F(-) levels to counteract the anion's toxicity. We show here that these 'Fluc' proteins, purified and reconstituted in liposomes and planar phospholipid bilayers, form constitutively open anion channels with extreme selectivity for F(-) over Cl(-). The active channel is a dimer of identical or homologous subunits arranged in antiparallel transmembrane orientation. This dual-topology assembly has not previously been seen in ion channels but is known in multidrug transporters of the SMR family, and is suggestive of an evolutionary antecedent of the inverted repeats found within the subunits of many membrane transport proteins. DOI:http://dx.doi.org/10.7554/eLife.01084.001.

  3. Relative transmembrane segment rearrangements during BK channel activation resolved by structurally assigned fluorophore–quencher pairing

    Science.gov (United States)

    Pantazis, Antonios

    2012-01-01

    Voltage-activated proteins can sense, and respond to, changes in the electric field pervading the cell membrane by virtue of a transmembrane helix bundle, the voltage-sensing domain (VSD). Canonical VSDs consist of four transmembrane helices (S1–S4) of which S4 is considered a principal component because it possesses charged residues immersed in the electric field. Membrane depolarization compels the charges, and by extension S4, to rearrange with respect to the field. The VSD of large-conductance voltage- and Ca-activated K+ (BK) channels exhibits two salient inconsistencies from the canonical VSD model: (1) the BK channel VSD possesses an additional nonconserved transmembrane helix (S0); and (2) it exhibits a “decentralized” distribution of voltage-sensing charges, in helices S2 and S3, in addition to S4. Considering these unique features, the voltage-dependent rearrangements of the BK VSD could differ significantly from the standard model of VSD operation. To understand the mode of operation of this unique VSD, we have optically tracked the relative motions of the BK VSD transmembrane helices during activation, by manipulating the quenching environment of site-directed fluorescent labels with native and introduced Trp residues. Having previously reported that S0 and S4 diverge during activation, in this work we demonstrate that S4 also diverges from S1 and S2, whereas S2, compelled by its voltage-sensing charged residues, moves closer to S1. This information contributes spatial constraints for understanding the BK channel voltage-sensing process, revealing the structural rearrangements in a non-canonical VSD. PMID:22802360

  4. Computing characterizations of drugs for ion channels and receptors using Markov models

    CERN Document Server

    Tveito, Aslak

    2016-01-01

    Flow of ions through voltage gated channels can be represented theoretically using stochastic differential equations where the gating mechanism is represented by a Markov model. The flow through a channel can be manipulated using various drugs, and the effect of a given drug can be reflected by changing the Markov model. These lecture notes provide an accessible introduction to the mathematical methods needed to deal with these models. They emphasize the use of numerical methods and provide sufficient details for the reader to implement the models and thereby study the effect of various drugs. Examples in the text include stochastic calcium release from internal storage systems in cells, as well as stochastic models of the transmembrane potential. Well known Markov models are studied and a systematic approach to including the effect of mutations is presented. Lastly, the book shows how to derive the optimal properties of a theoretical model of a drug for a given mutation defined in terms of a Markov model.

  5. A limited 4 Å radial displacement of the S4-S5 linker is sufficient for internal gate closing in Kv channels.

    Science.gov (United States)

    Faure, Élise; Starek, Greg; McGuire, Hugo; Bernèche, Simon; Blunck, Rikard

    2012-11-16

    Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3-4 Å is sufficient to prevent ion permeation through the pore.

  6. Molecular dynamics and brownian dynamics investigation of ion permeation and anesthetic halothane effects on a proton-gated ion channel.

    Science.gov (United States)

    Cheng, Mary Hongying; Coalson, Rob D; Tang, Pei

    2010-11-24

    Bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC) is activated to cation permeation upon lowering the solution pH. Its function can be modulated by anesthetic halothane. In the present work, we integrate molecular dynamics (MD) and Brownian dynamics (BD) simulations to elucidate the ion conduction, charge selectivity, and halothane modulation mechanisms in GLIC, based on recently resolved X-ray crystal structures of the open-channel GLIC. MD calculations of the potential of mean force (PMF) for a Na(+) revealed two energy barriers in the extracellular domain (R109 and K38) and at the hydrophobic gate of transmembrane domain (I233), respectively. An energy well for Na(+) was near the intracellular entrance: the depth of this energy well was modulated strongly by the protonation state of E222. The energy barrier for Cl(-) was found to be 3-4 times higher than that for Na(+). Ion permeation characteristics were determined through BD simulations using a hybrid MD/continuum electrostatics approach to evaluate the energy profiles governing the ion movement. The resultant channel conductance and a near-zero permeability ratio (P(Cl)/P(Na)) were comparable to experimental data. On the basis of these calculations, we suggest that a ring of five E222 residues may act as an electrostatic gate. In addition, the hydrophobic gate region may play a role in charge selectivity due to a higher dehydration energy barrier for Cl(-) ions. The effect of halothane on the Na(+) PMF was also evaluated. Halothane was found to perturb salt bridges in GLIC that may be crucial for channel gating and open-channel stability, but had no significant impact on the single ion PMF profiles.

  7. Li-Ion Electrolytes with Improved Safety and Tolerance to High-Voltage Systems

    Science.gov (United States)

    Smart, Marshall C.; Bugga, Ratnakumar V.; Prakash, Surya; Krause, Frederick C.

    2013-01-01

    Given that lithium-ion (Li-ion) technology is the most viable rechargeable energy storage device for near-term applications, effort has been devoted to improving the safety characteristics of this system. Therefore, extensive effort has been devoted to developing nonflammable electrolytes to reduce the flammability of the cells/battery. A number of promising electrolytes have been developed incorporating flame-retardant additives, and have been shown to have good performance in a number of systems. However, these electrolyte formulations did not perform well when utilizing carbonaceous anodes with the high-voltage materials. Thus, further development was required to improve the compatibility. A number of Li-ion battery electrolyte formulations containing a flame-retardant additive [i.e., triphenyl phosphate (TPP)] were developed and demonstrated in high-voltage systems. These electrolytes include: (1) formulations that incorporate varying concentrations of the flame-retardant additive (from 5 to 15%), (2) the use of mono-fluoroethylene carbonate (FEC) as a co-solvent, and (3) the use of LiBOB as an electrolyte additive intended to improve the compatibility with high-voltage systems. Thus, improved safety has been provided without loss of performance in the high-voltage, high-energy system.

  8. Voltage imaging to understand connections and functions of neuronal circuits

    Science.gov (United States)

    Antic, Srdjan D.; Empson, Ruth M.

    2016-01-01

    Understanding of the cellular mechanisms underlying brain functions such as cognition and emotions requires monitoring of membrane voltage at the cellular, circuit, and system levels. Seminal voltage-sensitive dye and calcium-sensitive dye imaging studies have demonstrated parallel detection of electrical activity across populations of interconnected neurons in a variety of preparations. A game-changing advance made in recent years has been the conceptualization and development of optogenetic tools, including genetically encoded indicators of voltage (GEVIs) or calcium (GECIs) and genetically encoded light-gated ion channels (actuators, e.g., channelrhodopsin2). Compared with low-molecular-weight calcium and voltage indicators (dyes), the optogenetic imaging approaches are 1) cell type specific, 2) less invasive, 3) able to relate activity and anatomy, and 4) facilitate long-term recordings of individual cells' activities over weeks, thereby allowing direct monitoring of the emergence of learned behaviors and underlying circuit mechanisms. We highlight the potential of novel approaches based on GEVIs and compare those to calcium imaging approaches. We also discuss how novel approaches based on GEVIs (and GECIs) coupled with genetically encoded actuators will promote progress in our knowledge of brain circuits and systems. PMID:27075539

  9. Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy

    Directory of Open Access Journals (Sweden)

    Stylianos Michalakis

    2018-03-01

    Full Text Available The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP or cyclic adenosine monophosphate (cAMP. Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN channels and voltage-gated potassium channels (KCN. In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

  10. Ion mass dependence for low energy channeling in single-wall nanotubes

    International Nuclear Information System (INIS)

    Zheng Liping; Zhu Zhiyuan; Li Yong; Zhu Dezhang; Xia Huihao

    2008-01-01

    An Monte Carlo (MC) simulation program has been used to study ion mass dependence for the low energy channeling of natural- and pseudo-Ar ions in single-wall nanotubes. The MC simulations show that the channeling critical angle Ψ C obeys the (E) -1/2 and the (M 1 ) -1/2 rules, where E is the incident energy and M 1 is the ion mass. The reason for this may be that the motion of the channeled (or de-channeled) ions should be correlated with both the incident energy E and the incident momentum (2M 1 E) 1/2 , in order to obey the conservation of energy and momentum

  11. Point mutations in the transmembrane region of the clic1 ion channel selectively modify its biophysical properties.

    Directory of Open Access Journals (Sweden)

    Stefania Averaimo

    Full Text Available Chloride intracellular Channel 1 (CLIC1 is a metamorphic protein that changes from a soluble cytoplasmic protein into a transmembrane protein. Once inserted into membranes, CLIC1 multimerises and is able to form chloride selective ion channels. Whilst CLIC1 behaves as an ion channel both in cells and in artificial lipid bilayers, its structure in the soluble form has led to some uncertainty as to whether it really is an ion channel protein. CLIC1 has a single putative transmembrane region that contains only two charged residues: arginine 29 (Arg29 and lysine 37 (Lys37. As charged residues are likely to have a key role in ion channel function, we hypothesized that mutating them to neutral alanine to generate K37A and R29A CLIC1 would alter the electrophysiological characteristics of CLIC1. By using three different electrophysiological approaches: i single channel Tip-Dip in artificial bilayers using soluble recombinant CLIC1, ii cell-attached and iii whole-cell patch clamp recordings in transiently transfected HEK cells, we determined that the K37A mutation altered the single-channel conductance while the R29A mutation affected the single-channel open probability in response to variation in membrane potential. Our results show that mutation of the two charged amino acids (K37 and R29 in the putative transmembrane region of CLIC1 alters the biophysical properties of the ion channel in both artificial bilayers and cells. Hence these charged residues are directly involved in regulating its ion channel activity. This strongly suggests that, despite its unusual structure, CLIC1 itself is able to form a chloride ion channel.

  12. Experimental investigation on lithium-ion battery thermal management based on flow boiling in mini-channel

    International Nuclear Information System (INIS)

    An, Zhoujian; Jia, Li; Li, Xuejiao; Ding, Yong

    2017-01-01

    Highlights: • A new type of BTM system based on flow boiling in mini-channel are presented. • Uniform temperature and volume distribution of battery module are obtained. • The temperatures of battery cell are maintained around 40 °C. • There exists an appropriate Re number range for boiling heat transfer in mini-channel. - Abstract: In order to guarantee the safety and prolong the lifetime of lithium-ion power battery within electric vehicles, thermal management system is essential. A new type of thermal management system based on flow boiling in mini-channel utilizing dielectric hydrofluoroether liquid which boiling point is 34 °C is proposed. The cooling experiments for battery module are carried out at different discharge rates and flow Re number. The cooling effect and the influence of battery cooling on the electrochemical characteristics are concerned. The experimental results show that the thermal management can efficiently reduce maximum temperature of battery module and surface maximum temperature difference. A relatively uniform temperature and voltage distributions are provided within the battery module at higher discharge rate benefit from the advantage of boiling heat transfer with uniform temperature distribution on cold plate. It is shown that the voltage decreases with the increase of Re number of fluid due to the reducing of temperature. There exist slight fluctuations of voltage distribution because of the non-uniformity of temperature distribution within the battery module at higher discharge rates. For different discharge rate, there also exists an appropriate Re number range during which the mode of heat transfer is mainly in boiling heat transfer mode and the cooling result can be greatly improved.

  13. Caution Is Required in Interpretation of Mutations in the Voltage Sensing Domain of Voltage Gated Channels as Evidence for Gating Mechanisms

    Directory of Open Access Journals (Sweden)

    Alisher M. Kariev

    2015-01-01

    Full Text Available The gating mechanism of voltage sensitive ion channels is generally considered to be the motion of the S4 transmembrane segment of the voltage sensing domains (VSD. The primary supporting evidence came from R→C mutations on the S4 transmembrane segment of the VSD, followed by reaction with a methanethiosulfonate (MTS reagent. The cys side chain is –SH (reactive form –S−; the arginine side chain is much larger, leaving space big enough to accommodate the MTS sulfonate head group. The cavity created by the mutation has space for up to seven more water molecules than were present in wild type, which could be displaced irreversibly by the MTS reagent. Our quantum calculations show there is major reorientation of three aromatic residues that face into the cavity in response to proton displacement within the VSD. Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain –OH. These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone. The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations.

  14. Calculating tracer currents through narrow ion channels: Beyond the independent particle model.

    Science.gov (United States)

    Coalson, Rob D; Jasnow, David

    2018-06-01

    Discrete state models of single-file ion permeation through a narrow ion channel pore are employed to analyze the ratio of forward to backward tracer current. Conditions under which the well-known Ussing formula for this ratio hold are explored in systems where ions do not move independently through the channel. Building detailed balance into the rate constants for the model in such a way that under equilibrium conditions (equal rate of forward vs. backward permeation events) the Nernst Equation is satisfied, it is found that in a model where only one ion can occupy the channel at a time, the Ussing formula is always obeyed for any number of binding sites, reservoir concentrations of the ions and electric potential difference across the membrane which the ion channel spans, independent of the internal details of the permeation pathway. However, numerical analysis demonstrates that when multiple ions can occupy the channel at once, the nonequilibrium forward/backward tracer flux ratio deviates from the prediction of the Ussing model. Assuming an appropriate effective potential experienced by ions in the channel, we provide explicit formulae for the rate constants in these models. © 2018 IOP Publishing Ltd.

  15. Distribution of voltage-dependent and intracellular Ca2+ channels in submucosal neurons from rat distal colon.

    Science.gov (United States)

    Rehn, Matthias; Bader, Sandra; Bell, Anna; Diener, Martin

    2013-09-01

    with various types of high-voltage activated Ca(v) channels and with IP3 receptors for intracellular Ca2+ signaling.

  16. An Improved Targeted cAMP Sensor to Study the Regulation of Adenylyl Cyclase 8 by Ca2+ Entry through Voltage-Gated Channels

    Science.gov (United States)

    Everett, Katy L.; Cooper, Dermot M. F.

    2013-01-01

    Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC) 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes. PMID:24086669

  17. An improved targeted cAMP sensor to study the regulation of adenylyl cyclase 8 by Ca2+ entry through voltage-gated channels.

    Directory of Open Access Journals (Sweden)

    Katy L Everett

    Full Text Available Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca(2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca(2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes.

  18. Divergent biophysical properties, gating mechanisms, and possible functions of the two skeletal muscle Ca(V)1.1 calcium channel splice variants.

    Science.gov (United States)

    Tuluc, Petronel; Flucher, Bernhard E

    2011-12-01

    Voltage-gated calcium channels are multi-subunit protein complexes that specifically allow calcium ions to enter the cell in response to membrane depolarization. But, for many years it seemed that the skeletal muscle calcium channel Ca(V)1.1 is the exception. The classical splice variant Ca(V)1.1a activates slowly, has a very small current amplitude and poor voltage sensitivity. In fact adult muscle fibers work perfectly well even in the absence of calcium influx. Recently a new splice variant of the skeletal muscle calcium channel Ca(V)1.1e has been characterized. The lack of the 19 amino acid exon 29 in this splice variant results in a rapidly activating calcium channel with high current amplitude and good voltage sensitivity. Ca(V)1.1e is the dominant channel in embryonic muscle, where the expression of this high calcium-conducting Ca(V)1.1 isoform readily explains developmental processes depending on L-type calcium currents. Moreover, the availability of these two structurally similar but functionally distinct channel variants facilitates the analysis of the molecular mechanisms underlying the unique current properties of the classical Ca(V)1.1a channel.

  19. Sensing charges of the Ciona intestinalis voltage-sensing phosphatase.

    Science.gov (United States)

    Villalba-Galea, Carlos A; Frezza, Ludivine; Sandtner, Walter; Bezanilla, Francisco

    2013-11-01

    Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.

  20. High quality ion channels recordings on an injection molded polymer chip

    DEFF Research Database (Denmark)

    Tanzi, Simone

    In this thesis we demonstrate high quality recordings of the ion channel activity across the cell membrane in a biological cell by employing the so called patch clamping technique on an injection molded polymer microfluidic device. Such recordings are traditionally made using glass micropipettes,...

  1. Superconcentrated electrolytes for a high-voltage lithium-ion battery

    Science.gov (United States)

    Wang, Jianhui; Yamada, Yuki; Sodeyama, Keitaro; Chiang, Ching Hua; Tateyama, Yoshitaka; Yamada, Atsuo

    2016-01-01

    Finding a viable electrolyte for next-generation 5 V-class lithium-ion batteries is of primary importance. A long-standing obstacle has been metal-ion dissolution at high voltages. The LiPF6 salt in conventional electrolytes is chemically unstable, which accelerates transition metal dissolution of the electrode material, yet beneficially suppresses oxidative dissolution of the aluminium current collector; replacing LiPF6 with more stable lithium salts may diminish transition metal dissolution but unfortunately encounters severe aluminium oxidation. Here we report an electrolyte design that can solve this dilemma. By mixing a stable lithium salt LiN(SO2F)2 with dimethyl carbonate solvent at extremely high concentrations, we obtain an unusual liquid showing a three-dimensional network of anions and solvent molecules that coordinate strongly to Li+ ions. This simple formulation of superconcentrated LiN(SO2F)2/dimethyl carbonate electrolyte inhibits the dissolution of both aluminium and transition metal at around 5 V, and realizes a high-voltage LiNi0.5Mn1.5O4/graphite battery that exhibits excellent cycling durability, high rate capability and enhanced safety. PMID:27354162

  2. Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.

    Science.gov (United States)

    Liu, Dong-Hai; Huang, Xu; Guo, Xin; Meng, Xiang-Min; Wu, Yi-Song; Lu, Hong-Li; Zhang, Chun-Mei; Kim, Young-chul; Xu, Wen-Xie

    2014-01-01

    Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV) was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV) to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

  3. Highly Sensitive and Patchable Pressure Sensors Mimicking Ion-Channel-Engaged Sensory Organs.

    Science.gov (United States)

    Chun, Kyoung-Yong; Son, Young Jun; Han, Chang-Soo

    2016-04-26

    Biological ion channels have led to much inspiration because of their unique and exquisite operational functions in living cells. Specifically, their extreme and dynamic sensing abilities can be realized by the combination of receptors and nanopores coupled together to construct an ion channel system. In the current study, we demonstrated that artificial ion channel pressure sensors inspired by nature for detecting pressure are highly sensitive and patchable. Our ion channel pressure sensors basically consisted of receptors and nanopore membranes, enabling dynamic current responses to external forces for multiple applications. The ion channel pressure sensors had a sensitivity of ∼5.6 kPa(-1) and a response time of ∼12 ms at a frequency of 1 Hz. The power consumption was recorded as less than a few μW. Moreover, a reliability test showed stability over 10 000 loading-unloading cycles. Additionally, linear regression was performed in terms of temperature, which showed no significant variations, and there were no significant current variations with humidity. The patchable ion channel pressure sensors were then used to detect blood pressure/pulse in humans, and different signals were clearly observed for each person. Additionally, modified ion channel pressure sensors detected complex motions including pressing and folding in a high-pressure range (10-20 kPa).

  4. NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel.

    Science.gov (United States)

    Paramonov, A S; Lyukmanova, E N; Myshkin, M Yu; Shulepko, M A; Kulbatskii, D S; Petrosian, N S; Chugunov, A O; Dolgikh, D A; Kirpichnikov, M P; Arseniev, A S; Shenkarev, Z O

    2017-03-01

    Voltage-gated Na + channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na + channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na + channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of 13 C, 15 N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na + channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 3 10 -helical conformation. Water accessibility of S3 helix, observed by the Mn 2+ titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. 15 N relaxation data revealed characteristic pattern of μs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K + channels. These results validate structural studies of isolated VSDs of Na + channels and show possible pitfalls in application of this 'divide and conquer' approach. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. TRANSMISSION ION CHANNELING IMAGES OF CRYSTAL DEFECTS

    NARCIS (Netherlands)

    KING, PJC; BREESE, MBH; WILSHAW, PR; SMULDERS, PJM; GRIME, GW

    This paper demonstrates how images of crystal defects can be produced using ion channeling. A focused, scanned beam of MeV protons from the University of Oxford Nuclear Microprobe has been used. With the beam aligned with a channeling direction of the crystal, protons transmitted through the thinned

  6. First high-voltage measurements using Ca{sup +} ions at the ALIVE experiment

    Energy Technology Data Exchange (ETDEWEB)

    König, K., E-mail: kkoenig@ikp.tu-darmstadt.de [Technische Universität Darmstadt, Institut für Kernphysik (Germany); Geppert, Ch. [Universität Mainz, Institut für Kernchemie (Germany); Krämer, J.; Maaß, B. [Technische Universität Darmstadt, Institut für Kernphysik (Germany); Otten, E. W. [Universität Mainz, Institut für Physik (Germany); Ratajczyk, T.; Nörtershäuser, W. [Technische Universität Darmstadt, Institut für Kernphysik (Germany)

    2017-11-15

    Many physics experiments depend on accurate high-voltage measurements to determine for example the exact retardation potential of an electron spectrometer as in the KATRIN experiment or the acceleration voltage of the ions at ISOL facilities. Until now only precision high-voltage dividers can be used to measure voltages up to 65 kV with an accuracy of 1 ppm. However, these dividers need frequent calibration and cross-checking and the direct traceability is not given. In this article we will describe the status of an experiment which aims to measure high voltages using collinear laser spectroscopy and which has the potential to provide a high-voltage standard and hence, a calibration source for precision high-voltage dividers on the 1 ppm level.

  7. Natural products as tools for studies of ligand-gated ion channels

    DEFF Research Database (Denmark)

    Strømgaard, Kristian

    2005-01-01

    Ligand-gated ion channels, or ionotropic receptors, constitute a group of membrane-bound proteins that regulate the flux of ions across the cell membrane. In the brain, ligand-gated ion channels mediate fast neurotransmission. They are crucial for normal brain function and involved in many diseases...

  8. Gold removal rate by ion sputtering as a function of ion-beam voltage and raster size using Auger electron spectroscopy. Final report

    International Nuclear Information System (INIS)

    Boehning, C.W.

    1983-01-01

    Gold removal rate was measured as a function of ion beam voltage and raster size using Auger electron spectroscopy (AES). Three different gold thicknesses were developed as standards. Two sputter rate calibration curves were generated by which gold sputter rate could be determined for variations in ion beam voltage or raster size

  9. The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

    Directory of Open Access Journals (Sweden)

    Abel Peter W

    2007-11-01

    Full Text Available Abstract Background Uterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv channels and large-conductance, calcium-activated potassium (BK channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium. Methods Functional assays which evaluated the effects of channel blockers and various contractile agonists were accomplished by quantifying contractility of isolated uterine smooth muscle obtained from nonpregnant mice as well as mice at various stages of pregnancy. Expression of Kv channel proteins in isolated uterine smooth muscle was evaluated by Western blots. Results The Kv channel blocker 4-aminopyridine (4-AP caused contractions in nonpregnant mouse myometrium (EC50 = 54 micromolar, maximal effect at 300 micromolar but this effect disappeared in pregnant mice; similarly, the Kv4.2/Kv4.3 blocker phrixotoxin-2 caused contractions in nonpregnant, but not pregnant, myometrium. Contractile responses to 4-AP were not dependent upon nerves, as neither tetrodotoxin nor storage of tissues at room temperature significantly altered these responses, nor were responses dependent upon the presence of the endometrium. Spontaneous contractions and contractions in response to 4-AP did not appear to be mediated by BK, as the BK channel-selective blockers iberiotoxin, verruculogen, or tetraethylammonium failed to affect either spontaneous contractions or 4-AP-elicited responses. A number of different Kv channel alpha subunit proteins were

  10. Simulation of biological ion channels with technology computer-aided design.

    Science.gov (United States)

    Pandey, Santosh; Bortei-Doku, Akwete; White, Marvin H

    2007-01-01

    Computer simulations of realistic ion channel structures have always been challenging and a subject of rigorous study. Simulations based on continuum electrostatics have proven to be computationally cheap and reasonably accurate in predicting a channel's behavior. In this paper we discuss the use of a device simulator, SILVACO, to build a solid-state model for KcsA channel and study its steady-state response. SILVACO is a well-established program, typically used by electrical engineers to simulate the process flow and electrical characteristics of solid-state devices. By employing this simulation program, we have presented an alternative computing platform for performing ion channel simulations, besides the known methods of writing codes in programming languages. With the ease of varying the different parameters in the channel's vestibule and the ability of incorporating surface charges, we have shown the wide-ranging possibilities of using a device simulator for ion channel simulations. Our simulated results closely agree with the experimental data, validating our model.

  11. Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid

    Directory of Open Access Journals (Sweden)

    Galyna Maleeva

    2017-05-01

    Full Text Available Niflumic acid (NFA is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels. Here we present evidence that NFA is an efficient blocker of chloride-permeable glycine receptors (GlyRs with subunit heterogeneity of action. Using the whole-cell configuration of patch-clamp recordings and molecular modeling, we analyzed the action of NFA on homomeric α1ΔIns, α2B, α3L, and heteromeric α1β and α2β GlyRs expressed in CHO cells. NFA inhibited glycine-induced currents in a voltage-dependent manner and its blocking potency in α2 and α3 GlyRs was higher than that in α1 GlyR. The Woodhull analysis suggests that NFA blocks α1 and α2 GlyRs at the fractional electrical distances of 0.16 and 0.65 from the external membrane surface, respectively. Thus, NFA binding site in α1 GlyR is closer to the external part of the membrane, while in α2 GlyR it is significantly deeper in the pore. Mutation G254A at the cytoplasmic part of the α1 GlyR pore-lining TM2 helix (level 2′ increased the NFA blocking potency, while incorporation of the β subunit did not have a significant effect. The Hill plot analysis suggests that α1 and α2 GlyRs are preferably blocked by two and one NFA molecules, respectively. Molecular modeling using Monte Carlo energy minimizations provides the structural rationale for the experimental data and proposes more than one interaction site along the pore where NFA can suppress the ion permeation.

  12. Ozone synthesis improves by increasing number density of plasma channels and lower voltage in a nonthermal plasma

    Science.gov (United States)

    Arif Malik, Muhammad; Hughes, David

    2016-04-01

    Improvements in ozone synthesis from air and oxygen by increasing the number density of plasma channels and lower voltage for the same specific input energy (SIE) were explored in a nonthermal plasma based on a sliding discharge. The number of plasma channels and energy per pulse increased in direct proportion to the increase in the effective length of the anode (the high voltage electrode). Decreasing the discharge gap increased the energy per pulse for the same length and allowed the installation of more electrode pairs in the same space. It allowed the increase of the number of plasma channels in the same space to achieve the same SIE at a lower peak voltage with less energy per plasma channel. The ozone concentration gradually increased to ~1500 ppmv (140 to 50 g kWh-1) from air and to ~6000 ppmv (400 to 200 g kWh-1) from oxygen with a gradual increase in the SIE to ~200 J L-1, irrespective of the variations in electrode geometry, applied voltage or flow rate of the feed gas. A gradual increase in SIE beyond 200 J L-1 gradually increased the ozone concentration to a certain maximum value followed by a decline, but the rate of increase and the maximum value was higher for the greater number of plasma channels and lower peak voltage combination. The maximum ozone concentration was ~5000 ppmv (~30 g kWh-1) from air and ~22 000 ppmv (~80 g kWh-1) from oxygen. The results are explained on the basis of characteristics of the plasma and ozone synthesis mechanism.

  13. Ca2+-dependent K+ Channels in Exocrine Salivary Glands

    Science.gov (United States)

    Catalán, Marcelo A.; Peña-Munzenmayer, Gaspar; Melvin, James E.

    2014-01-01

    In the last 15 years, remarkable progress has been realized in identifying the genes that encode the ion-transporting proteins involved in exocrine gland function, including salivary glands. Among these proteins, Ca2+-dependent K+ channels take part in key functions including membrane potential regulation, fluid movement and K+ secretion in exocrine glands. Two K+ channels have been identified in exocrine salivary glands: 1) a Ca2+-activated K+ channel of intermediate single channel conductance encoded by the KCNN4 gene; and, 2) a voltage- and Ca2+-dependent K+ channel of large single channel conductance encoded by the KCNMA1 gene. This review focuses on the physiological roles of Ca2+-dependent K+ channels in exocrine salivary glands. We also discuss interesting recent findings on the regulation of Ca2+-dependent K+ channels by protein-protein interactions that may significantly impact exocrine gland physiology. PMID:24559652

  14. Active stabilization of ion trap radiofrequency potentials

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, K. G.; Wong-Campos, J. D.; Restelli, A.; Landsman, K. A.; Neyenhuis, B.; Mizrahi, J.; Monroe, C. [Joint Quantum Institute and University of Maryland Department of Physics, College Park, Maryland 20742 (United States)

    2016-05-15

    We actively stabilize the harmonic oscillation frequency of a laser-cooled atomic ion confined in a radiofrequency (rf) Paul trap by sampling and rectifying the high voltage rf applied to the trap electrodes. We are able to stabilize the 1 MHz atomic oscillation frequency to be better than 10 Hz or 10 ppm. This represents a suppression of ambient noise on the rf circuit by 34 dB. This technique could impact the sensitivity of ion trap mass spectrometry and the fidelity of quantum operations in ion trap quantum information applications.

  15. Binding of ArgTX-636 in the NMDA receptor ion channel

    DEFF Research Database (Denmark)

    Poulsen, Mette H; Andersen, Jacob; Christensen, Rune

    2015-01-01

    of NMDAR activity and have therapeutic potential for treatment of a variety of brain diseases or as pharmacological tools for studies of the neurobiological role of NMDARs. We have performed a kinetic analysis of the blocking mechanism of the prototypical polyamine toxin NMDAR ion channel blocker...

  16. Monitoring Ion Activities In and Around Cells Using Ion-Selective Liquid-Membrane Microelectrodes

    Directory of Open Access Journals (Sweden)

    Mark D. Parker

    2013-01-01

    Full Text Available Determining the effective concentration (i.e., activity of ions in and around living cells is important to our understanding of the contribution of those ions to cellular function. Moreover, monitoring changes in ion activities in and around cells is informative about the actions of the transporters and/or channels operating in the cell membrane. The activity of an ion can be measured using a glass microelectrode that includes in its tip a liquid-membrane doped with an ion-selective ionophore. Because these electrodes can be fabricated with tip diameters that are less than 1 μm, they can be used to impale single cells in order to monitor the activities of intracellular ions. This review summarizes the history, theory, and practice of ion-selective microelectrode use and brings together a number of classic and recent examples of their usefulness in the realm of physiological study.

  17. Creation and dynamical co-evolution of electron and ion channel transport barriers

    International Nuclear Information System (INIS)

    Newman, D.E.

    2002-01-01

    A wide variety of magnetic confinement devices have found transitions to an enhanced confinement regime. Simple dynamical models have been able to capture much of the dynamics of these barriers however an open question has been the disconnected nature of the electron thermal transport channel sometimes observed in the presence of a standard ('ion channel' barrier. By adding to simple barrier model an evolution equation for electron fluctuations we can investigate the interaction between the formation of the standard ion channel barrier and the somewhat less common electron channel barrier. Barrier formation in the electron channel is even more sensitive to the alignment of the various gradients making up the sheared radial electric field than the ion barrier is. Electron channel heat transport is found to significantly increase after the formation of the ion channel barrier but before the electron channel barrier is formed. This increased transport is important in the barrier evolution. (author)

  18. A new functional role for mechanistic/mammalian target of rapamycin complex 1 (mTORC1 in the circadian regulation of L-type voltage-gated calcium channels in avian cone photoreceptors.

    Directory of Open Access Journals (Sweden)

    Cathy Chia-Yu Huang

    Full Text Available In the retina, the L-type voltage-gated calcium channels (L-VGCCs are responsible for neurotransmitter release from photoreceptors and are under circadian regulation. Both the current densities and protein expression of L-VGCCs are significantly higher at night than during the day. However, the underlying mechanisms of circadian regulation of L-VGCCs in the retina are not completely understood. In this study, we demonstrated that the mechanistic/mammalian target of rapamycin complex (mTORC signaling pathway participated in the circadian phase-dependent modulation of L-VGCCs. The activities of the mTOR cascade, from mTORC1 to its downstream targets, displayed circadian oscillations throughout the course of a day. Disruption of mTORC1 signaling dampened the L-VGCC current densities, as well as the protein expression of L-VGCCs at night. The decrease of L-VGCCs at night by mTORC1 inhibition was in part due to a reduction of L-VGCCα1 subunit translocation from the cytosol to the plasma membrane. Finally, we showed that mTORC1 was downstream of the phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT signaling pathway. Taken together, mTORC1 signaling played a role in the circadian regulation of L-VGCCs, in part through regulation of ion channel trafficking and translocation, which brings to light a new functional role for mTORC1: the modulation of ion channel activities.

  19. Laser-Driven Ion Acceleration from Plasma Micro-Channel Targets

    Science.gov (United States)

    Zou, D. B.; Pukhov, A.; Yi, L. Q.; Zhou, H. B.; Yu, T. P.; Yin, Y.; Shao, F. Q.

    2017-02-01

    Efficient energy boost of the laser-accelerated ions is critical for their applications in biomedical and hadron research. Achiev-able energies continue to rise, with currently highest energies, allowing access to medical therapy energy windows. Here, a new regime of simultaneous acceleration of ~100 MeV protons and multi-100 MeV carbon-ions from plasma micro-channel targets is proposed by using a ~1020 W/cm2 modest intensity laser pulse. It is found that two trains of overdense electron bunches are dragged out from the micro-channel and effectively accelerated by the longitudinal electric-field excited in the plasma channel. With the optimized channel size, these “superponderomotive” energetic electrons can be focused on the front surface of the attached plastic substrate. The much intense sheath electric-field is formed on the rear side, leading to up to ~10-fold ionic energy increase compared to the simple planar geometry. The analytical prediction of the optimal channel size and ion maximum energies is derived, which shows good agreement with the particle-in-cell simulations.

  20. Angular distributions of ions channeled in the Si crystals

    International Nuclear Information System (INIS)

    Petrovic, S.; Korica, S.; Kokkoris, M.; Neskovic, N.

    2002-01-01

    In this study we analyze the angular distributions of Ne 10+ ions channeled in the Si crystals. The ion energy is 60 MeV and the crystal thickness is varied from 286 to 3435 nm. This thickness range corresponds to the reduced crystal thickness range from 0.5 to 6, i.e. from the second to the twelfth rainbow cycle. The angular distributions were obtained via the numerical solution of the ion equations of motion and the computer simulation method. The analysis shows that the angular distribution has a periodic behavior. We also analyze the transmission patterns corresponding to the angular distributions. These patterns should be compared to the experimental patterns obtainable by a two-dimensional position sensitive detector. We demonstrate that, when the ion beam divergence is sufficiently large, i.e. much larger than the critical angle for channeling, the channeling star effect occurs in the transmission patterns