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Sample records for calcium channel gating

  1. Voltage-Gated Calcium Channels in Nociception

    Science.gov (United States)

    Yasuda, Takahiro; Adams, David J.

    Voltage-gated calcium channels (VGCCs) are a large and functionally diverse group of membrane ion channels ubiquitously expressed throughout the central and peripheral nervous systems. VGCCs contribute to various physiological processes and transduce electrical activity into other cellular functions. This chapter provides an overview of biophysical properties of VGCCs, including regulation by auxiliary subunits, and their physiological role in neuronal functions. Subsequently, then we focus on N-type calcium (Cav2.2) channels, in particular their diversity and specific antagonists. We also discuss the role of N-type calcium channels in nociception and pain transmission through primary sensory dorsal root ganglion neurons (nociceptors). It has been shown that these channels are expressed predominantly in nerve terminals of the nociceptors and that they control neurotransmitter release. To date, important roles of N-type calcium channels in pain sensation have been elucidated genetically and pharmacologically, indicating that specific N-type calcium channel antagonists or modulators are particularly useful as therapeutic drugs targeting chronic and neuropathic pain.

  2. Regulation of voltage-gated calcium channels by proteolysis

    Science.gov (United States)

    Kathryn, ABELE; Jian, YANG

    2015-01-01

    Voltage gated calcium channels (VGCCs) are multi-subunit membrane proteins present in a variety of tissues and control many essential physiological processes. Due to their vital importance, VGCCs are regulated by a myriad of proteins and signaling pathways. Here we review the literature on the regulation of VGCCs by proteolysis of the pore-forming α1 subunit, Cavα1. This form of regulation modulates channel function and degradation and affects cellular gene expression and excitability. L-type Ca2+ channels are proteolyzed in two ways, depending on tissue localization. In the heart and skeletal muscle, the distal C-terminus of Cavα1 is cleaved and acts as an autoinhibitor when it reassociates with the proximal C-terminus. Relief of this autoinhibition underlies the β-adrenergic stimulation-induced enhancement of cardiac and skeletal muscle calcium currents, part of the “fight or flight” response. Proteolysis of the distal C-terminus of L-type channels also occurs in the brain and is probably catalyzed by a calpain-like protease. In some brain regions, the entire C-terminus of L-type Ca2+ channels can be cleaved by an unknown protease and translocates to the nucleus acting as a transcription factor. The distal C-terminus of P/Q-channel Cavα1 is also proteolyzed and translocates to the nucleus. Truncated forms of the PQ-channel Cavα1 are produced by many disease-causing mutations and interfere with the function of full-length channels. Truncated forms of N-type channel Cavα1, generated by mutagenesis, affect the expression of full-length channels. New forms of proteolysis of VGCC subunits remain to be discovered and may represent a fruitful area of VGCC research. PMID:23090491

  3. Plasma Membrane Cyclic Nucleotide Gated Calcium Channels Control Land Plant Thermal Sensing and Acquired Thermotolerance

    National Research Council Canada - National Science Library

    Andrija Finka; America Farinia Henriquez Cuendet; Frans J.M. Maathuis; Younousse Saidi; Pierre Goloubinoff

    2012-01-01

    .... Here, we found that the cyclic nucleotide gated calcium channel (CNGC) CNGCb gene from Physcomitrella patens and its Arabidopsis thaliana ortholog CNGC2, encode a component of cyclic nucleotide gated Ca²...

  4. Voltage-Gated Calcium Channel Antagonists and Traumatic Brain Injury

    Directory of Open Access Journals (Sweden)

    Bruce Lyeth

    2013-06-01

    Full Text Available Traumatic brain injury (TBI is a leading cause of death and disability in the United States. Despite more than 30 years of research, no pharmacological agents have been identified that improve neurological function following TBI. However, several lines of research described in this review provide support for further development of voltage gated calcium channel (VGCC antagonists as potential therapeutic agents. Following TBI, neurons and astrocytes experience a rapid and sometimes enduring increase in intracellular calcium ([Ca2+]i. These fluxes in [Ca2+]i drive not only apoptotic and necrotic cell death, but also can lead to long-term cell dysfunction in surviving cells. In a limited number of in vitro experiments, both L-type and N-type VGCC antagonists successfully reduced calcium loads as well as neuronal and astrocytic cell death following mechanical injury. In rodent models of TBI, administration of VGCC antagonists reduced cell death and improved cognitive function. It is clear that there is a critical need to find effective therapeutics and rational drug delivery strategies for the management and treatment of TBI, and we believe that further investigation of VGCC antagonists should be pursued before ruling out the possibility of successful translation to the clinic.

  5. How voltage-gated calcium channels gate forms of homeostatic synaptic plasticity

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    C. Andrew eFrank

    2014-02-01

    Full Text Available Throughout life, animals face a variety of challenges such as developmental growth, the presence of toxins, or changes in temperature. Neuronal circuits and synapses respond to challenges by executing an array of neuroplasticity paradigms. Some paradigms allow neurons to up- or downregulate activity outputs, while countervailing ones ensure that outputs remain within appropriate physiological ranges. A growing body of evidence suggests that homeostatic synaptic plasticity (HSP is critical in the latter case. Voltage-gated calcium channels gate forms of HSP. Presynaptically, the aggregate data show that when synapse activity is weakened, homeostatic signaling systems can act to correct impairments, in part by increasing calcium influx through presynaptic CaV2-type channels. Increased calcium influx is often accompanied by parallel increases in the size of active zones and the size of the readily releasable pool of presynaptic vesicles. These changes coincide with homeostatic enhancements of neurotransmitter release. Postsynaptically, there is a great deal of evidence that reduced network activity and loss of calcium influx through CaV1-type calcium channels also results in adaptive homeostatic signaling. Some adaptations drive presynaptic enhancements of vesicle pool size and turnover rate via retrograde signaling, as well as de novo insertion of postsynaptic neurotransmitter receptors. Enhanced calcium influx through CaV1 after network activation or single cell stimulation can elicit the opposite response – homeostatic depression via removal of excitatory receptors.There exist intriguing links between HSP and calcium channelopathies – such as forms of epilepsy, migraine, ataxia, and myasthenia. The episodic nature of some of these disorders suggests alternating periods of stable and unstable function. Uncovering information about how calcium channels are regulated in the context of HSP could be relevant toward understanding these and other

  6. 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 o...

  7. 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 Ca2+ entering the cilium through voltage-gated Ca2+ (CaV) channels that are found exclusively in the cilia. As ciliary Ca2+ levels return to normal, the cell pivots and swims forward in a new direction. Thus, the activation of the CaV channels causes cells to make a turn in their swimming paths. For 45 years, the physiological characteristics of the Paramecium ciliary CaV channels have been known, but the proteins were not identified until recently, when the P. tetraurelia ciliary membrane proteome was determined. Three CaVα1 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 CaV 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 CaV channel activity do not express any of the three CaV1 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 CaV channels with backward swimming through ciliary reversal. The PwB protein, found in endoplasmic reticulum fractions, co-immunoprecipitates with the CaV1c 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.

  8. LRRK2 regulates voltage-gated calcium channel function.

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    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

  9. 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 arteriol...... publication, 10 November 2010; doi:10.1038/ki.2010.429....

  10. 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 Impact factor: 3.498, year: 2016

  11. Nuclear-localized cyclic nucleotide-gated channels mediate symbiotic calcium oscillations.

    Science.gov (United States)

    Charpentier, Myriam; Sun, Jongho; Vaz Martins, Teresa; Radhakrishnan, Guru V; Findlay, Kim; Soumpourou, Eleni; Thouin, Julien; Véry, Anne-Aliénor; Sanders, Dale; Morris, Richard J; Oldroyd, Giles E D

    2016-05-27

    Nuclear-associated Ca(2+) oscillations mediate plant responses to beneficial microbial partners--namely, nitrogen-fixing rhizobial bacteria that colonize roots of legumes and arbuscular mycorrhizal fungi that colonize roots of the majority of plant species. A potassium-permeable channel is known to be required for symbiotic Ca(2+) oscillations, but the calcium channels themselves have been unknown until now. We show that three cyclic nucleotide-gated channels in Medicago truncatula are required for nuclear Ca(2+) oscillations and subsequent symbiotic responses. These cyclic nucleotide-gated channels are located at the nuclear envelope and are permeable to Ca(2+) We demonstrate that the cyclic nucleotide-gated channels form a complex with the postassium-permeable channel, which modulates nuclear Ca(2+) release. These channels, like their counterparts in animal cells, might regulate multiple nuclear Ca(2+) responses to developmental and environmental conditions. Copyright © 2016, American Association for the Advancement of Science.

  12. The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential

    Science.gov (United States)

    Zamponi, Gerald W.; Striessnig, Joerg; Koschak, Alexandra

    2015-01-01

    Voltage-gated calcium channels are required for many key functions in the body. In this review, the different subtypes of voltage-gated calcium channels are described and their physiologic roles and pharmacology are outlined. We describe the current uses of drugs interacting with the different calcium channel subtypes and subunits, as well as specific areas in which there is strong potential for future drug development. Current therapeutic agents include drugs targeting L-type CaV1.2 calcium channels, particularly 1,4-dihydropyridines, which are widely used in the treatment of hypertension. T-type (CaV3) channels are a target of ethosuximide, widely used in absence epilepsy. The auxiliary subunit α2δ-1 is the therapeutic target of the gabapentinoid drugs, which are of value in certain epilepsies and chronic neuropathic pain. The limited use of intrathecal ziconotide, a peptide blocker of N-type (CaV2.2) calcium channels, as a treatment of intractable pain, gives an indication that these channels represent excellent drug targets for various pain conditions. We describe how selectivity for different subtypes of calcium channels (e.g., CaV1.2 and CaV1.3 L-type channels) may be achieved in the future by exploiting differences between channel isoforms in terms of sequence and biophysical properties, variation in splicing in different target tissues, and differences in the properties of the target tissues themselves in terms of membrane potential or firing frequency. Thus, use-dependent blockers of the different isoforms could selectively block calcium channels in particular pathologies, such as nociceptive neurons in pain states or in epileptic brain circuits. Of important future potential are selective CaV1.3 blockers for neuropsychiatric diseases, neuroprotection in Parkinson’s disease, and resistant hypertension. In addition, selective or nonselective T-type channel blockers are considered potential therapeutic targets in epilepsy, pain, obesity, sleep, and

  13. Spatial distribution of calcium-gated chloride channels in olfactory cilia.

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    French, Donald A; Badamdorj, Dorjsuren; Kleene, Steven J

    2010-12-30

    In vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 µm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity. To determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29% of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia. On average, the two types of olfactory transduction channel are concentrated in the same region of the cilium. This may contribute to the efficient detection of weak stimuli.

  14. T-type calcium channel: a privileged gate for calcium entry and control of adrenal steroidogenesis

    Directory of Open Access Journals (Sweden)

    Michel Florian Rossier

    2016-05-01

    Full Text Available Intracellular calcium plays a crucial role in modulating a variety of functions such as muscle contraction, hormone secretion, gene expression or cell growth. Calcium signaling has been however shown to be more complex than initially thought. Indeed, it is confined within cell microdomains and different calcium channels are associated with different functions, as shown by various channelopathies.Sporadic mutations on voltage-operated L-type calcium channels in adrenal glomerulosa cells have been shown recently to be the second most prevalent genetic abnormalities present in human aldosterone-producing adenoma. The observed modification of the threshold of activation of the mutated channels not only provides an explanation for this gain of function but reminds us on the importance of maintaining adequate electrophysiological characteristics to make channels able to exert specific cellular functions. Indeed, the contribution to steroid production of the various calcium channels expressed in adrenocortical cells is not equal and the reason has been investigated for a long time. Given the very negative resting potential of these cells, and the small membrane depolarization induced by their physiological agonists, low threshold T-type calcium channels are particularly well suited for responding under these conditions and conveying calcium into the cell, at the right place for controlling steroidogenesis. In contrast, high threshold L-type channels are normally activated by much stronger cell depolarizations. The fact that dihydropyridine calcium antagonists, specific for L-type channels, are poorly efficient for reducing aldosterone secretion either in vivo or in vitro, strongly supports the view that these two types of channels differently affect steroid biosynthesis.Whether a similar analysis is transposable to fasciculata cells and cortisol secretion is one of the questions addressed in the present review. No similar mutations on L-type or T

  15. Voltage gated calcium channels negatively regulate protective immunity to Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Shashank Gupta

    Full Text Available Mycobacterium tuberculosis modulates levels and activity of key intracellular second messengers to evade protective immune responses. Calcium release from voltage gated calcium channels (VGCC regulates immune responses to pathogens. In this study, we investigated the roles of VGCC in regulating protective immunity to mycobacteria in vitro and in vivo. Inhibiting L-type or R-type VGCC in dendritic cells (DCs either using antibodies or by siRNA increased calcium influx in an inositol 1,4,5-phosphate and calcium release calcium activated channel dependent mechanism that resulted in increased expression of genes favoring pro-inflammatory responses. Further, VGCC-blocked DCs activated T cells that in turn mediated killing of M. tuberculosis inside macrophages. Likewise, inhibiting VGCC in infected macrophages and PBMCs induced calcium influx, upregulated the expression of pro-inflammatory genes and resulted in enhanced killing of intracellular M. tuberculosis. Importantly, compared to healthy controls, PBMCs of tuberculosis patients expressed higher levels of both VGCC, which were significantly reduced following chemotherapy. Finally, blocking VGCC in vivo in M. tuberculosis infected mice using specific antibodies increased intracellular calcium and significantly reduced bacterial loads. These results indicate that L-type and R-type VGCC play a negative role in M. tuberculosis infection by regulating calcium mobilization in cells that determine protective immunity.

  16. Single calcium channel domain gating of synaptic vesicle fusion at fast synapses; analysis by graphic modeling

    Science.gov (United States)

    Stanley, Elise F

    2015-01-01

    At fast-transmitting presynaptic terminals Ca2+ enter through voltage gated calcium channels (CaVs) and bind to a synaptic vesicle (SV) -associated calcium sensor (SV-sensor) to gate fusion and discharge. An open CaV generates a high-concentration plume, or nanodomain of Ca2+ that dissipates precipitously with distance from the pore. At most fast synapses, such as the frog neuromuscular junction (NMJ), the SV sensors are located sufficiently close to individual CaVs to be gated by single nanodomains. However, at others, such as the mature rodent calyx of Held (calyx of Held), the physiology is more complex with evidence that CaVs that are both close and distant from the SV sensor and it is argued that release is gated primarily by the overlapping Ca2+ nanodomains from many CaVs. We devised a 'graphic modeling' method to sum Ca2+ from individual CaVs located at varying distances from the SV-sensor to determine the SV release probability and also the fraction of that probability that can be attributed to single domain gating. This method was applied first to simplified, low and high CaV density model release sites and then to published data on the contrasting frog NMJ and the rodent calyx of Held native synapses. We report 3 main predictions: the SV-sensor is positioned very close to the point at which the SV fuses with the membrane; single domain-release gating predominates even at synapses where the SV abuts a large cluster of CaVs, and even relatively remote CaVs can contribute significantly to single domain-based gating. PMID:26457441

  17. Functional and pharmacological consequences of the distribution of voltage-gated calcium channels in the renal blood vessels

    DEFF Research Database (Denmark)

    Hansen, P B L

    2013-01-01

    Calcium channel blockers are widely used to treat hypertension because they inhibit voltage-gated calcium channels that mediate transmembrane calcium influx in, for example, vascular smooth muscle and cardiomyocytes. The calcium channel family consists of several subfamilies, of which the L......-type is usually associated with vascular contractility. However, the L-, T- and P-/Q-types of calcium channels are present in the renal vasculature and are differentially involved in controlling vascular contractility, thereby contributing to regulation of kidney function and blood pressure. In the preglomerular...... vascular bed, all the three channel families are present. However, the T-type channel is the only channel in cortical efferent arterioles which is in contrast to the juxtamedullary efferent arteriole, and that leads to diverse functional effects of L- and T-type channel inhibition. Furthermore...

  18. GABA(A) Increases Calcium in Subventricular Zone Astrocyte-Like Cells Through L- and T-Type Voltage-Gated Calcium Channels

    DEFF Research Database (Denmark)

    Young, Stephanie Z; Platel, Jean-Claude; Nielsen, Jakob V

    2010-01-01

    induced Ca(2+) increases in 40-50% of SVZ astrocytes. GABA(A)-induced Ca(2+) increases were prevented with nifedipine and mibefradil, blockers of L- and T-type voltage-gated calcium channels (VGCC). The L-type Ca(2+) channel activator BayK 8644 increased the percentage of GABA(A)-responding astrocyte...

  19. Functional and pharmacological consequences of the distribution of voltage-gated calcium channels in the renal blood vessels.

    Science.gov (United States)

    Hansen, P B L

    2013-04-01

    Calcium channel blockers are widely used to treat hypertension because they inhibit voltage-gated calcium channels that mediate transmembrane calcium influx in, for example, vascular smooth muscle and cardiomyocytes. The calcium channel family consists of several subfamilies, of which the L-type is usually associated with vascular contractility. However, the L-, T- and P-/Q-types of calcium channels are present in the renal vasculature and are differentially involved in controlling vascular contractility, thereby contributing to regulation of kidney function and blood pressure. In the preglomerular vascular bed, all the three channel families are present. However, the T-type channel is the only channel in cortical efferent arterioles which is in contrast to the juxtamedullary efferent arteriole, and that leads to diverse functional effects of L- and T-type channel inhibition. Furthermore, by different mechanisms, T-type channels may contribute to both constriction and dilation of the arterioles. Finally, P-/Q-type channels are involved in the regulation of human intrarenal arterial contractility. The calcium blockers used in the clinic affect not only L-type but also P-/Q- and T-type channels. Therefore, the distinct effect obtained by inhibiting a given subtype or set of channels under experimental settings should be considered when choosing a calcium blocker for treatment. T-type channels seem to be crucial for regulating the GFR and the filtration fraction. Use of blockers is expected to lead to preferential efferent vasodilation, reduction of glomerular pressure and proteinuria. Therefore, renovascular T-type channels might provide novel therapeutic targets, and may have superior renoprotective effects compared to conventional calcium blockers. Acta Physiologica © 2013 Scandinavian Physiological Society.

  20. H2O2augments 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.

  1. Electromagnetic fields act via activation of voltage-gated calcium channels to produce beneficial or adverse effects

    OpenAIRE

    Pall, Martin L

    2013-01-01

    The direct targets of extremely low and microwave frequency range electromagnetic fields (EMFs) in producing non-thermal effects have not been clearly established. However, studies in the literature, reviewed here, provide substantial support for such direct targets. Twenty-three studies have shown that voltage-gated calcium channels (VGCCs) produce these and other EMF effects, such that the L-type or other VGCC blockers block or greatly lower diverse EMF effects. Furthermore, the voltage-gat...

  2. Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels.

    Science.gov (United States)

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

    Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca(2+)]E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca(2+)]I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca(2+)]E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca(2+)]I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca(2+)]I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca(2+)]I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Clinical features of neuromuscular disorders in patients with N-type voltage-gated calcium channel antibodies

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    Andreas Totzeck

    2016-09-01

    Full Text Available Neuromuscular junction disorders affect the pre- or postsynaptic nerve to muscle transmission due to autoimmune antibodies. Members of the group like myasthenia gravis and Lambert-Eaton syndrome have pathophysiologically distinct characteristics. However, in practice, distinction may be difficult. We present a series of three patients with a myasthenic syndrome, dropped-head syndrome, bulbar and respiratory muscle weakness and positive testing for anti-N-type voltage-gated calcium channel antibodies. In two cases anti-acetylcholin receptor antibodies were elevated, anti-P/Q-type voltage-gated calcium channel antibodies were negative. All patients initially responded to pyridostigmine with a non-response in the course of the disease. While one patient recovered well after treatment with intravenous immunoglobulins, 3,4-diaminopyridine, steroids and later on immunosuppression with mycophenolate mofetil, a second died after restriction of treatment due to unfavorable cancer diagnosis, the third patient declined treatment. Although new antibodies causing neuromuscular disorders were discovered, clinical distinction has not yet been made. Our patients showed features of pre- and postsynaptic myasthenic syndrome as well as severe dropped-head syndrome and bulbar and axial muscle weakness, but only anti-N-type voltage-gated calcium channel antibodies were positive. When administered, one patient benefited from 3,4-diaminopyridine. We suggest that this overlap-syndrome should be considered especially in patients with assumed seronegative myasthenia gravis and lack of improvement under standard therapy.

  4. Small-cell lung cancer with voltage-gated calcium channel antibody-positive paraneoplastic limbic encephalitis: a case report.

    Science.gov (United States)

    Kaira, Kyoichi; Okamura, Takashi; Takahashi, Hiroki; Horiguchi, Norio; Sunaga, Noriaki; Hisada, Takeshi; Yamada, Masanobu

    2014-04-08

    Paraneoplastic limbic encephalitis is a rare neurological syndrome and clinically characterized by cognitive dysfunction, memory impairment, seizures and psychiatric symptoms. Paraneoplastic limbic encephalitis is most frequently found in small-cell lung cancer, among various malignancies, and antineuronal antibodies are related to the autoimmune mechanism. We experienced a rare case of a patient with small-cell lung cancer with anti-voltage-gated calcium channel antibody-positive paraneoplastic limbic encephalitis. A 61-year-old Japanese man with a history of smoking cigarettes presented with seizure, confusion and personality change in acute onset. Brain magnetic resonance imaging showed high signal intensity on T2-weighted image in his right temporal lobe, suggestive of limbic encephalitis. A mediastinoscopy of the lymph node revealed small-cell lung carcinoma, and he was staged as having limited stage disease. Antibodies against P/Q-type and N-type voltage-gated calcium channel were positive and Hu antibody was negative. He was started on chemotherapy of carboplatin plus etoposide with concurrent thoracic radiotherapy. Neurological symptoms were gradually improved after systemic chemotherapy. We should be alert to the potential of malignant neoplasms associated with paraneoplastic limbic encephalitis when we examine a patient with cancer with neurological disorders such as personality change, disorientation, unconsciousness and memory loss. A clinical marker such as voltage-gated calcium channel antibody may help our diagnosis in clinical practice.

  5. Alcohol Dependence Disrupts Amygdalar L-Type Voltage-Gated Calcium Channel Mechanisms.

    Science.gov (United States)

    Varodayan, Florence P; de Guglielmo, Giordano; Logrip, Marian L; George, Olivier; Roberto, Marisa

    2017-04-26

    L-type voltage-gated calcium channels (LTCCs) are implicated in several psychiatric disorders that are comorbid with alcoholism and involve amygdala dysfunction. Within the amygdala, the central nucleus (CeA) is critical in acute alcohol's reinforcing actions, and its dysregulation in human alcoholics drives their negative emotional state and motivation to drink. Here we investigated the specific role of CeA LTCCs in the effects of acute alcohol at the molecular, cellular physiology, and behavioral levels, and their potential neuroadaptation in alcohol-dependent rats. Alcohol increases CeA activity (neuronal firing rates and GABA release) in naive rats by engaging LTCCs, and intra-CeA LTCC blockade reduces alcohol intake in nondependent rats. Alcohol dependence reduces CeA LTCC membrane abundance and disrupts this LTCC-based mechanism; instead, corticotropin-releasing factor type 1 receptors (CRF1s) mediate alcohol's effects on CeA activity and drive the escalated alcohol intake of alcohol-dependent rats. Collectively, our data indicate that alcohol dependence functionally alters the molecular mechanisms underlying the CeA's response to alcohol (from LTCC- to CRF1-driven). This mechanistic switch contributes to and reflects the prominent role of the CeA in the negative emotional state that drives excessive drinking.SIGNIFICANCE STATEMENT The central amygdala (CeA) plays a critical role in the development of alcohol dependence. As a result, much preclinical alcohol research aims to identify relevant CeA neuroadaptions that promote the transition to dependence. Here we report that acute alcohol increases CeA neuronal activity in naive rats by engaging L-type calcium channels (LTCCs) and that intra-CeA LTCC blockade reduces alcohol intake in nondependent rats. Alcohol dependence disrupts this LTCC-based mechanism; instead, corticotropin-releasing factor type 1 receptors (CRF1s) mediate alcohol's effects on CeA activity and drive the escalated alcohol intake of alcohol

  6. 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.

  7. Expression and cellular localization of the voltage-gated calcium channel α2δ3 in the rodent retina

    Science.gov (United States)

    Müller, Luis Pérez de Sevilla; Sargoy, Allison; Fernández-Sánchez, Laura; Rodriguez, Allen; Liu, Janelle; Cuenca, Nicolás; Brecha, Nicholas

    2015-01-01

    High voltage activated calcium channels are hetero-oligomeric protein complexes that mediate multiple cellular processes including the influx of extracellular Ca2+, neurotransmitter release, gene transcription and synaptic plasticity. These channels consist of a primary α1 pore-forming subunit, which is associated with an extracellular α2δ subunit and an intracellular β auxiliary subunit, which alter the gating properties and trafficking of the calcium channel. The cellular localization of the α2δ3 subunit in the mouse and rat retina is unknown. In this study, using RT-PCR a single band at ~305 bp corresponding to the predicted size of the α2δ3 subunit fragment was in mouse and rat retina and brain homogenates. Western blotting of rodent retina and brain homogenates showed a single 123 kDa band. Immunohistochemistry using an affinity purified antibody to the α2δ3 subunit revealed immunoreactive cell bodies in the ganglion cell layer (GCL) and inner nuclear layer (INL), and immunoreactive processes in the inner plexiform layer (IPL) and the outer plexiform layer (OPL). α2δ3 immunoreactivity was localized to multiple cell types, including ganglion, amacrine and bipolar cells, and photoreceptors, but not by horizontal cells. The expression of the α2δ3 calcium channel subunit to multiple cell types suggests this subunit participates widely in Ca channel-mediated signaling in the retina. PMID:25631988

  8. Large conductance, calcium- and voltage-gated potassium (BK) channels: regulation by cholesterol.

    Science.gov (United States)

    Dopico, Alejandro M; Bukiya, Anna N; Singh, Aditya K

    2012-08-01

    Cholesterol (CLR) is an essential component of eukaryotic plasma membranes. CLR regulates the membrane physical state, microdomain formation and the activity of membrane-spanning proteins, including ion channels. Large conductance, voltage- and Ca²⁺-gated K⁺ (BK) channels link membrane potential to cell Ca²⁺ homeostasis. Thus, they control many physiological processes and participate in pathophysiological mechanisms leading to human disease. Because plasmalemma BK channels cluster in CLR-rich membrane microdomains, a major driving force for studying BK channel-CLR interactions is determining how membrane CLR controls the BK current phenotype, including its pharmacology, channel sorting, distribution, and role in cell physiology. Since both BK channels and CLR tissue levels play a pathophysiological role in human disease, identifying functional and structural aspects of the CLR-BK channel interaction may open new avenues for therapeutic intervention. Here, we review the studies documenting membrane CLR-BK channel interactions, dissecting out the many factors that determine the final BK current response to changes in membrane CLR content. We also summarize work in reductionist systems where recombinant BK protein is studied in artificial lipid bilayers, which documents a direct inhibition of BK channel activity by CLR and builds a strong case for a direct interaction between CLR and the BK channel-forming protein. Bilayer lipid-mediated mechanisms in CLR action are also discussed. Finally, we review studies of BK channel function during hypercholesterolemia, and underscore the many consequences that the CLR-BK channel interaction brings to cell physiology and human disease. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Role for voltage gated calcium channels in calcitonin gene-related peptide release in the rat trigeminovascular system

    DEFF Research Database (Denmark)

    Amrutkar, D V; Ploug, K B; Olesen, J

    2011-01-01

    Clinical and genetic studies have suggested a role for voltage gated calcium channels (VGCCs) in the pathogenesis of migraine. Release of calcitonin gene-related peptide (CGRP) from trigeminal neurons has also been implicated in migraine. The VGCCs are located presynaptically on neurons and are i......Clinical and genetic studies have suggested a role for voltage gated calcium channels (VGCCs) in the pathogenesis of migraine. Release of calcitonin gene-related peptide (CGRP) from trigeminal neurons has also been implicated in migraine. The VGCCs are located presynaptically on neurons...... and are involved in the release of these peptides to different stimuli. We have examined the presence and importance of VGCCs in controlling the CGRP release from rat dura mater, freshly isolated trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC). Each of the four VGCCs, P/Q-, N-, and L- and T...... the potassium induced CGRP release. In the absence of calcium ions (Ca2+) and in the presence of a cocktail of blockers, the stimulated CGRP release from dura mater was reduced almost to the same level as basal CGRP release. In the TG ¿-conotoxin GVIA inhibited the potassium induced CGRP release significantly...

  10. Role for voltage gated calcium channels in calcitonin gene-related peptide release in the rat trigeminovascular system

    DEFF Research Database (Denmark)

    Amrutkar, D V; Ploug, K B; Olesen, J

    2011-01-01

    Clinical and genetic studies have suggested a role for voltage gated calcium channels (VGCCs) in the pathogenesis of migraine. Release of calcitonin gene-related peptide (CGRP) from trigeminal neurons has also been implicated in migraine. The VGCCs are located presynaptically on neurons and are i......Clinical and genetic studies have suggested a role for voltage gated calcium channels (VGCCs) in the pathogenesis of migraine. Release of calcitonin gene-related peptide (CGRP) from trigeminal neurons has also been implicated in migraine. The VGCCs are located presynaptically on neurons...... and are involved in the release of these peptides to different stimuli. We have examined the presence and importance of VGCCs in controlling the CGRP release from rat dura mater, freshly isolated trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC). Each of the four VGCCs, P/Q-, N-, and L- and T...... the potassium induced CGRP release. In the absence of calcium ions (Ca2+) and in the presence of a cocktail of blockers, the stimulated CGRP release from dura mater was reduced almost to the same level as basal CGRP release. In the TG ω-conotoxin GVIA inhibited the potassium induced CGRP release significantly...

  11. Lung adenocarcinoma with Lambert–Eaton myasthenic syndrome indicated by voltage-gated calcium channel: a case report

    Directory of Open Access Journals (Sweden)

    Arai Hiromasa

    2012-09-01

    Full Text Available Abstract Introduction Lambert–Eaton myasthenic syndrome is a rare disorder and it is known as a paraneoplastic neurological syndrome. Small cell lung cancer often accompanies this syndrome. Lambert–Eaton myasthenic syndrome associated with lung adenocarcinoma is extremely rare; there are only a few reported cases worldwide. Case presentation A 75-year-old Japanese man with a past history of chronic rheumatoid arthritis and Sjögren syndrome was diagnosed with Lambert–Eaton myasthenic syndrome by electromyography and serum anti-P/Q-type voltage-gated calcium channel antibody level preceding the diagnosis of lung cancer. A chest computed tomography to screen for malignant lesions revealed an abnormal shadow in the lung. Although a histopathological examination by bronchoscopic study could not reveal the malignancy, lung cancer was mostly suspected after the results of a chest computed tomography and [18F]-fluorodeoxyglucose positron emission tomography. An intraoperative diagnosis based on the frozen section obtained by tumor biopsy was adenocarcinoma so the patient underwent a lobectomy of the right lower lobe and lymph node dissection with video-assisted thoracoscopic surgery. The permanent pathological examination was the same as the frozen diagnosis (pT2aN1M0: Stage IIa: TNM staging 7th edition. Immunohistochemistry revealed that most of the cancer cells were positive for P/Q-type voltage-gated calcium channel. Conclusions Our case is a rare combination of Lambert–Eaton myasthenic syndrome associated with lung adenocarcinoma, rheumatoid arthritis and Sjögren syndrome, and to the best of our knowledge it is the first report that indicates the presence of voltage-gated calcium channel in lung adenocarcinoma by immunostaining.

  12. Fetal calcium regulates branching morphogenesis in the developing human and mouse lung: involvement of voltage-gated calcium channels.

    Directory of Open Access Journals (Sweden)

    Sarah C Brennan

    Full Text Available Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9-17 of human gestation, embryonic days (E11.5-16.5 in mouse in a hypercalcaemic environment (~1.7 in the fetus vs. ~1.1-1.3 mM for an adult. Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca(2+ channels (VGCC, inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (CaV1.2 and CaV1.3, P/Q type (CaV2.1, N-type (CaV2.2, R-type (CaV2.3, and T-type (CaV3.2 and CaV3.3 VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(3, demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to

  13. Physiology and Evolution of Voltage-Gated Calcium Channels in Early Diverging Animal Phyla: Cnidaria, Placozoa, Porifera and Ctenophora

    Science.gov (United States)

    Senatore, Adriano; Raiss, Hamad; Le, Phuong

    2016-01-01

    Voltage-gated calcium (Cav) channels serve dual roles in the cell, where they can both depolarize the membrane potential for electrical excitability, and activate transient cytoplasmic Ca2+ signals. In animals, Cav channels play crucial roles including driving muscle contraction (excitation-contraction coupling), gene expression (excitation-transcription coupling), pre-synaptic and neuroendocrine exocytosis (excitation-secretion coupling), regulation of flagellar/ciliary beating, and regulation of cellular excitability, either directly or through modulation of other Ca2+-sensitive ion channels. In recent years, genome sequencing has provided significant insights into the molecular evolution of Cav channels. Furthermore, expanded gene datasets have permitted improved inference of the species phylogeny at the base of Metazoa, providing clearer insights into the evolution of complex animal traits which involve Cav channels, including the nervous system. For the various types of metazoan Cav channels, key properties that determine their cellular contribution include: Ion selectivity, pore gating, and, importantly, cytoplasmic protein-protein interactions that direct sub-cellular localization and functional complexing. It is unclear when these defining features, many of which are essential for nervous system function, evolved. In this review, we highlight some experimental observations that implicate Cav channels in the physiology and behavior of the most early-diverging animals from the phyla Cnidaria, Placozoa, Porifera, and Ctenophora. Given our limited understanding of the molecular biology of Cav channels in these basal animal lineages, we infer insights from better-studied vertebrate and invertebrate animals. We also highlight some apparently conserved cellular functions of Cav channels, which might have emerged very early on during metazoan evolution, or perhaps predated it. PMID:27867359

  14. Physiology and evolution of voltage-gated calcium channels in early diverging animal phyla: Cnidaria, Placozoa, Porifera and Ctenophora

    Directory of Open Access Journals (Sweden)

    Adriano Senatore

    2016-11-01

    Full Text Available Voltage-gated calcium (Cav channels serve dual roles in the cell, where they can both depolarize the membrane potential for electrical excitability, and activate transient cytoplasmic Ca2+ signals. In animals, Cav channels play crucial roles including driving muscle contraction (excitation-contraction coupling, gene expression (excitation-transcription coupling, pre-synaptic and neuroendocrine exocytosis (excitation-secretion coupling, regulation of flagellar/ciliary beating, and regulation of cellular excitability, either directly or through modulation of other Ca2+-sensitive ion channels. In recent years, genome sequencing has provided significant insights into the molecular evolution of Cav channels. Furthermore, expanded gene datasets have permitted improved inference of the species phylogeny at the base of Metazoa, providing clearer insights into the evolution of complex animal traits which involve Cav channels, including the nervous system. For the various types of metazoan Cav channels, key properties that determine their cellular contribution include: ion selectivity, pore gating, and, importantly, cytoplasmic protein-protein interactions that direct sub-cellular localization and functional complexing. It is unclear when many of these defining features, many of which are essential for nervous system function, evolved. In this review, we highlight some experimental observations that implicate Cav channels in the physiology and behavior of the most early-diverging animals from the phyla Cnidaria, Placozoa, Porifera and Ctenophora. Given our limited understanding of the molecular biology of Cav channels in these basal animal lineages, we infer insights from better-studied vertebrate and invertebrate animals. We also highlight some apparently conserved cellular functions of Cav channels, which might have emerged very early on during metazoan evolution, or perhaps predated it.

  15. Physiology and Evolution of Voltage-Gated Calcium Channels in Early Diverging Animal Phyla: Cnidaria, Placozoa, Porifera and Ctenophora.

    Science.gov (United States)

    Senatore, Adriano; Raiss, Hamad; Le, Phuong

    2016-01-01

    Voltage-gated calcium (Cav) channels serve dual roles in the cell, where they can both depolarize the membrane potential for electrical excitability, and activate transient cytoplasmic Ca(2+) signals. In animals, Cav channels play crucial roles including driving muscle contraction (excitation-contraction coupling), gene expression (excitation-transcription coupling), pre-synaptic and neuroendocrine exocytosis (excitation-secretion coupling), regulation of flagellar/ciliary beating, and regulation of cellular excitability, either directly or through modulation of other Ca(2+)-sensitive ion channels. In recent years, genome sequencing has provided significant insights into the molecular evolution of Cav channels. Furthermore, expanded gene datasets have permitted improved inference of the species phylogeny at the base of Metazoa, providing clearer insights into the evolution of complex animal traits which involve Cav channels, including the nervous system. For the various types of metazoan Cav channels, key properties that determine their cellular contribution include: Ion selectivity, pore gating, and, importantly, cytoplasmic protein-protein interactions that direct sub-cellular localization and functional complexing. It is unclear when these defining features, many of which are essential for nervous system function, evolved. In this review, we highlight some experimental observations that implicate Cav channels in the physiology and behavior of the most early-diverging animals from the phyla Cnidaria, Placozoa, Porifera, and Ctenophora. Given our limited understanding of the molecular biology of Cav channels in these basal animal lineages, we infer insights from better-studied vertebrate and invertebrate animals. We also highlight some apparently conserved cellular functions of Cav channels, which might have emerged very early on during metazoan evolution, or perhaps predated it.

  16. Seeing the forest through the trees: towards a unified view on physiological calcium regulation of voltage-gated sodium channels.

    Science.gov (United States)

    Van Petegem, Filip; Lobo, Paolo A; Ahern, Christopher A

    2012-12-05

    Voltage-gated sodium channels (Na(V)s) underlie the upstroke of the action potential in the excitable tissues of nerve and muscle. After opening, Na(V)s rapidly undergo inactivation, a crucial process through which sodium conductance is negatively regulated. Disruption of inactivation by inherited mutations is an established cause of lethal cardiac arrhythmia, epilepsy, or painful syndromes. Intracellular calcium ions (Ca(2+)) modulate sodium channel inactivation, and multiple players have been suggested in this process, including the cytoplasmic Na(V) C-terminal region including two EF-hands and an IQ motif, the Na(V) domain III-IV linker, and calmodulin. Calmodulin can bind to the IQ domain in both Ca(2+)-bound and Ca(2+)-free conditions, but only to the DIII-IV linker in a Ca(2+)-loaded state. The mechanism of Ca(2+) regulation, and its composite effect(s) on channel gating, has been shrouded in much controversy owing to numerous apparent experimental inconsistencies. Herein, we attempt to summarize these disparate data and propose a novel, to our knowledge, physiological mechanism whereby calcium ions promote sodium current facilitation due to Ca(2+) memory at high-action-potential frequencies where Ca(2+) levels may accumulate. The available data suggest that this phenomenon may be disrupted in diseases where cytoplasmic calcium ion levels are chronically high and where targeted phosphorylation may decouple the Ca(2+) regulatory machinery. Many Na(V) disease mutations associated with electrical dysfunction are located in the Ca(2+)-sensing machinery and misregulation of Ca(2+)-dependent channel modulation is likely to contribute to disease phenotypes. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. Altered voltage-gated calcium channels in rat inferior colliculus neurons contribute to alcohol withdrawal seizures

    Science.gov (United States)

    N’Gouemo, Prosper

    2015-01-01

    We have previously reported that enhanced susceptibility to alcohol withdrawal seizures (AWS) parallels the enhancement of the current density of high-threshold voltage-gated Ca2+ (CaV) channels in rat inferior colliculus (IC) neurons. However, whether this increased current density is a cause or consequence of AWS is unclear. Here, I report changes in the current density of CaV channels in IC neurons during the course of alcohol withdrawal and the potential anticonvulsant effect of intra-IC infusions of L- and P-type CaV channel antagonists. Whole-cell currents were activated by depolarizing pulses using barium as the charge carrier. Currents and seizure susceptibility were evaluated in control animals 3 h after alcohol intoxication, as well as 3 h (before AWS), 24 h (when AWS susceptibility is maximal), and 48 h (when AWS susceptibility is no longer present) after alcohol withdrawal. Nifedipine, nimodipine (L-type antagonists) or ω-agatoxin TK (P-type antagonist) were infused intra-IC to probe the role of CaV channels in the pathogenesis of AWS. CaV current density and conductance in IC neurons were significantly increased 3 and 24 h after alcohol withdrawal compared with the control group or the group tested 3 h following ethanol intoxication. Blockade of L-type CaV channels within the IC completely suppressed AWS, and inhibition of P-type channels reduced AWS severity. These findings suggest that the enhancement of CaV currents in IC neurons occurs prior to AWS onset and that alterations in L- and P-type CaV channels in these neurons may underlie the pathogenesis of AWS. PMID:25914156

  18. Dehydroepiandrosterone (DHEA) inhibits voltage-gated T-type calcium channels.

    Science.gov (United States)

    Chevalier, M; Gilbert, G; Lory, P; Marthan, R; Quignard, J F; Savineau, J P

    2012-06-01

    Dehydroepiandrosterone (DHEA) and its sulfated form, DHEAS, are the most abundant steroid hormones in the mammalian blood flow. DHEA may have beneficial effects in various pathophysiological conditions such as cardiovascular diseases or deterioration of the sense of well-being. However to date, the cellular mechanism underlying DHEA action remains elusive and may involve ion channel modulation. In this study, we have characterized the effect of DHEA on T-type voltage-activated calcium channels (T-channels), which are involved in several cardiovascular and neuronal diseases. Using the whole-cell patch-clamp technique, we demonstrate that DHEA inhibits the three recombinant T-channels (Ca(V)3.1, Ca(V)3.2 and Ca(V)3.3) expressed in NG108-15 cell line, as well as native T-channels in pulmonary artery smooth muscle cells. This effect of DHEA is both concentration (IC(50) between 2 and 7μM) and voltage-dependent and results in a significant shift of the steady-state inactivation curves toward hyperpolarized potentials. Consequently, DHEA reduces window T-current and inhibits membrane potential oscillations induced by Ca(V)3 channels. DHEA inhibition is not dependent on the activation of nuclear androgen or estrogen receptors and implicates a PTX-sensitive Gi protein pathway. Functionally, DHEA and the T-type inhibitor NNC 55-0396 inhibited KCl-induced contraction of pulmonary artery rings and their effect was not cumulative. Altogether, the present data demonstrate that DHEA inhibits T-channels by a Gi protein dependent pathway. DHEA-induced alteration in T-channel activity could thus account for its therapeutic action and/or physiological effects. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Differential effects of voltage-gated calcium channel blockers on calcium channel alpha-2-delta-1 subunit protein-mediated nociception.

    Science.gov (United States)

    Chang, E; Chen, X; Kim, M; Gong, N; Bhatia, S; Luo, Z D

    2015-05-01

    Overexpression of the voltage-gated calcium channel (VGCC) alpha-2-delta1 subunit protein (Cav α2 δ1 ) has been shown to cause pain states. However, whether VGCC are involved in pain states driven by abnormal Cav α2 δ1 expression is not known. Intrathecal injection of N-, P/Q- and L-type VGCC blockers were tested in two models: a transgenic neuronal Cav α2 δ1 overexpression (TG) model with behavioural hypersensitivity and a spinal nerve ligation (SNL) model with Cav α2 δ1 overexpression in sensory pathways and neuropathy pain states. The nociceptive response to mechanical stimuli was significantly attenuated in both models with ω-conotoxin GVIA (an N-type VGCC blocker) and nifedipine (an L-type VGCC blocker), in which ω-conotoxin GVIA appeared more potent than nifedipine. Treatments with ω-agatoxin IVA (P-VGCC blocker), but not ω-conotoxin MVIIC (Q-VGCC blocker) had similar potency in the TG model as the N-type VGCC blocker, while both ω-agatoxin IVA and ω-conotoxin MVIIC had minimal effects in the SNL model compared with controls. These findings suggest that, at the spinal level, N- and L-type VGCC are likely involved in behavioural hypersensitivity states driven by Cav α2 δ1 overexpression. Q-type VGCC has minimal effects in both models. The anti-nociceptive effects of P-type VGCC blocker in the Cav α2 δ1 TG mice, but minimally at the SNL model with presynaptic Cav α2 δ1 up-regulation, suggest that its potential action site(s) is at the post-synaptic and/or supraspinal level. These findings support that N-, L- and P/Q-type VGCC have differential contributions to behavioural hypersensitivity modulated by Cav α2 δ1 dysregulation at the spinal cord level. © 2014 European Pain Federation - EFIC®

  20. Biophysics of BK Channel Gating.

    Science.gov (United States)

    Pantazis, A; Olcese, R

    2016-01-01

    BK channels are universal regulators of cell excitability, given their exceptional unitary conductance selective for K(+), joint activation mechanism by membrane depolarization and intracellular [Ca(2+)] elevation, and broad expression pattern. In this chapter, we discuss the structural basis and operational principles of their activation, or gating, by membrane potential and calcium. We also discuss how the two activation mechanisms interact to culminate in channel opening. As members of the voltage-gated potassium channel superfamily, BK channels are discussed in the context of archetypal family members, in terms of similarities that help us understand their function, but also seminal structural and biophysical differences that confer unique functional properties. © 2016 Elsevier Inc. All rights reserved.

  1. The L-Type Voltage-Gated Calcium Channel Ca [subscript V] 1.2 Mediates Fear Extinction and Modulates Synaptic Tone in the Lateral Amygdala

    Science.gov (United States)

    Temme, Stephanie J.; Murphy, Geoffrey G.

    2017-01-01

    L-type voltage-gated calcium channels (LVGCCs) have been implicated in both the formation and the reduction of fear through Pavlovian fear conditioning and extinction. Despite the implication of LVGCCs in fear learning and extinction, studies of the individual LVGCC subtypes, Ca[subscript V]1.2 and Ca[subscript V] 1.3, using transgenic mice have…

  2. Electromagnetic fields act via activation of voltage-gated calcium channels to produce beneficial or adverse effects.

    Science.gov (United States)

    Pall, Martin L

    2013-08-01

    The direct targets of extremely low and microwave frequency range electromagnetic fields (EMFs) in producing non-thermal effects have not been clearly established. However, studies in the literature, reviewed here, provide substantial support for such direct targets. Twenty-three studies have shown that voltage-gated calcium channels (VGCCs) produce these and other EMF effects, such that the L-type or other VGCC blockers block or greatly lower diverse EMF effects. Furthermore, the voltage-gated properties of these channels may provide biophysically plausible mechanisms for EMF biological effects. Downstream responses of such EMF exposures may be mediated through Ca(2+) /calmodulin stimulation of nitric oxide synthesis. Potentially, physiological/therapeutic responses may be largely as a result of nitric oxide-cGMP-protein kinase G pathway stimulation. A well-studied example of such an apparent therapeutic response, EMF stimulation of bone growth, appears to work along this pathway. However, pathophysiological responses to EMFs may be as a result of nitric oxide-peroxynitrite-oxidative stress pathway of action. A single such well-documented example, EMF induction of DNA single-strand breaks in cells, as measured by alkaline comet assays, is reviewed here. Such single-strand breaks are known to be produced through the action of this pathway. Data on the mechanism of EMF induction of such breaks are limited; what data are available support this proposed mechanism. Other Ca(2+) -mediated regulatory changes, independent of nitric oxide, may also have roles. This article reviews, then, a substantially supported set of targets, VGCCs, whose stimulation produces non-thermal EMF responses by humans/higher animals with downstream effects involving Ca(2+) /calmodulin-dependent nitric oxide increases, which may explain therapeutic and pathophysiological effects. © 2013 The Author. Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular

  3. 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 Ca2+ 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.

  4. Calcium channel blocker overdose

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/002580.htm Calcium-channel blocker overdose To use the sharing features on this page, please enable JavaScript. Calcium-channel blockers are a type of medicine used to ...

  5. S-petasin and butterbur lactones dilate vessels through blockage of voltage gated calcium channels and block DNA synthesis.

    Science.gov (United States)

    Sheykhzade, Majid; Smajilovic, Sanela; Issa, Ali; Haunso, Stig; Christensen, Søren Brøgger; Tfelt-Hansen, Jacob

    2008-09-28

    Eremophilanlactones isolated from roots of Petasites hybridus (L.) G.M. et Sch. (Asteraceae) and S-petasin have vasodilatory effects with pD(2) -log (EC(50)) values of 6.01+/-0.08, 5.24+/-0.10, 4.74+/-0.13, and 5.43+/-0.06 for S-petasin, the (Z)-3-methylthioacrylic ester of 2beta-hydroxy-8betaH-7(11)-eremophilene-12,8-olide, the angelic ester of 2beta-hydroxy-8alphaH-7(11)-eremophilene-12,8-olide, and the angelic ester of 2beta-hydroxy-8betaH-7(11)-eremophilene-12,8-olide, respectively, in the mesenteric arteries. The pD(2) values were somewhat lower for all compounds in aortic segments. The vasodilation was caused by a blockage of the voltage gated calcium channels. S-petasin, (Z)-3-methylthioacrylic ester of 2beta-hydroxy-8betaH-7(11)-eremophilene-12,8-olide, and the angelic ester of 2beta-hydroxy-8alphaH-7(11)-eremophilene-12,8-olide displayed similar potencies in inhibiting DNA synthesis in cardiomyocytes and vascular smooth muscle cells.

  6. Targeting voltage-gated calcium channels: developments in peptide and small-molecule inhibitors for the treatment of neuropathic pain

    Science.gov (United States)

    Vink, S; Alewood, PF

    2012-01-01

    Chronic pain affects approximately 20% of people worldwide and places a large economic and social burden on society. Despite the availability of a range of analgesics, this condition is inadequately treated, with complete alleviation of symptoms rarely occurring. In the past 30 years, the voltage-gated calcium channels (VGCCs) have been recognized as potential targets for analgesic development. Although the majority of the research has been focused on Cav2.2 in particular, other VGCC subtypes such as Cav3.2 have recently come to the forefront of analgesic research. Venom peptides from marine cone snails have been proven to be a valuable tool in neuroscience, playing a major role in the identification and characterization of VGCC subtypes and producing the first conotoxin-based drug on the market, the ω-conotoxin, ziconotide. This peptide potently and selectively inhibits Cav2.2, resulting in analgesia in chronic pain states. However, this drug is only available via intrathecal administration, and adverse effects and a narrow therapeutic window have limited its use in the clinic. Other Cav2.2 inhibitors are currently in development and offer the promise of an improved route of administration and safety profile. This review assesses the potential of targeting VGCCs for analgesic development, with a main focus on conotoxins that block Cav2.2 and the developments made to transform them into therapeutics. PMID:22725651

  7. A Rare Case of Cerebellar Ataxia Due to Voltage-Gated Calcium Channel and Glutamic Acid Decarboxylase Autoantibodies.

    Science.gov (United States)

    Annunziata, Giuseppe; Lobo, Pamela; Carbuccia, Cristian

    2017-11-27

    BACKGROUND Autoimmune cerebellar ataxia can be paraneoplastic in nature or can occasionally present without evidence of an ongoing malignancy. The detection of specific autoantibodies has been statistically linked to different etiologies. CASE REPORT A 55-year-old African-American woman with hypertension and a past history of morbid obesity and uncontrolled diabetes status post gastric bypass four years prior to the visit (with significantly improved body mass index and hemoglobin A1c controlled at the time of the clinical encounter) presented to the office complaining of gradual onset of unsteadiness and recurrent falls for the past three years, as well as difficulties coordinating routine daily activities. The neurologic exam showed moderate dysarthria and ataxic gait with bilateral dysmetria and positive Romberg test. Routine laboratory test results were only remarkable for a mild elevation of erythrocyte sedimentation rate, and most laboratory and imaging tests for common causes of ataxia failed to demonstrate an etiology. Upon further workup, evidence of anti-voltage-gated calcium channel and anti-glutamic acid decarboxylase antibody was demonstrated. She was then treated with intravenous immunoglobulins with remarkable clinical improvement. CONCLUSIONS We present a case of antibody-mediated ataxia not associated with malignancy. While ataxia is rarely related to autoantibodies, in such cases it is critical to understand the etiology of this disabling condition in order to treat it correctly. Clinicians should be aware of the possible association with specific autoantibodies and the necessity to rule out an occult malignancy in such cases.

  8. Enigma homolog 1 scaffolds protein kinase D1 to regulate the activity of the cardiac L-type voltage-gated calcium channel.

    Science.gov (United States)

    Maturana, Andrés D; Wälchli, Sébastien; Iwata, Miki; Ryser, Stephan; Van Lint, Johannes; Hoshijima, Masahiko; Schlegel, Werner; Ikeda, Yasuhiro; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2008-06-01

    In cardiomyocytes, protein kinase D1 (PKD1) plays a central role in the response to stress signals. From a yeast two-hybrid assay, we have identified Enigma Homolog 1 (ENH1) as a new binding partner of PKD1. Since in neurons, ENH1, associated with protein kinase Cepsilon, was shown to modulate the activity of N-type calcium channels, and the pore-forming subunit of the cardiac L-type voltage-gated calcium channel, alpha1C, possesses a potential phosphorylation site for PKD1, we studied here a possible role of ENH1 and PKD1 in the regulation of the cardiac L-type voltage-gated calcium channel. PKD1-interacting proteins were searched by yeast two-hybrid screening. In vivo protein interactions in cardiomyocytes isolated from heart ventricles of newborn rats were tested by co-immunoprecipitation. Small interfering RNA and a dominant negative mutant of PKD1 were delivered into cardiomyocytes by use of an adenovirus. Calcium currents were measured by the patch-clamp technique. Both ENH1 and PKD1 interact with alpha1C in cardiomyocytes. This interaction is increased upon stimulation. Silencing of ENH1 prevented the binding of PKD1 to alpha1C. Moreover, a dominant negative mutant of PKD1 or the silencing of ENH1 inhibited the alpha-adrenergic-induced increase of L-type calcium currents. We found a new binding partner, ENH1, and a new target, alpha1C, for PKD1 in neonatal rat cardiomyocytes. We propose a model where ENH1 scaffolds PKD1 to alpha1C in order to form a signalling complex that regulates the activity of cardiac L-type voltage-gated Ca(2+) channels.

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

    NARCIS (Netherlands)

    Majoie, H.J.; Baets, M.H.V. de; Renier, W.O.; Lang, B.; Vincent, A.

    2006-01-01

    OBJECTIVE: To determine the prevalence of antibodies to ion channels in patients with long standing epilepsy. BACKGROUND: 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,

  10. Comprehensive behavioral analysis of voltage-gated calcium channel beta-anchoring and -regulatory protein knockout mice

    Science.gov (United States)

    Nakao, Akito; Miki, Takafumi; Shoji, Hirotaka; Nishi, Miyuki; Takeshima, Hiroshi; Miyakawa, Tsuyoshi; Mori, Yasuo

    2015-01-01

    Calcium (Ca2+) influx through voltage-gated Ca2+ channels (VGCCs) induces numerous intracellular events such as neuronal excitability, neurotransmitter release, synaptic plasticity, and gene regulation. It has been shown that genes related to Ca2+ signaling, such as the CACNA1C, CACNB2, and CACNA1I genes that encode VGCC subunits, are associated with schizophrenia and other psychiatric disorders. Recently, VGCC beta-anchoring and -regulatory protein (BARP) was identified as a novel regulator of VGCC activity via the interaction of VGCC β subunits. To examine the role of the BARP in higher brain functions, we generated BARP knockout (KO) mice and conducted a comprehensive battery of behavioral tests. BARP KO mice exhibited greatly reduced locomotor activity, as evidenced by decreased vertical activity, stereotypic counts in the open field test, and activity level in the home cage, and longer latency to complete a session in spontaneous T-maze alteration test, which reached “study-wide significance.” Acoustic startle response was also reduced in the mutants. Interestingly, they showed multiple behavioral phenotypes that are seemingly opposite to those seen in the mouse models of schizophrenia and its related disorders, including increased working memory, flexibility, prepulse inhibition, and social interaction, and decreased locomotor activity, though many of these phenotypes are statistically weak and require further replications. These results demonstrate that BARP is involved in the regulation of locomotor activity and, possibly, emotionality. The possibility was also suggested that BARP KO mice may serve as a unique tool for investigating the pathogenesis/pathophysiology of schizophrenia and related disorders. Further evaluation of the molecular and physiological phenotypes of the mutant mice would provide new insights into the role of BARP in higher brain functions. PMID:26136667

  11. Protein kinase D regulates the human cardiac L-type voltage-gated calcium channel through serine 1884.

    Science.gov (United States)

    Aita, Yusuke; Kurebayashi, Nagomi; Hirose, Shigehisa; Maturana, Andrés D

    2011-12-15

    Protein kinase D (PKD) regulates the activity of the L-type calcium channel in rat ventricular cardiomyocytes. However, the functional target residues of PKD on the L-type calcium channel remain to be identified. Our aim was to identify the functional phosphorylation sites of PKD on the human L-type calcium channel. The pore subunit of the human CaV1.2 (hCaV1.2) was stably expressed in HEK293 cells. Both the expression of a dominant-negative mutant of PKD and the mutation of serine 1884 but not serine 1930, putative targets of PKD, strongly reduced L-type calcium currents and single channel activity without affecting the channel's expression at the plasma membrane. Our results suggest that serine 1884 is essential for the regulation of hCaV1.2 by PKD. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Short-Term Facilitation at a Detonator Synapse Requires the Distinct Contribution of Multiple Types of Voltage-Gated Calcium Channels.

    Science.gov (United States)

    Chamberland, Simon; Evstratova, Alesya; Tóth, Katalin

    2017-05-10

    Neuronal calcium elevations are shaped by several key parameters, including the properties, density, and the spatial location of voltage-gated calcium channels (VGCCs). These features allow presynaptic terminals to translate complex firing frequencies and tune the amount of neurotransmitter released. Although synchronous neurotransmitter release relies on both P/Q- and N-type VGCCs at hippocampal mossy fiber-CA3 synapses, the specific contribution of VGCCs to calcium dynamics, neurotransmitter release, and short-term facilitation remains unknown. Here, we used random-access two-photon calcium imaging together with electrophysiology in acute mouse hippocampal slices to dissect the roles of P/Q- and N-type VGCCs. Our results show that N-type VGCCs control glutamate release at a limited number of release sites through highly localized Ca 2+ elevations and support short-term facilitation by enhancing multivesicular release. In contrast, Ca 2+ entry via P/Q-type VGCCs promotes the recruitment of additional release sites through spatially homogeneous Ca 2+ elevations. Altogether, our results highlight the specialized contribution of P/Q- and N-types VGCCs to neurotransmitter release. SIGNIFICANCE STATEMENT In presynaptic terminals, neurotransmitter release is dynamically regulated by the transient opening of different types of voltage-gated calcium channels. Hippocampal giant mossy fiber terminals display extensive short-term facilitation during repetitive activity, with a large several fold postsynaptic response increase. Though, how giant mossy fiber terminals leverage distinct types of voltage-gated calcium channels to mediate short-term facilitation remains unexplored. Here, we find that P/Q- and N-type VGCCs generate different spatial patterns of calcium elevations in giant mossy fiber terminals and support short-term facilitation through specific participation in two mechanisms. Whereas N-type VGCCs contribute only to the synchronization of multivesicular release

  13. Calcium channel blocker poisoning

    Directory of Open Access Journals (Sweden)

    Miran Brvar

    2005-04-01

    Full Text Available Background: Calcium channel blockers act at L-type calcium channels in cardiac and vascular smooth muscles by preventing calcium influx into cells with resultant decrease in vascular tone and cardiac inotropy, chronotropy and dromotropy. Poisoning with calcium channel blockers results in reduced cardiac output, bradycardia, atrioventricular block, hypotension and shock. The findings of hypotension and bradycardia should suggest poisoning with calcium channel blockers.Conclusions: Treatment includes immediate gastric lavage and whole-bowel irrigation in case of ingestion of sustainedrelease products. All patients should receive an activated charcoal orally. Specific treatment includes calcium, glucagone and insulin, which proved especially useful in shocked patients. Supportive care including the use of catecholamines is not always effective. In the setting of failure of pharmacological therapy transvenous pacing, balloon pump and cardiopulmonary by-pass may be necessary.

  14. Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating

    Science.gov (United States)

    Vais, Horia; Foskett, J. Kevin; Ullah, Ghanim; Pearson, John E.

    2012-01-01

    The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channel plays a central role in the generation and modulation of intracellular Ca2+ signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP3, free Ca2+, free ATP4−) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP3R channel activity by free Ca2+ in the ER lumen ([Ca2+]ER) remains poorly understood because of limitations of Ca2+ flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca2+]ER on homotetrameric rat type 3 InsP3R channel activity. In optimal [Ca2+]i and subsaturating [InsP3], jumps of [Ca2+]ER from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP3 but restored when [Ca2+]ER was raised to 1.1 mM. In suboptimal [Ca2+]i, jumps of [Ca2+]ER (70 nM to 300 µM) enhanced channel activity. Thus, [Ca2+]ER effects on channel activity exhibited a biphasic dependence on [Ca2+]i. In addition, the effect of high [Ca2+]ER was attenuated when a voltage was applied to oppose Ca2+ flux through the channel. These observations can be accounted for by Ca2+ flux driven through the open InsP3R channel by [Ca2+]ER, raising local [Ca2+]i around the channel to regulate its activity through its cytoplasmic regulatory Ca2+-binding sites. Importantly, [Ca2+]ER regulation of InsP3R channel activity depended on cytoplasmic Ca2+-buffering conditions: it was more pronounced when [Ca2+]i was weakly buffered but completely abolished in strong Ca2+-buffering conditions. With strong cytoplasmic buffering and Ca2+ flux sufficiently reduced by applied voltage, both activation and inhibition of InsP3R channel gating by physiological levels of [Ca2+]ER were completely abolished

  15. Impact of phosphomimetic and non-phosphorylatable mutations of phospholemman on L-type calcium channels gating in HEK 293T cells.

    Science.gov (United States)

    Guo, Kai; Wang, Yue-Peng; Zhou, Zhi-Wen; Jiang, Yi-Bo; Li, Wei; Chen, Xiao-Meng; Li, Yi-Gang

    2015-03-01

    Phospholemman (PLM) is an important phosphorylation substrate for protein kinases A and C in the heart. Until now, the association between PLM phosphorylation status and L-type calcium channels (LTCCs) gating has not been fully understood. We investigated the kinetics of LTCCs in HEK 293T cells expressing phosphomimetic or nonphosphorylatable PLM mutants. The LTCCs gating was measured in HEK 293T cells transfected with LTCC and wild-type (WT) PLM, phosphomimetic or nonphosphorylatable PLM mutants: 6263AA, 6869AA, AAAA, 6263DD, 6869DD or DDDD. WT PLM significantly slowed LTCCs activation and deactivation while enhanced voltage-dependent inactivation (VDI). PLM mutants 6869DD and DDDD significantly increased the peak of the currents. 6263DD accelerated channel activation, while 6263AA slowed it more than WT PLM. 6869DD significantly enhanced PLM-induced increase of VDI. AAAA slowed the channel activation more than 6263AA, and DDDD accelerated the channel VDI more than 6869DD. Our results demonstrate that phosphomimetic PLM could stimulate LTCCs and alter their dynamics, while PLM nonphosphorylatable mutant produced the opposite effects. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  16. Inactivation of gating currents of L-type calcium channels. Specific role of the alpha 2 delta subunit.

    Science.gov (United States)

    Shirokov, R; Ferreira, G; Yi, J; Ríos, E

    1998-06-01

    In studies of gating currents of rabbit cardiac Ca channels expressed as alpha 1C/beta 2a or alpha 1C/beta 2a/alpha 2 delta subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489-517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863-895). Charge 1 (voltage of half-maximal transfer, V1/2 approximately 0 mV) gates noninactivated channels, while charge 2 (V1/2 approximately -90 mV) is generated in inactivated channels. In alpha 1C/beta 2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant tau approximately 8, while the available charge 2 decreased upon recovery from inactivation (at -200 mV) with tau approximately 0.3 s. These processes therefore are much slower than charge movement, which takes charge movement and that of changes in their availability, which was even wider in the presence of alpha 2 delta, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the alpha 2 delta subunit did not change the V1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to tau approximately 2 s), while slowing the reduction of charge 2 upon recovery (tau approximately 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489-498) and inactivation as discrete modal change. The effects of alpha 2 delta are reproduced assuming that the subunit lowers the free energy of the inactivated mode.

  17. 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 (CaV) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (CaVβ). 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:CaVβ interactions and reduce the entropic penalty associated with AID binding to CaVβ. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the CaVα1:CaVβ interaction that modulate CaV function in an CaVβ 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 CaV modulator design.

  18. Role of voltage-gated sodium, potassium and calcium channels in the development of cocaine-associated cardiac arrhythmias

    Science.gov (United States)

    O'Leary, Michael E; Hancox, Jules C

    2010-01-01

    Cocaine is a highly active stimulant that alters dopamine metabolism in the central nervous system resulting in a feeling of euphoria that with time can lead to addictive behaviours. Cocaine has numerous deleterious effects in humans including seizures, vasoconstriction, ischaemia, increased heart rate and blood pressure, cardiac arrhythmias and sudden death. The cardiotoxic effects of cocaine are indirectly mediated by an increase in sympathomimetic stimulation to the heart and coronary vasculature and by a direct effect on the ion channels responsible for maintaining the electrical excitability of the heart. The direct and indirect effects of cocaine work in tandem to disrupt the co-ordinated electrical activity of the heart and have been associated with life-threatening cardiac arrhythmias. This review focuses on the direct effects of cocaine on cardiac ion channels, with particular focus on sodium, potassium and calcium channels, and on the contributions of these channels to cocaine-induced arrhythmias. Companion articles in this edition of the journal examine the epidemiology of cocaine use (Wood & Dargan [1]) and the treatment of cocaine-associated arrhythmias (Hoffmann [2]). PMID:20573078

  19. Assay for calcium channels

    Energy Technology Data Exchange (ETDEWEB)

    Glossmann, H.; Ferry, D.R.

    1985-01-01

    This chapter focuses on biochemical assays for Ca/sup 2 +/-selective channels in electrically excitable membranes which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydropyridines, diltiazen (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives which block calcium channels with ED/sub 50/ values in the nanomolar range. Although tritiated d-cis-diltiazem and verapamil can be used to label calcium channels, the 1,4-dihydropyridines offer numerous advantages. The various sections cover tissue specificity of channel labeling, the complex interactions of divalent cations with the (/sup 3/H)nimodipine-labeled calcium channels, and the allosteric regulation of (/sup 3/H)nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers. A comparison of the properties of different tritiated 1,4-dihydropyridine radioligands and the iodinated channel probe (/sup 125/I)iodipine is given.

  20. Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25

    Directory of Open Access Journals (Sweden)

    Medler Kathryn F

    2006-03-01

    Full Text Available Abstract Background Taste receptor cells are responsible for transducing chemical stimuli from the environment and relaying information to the nervous system. Bitter, sweet and umami stimuli utilize G-protein coupled receptors which activate the phospholipase C (PLC signaling pathway in Type II taste cells. However, it is not known how these cells communicate with the nervous system. Previous studies have shown that the subset of taste cells that expresses the T2R bitter receptors lack voltage-gated Ca2+ channels, which are normally required for synaptic transmission at conventional synapses. Here we use two lines of transgenic mice expressing green fluorescent protein (GFP from two taste-specific promoters to examine Ca2+ signaling in subsets of Type II cells: T1R3-GFP mice were used to identify sweet- and umami-sensitive taste cells, while TRPM5-GFP mice were used to identify all cells that utilize the PLC signaling pathway for transduction. Voltage-gated Ca2+ currents were assessed with Ca2+ imaging and whole cell recording, while immunocytochemistry was used to detect expression of SNAP-25, a presynaptic SNARE protein that is associated with conventional synapses in taste cells. Results Depolarization with high K+ resulted in an increase in intracellular Ca2+ in a small subset of non-GFP labeled cells of both transgenic mouse lines. In contrast, no depolarization-evoked Ca2+ responses were observed in GFP-expressing taste cells of either genotype, but GFP-labeled cells responded to the PLC activator m-3M3FBS, suggesting that these cells were viable. Whole cell recording indicated that the GFP-labeled cells of both genotypes had small voltage-dependent Na+ and K+ currents, but no evidence of Ca2+ currents. A subset of non-GFP labeled taste cells exhibited large voltage-dependent Na+ and K+ currents and a high threshold voltage-gated Ca2+ current. Immunocytochemistry indicated that SNAP-25 was expressed in a separate population of taste cells

  1. Voltage-gated Proton Channels

    Science.gov (United States)

    DeCoursey, Thomas E.

    2014-01-01

    Voltage-gated proton channels, HV1, have vaulted from the realm of the esoteric into the forefront of a central question facing ion channel biophysicists, namely the mechanism by which voltage-dependent gating occurs. This transformation is the result of several factors. Identification of the gene in 2006 revealed that proton channels are homologues of the voltage-sensing domain of most other voltage-gated ion channels. Unique, or at least eccentric, properties of proton channels include dimeric architecture with dual conduction pathways, perfect proton selectivity, a single-channel conductance ~103 smaller than most ion channels, voltage-dependent gating that is strongly modulated by the pH gradient, ΔpH, and potent inhibition by Zn2+ (in many species) but an absence of other potent inhibitors. The recent identification of HV1 in three unicellular marine plankton species has dramatically expanded the phylogenetic family tree. Interest in proton channels in their own right has increased as important physiological roles have been identified in many cells. Proton channels trigger the bioluminescent flash of dinoflagellates, facilitate calcification by coccolithophores, regulate pH-dependent processes in eggs and sperm during fertilization, secrete acid to control the pH of airway fluids, facilitate histamine secretion by basophils, and play a signaling role in facilitating B-cell receptor mediated responses in B lymphocytes. The most elaborate and best-established functions occur in phagocytes, where proton channels optimize the activity of NADPH oxidase, an important producer of reactive oxygen species. Proton efflux mediated by HV1 balances the charge translocated across the membrane by electrons through NADPH oxidase, minimizes changes in cytoplasmic and phagosomal pH, limits osmotic swelling of the phagosome, and provides substrate H+ for the production of H2O2 and HOCl, reactive oxygen species crucial to killing pathogens. PMID:23798303

  2. N- and L-type voltage-gated calcium channels mediate fast calcium transients in axonal shafts of mouse peripheral nerve.

    Directory of Open Access Journals (Sweden)

    Ruxandra eBarzan

    2016-06-01

    Full Text Available In the peripheral nervous system a vast number of axons are accommodated within fiber bundles that constitute peripheral nerves. A major function of peripheral axons is to propagate action potentials along their length, and hence they are equipped with Na+ and K+ channels, which ensure successful generation, conduction and termination of each action potential. However little is known about Ca2+ ion channels expressed along peripheral axons and their possible functional significance. The goal of the present study was to test whether voltage-gated Ca2+ channels (VGCCs are present along peripheral nerve axons in situ and mediate rapid activity-dependent Ca2+ elevations under physiological circumstances. To address this question we used mouse sciatic nerve slices, Ca2+ indicator Oregon Green BAPTA-1, and 2-photon Ca2+ imaging in fast line scan mode (500 Hz. We report that transient increases in intra-axonal Ca2+ concentration take place along peripheral nerve axons in situ when axons are stimulated electrically with single pulses. Furthermore, we show for the first time that Ca2+ transients in peripheral nerves are fast, i.e. occur in a millisecond time-domain. Combining Ca2+ imaging and pharmacology with specific blockers of different VGCCs subtypes we demonstrate that Ca2+ transients in peripheral nerves are mediated mainly by N-type and L-type VGCCs. Discovery of fast Ca2+ entry into the axonal shafts through VGCCs in peripheral nerves suggests that Ca2+ may be involved in regulation of action potential propagation and/or properties in this system, or mediate neurotransmitter release along peripheral axons as it occurs in the optic nerve and white matter of the central nervous system.

  3. Immunolocalization of a voltage-gated calcium channel β subunit in the tentacles and cnidocytes of the Portuguese man-of-war, Physalia physalis.

    Science.gov (United States)

    Bouchard, Christelle; Anderson, Peter A V

    2014-12-01

    This study investigated the localization of a voltage-gated calcium channel (VGCC) β subunit in the tentacles and cnidocytes of the Portuguese man-of-war using confocal immunocytochemistry. An antibody specific to the Ca(2+) channel β subunit of the Portuguese-man-of-war (PpCaVβ) was generated, and characterized by Western immunoblotting. The antibody labeling was widespread in the ectoderm of cnidosacs of the tentacles. The binding of the antibody on isolated cnidocytes was distributed at the base of the cell and appeared as multiple strong fluorescent plaques located around the basal hemisphere of the cell. The distribution of PpCaVβ in the cnidocyte is consistent with previous studies on other hydrozoans that demonstrated that cnidocytes convey sensory information to other cnidocytes through chemical synapses in which the cnidocyte is pre-synaptic to elements of the animal's nervous system. Importantly and surprisingly, PpCaVβ did not localize to the apical surface of the cnidocyte where the exocytotic events involved in cnidocyst discharge are thought to take place. © 2014 Marine Biological Laboratory.

  4. Functional importance of T-type voltage-gated calcium channels in the cardiovascular and renal system

    DEFF Research Database (Denmark)

    Hansen, Pernille B L

    2015-01-01

    to the conclusion that Cav3.1 and Cav3.2 channels have important, but different, functions in mice. T-type Cav3.1 channels affect heart rate, whereas Cav3.2 channels are involved in cardiac hypertrophy. In the vascular system, Cav3.2 activation leads to dilation of blood vessels, whereas Cav3.1 channels are mainly...

  5. Deltamethrin affects the expression of voltage-gated calcium channel α1 subunits and the locomotion, egg-laying, foraging behavior of Caenorhabditis elegans.

    Science.gov (United States)

    Zeng, Rune; Yu, Xing; Tan, Xing; Ye, Shan; Ding, Zhong

    2017-05-01

    Deltamethrin belongs to the class of synthetic pyrethroids, which are being widely used as insecticides in agricultural practices. Voltage-gated sodium channels (VGSCs) are the primary targets of these chemicals for toxicity to insects. Caenorhabditis elegans (C. elegans) does not have VGSCs but is susceptible to deltamethrin. Recent findings have suggested that pyrethroids can affect voltage-gated calcium channels (VGCCs). However, it remains elusive whether deltamethrin induces toxicity to C. elegans via modulating the activity of VGCCs. To identify the potential target of deltamethrin, we exposed C. elegans to different concentrations of deltamethrin and Ca(2+) channel blockers for different times, characterized the behavioral toxicity of deltamethrin on C. elegans, and determined the expression of egl-19, unc-2, and cca-1, which encode the α1-subunit of the L-, R/N/P/Q-, and T-type VGCC, respectively. We found that deltamethrin inhibited the locomotion, egg-laying and foraging ability of C. elegans in a concentration dependent manner. We also showed that body length of worms on agar plates containing 200mgL(-1) deltamethrin for 12h was not significantly different from controls, whereas the cholinesterase inhibitor carbofuran caused hypercontraction which is a characteristic of organophosphates and carbamates, suggesting that deltamethrin's mode of action is distinct from those nematicides. In addition, unc-2 was significantly up-regulated following 0.05mgL(-1) deltamethrin exposure for 24h; while egl-19 and cca-1 were significantly up-regulated following 5 and 50mgL(-1) deltamethrin exposure for 24h. Further tests of worms' sensitivity and expression of three α1-subunits of VGCC to Ca(2+) channel blockers indicate that deltamethrin may induce toxic behavior C. elegans via modulation of the expression of the α1-subunits of VGCC. This study provides insights into the linkage between deltamethrin-induced toxic behavior and the regulation of α1-subunits of VGCC

  6. Mechanisms of NMDA Receptor- and Voltage-Gated L-Type Calcium Channel-Dependent Hippocampal LTP Critically Rely on Proteolysis That Is Mediated by Distinct Metalloproteinases.

    Science.gov (United States)

    Wiera, Grzegorz; Nowak, Daria; van Hove, Inge; Dziegiel, Piotr; Moons, Lieve; Mozrzymas, Jerzy W

    2017-02-01

    Long-term potentiation (LTP) is widely perceived as a memory substrate and in the hippocampal CA3-CA1 pathway, distinct forms of LTP depend on NMDA receptors (nmdaLTP) or L-type voltage-gated calcium channels (vdccLTP). LTP is also known to be effectively regulated by extracellular proteolysis that is mediated by various enzymes. Herein, we investigated whether in mice hippocampal slices these distinct forms of LTP are specifically regulated by different metalloproteinases (MMPs). We found that MMP-3 inhibition or knock-out impaired late-phase LTP in the CA3-CA1 pathway. Interestingly, late-phase LTP was also decreased by MMP-9 blockade. When both MMP-3 and MMP-9 were inhibited, both early- and late-phase LTP was impaired. Using immunoblotting, in situ zymography, and immunofluorescence, we found that LTP induction was associated with an increase in MMP-3 expression and activity in CA1 stratum radiatum. MMP-3 inhibition and knock-out prevented the induction of vdccLTP, with no effect on nmdaLTP. L-type channel-dependent LTP is known to be impaired by hyaluronic acid digestion. We found that slice treatment with hyaluronidase occluded the effect of MMP-3 blockade on LTP, further confirming a critical role for MMP-3 in this form of LTP. In contrast to the CA3-CA1 pathway, LTP in the mossy fiber-CA3 projection did not depend on MMP-3, indicating the pathway specificity of the actions of MMPs. Overall, our study indicates that the activation of perisynaptic MMP-3 supports L-type channel-dependent LTP in the CA1 region, whereas nmdaLTP depends solely on MMP-9. Various types of long-term potentiation (LTP) are correlated with distinct phases of memory formation and retrieval, but the underlying molecular signaling pathways remain poorly understood. Extracellular proteases have emerged as key players in neuroplasticity phenomena. The present study found that L-type calcium channel-dependent LTP in the CA3-CA1 hippocampal projection is critically regulated by the activity

  7. Cisplatin alters the function and expression of N-type voltage-gated calcium channels in the absence of morphological damage of sensory neurons.

    Science.gov (United States)

    Leo, Markus; Schmitt, Linda-Isabell; Jastrow, Holger; Thomale, Jürgen; Kleinschnitz, Christoph; Hagenacker, Tim

    2017-01-01

    Platinum-based chemotherapeutic agents, such as cisplatin, are still frequently used for treating various types of cancer. Besides its high effectiveness, cisplatin has several serious side effects. One of the most common side effects is dorsal root ganglion (DRG) neurotoxicity. However, the mechanisms underlying this neurotoxicity are still unclear and controversially discussed. Cisplatin-mediated modulation of voltage-gated calcium channels (VGCCs) in the DRG neurons has been shown to alter intracellular calcium homeostasis, a process critical for the induction of neurotoxicity. Using the whole-cell patch-clamp technique, immunostaining, behavioural experiments and electron microscopy (EM) of rat DRGs, we here demonstrate that cisplatin-induced neurotoxicity is due to functional alteration of VGCC, but not due to morphological damage. In vitro application of cisplatin (0.5 µM) increased N-type VGCC currents ( ICa(V)) in small DRG neurons. Repetitive in vivo administration of cisplatin (1.5 mg/kg, cumulative 12 mg/kg) increased the protein level of N-type VGCC over 26 days, with the protein level being increased for at least 14 days after the final cisplatin administration. Behavioural studies revealed that N-type VGCCs are crucial for inducing symptoms of cisplatin-related neuropathic pain, such as thermal and mechanical hyperalgesia. EM and histology showed no evidence of any structural damage, apoptosis or necrosis in DRG cells after cisplatin exposure for 26 days. Furthermore, no nuclear DNA damage in sensory neurons was observed. Here, we provide evidence for a mainly functionally driven induction of neuropathic pain by cisplatin.

  8. Cisplatin-induced neuropathic pain is mediated by upregulation of N-type voltage-gated calcium channels in dorsal root ganglion neurons.

    Science.gov (United States)

    Leo, Markus; Schmitt, Linda-Isabell; Erkel, Martin; Melnikova, Margarita; Thomale, Jürgen; Hagenacker, Tim

    2017-02-01

    Cisplatin is important in the treatment of various types of cancer. Although it is highly effective, it also has severe side effects, with neurotoxicity in dorsal root ganglion (DRG) neurons being one of the most common. The key mechanisms of neurotoxicity are still controversially discussed; however, disturbances of the calcium homeostasis in DRG neurons have been suggested to mediate cisplatin neurotoxicity. By using the whole-cell patch-clamp technique, immunostaining and behavioral experiments with Sprague-Dawley rats, we examined the influence of short- and long-term exposure to cisplatin on voltage-gated calcium channel (VGCC) currents (ICa(V)) in small DRG neurons. In vitro exposure to cisplatin reduced ICa(V) in a concentration-dependent manner (0.01-50μM; 13.8-77.3%; IC50 5.07μM). Subtype-specific measurements of VGCCs showed differential effects on ICa(V). While the ICa(V) of P/Q-, L- and T-type VGCCs were reduced, ICa(V) of N-type VGCCs were increased by 30.3% during depolarization to 0mV. Exposure of DRG neurons to cisplatin (0.5 or 5μM) for 24-48h in vitro significantly increased a CaMK II-mediated ICa(V) current density. Immunostaining and western blot analysis revealed an increase of N-type VGCC protein level in DRG neurons 24h after cisplatin exposure. Cisplatin-mediated activation of caspase-3 was prevented by inhibition of N-type VGCCs using Ɯ-conotoxin MVIIA. Behavioral experiments showed that Ɯ-conotoxin MVIIA treatment prevented neuropathic syndromes in vivo by inhibiting upregulation of the N-type protein level. Here we show evidence for the first time for a crucial role of N-type VGCC in the genesis of cisplatin-induced polyneuropathy. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Estradiol inhibits depolarization-evoked exocytosis in PC12 cells via N-type voltage-gated calcium channels.

    NARCIS (Netherlands)

    Adams, K.L.; Maxson, M.M.; Mellander, L.; Westerink, R.H.S.|info:eu-repo/dai/nl/239425952; Ewing, A.G.

    2010-01-01

    Fast neuromodulatory effects of 17-β-estradiol (E2) on cytosolic calcium concentration ([Ca(2+)](i)) have been reported in many cell types, but little is known about its direct effects on vesicular neurotransmitter secretion (exocytosis). We examined the effects of E2 on depolarization-evoked

  10. A blocker of N- and T-type voltage-gated calcium channels attenuates ethanol-induced intoxication, place preference, self-administration, and reinstatement.

    Science.gov (United States)

    Newton, Philip M; Zeng, Lily; Wang, Victoria; Connolly, Jacklyn; Wallace, Melisa J; Kim, Chanki; Shin, Hee-Sup; Belardetti, Francesco; Snutch, Terrance P; Messing, Robert O

    2008-11-05

    There is a clear need for new therapeutics to treat alcoholism. Here, we test our hypothesis that selective inhibitors of neuronal calcium channels will reduce ethanol consumption and intoxication, based on our previous studies using knock-out mice and cell culture systems. We demonstrate that pretreatment with the novel mixed N-type and T-type calcium channel antagonist 1-(6,6-bis(4-fluorophenyl)hexyl)-4-(3,4,5-trimethoxybenzyl)piperazine (NP078585) reduced ethanol intoxication. NP078585 also attenuated the reinforcing and rewarding properties of ethanol, measured by operant self-administration and the expression of an ethanol conditioned place preference, and abolished stress-induced reinstatement of ethanol seeking. NP078585 did not affect alcohol responses in mice lacking N-type calcium channels. These results suggest that selective calcium channel inhibitors may be useful in reducing acute ethanol intoxication and alcohol consumption by human alcoholics.

  11. The β4subunit of the voltage-gated calcium channel (Cacnb4) regulates the rate of cell proliferation in Chinese Hamster Ovary cells.

    Science.gov (United States)

    Rima, Mohamad; Daghsni, Marwa; De Waard, Stephan; Gaborit, Nathalie; Fajloun, Ziad; Ronjat, Michel; Mori, Yasuo; Brusés, Juan L; De Waard, Michel

    2017-08-01

    The β subunits of Voltage-Gated Calcium Channel (VGCC) are cytosolic proteins that interact with the VGCC pore -forming subunit and participate in the trafficking of the channel to the cell membrane and in ion influx regulation. β subunits also exert functions independently of their binding to VGCC by translocation to the cell nucleus including the control of gene expression. Mutations of the neuronal Cacnb4 (β 4 ) subunit are linked to human neuropsychiatric disorders including epilepsy and intellectual disabilities. It is believed that the pathogenic phenotype induced by these mutations is associated with channel-independent functions of the β 4 subunit. In this report, we investigated the role of β 4 subunit in cell proliferation and cell cycle progression and examined whether these functions could be altered by a pathogenic mutation. To this end, stably transfected Chinese Hamster Ovary (CHO-K1) cells expressing either rat full-length β 4 or the rat C-terminally truncated epileptic mutant variant β 1-481 were generated. The subcellular localization of both proteins differed significantly. Full-length β 4 localizes almost exclusively in the cell nucleus and concentrates into the nucleolar compartment, while the C-terminal-truncated β 1-481 subunit was less concentrated within the nucleus and absent from the nucleoli. Cell proliferation was found to be reduced by the expression of β 4 , while it was unaffected by the epileptic mutant. Also, full-length β 4 interfered with cell cycle progression by presumably preventing cells from entering the S-phase via a mechanism that partially involves endogenous B56δ, a regulatory subunit of the phosphatase 2A (PP2A) that binds β 4 but not β 1-481 . Analysis of β 4 subcellular distribution during the cell cycle revealed that the protein is highly expressed in the nucleus at the G1/S transition phase and that it is translocated out of the nucleus during chromatin condensation and cell division. These results

  12. A Blocker of N- and T-type Voltage-Gated Calcium Channels Attenuates Ethanol-Induced Intoxication, Place Preference, Self-Administration, and Reinstatement

    OpenAIRE

    Newton, Philip M.; Zeng, Lily; Wang, Victoria; Connolly, Jacklyn; Wallace, Melisa Joellan; Kim, Chanki; Shin, Hee-sup; Belardetti, Francesco; Snutch, Terrance P; Messing, Robert O.

    2008-01-01

    There is a clear need for new therapeutics to treat alcoholism. Here, we test our hypothesis that selective inhibitors of neuronal calcium channels will reduce ethanol consumption and intoxication, based on our previous studies using knock-out mice and cell culture systems. We demonstrate that pretreatment with the novel mixed N-type and T-type calcium channel antagonist 1-(6,6-bis(4-fluorophenyl)hexyl)-4-(3,4,5-trimethoxybenzyl)piperazine (NP078585) reduced ethanol intoxication. NP078585 als...

  13. Voltage-gated Potassium Channels as Therapeutic Drug Targets

    OpenAIRE

    Wulff, Heike; Castle, Neil A.; Pardo, Luis A.

    2009-01-01

    The human genome contains 40 voltage-gated potassium channels (KV) which are involved in diverse physiological processes ranging from repolarization of neuronal or cardiac action potentials, over regulating calcium signaling and cell volume, to driving cellular proliferation and migration. KV channels offer tremendous opportunities for the development of new drugs for cancer, autoimmune diseases and metabolic, neurological and cardiovascular disorders. This review first discusses pharmacologi...

  14. VKCDB: Voltage-gated potassium channel database

    Directory of Open Access Journals (Sweden)

    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.

  15. α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor activation protects against phencyclidine-induced caspase-3 activity by activating voltage-gated calcium channels.

    Science.gov (United States)

    Timpe, Jennifer M; Wang, Cheng Z; Kim, Jisoo; Johnson, Kenneth M

    2014-12-01

    Phencyclidine (PCP) is a noncompetitive, open channel blocker of the N-methyl-D-aspartate (NMDA) receptor-ion channel complex. When administered to immature animals, it is known to cause apoptotic neurodegeneration in several regions, and this is followed by olanzapine-sensitive, schizophrenia-like behaviors in late adolescence and adulthood. Clarification of its mechanism of action could yield data that would help to inform the treatment of schizophrenia. In our initial experiments, we found that α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) inhibited PCP-induced apoptosis in organotypic neonatal rat brain slices in a concentration-dependent and 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive manner. Calcium signaling pathways are widely implicated in apoptosis, and PCP prevents calcium influx through NMDA receptor channels. We therefore hypothesized that AMPA could protect against this effect by activation of voltage-dependent calcium channels (VDCCs). In support of this hypothesis, pretreatment with the calcium channel blocker cadmium chloride eliminated AMPA-mediated protection against PCP. Furthermore, the L-type VDCC inhibitor nifedipine (10 µM) fully abrogated the effects of AMPA, suggesting that L-type VDCCs are required for AMPA-mediated protection against PCP-induced neurotoxicity. Whereas the P/Q-type inhibitor ω-agatoxin TK (200 nM) reduced AMPA protection by 51.7%, the N-type VDCC inhibitor ω-conotoxin (2 µM) had no effect. Decreased AMPA-mediated protection following cotreatment with K252a, a TrkB inhibitor, suggests that brain-derived neurotrophic factor signaling plays an important role. By analogy, these results suggest that activation of L-type, and to a lesser extent P/Q-type, VDCCs might be advantageous in treating conditions associated with diminished NMDAergic activity during early development. © 2014 Wiley Periodicals, Inc.

  16. Hysteresis in voltage-gated channels.

    Science.gov (United States)

    Villalba-Galea, Carlos A

    2017-03-04

    Ion channels constitute a superfamily of membrane proteins found in all living creatures. Their activity allows fast translocation of ions across the plasma membrane down the ion's transmembrane electrochemical gradient, resulting in a difference in electrical potential across the plasma membrane, known as the membrane potential. A group within this superfamily, namely voltage-gated channels, displays activity that is sensitive to the membrane potential. The activity of voltage-gated channels is controlled by the membrane potential, while the membrane potential is changed by these channels' activity. This interplay produces variations in the membrane potential that have evolved into electrical signals in many organisms. These signals are essential for numerous biological processes, including neuronal activity, insulin release, muscle contraction, fertilization and many others. In recent years, the activity of the voltage-gated channels has been observed not to follow a simple relationship with the membrane potential. Instead, it has been shown that the activity of voltage-gated channel displays hysteresis. In fact, a growing number of evidence have demonstrated that the voltage dependence of channel activity is dynamically modulated by activity itself. In spite of the great impact that this property can have on electrical signaling, hysteresis in voltage-gated channels is often overlooked. Addressing this issue, this review provides examples of voltage-gated ion channels displaying hysteretic behavior. Further, this review will discuss how Dynamic Voltage Dependence in voltage-gated channels can have a physiological role in electrical signaling. Furthermore, this review will elaborate on the current thoughts on the mechanism underlying hysteresis in voltage-gated channels.

  17. Calcium Signalling through Ligand-Gated Ion Channels such as P2X1 Receptors in the Platelet and other Non-Excitable Cells.

    Science.gov (United States)

    Mahaut-Smith, Martyn P; Taylor, Kirk A; Evans, Richard J

    2016-01-01

    Ligand-gated ion channels on the cell surface are directly activated by the binding of an agonist to their extracellular domain and often referred to as ionotropic receptors. P2X receptors are ligand-gated non-selective cation channels with significant permeability to Ca(2+) whose principal physiological agonist is ATP. This chapter focuses on the mechanisms by which P2X1 receptors, a ubiquitously expressed member of the family of ATP-gated channels, can contribute to cellular responses in non-excitable cells. Much of the detailed information on the contribution of P2X1 to Ca(2+) signalling and downstream functional events has been derived from the platelet. The underlying primary P2X1-generated signalling event in non-excitable cells is principally due to Ca(2+) influx, although Na(+) entry will also occur along with membrane depolarization. P2X1 receptor stimulation can lead to additional Ca(2+) mobilization via a range of routes such as amplification of G-protein-coupled receptor-dependent Ca(2+) responses. This chapter also considers the mechanism by which cells generate extracellular ATP for autocrine or paracrine activation of P2X1 receptors. For example cytosolic ATP efflux can result from opening of pannexin anion-permeable channels or following damage to the cell membrane. Alternatively, ATP stored in specialised secretory vesicles can undergo quantal release via the process of exocytosis. Examples of physiological or pathophysiological roles of P2X1-dependent signalling in non-excitable cells are also discussed, such as thrombosis and immune responses.

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

    Directory of Open Access Journals (Sweden)

    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

  19. Reptation theory of ion channel gating.

    Science.gov (United States)

    Millhauser, G L

    1990-04-01

    Reptation theory is a highly successful approach for describing polymer dynamics in entangled systems. In turn, this molecular process is the basis of viscoelasticity. We apply a modified version of reptation dynamics to develop an actual physical model of ion channel gating. We show that at times longer than microseconds these dynamics predict an alpha-helix-screw motion for the amphipathic protein segment that partially lines the channel pore. Such motion has been implicated in several molecular mechanics studies of both voltage-gated and transmitter-gated channels. The experimental probability density function (pdf) for this process follows t-3/2 which has been observed in several experimental systems. Reptation theory predicts that channel gating will occur on the millisecond time scale and this is consistent with experimental results from single-channel recording. We examine the consequences of reptation over random barriers and we show that, to first order, the pdf remains unchanged. In the case of a charged helix undergoing reptation in the presence of a transmembrane potential we show that the tail of the pdf will be exponential. We provide a list of practical experimental predictions to test the validity of this physical theory.

  20. Reptation theory of ion channel gating.

    OpenAIRE

    Millhauser, G L

    1990-01-01

    Reptation theory is a highly successful approach for describing polymer dynamics in entangled systems. In turn, this molecular process is the basis of viscoelasticity. We apply a modified version of reptation dynamics to develop an actual physical model of ion channel gating. We show that at times longer than microseconds these dynamics predict an alpha-helix-screw motion for the amphipathic protein segment that partially lines the channel pore. Such motion has been implicated in several mole...

  1. Long-term habituation of the gill-withdrawal reflex in Aplysia requires gene transcription, calcineurin and L-type voltage-gated calcium channels

    Directory of Open Access Journals (Sweden)

    Joseph eEsdin

    2010-11-01

    Full Text Available Although habituation is possibly the simplest form of learning, we still do not fully understand the neurobiological basis of habituation in any organism. To advance the goal of a comprehensive understanding of habituation, we have studied long-term habituation (LTH of the gill-withdrawal reflex (GWR in the marine snail Aplysia californica. Previously, we showed that habituation of the GWR in a reduced preparation lasts for up to 12 hr, and depends on protein synthesis, as well as activation of protein phosphatases 1 and 2A and postsynaptic glutamate receptors. Here, we have used the reduced preparation to further analyze the mechanisms of LTH in Aplysia. We found that LTH of the GWR depends on RNA synthesis because it was blocked by both the irreversible transcriptional inhibitor actinomycin-D and the reversible transcriptional inhibitor, 5,6-dichlorobenzimidazole riboside (DRB. In addition, LTH requires activation of protein phosphatase 2B (calcineurin, because it was disrupted by ascomycin. Finally, LTH was blocked by nitrendipine, which indicates that activation of L-type voltage-gated Ca2+ channels is required for this form of learning. Together with our previous results, the present results indicate that exclusively presynaptic mechanisms, although possibly sufficient for short-term habituation, are insufficient for LTH. Rather, LTH must involve postsynaptic, as well as presynaptic, mechanisms.

  2. Voltage-gated Potassium Channels as Therapeutic Drug Targets

    Science.gov (United States)

    Wulff, Heike; Castle, Neil A.; Pardo, Luis A.

    2009-01-01

    The human genome contains 40 voltage-gated potassium channels (KV) which are involved in diverse physiological processes ranging from repolarization of neuronal or cardiac action potentials, over regulating calcium signaling and cell volume, to driving cellular proliferation and migration. KV channels offer tremendous opportunities for the development of new drugs for cancer, autoimmune diseases and metabolic, neurological and cardiovascular disorders. This review first discusses pharmacological strategies for targeting KV channels with venom peptides, antibodies and small molecules and then highlights recent progress in the preclinical and clinical development of drugs targeting KV1.x, KV7.x (KCNQ), KV10.1 (EAG1) and KV11.1 (hERG) channels. PMID:19949402

  3. Philosophy of voltage-gated proton channels.

    Science.gov (United States)

    DeCoursey, Thomas E; Hosler, Jonathan

    2014-03-06

    In this review, voltage-gated proton channels are considered from a mainly teleological perspective. Why do proton channels exist? What good are they? Why did they go to such lengths to develop several unique hallmark properties such as extreme selectivity and ΔpH-dependent gating? Why is their current so minuscule? How do they manage to be so selective? What is the basis for our belief that they conduct H(+) and not OH(-)? Why do they exist in many species as dimers when the monomeric form seems to work quite well? It is hoped that pondering these questions will provide an introduction to these channels and a way to logically organize their peculiar properties as well as to understand how they are able to carry out some of their better-established biological functions.

  4. Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF aggregate is sufficient to cause cell death.

    Directory of Open Access Journals (Sweden)

    Makoto Takahashi

    Full Text Available The human α1A voltage-dependent calcium channel (Cav2.1 is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C-tail contains a small poly-glutamine (Q tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6. A recent study has shown that a 75-kDa C-terminal fragment (CTF containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (rCTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12 cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range than with Q13 (normal-length. Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB and phosphorylated-CREB (p-CREB in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

  5. Spinal morphine but not ziconotide or gabapentin analgesia is affected by alternative splicing of voltage-gated calcium channel CaV2.2 pre-mRNA.

    Science.gov (United States)

    Jiang, Yu-Qiu; Andrade, Arturo; Lipscombe, Diane

    2013-12-26

    Presynaptic voltage-gated calcium Ca(V)2.2 channels play a privileged role in spinal level sensitization following peripheral nerve injury. Direct and indirect inhibitors of Ca(V)2.2 channel activity in spinal dorsal horn are analgesic in chronic pain states. Ca(V)2.2 channels represent a family of splice isoforms that are expressed in different combinations according to cell-type. A pair of mutually exclusive exons in the Ca(V)2.2 encoding Cacna1b gene, e37a and e37b, differentially influence morphine analgesia. In mice that lack exon e37a, which is enriched in nociceptors, the analgesic efficacy of intrathecal morphine against noxious thermal stimuli is reduced. Here we ask if sequences unique to e37a influence: the development of abnormal thermal and mechanical sensitivity associated with peripheral nerve injury; and the actions of two other classes of analgesics that owe part or all of their efficacy to Ca(V)2.2 channel inhibition. We find that: i) the analgesic efficacy of morphine, but not ziconotide or gabapentin, is reduced in mice lacking e37a, ii) the induction and maintenance of behaviors associated with sensitization that accompany peripheral nerve injury, do not require e37a-specific sequence, iii) intrathecal morphine, but not ziconotide or gabapentin analgesia to thermal stimuli is significantly lower in wild-type mice after peripheral nerve injury, iv) the analgesic efficacy of ziconotide and gabapentin to mechanical stimuli is reduced following nerve injury, and iv) intrathecal morphine analgesia to thermal stimuli in mice lacking e37a is not further reduced by peripheral nerve injury. Our findings show that the analgesic action of morphine, but not ziconotide or gabapentin, to thermal stimuli is linked to which Cacna1b exon, e37a or e37b, is selected during alternative pre-mRNA splicing.

  6. Voltage-gated lipid ion channels

    DEFF Research Database (Denmark)

    Blicher, Andreas; Heimburg, Thomas Rainer

    2013-01-01

    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......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...

  7. Harnessing the Flow of Excitation: TRP, Voltage-Gated Na(+), and Voltage-Gated Ca(2+) Channels in Contemporary Medicine.

    Science.gov (United States)

    Frolov, Roman V; Weckström, Matti

    2016-01-01

    Cellular signaling in both excitable and nonexcitable cells involves several classes of ion channels. Some of them are of minor importance, with very specialized roles in physiology, but here we concentrate on three major channel classes: TRP (transient receptor potential channels), voltage-gated sodium channels (Nav), and voltage-gated calcium channels (Cav). Here, we first propose a conceptual framework binding together all three classes of ion channels, a "flow-of-excitation model" that takes into account the inputs mediated by TRP and other similar channels, the outputs invariably provided by Cav channels, and the regenerative transmission of signals in the neural networks, for which Nav channels are responsible. We use this framework to examine the function, structure, and pharmacology of these channel classes both at cellular and also at whole-body physiological level. Building on that basis we go through the pathologies arising from the direct or indirect malfunction of the channels, utilizing ion channel defects, the channelopathies. The pharmacological interventions affecting these channels are numerous. Part of those are well-established treatments, like treatment of hypertension or some forms of epilepsy, but many other are deeply problematic due to poor drug specificity, ion channel diversity, and widespread expression of the channels in tissues other than those actually targeted. © 2016 Elsevier Inc. All rights reserved.

  8. Suggestive evidence for association between L-type voltage-gated calcium channel (CACNA1C) gene haplotypes and bipolar disorder in Latinos: a family-based association study

    Science.gov (United States)

    Gonzalez, Suzanne; Xu, Chun; Ramirez, Mercedes; Zavala, Juan; Armas, Regina; Contreras, Salvador A; Contreras, Javier; Dassori, Albana; Leach, Robin J; Flores, Deborah; Jerez, Alvaro; Raventós, Henriette; Ontiveros, Alfonso; Nicolini, Humberto; Escamilla, Michael

    2013-01-01

    Objectives Through recent genome-wide association studies (GWAS), several groups have reported significant association between variants in the alpha 1C subunit of the L-type voltage-gated calcium channel (CACNA1C) and bipolar disorder (BP) in European and European-American cohorts. We performed a family-based association study to determine whether CACNA1C is associated with BP in the Latino population. Methods This study consisted of 913 individuals from 215 Latino pedigrees recruited from the United States, Mexico, Guatemala, and Costa Rica. The Illumina GoldenGate Genotyping Assay was used to genotype 58 single-nucleotide polymorphisms (SNPs) that spanned a 602.9 kb region encompassing the CACNA1C gene including two SNPs (rs7297582 and rs1006737) previously shown to associate with BP. Individual SNP and haplotype association analyses were performed using Family-Based Association Test (version 2.0.3) and Haploview (version 4.2) software. Results An eight-locus haplotype block that included these two markers showed significant association with BP (global marker permuted p = 0.0018) in the Latino population. For individual SNPs, this sample had insufficient power (10%) to detect associations with SNPs with minor effect (odds ratio = 1.15). Conclusions Although we were not able to replicate findings of association between individual CACNA1C SNPs rs7297582 and rs1006737 and BP, we were able to replicate the GWAS signal reported for CACNA1C through a haplotype analysis that encompassed these previously reported significant SNPs. These results provide additional evidence that CACNA1C is associated with BP and provides the first evidence that variations in this gene might play a role in the pathogenesis of this disorder in the Latino population. PMID:23437964

  9. Lack of direct evidence for a functional role of voltage-operated calcium channels in juxtaglomerular cells

    DEFF Research Database (Denmark)

    Kurtz, A; Skott, O; Chegini, S

    1990-01-01

    In this study we have examined the role of voltage-gated calcium channels in the regulation of calcium in juxtaglomerular cells. Using a combination of patch-clamp and single-cell calcium measurement we obtained evidence neither for voltage-operated calcium currents nor for changes of the intrace...

  10. Acute exposure to low-level CW and GSM-modulated 900 MHz radiofrequency does not affect Ba 2+ currents through voltage-gated calcium channels in rat cortical neurons.

    Science.gov (United States)

    Platano, Daniela; Mesirca, Pietro; Paffi, Alessandra; Pellegrino, Monica; Liberti, Micaela; Apollonio, Francesca; Bersani, Ferdinando; Aicardi, Giorgio

    2007-12-01

    We have studied the non-thermal effects of radiofrequency (RF) electromagnetic fields (EMFs) on Ba(2+) currents (I Ba 2+) through voltage-gated calcium channels (VGCC), recorded in primary cultures of rat cortical neurons using the patch-clamp technique. To assess whether low-level acute RF field exposure could modify the amplitude and/or the voltage-dependence of I Ba 2+, Petri dishes containing cultured neurons were exposed for 1-3 periods of 90 s to 900 MHz RF-EMF continuous wave (CW) or amplitude-modulated according to global system mobile communication standard (GSM) during whole-cell recording. The specific absorption rates (SARs) were 2 W/kg for CW and 2 W/kg (time average value) for GSM-modulated signals, respectively. The results obtained indicate that single or multiple acute exposures to either CW or GSM-modulated 900 MHz RF-EMFs do not significantly alter the current amplitude or the current-voltage relationship of I Ba 2+, through VGCC. (c) 2007 Wiley-Liss, Inc.

  11. 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.

  12. Calmodulin regulates Cav3 T-type channels at their gating brake.

    Science.gov (United States)

    Chemin, Jean; Taiakina, Valentina; Monteil, Arnaud; Piazza, Michael; Guan, Wendy; Stephens, Robert F; Kitmitto, Ashraf; Pang, Zhiping P; Dolphin, Annette C; Perez-Reyes, Edward; Dieckmann, Thorsten; Guillemette, Joseph Guy; Spafford, J David

    2017-09-25

    Calcium (Cav1 and Cav2) and sodium channels possess homologous CaM-binding motifs, known as IQ motifs in their C-termini, which associate with calmodulin (CaM), a universal calcium sensor. Cav3 T-type channels, which serve as pacemakers of the mammalian brain and heart, lack a C-terminal IQ motif. We illustrate that T-type channels associate with CaM using co-immunoprecipitation experiments and single particle cryo-electron microscopy. We demonstrate that protostome invertebrate (LCav3) and human Cav3.1, Cav3.2, and Cav3.3 T-type channels specifically associate with CaM at helix 2 of the gating brake in the I-II linker of the channels. Isothermal titration calorimetry results revealed that the gating brake and CaM bind each other with high nanomolar affinity. We show that the gating brake assumes a helical conformation upon binding CaM, with associated conformational changes to both CaM lobes as indicated by amide chemical shifts of the amino acids of CaM in 1H-15N HSQC NMR spectra. Intact Ca2+-binding sites on CaM and an intact gating brake sequence (first 39 aa of the I-II linker) were required in Cav3.2 channels to prevent the runaway gating phenotype, a hyperpolarizing shift in voltage sensitivities and faster gating kinetics. We conclude that the presence of high-nanomolar affinity binding sites for CaM at its universal gating brake and its unique form of regulation via the tuning of the voltage range of activity could influence the participation of Cav3 T-type channels in heart and brain rhythms. Our findings may have implications for arrhythmia disorders arising from mutations in the gating brake or CaM. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  13. Glucose Stimulates GLP-1 Secretion from Isolated Perfused Rat Small Intestine by SGLT1 and GLUT2 Mediated Uptake, Causing V-gated Calcium Channel Activation

    DEFF Research Database (Denmark)

    Kuhre, Rune Ehrenreich; Frost, Charlotte Rasmussen; Svendsen, Berit

    2014-01-01

    inhibition (1 mM phloretin) (Glu. 13.94 ± 2.5 vs. 30.90 ± 10.9 pM, Glu.+PT: 12.44 ± 1.3 vs. 17.18 ± 3.4 pM, P > 0.0001, n = 6), and KATP-channel closure by two sulfonylurea drugs stimulated GLP-1 release (tolbutamide: 14.7 ± 0.2 vs. 28.6 ± 5.2 pM; gliclazide: 15.2 ± 0.3 vs. 24.6 ± 2.4 pM, P

  14. Excitability Constraints on Voltage-Gated Sodium Channels

    OpenAIRE

    Brenner, Michael; Angelino, Elaine

    2007-01-01

    We study how functional constraints bound and shape evolution through an analysis of mammalian voltage-gated sodium channels. The primary function of sodium channels is to allow the propagation of action potentials. Since Hodgkin and Huxley, mathematical models have suggested that sodium channel properties need to be tightly constrained for an action potential to propagate. There are nine mammalian genes encoding voltage-gated sodium channels, many of which are more than approximately 90% ide...

  15. Deletion of cytosolic gating ring decreases gate and voltage sensor coupling in BK channels.

    Science.gov (United States)

    Zhang, Guohui; Geng, Yanyan; Jin, Yakang; Shi, Jingyi; McFarland, Kelli; Magleby, Karl L; Salkoff, Lawrence; Cui, Jianmin

    2017-03-06

    Large conductance Ca 2+ -activated K + channels (BK channels) gate open in response to both membrane voltage and intracellular Ca 2+ The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca 2+ sensor. How these voltage and Ca 2+ sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca 2+ activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca 2+ sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel's β1 and β2 subunits. © 2017 Zhang et al.

  16. The L-type Voltage-Gated Calcium Channel co-localizes with Syntaxin 1A in nano-clusters at the plasma membrane.

    Science.gov (United States)

    Sajman, Julia; Trus, Michael; Atlas, Daphne; Sherman, Eilon

    2017-09-12

    The secretory signal elicited by membrane depolarization traverses from the Ca(2+)-bound α11.2 pore-forming subunit of the L-type Ca(2+)-channel (Cav1.2) to syntaxin 1 A (Sx1A) via an intra-membrane signaling mechanism. Here, we report the use of two-color Photo-Activated-Localization-Microscopy (PALM) to determine the relation between Cav1.2 and Sx1A in single-molecule detail. We observed nanoscale co-clusters of PAmCherry-tagged Sx1A and Dronpa-tagged α11.2 at a ~1:1 ratio. PAmCherry-tagged Sx1A(C145A), or PAmCherry-tagged Sx2, an inactive Cav1.2 modulator, in which Cys145 is a Ser residue, showed no co-clustering. These results are  consistent with the crucial role of the single cytosolic Sx1ACys145 in clustering with Cav1.2. Cav1.2 and the functionally inactive transmembrane-domain double mutant Sx1A(C271V/C272V) engendered clusters with a ~2:1 ratio. A higher extent of co-clustering, which coincides with compromised depolarization-evoked transmitter-release, was observed also by oxidation of Sx1ACys271 and Cys272. Our super-resolution-imaging results set the stage for studying co-clustering of the channel with other exocytotic proteins at a single-molecule level.

  17. Phylogeny unites animal sodium leak channels with fungal calcium channels in an ancient, voltage-insensitive clade.

    Science.gov (United States)

    Liebeskind, Benjamin J; Hillis, David M; Zakon, Harold H

    2012-12-01

    Proteins in the superfamily of voltage-gated ion channels mediate behavior across the tree of life. These proteins regulate the movement of ions across cell membranes by opening and closing a central pore that controls ion flow. The best-known members of this superfamily are the voltage-gated potassium, calcium (Ca(v)), and sodium (Na(v)) channels, which underlie impulse conduction in nerve and muscle. Not all members of this family are opened by changes in voltage, however. NALCN (NA(+) leak channel nonselective) channels, which encode a voltage-insensitive "sodium leak" channel, have garnered a growing interest. This study examines the phylogenetic relationship among Na(v)/Ca(v) voltage-gated and voltage-insensitive channels in the eukaryotic group Opisthokonta, which includes animals, fungi, and their unicellular relatives. We show that NALCN channels diverged from voltage-gated channels before the divergence of fungi and animals and that the closest relatives of NALCN channels are fungal calcium channels, which they functionally resemble.

  18. Study on effective MOSFET channel length extracted from gate capacitance

    Science.gov (United States)

    Tsuji, Katsuhiro; Terada, Kazuo; Fujisaka, Hisato

    2018-01-01

    The effective channel length (L GCM) of metal–oxide–semiconductor field-effect transistors (MOSFETs) is extracted from the gate capacitances of actual-size MOSFETs, which are measured by charge-injection-induced-error-free charge-based capacitance measurement (CIEF CBCM). To accurately evaluate the capacitances between the gate and the channel of test MOSFETs, the parasitic capacitances are removed by using test MOSFETs having various channel sizes and a source/drain reference device. A strong linear relationship between the gate-channel capacitance and the design channel length is obtained, from which L GCM is extracted. It is found that L GCM is slightly less than the effective channel length (L CRM) extracted from the measured MOSFET drain current. The reason for this is discussed, and it is found that the capacitance between the gate electrode and the source and drain regions affects this extraction.

  19. The genetic background affects the vascular response in T-type calcium channels 3.2 deficient mice

    DEFF Research Database (Denmark)

    Svenningsen, Per; Hansen, Pernille B L

    2016-01-01

    Voltage-gated calcium channels (Cav ) are important regulators of vascular tone and are attractive targets for pharmacological treatment of hypertension. The clinical used calcium blockers are often not selective for one channel but affect several types of calcium channels (Hansen 2015). L......-type channels are the dominant Ca(2+) entry pathway in vascular smooth muscle cells, however, T-type calcium channels are also expressed in the cardiovascular system where they play a functional role in the regulation of both contraction and vasodilation in (Chen et al. 2003; Hansen et al. 2001). This article...... is protected by copyright. All rights reserved....

  20. Altered expression of the voltage-gated calcium channel subunit alpha(2)delta-1: A comparison between two experimental models of epilepsy and a sensory nerve ligation model of neuropathic pain

    Czech Academy of Sciences Publication Activity Database

    Nieto-Rostro, M.; Sandhu, G.; Bauer, C. S.; Jiruška, Přemysl; Jefferys, J. G. R.; Dolphin, A. C.

    2014-01-01

    Roč. 283, Dec (2014), s. 124-137 ISSN 0306-4522 R&D Projects: GA MZd(CZ) NT14489 Institutional support: RVO:67985823 Keywords : calcium channel * dorsal root ganglion (DRG) * alpha2delta subunit * epilepsy * neuropathic pain * reactive gliosis Subject RIV: FH - Neurology Impact factor: 3.357, year: 2014

  1. Molecular and functional characterization of voltage-gated sodium channels in human sperm.

    Science.gov (United States)

    Pinto, Francisco M; Ravina, Cristina G; Fernández-Sánchez, Manuel; Gallardo-Castro, Manuel; Cejudo-Román, Antonio; Candenas, Luz

    2009-07-16

    We have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility. Freshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA). Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2. The mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9) were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels. This research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function.

  2. Molecular and functional characterization of voltage-gated sodium channels in human sperm

    Directory of Open Access Journals (Sweden)

    Cejudo-Román Antonio

    2009-07-01

    Full Text Available Abstract Background We have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility. Methods Freshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA. Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2. Results The mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9 were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels. Conclusion This research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function.

  3. Calcium signalling through L-type calcium channels: role in pathophysiology of spinal nociceptive transmission.

    Science.gov (United States)

    Roca-Lapirot, Olivier; Radwani, Houda; Aby, Franck; Nagy, Frédéric; Landry, Marc; Fossat, Pascal

    2017-02-18

    L-type voltage-gated calcium channels are ubiquitous channels in the CNS. L-type calcium channels (LTCs) are mostly post-synaptic channels regulating neuronal firing and gene expression. They play a role in important physio-pathological processes such as learning and memory, Parkinson's disease, autism and, as recognized more recently, in the pathophysiology of pain processes. Classically, the fundamental role of these channels in cardiovascular functions has limited the use of classical molecules to treat LTC-dependent disorders. However, when applied locally in the dorsal horn of the spinal cord, the three families of LTC pharmacological blockers - dihydropyridines (nifedipine), phenylalkylamines (verapamil) and benzothiazepines (diltiazem) - proved effective in altering short-term sensitization to pain, inflammation-induced hyperexcitability and neuropathy-induced allodynia. Two subtypes of LTCs, Cav 1.2 and Cav 1.3, are expressed in the dorsal horn of the spinal cord, where Cav 1.2 channels are localized mostly in the soma and proximal dendritic shafts, and Cav 1.3 channels are more distally located in the somato-dendritic compartment. Together with their different kinetics and pharmacological properties, this spatial distribution contributes to their separate roles in shaping short- and long-term sensitization to pain. Cav 1.3 channels sustain the expression of plateau potentials, an input/output amplification phenomenon that contributes to short-term sensitization to pain such as prolonged after-discharges, dynamic receptive fields and windup. The Cav 1.2 channels support calcium influx that is crucial for the excitation-transcription coupling underlying nerve injury-induced dorsal horn hyperexcitability. These subtype-specific cellular mechanisms may have different consequences in the development and/or the maintenance of pathological pain. Recent progress in developing more specific compounds for each subunit will offer new opportunities to modulate LTCs

  4. Computational study of a calcium release-activated calcium channel

    Science.gov (United States)

    Talukdar, Keka; Shantappa, Anil

    2016-05-01

    The naturally occurring proteins that form hole in membrane are commonly known as ion channels. They play multiple roles in many important biological processes. Deletion or alteration of these channels often leads to serious problems in the physiological processes as it controls the flow of ions through it. The proper maintenance of the flow of ions, in turn, is required for normal health. Here we have investigated the behavior of a calcium release-activated calcium ion channel with pdb entry 4HKR in Drosophila Melanogaster. The equilibrium energy as well as molecular dynamics simulation is performed first. The protein is subjected to molecular dynamics simulation to find their energy minimized value. Simulation of the protein in the environment of water and ions has given us important results too. The solvation energy is also found using Charmm potential.

  5. 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.

  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. Gender specific hippocampal whole genome transcriptome data from mice lacking the Cav2.3 R-type or Cav3.2 T-type voltage-gated calcium channel

    Directory of Open Access Journals (Sweden)

    Anna Papazoglou

    2017-06-01

    Full Text Available Voltage-gated Ca2+ channels are of central relevance in mediating numerous intracellular and transcellular processes including excitation-contraction coupling, excitation secretion-coupling, hormone and neurotransmitter release and gene expression. The Cav2.3 R-type Ca2+ channel is a high-voltage activated channel which plays a crucial role in neurotransmitter release, long-term potentiation and hormone release. Furthermore, Cav2.3 R-type channels were reported to be involved in ictogenesis, epileptogenesis, fear behavior, sleep, pre-and postsynaptic integration and rhythmicity within the hippocampus. Cav3 T-type Ca2+ channels are low-voltage activated and also widely expressed throughout the brain enabling neurons to switch between different firing patterns and to modulate burst activity. Disruption of T-type Ca2+ current has been related to sleep disorders, epilepsy, Parkinson׳s disease, depression, schizophrenia and pain. Cav3.2 ablation was further attributed to elevated anxiety and hippocampal alterations resulting in impaired long-term potentiation and memory. Given the importance of Cav2.3 and Cav3.2 voltage-gated Ca2+ channels within the CNS, particularly the hippocampus, we collected gender specific microarray transcriptome data of murine hippocampal RNA probes using the Affymetrix Exon Expression Chip Mouse Gene 1.0 ST v1. Information presented here includes transcriptome data from Cav2.3+/+, Cav2.3+/−, Cav2.3−/−, Cav3.2+/+, Cav3.2+/− and Cav3.2−/− mice from both genders, the protocol and list of primers used for genotyping animals, the hippocampal RNA isolation procedure and quality controls.

  8. Chondroitin sulfates act as extracellular gating modifiers on voltage-dependent ion channels.

    Science.gov (United States)

    Vigetti, Davide; Andrini, Olga; Clerici, Moira; Negrini, Daniela; Passi, Alberto; Moriondo, Andrea

    2008-01-01

    Chondroitin sulfates are glycosaminoglycans bound to core proteins of proteoglycans in the extracellular matrix and perineuronal nets surrounding many types of neurones. Chondroitin 4- and chondroitin 6- sulfate can bind calcium ions with different affinities, depending on their sulfation position. Extracellular calcium plays a key role in determining the transmembrane potential sensed by voltage-operated ion channels (VOCs) by means of the "surface screening effect" theory (Gouy-Chapman-Stern theory). We wanted to test whether chondroitin sulfates at physiological concentration can effectively modulate the gating properties of VOCs. We recorded in patch-clamp experiments the shift of h and voltage-dependent calcium currents activation curves of Xenopus laevis photoreceptors perfused with chondroitin sulfate solutions in physiological extracellular calcium concentration. We found that chondroitin 4- and 6- sulfate, with different capabilities, can shift the activation curve of h and voltage-dependent calcium channels, compatibly with the surface screening effect theory. We conclude that chondroitin sulfates can alter VOCs gating by modulating the calcium concentration in the extracellular microenvironment. This phenomenon may explain why alterations in the chondroitin sulfation and abundance in the extracellular matrix are found along with altered neuronal function in pathological conditions. Copyright 2008 S. Karger AG, Basel.

  9. Discovery and Development of Calcium Channel Blockers

    Directory of Open Access Journals (Sweden)

    Théophile Godfraind

    2017-05-01

    Full Text Available In the mid 1960s, experimental work on molecules under screening as coronary dilators allowed the discovery of the mechanism of calcium entry blockade by drugs later named calcium channel blockers. This paper summarizes scientific research on these small molecules interacting directly with L-type voltage-operated calcium channels. It also reports on experimental approaches translated into understanding of their therapeutic actions. The importance of calcium in muscle contraction was discovered by Sidney Ringer who reported this fact in 1883. Interest in the intracellular role of calcium arose 60 years later out of Kamada (Japan and Heibrunn (USA experiments in the early 1940s. Studies on pharmacology of calcium function were initiated in the mid 1960s and their therapeutic applications globally occurred in the the 1980s. The first part of this report deals with basic pharmacology in the cardiovascular system particularly in isolated arteries. In the section entitled from calcium antagonists to calcium channel blockers, it is recalled that drugs of a series of diphenylpiperazines screened in vivo on coronary bed precontracted by angiotensin were initially named calcium antagonists on the basis of their effect in depolarized arteries contracted by calcium. Studies on arteries contracted by catecholamines showed that the vasorelaxation resulted from blockade of calcium entry. Radiochemical and electrophysiological studies performed with dihydropyridines allowed their cellular targets to be identified with L-type voltage-operated calcium channels. The modulated receptor theory helped the understanding of their variation in affinity dependent on arterial cell membrane potential and promoted the terminology calcium channel blocker (CCB of which the various chemical families are introduced in the paper. In the section entitled tissue selectivity of CCBs, it is shown that characteristics of the drug, properties of the tissue, and of the stimuli are

  10. Voltage Gated Ion Channel Function: Gating, Conduction, and the Role of Water and Protons

    Science.gov (United States)

    Kariev, Alisher M.; Green, Michael E.

    2012-01-01

    Ion channels, which are found in every biological cell, regulate the concentration of electrolytes, and are responsible for multiple biological functions, including in particular the propagation of nerve impulses. The channels with the latter function are gated (opened) by a voltage signal, which allows Na+ into the cell and K+ out. These channels have several positively charged amino acids on a transmembrane domain of their voltage sensor, and it is generally considered, based primarily on two lines of experimental evidence, that these charges move with respect to the membrane to open the channel. At least three forms of motion, with greatly differing extents and mechanisms of motion, have been proposed. There is a “gating current”, a capacitative current preceding the channel opening, that corresponds to several charges (for one class of channel typically 12–13) crossing the membrane field, which may not require protein physically crossing a large fraction of the membrane. The coupling to the opening of the channel would in these models depend on the motion. The conduction itself is usually assumed to require the “gate” of the channel to be pulled apart to allow ions to enter as a section of the protein partially crosses the membrane, and a selectivity filter at the opposite end of the channel determines the ion which is allowed to pass through. We will here primarily consider K+ channels, although Na+ channels are similar. We propose that the mechanism of gating differs from that which is generally accepted, in that the positively charged residues need not move (there may be some motion, but not as gating current). Instead, protons may constitute the gating current, causing the gate to open; opening consists of only increasing the diameter at the gate from approximately 6 Å to approximately 12 Å. We propose in addition that the gate oscillates rather than simply opens, and the ion experiences a barrier to its motion across the channel that is tuned

  11. Allosteric gating mechanism underlies the flexible gating of KCNQ1 potassium channels

    Science.gov (United States)

    Osteen, Jeremiah D.; Barro-Soria, Rene; Robey, Seth; Sampson, Kevin J.; Kass, Robert S.; Larsson, H. Peter

    2012-01-01

    KCNQ1 (Kv7.1) is a unique member of the superfamily of voltage-gated K+ channels in that it displays a remarkable range of gating behaviors tuned by coassembly with different β subunits of the KCNE family of proteins. To better understand the basis for the biophysical diversity of KCNQ1 channels, we here investigate the basis of KCNQ1 gating in the absence of β subunits using voltage-clamp fluorometry (VCF). In our previous study, we found the kinetics and voltage dependence of voltage-sensor movements are very similar to those of the channel gate, as if multiple voltage-sensor movements are not required to precede gate opening. Here, we have tested two different hypotheses to explain KCNQ1 gating: (i) KCNQ1 voltage sensors undergo a single concerted movement that leads to channel opening, or (ii) individual voltage-sensor movements lead to channel opening before all voltage sensors have moved. Here, we find that KCNQ1 voltage sensors move relatively independently, but that the channel can conduct before all voltage sensors have activated. We explore a KCNQ1 point mutation that causes some channels to transition to the open state even in the absence of voltage-sensor movement. To interpret these results, we adopt an allosteric gating scheme wherein KCNQ1 is able to transition to the open state after zero to four voltage-sensor movements. This model allows for widely varying gating behavior, depending on the relative strength of the opening transition, and suggests how KCNQ1 could be controlled by coassembly with different KCNE family members. PMID:22509038

  12. 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

  13. Gating the Selectivity Filter in ClC Chloride Channels

    Science.gov (United States)

    Dutzler, Raimund; Campbell, Ernest B.; MacKinnon, Roderick

    2003-04-01

    ClC channels conduct chloride (Cl-) ions across cell membranes and thereby govern the electrical activity of muscle cells and certain neurons, the transport of fluid and electrolytes across epithelia, and the acidification of intracellular vesicles. The structural basis of ClC channel gating was studied. Crystal structures of wild-type and mutant Escherichia coli ClC channels bound to a monoclonal Fab fragment reveal three Cl- binding sites within the 15-angstrom neck of an hourglass-shaped pore. The Cl- binding site nearest the extracellular solution can be occupied either by a Cl- ion or by a glutamate carboxyl group. Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays. These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl- ion.

  14. Gating motions in voltage-gated potassium channels revealed by coarse-grained molecular dynamics simulations

    NARCIS (Netherlands)

    Treptow, W.; Marrink, S.J.; Tarek, M.

    2008-01-01

    Voltage-gated potassium (Kv) channels are ubiquitous transmembrane proteins involved in electric signaling of excitable tissues. A fundamental property of these channels is the ability to open or close in response to changes in the membrane potential. To date, their structure-based activation

  15. Voltage-gated proton channel is expressed on phagosomes

    Energy Technology Data Exchange (ETDEWEB)

    Okochi, Yoshifumi; Sasaki, Mari [Department of Integrative Physiology, Graduate School of Medicine, Osaka University, 2-2, Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Developmental Neurophysiology, Okazaki Institute for Integrative Bioscience, Higashiyama 5-1, Myodaiji-cho, Okazaki, Aichi 444-8787 (Japan); Iwasaki, Hirohide [Department of Developmental Neurophysiology, Okazaki Institute for Integrative Bioscience, Higashiyama 5-1, Myodaiji-cho, Okazaki, Aichi 444-8787 (Japan); Okamura, Yasushi, E-mail: yokamura@phys2.med.osaka-u.ac.jp [Department of Integrative Physiology, Graduate School of Medicine, Osaka University, 2-2, Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Developmental Neurophysiology, Okazaki Institute for Integrative Bioscience, Higashiyama 5-1, Myodaiji-cho, Okazaki, Aichi 444-8787 (Japan)

    2009-05-01

    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.

  16. 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.

  17. 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.

  18. Excitability constraints on voltage-gated sodium channels.

    Directory of Open Access Journals (Sweden)

    Elaine Angelino

    2007-09-01

    Full Text Available We study how functional constraints bound and shape evolution through an analysis of mammalian voltage-gated sodium channels. The primary function of sodium channels is to allow the propagation of action potentials. Since Hodgkin and Huxley, mathematical models have suggested that sodium channel properties need to be tightly constrained for an action potential to propagate. There are nine mammalian genes encoding voltage-gated sodium channels, many of which are more than approximately 90% identical by sequence. This sequence similarity presumably corresponds to similarity of function, consistent with the idea that these properties must be tightly constrained. However, the multiplicity of genes encoding sodium channels raises the question: why are there so many? We demonstrate that the simplest theoretical constraints bounding sodium channel diversity--the requirements of membrane excitability and the uniqueness of the resting potential--act directly on constraining sodium channel properties. We compare the predicted constraints with functional data on mammalian sodium channel properties collected from the literature, including 172 different sets of measurements from 40 publications, wild-type and mutant, under a variety of conditions. The data from all channel types, including mutants, obeys the excitability constraint; on the other hand, channels expressed in muscle tend to obey the constraint of a unique resting potential, while channels expressed in neuronal tissue do not. The excitability properties alone distinguish the nine sodium channels into four different groups that are consistent with phylogenetic analysis. Our calculations suggest interpretations for the functional differences between these groups.

  19. Sialic Acids Attached to O-Glycans Modulate Voltage-gated Potassium Channel Gating*

    Science.gov (United States)

    Schwetz, Tara A.; Norring, Sarah A.; Ednie, Andrew R.; Bennett, Eric S.

    2011-01-01

    Neuronal, cardiac, and skeletal muscle action potentials are produced and conducted through the highly regulated activity of several types of voltage-gated ion channels. Voltage-gated potassium (Kv) channels are responsible for action potential repolarization. Glycans can be attached to glycoproteins through N- and O-linkages. Previous reports described the impact of N-glycans on voltage-gated ion channel function. Here, we show that sialic acids attached through O-linkages modulate gating of Kv2.1, Kv4.2, and Kv4.3. The conductance-voltage (G-V) relationships for each isoform were shifted uniquely by a depolarizing 8–16 mV under conditions of reduced sialylation. The data indicate that sialic acids modulate Kv channel activation through apparent electrostatic mechanisms that promote channel activity. Voltage-dependent steady-state inactivation was unaffected by changes in sialylation. N-Linked sialic acids cannot be responsible for the G-V shifts because Kv4.2 and Kv4.3 cannot be N-glycosylated, and immunoblot analysis confirmed Kv2.1 is not N-glycosylated. Glycosidase gel shift analysis suggested that Kv2.1, Kv4.2, and Kv4.3 were O-glycosylated and sialylated. To confirm this, azide-modified sugar residues involved specifically in O-glycan and sialic acid biosynthesis were shown to incorporate into all three Kv channel isoforms using Cu(I)-catalyzed cycloaddition chemistry. Together, the data indicate that sialic acids attached to O-glycans uniquely modulate gating of three Kv channel isoforms that are not N-glycosylated. These data provide the first evidence that external O-glycans, with core structures distinct from N-glycans in type and number of sugar residues, can modulate Kv channel function and thereby contribute to changes in electrical signaling that result from regulated ion channel expression and/or O-glycosylation. PMID:21115483

  20. Ankyrin Repeats Convey Force to Gate the NOMPC Mechanotransduction Channel.

    Science.gov (United States)

    Zhang, Wei; Cheng, Li E; Kittelmann, Maike; Li, Jiefu; Petkovic, Maja; Cheng, Tong; Jin, Peng; Guo, Zhenhao; Göpfert, Martin C; Jan, Lily Yeh; Jan, Yuh Nung

    2015-09-10

    How metazoan mechanotransduction channels sense mechanical stimuli is not well understood. The NOMPC channel in the transient receptor potential (TRP) family, a mechanotransduction channel for Drosophila touch sensation and hearing, contains 29 Ankyrin repeats (ARs) that associate with microtubules. These ARs have been postulated to act as a tether that conveys force to the channel. Here, we report that these N-terminal ARs form a cytoplasmic domain essential for NOMPC mechanogating in vitro, mechanosensitivity of touch receptor neurons in vivo, and touch-induced behaviors of Drosophila larvae. Duplicating the ARs elongates the filaments that tether NOMPC to microtubules in mechanosensory neurons. Moreover, microtubule association is required for NOMPC mechanogating. Importantly, transferring the NOMPC ARs to mechanoinsensitive voltage-gated potassium channels confers mechanosensitivity to the chimeric channels. These experiments strongly support a tether mechanism of mechanogating for the NOMPC channel, providing insights into the basis of mechanosensitivity of mechanotransduction channels. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Ryanodine receptor gating controls generation of diastolic calcium waves in cardiac myocytes

    Science.gov (United States)

    Petrovič, Pavol; Valent, Ivan; Cocherová, Elena; Pavelková, Jana

    2015-01-01

    The role of cardiac ryanodine receptor (RyR) gating in the initiation and propagation of calcium waves was investigated using a mathematical model comprising a stochastic description of RyR gating and a deterministic description of calcium diffusion and sequestration. We used a one-dimensional array of equidistantly spaced RyR clusters, representing the confocal scanning line, to simulate the formation of calcium sparks. Our model provided an excellent description of the calcium dependence of the frequency of diastolic calcium sparks and of the increased tendency for the production of calcium waves after a decrease in cytosolic calcium buffering. We developed a hypothesis relating changes in the propensity to form calcium waves to changes of RyR gating and tested it by simulation. With a realistic RyR gating model, increased ability of RyR to be activated by Ca2+ strongly increased the propensity for generation of calcium waves at low (0.05–0.1-µM) calcium concentrations but only slightly at high (0.2–0.4-µM) calcium concentrations. Changes in RyR gating altered calcium wave formation by changing the calcium sensitivity of spontaneous calcium spark activation and/or the average number of open RyRs in spontaneous calcium sparks. Gating changes that did not affect RyR activation by Ca2+ had only a weak effect on the propensity to form calcium waves, even if they strongly increased calcium spark frequency. Calcium waves induced by modulating the properties of the RyR activation site could be suppressed by inhibiting the spontaneous opening of the RyR. These data can explain the increased tendency for production of calcium waves under conditions when RyR gating is altered in cardiac diseases. PMID:26009544

  2. Signal processing by T-type calcium channel interactions in the cerebellum.

    Science.gov (United States)

    Engbers, Jordan D T; Anderson, Dustin; Zamponi, Gerald W; Turner, Ray W

    2013-11-27

    T-type calcium channels of the Cav3 family are unique among voltage-gated calcium channels due to their low activation voltage, rapid inactivation, and small single channel conductance. These special properties allow Cav3 calcium channels to regulate neuronal processing in the subthreshold voltage range. Here, we review two different subthreshold ion channel interactions involving Cav3 channels and explore the ability of these interactions to expand the functional roles of Cav3 channels. In cerebellar Purkinje cells, Cav3 and intermediate conductance calcium-activated potassium (IKCa) channels form a novel complex which creates a low voltage-activated, transient outward current capable of suppressing temporal summation of excitatory postsynaptic potentials (EPSPs). In large diameter neurons of the deep cerebellar nuclei, Cav3-mediated calcium current (I T) and hyperpolarization-activated cation current (I H) are activated during trains of inhibitory postsynaptic potentials. These currents have distinct, and yet synergistic, roles in the subthreshold domain with I T generating a rebound burst and I H controlling first spike latency and rebound spike precision. However, by shortening the membrane time constant the membrane returns towards resting value at a faster rate, allowing I H to increase the efficacy of I T and increase the range of burst frequencies that can be generated. The net effect of Cav3 channels thus depends on the channels with which they are paired. When expressed in a complex with a KCa channel, Cav3 channels reduce excitability when processing excitatory inputs. If functionally coupled with an HCN channel, the depolarizing effect of Cav3 channels is accentuated, allowing for efficient inversion of inhibitory inputs to generate a rebound burst output. Therefore, signal processing relies not only on the activity of individual subtypes of channels but also on complex interactions between ion channels whether based on a physical complex or by indirect

  3. Signal processing by T-type calcium channel interactions in the cerebellum

    Directory of Open Access Journals (Sweden)

    Jordan D.T. Engbers

    2013-11-01

    Full Text Available T-type calcium channels of the Cav3 family are unique among voltage-gated calcium channels due to their low activation voltage, rapid inactivation, and small single channel conductance. These special properties allow Cav3 calcium channels to regulate neuronal processing in the subthreshold voltage range. Here, we review two different subthreshold ion channel interactions involving Cav3 channels and explore the ability of these interactions to expand the functional roles of Cav3 channels. In cerebellar Purkinje cells, Cav3 and intermediate conductance calcium-activated potassium (IKCa channels form a novel complex which creates a low voltage-activated, transient outward current capable of suppressing temporal summation of excitatory postsynaptic potentials (EPSPs. In large diameter neurons of the deep cerebellar nuclei, Cav3-mediated calcium current (IT and hyperpolarization-activated cation current (IH are activated during trains of IPSPs. These currents have distinct, and yet synergistic, roles in the subthreshold domain with IT generating a rebound burst and IH controlling first spike latency and rebound spike precision. However, by shortening the membrane time constant the membrane returns towards resting value at a faster rate, allowing IH to increase the efficacy of IT, and increase the range of burst frequencies that can be generated. The net effect of Cav3 channels thus depends on the channels with which they are paired. When expressed in a complex with a KCa channel, Cav3 channels reduce excitability when processing excitatory inputs. If functionally coupled with an HCN channel, the depolarizing effect of Cav3 channels is accentuated, allowing for efficient inversion of inhibitory inputs to generate a rebound burst output. Therefore, signal processing relies not only on the activity of individual subtypes of channels but also on complex interactions between ion channels whether based on a physical complex or by indirect effects on

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

    Directory of Open Access Journals (Sweden)

    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. Spontaneous and CRH-Induced Excitability and Calcium Signaling in Mice Corticotrophs Involves Sodium, Calcium, and Cation-Conducting Channels.

    Science.gov (United States)

    Zemkova, Hana; Tomić, Melanija; Kucka, Marek; Aguilera, Greti; Stojilkovic, Stanko S

    2016-04-01

    Transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the proopiomelanocortin gene's regulatory elements are a useful model for studying corticotrophs. Using these mice, we studied the ion channels and mechanisms controlling corticotroph excitability. Corticotrophs were either quiescent or electrically active, with a 22-mV difference in the resting membrane potential (RMP) between the 2 groups. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in active cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory but not inhibitory action of CRH on electrical activity was mimicked by cAMP independently of the presence or absence of arachidonic acid. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling.

  6. A Calcium-Dependent Plasticity Rule for HCN Channels Maintains Activity Homeostasis and Stable Synaptic Learning

    Science.gov (United States)

    Honnuraiah, Suraj; Narayanan, Rishikesh

    2013-01-01

    Theoretical and computational frameworks for synaptic plasticity and learning have a long and cherished history, with few parallels within the well-established literature for plasticity of voltage-gated ion channels. In this study, we derive rules for plasticity in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and assess the synergy between synaptic and HCN channel plasticity in establishing stability during synaptic learning. To do this, we employ a conductance-based model for the hippocampal pyramidal neuron, and incorporate synaptic plasticity through the well-established Bienenstock-Cooper-Munro (BCM)-like rule for synaptic plasticity, wherein the direction and strength of the plasticity is dependent on the concentration of calcium influx. Under this framework, we derive a rule for HCN channel plasticity to establish homeostasis in synaptically-driven firing rate, and incorporate such plasticity into our model. In demonstrating that this rule for HCN channel plasticity helps maintain firing rate homeostasis after bidirectional synaptic plasticity, we observe a linear relationship between synaptic plasticity and HCN channel plasticity for maintaining firing rate homeostasis. Motivated by this linear relationship, we derive a calcium-dependent rule for HCN-channel plasticity, and demonstrate that firing rate homeostasis is maintained in the face of synaptic plasticity when moderate and high levels of cytosolic calcium influx induced depression and potentiation of the HCN-channel conductance, respectively. Additionally, we show that such synergy between synaptic and HCN-channel plasticity enhances the stability of synaptic learning through metaplasticity in the BCM-like synaptic plasticity profile. Finally, we demonstrate that the synergistic interaction between synaptic and HCN-channel plasticity preserves robustness of information transfer across the neuron under a rate-coding schema. Our results establish specific physiological roles

  7. Voltage-gated proton (H(v)1) channels, a singular voltage sensing domain.

    Science.gov (United States)

    Castillo, Karen; Pupo, Amaury; Baez-Nieto, David; Contreras, Gustavo F; Morera, Francisco J; Neely, Alan; Latorre, Ramon; Gonzalez, Carlos

    2015-11-14

    The main role of voltage-gated proton channels (Hv1) is to extrude protons from the intracellular milieu when, mediated by different cellular processes, the H(+) concentration increases. Hv1 are exquisitely selective for protons and their structure is homologous to the voltage sensing domain (VSD) of other voltage-gated ion channels like sodium, potassium, and calcium channels. In clear contrast to the classical voltage-dependent channels, Hv1 lacks a pore domain and thus permeation necessarily occurs through the voltage sensing domain. Hv1 channels are activated by depolarizing voltages, and increases in internal proton concentration. It has been proposed that local conformational changes of the transmembrane segment S4, driven by depolarization, trigger the molecular rearrangements that open Hv1. However, it is still unclear how the electromechanical coupling is achieved between the VSD and the potential pore, allowing the proton flux from the intracellular to the extracellular side. Here we provide a revised view of voltage activation in Hv1 channels, offering a comparative scenario with other voltage sensing channels domains. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. 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

  9. Analysis of gate underlap channel double gate MOS transistor for electrical detection of bio-molecules

    Science.gov (United States)

    Ajay; Narang, Rakhi; Saxena, Manoj; Gupta, Mridula

    2015-12-01

    In this paper, an analytical model for gate drain underlap channel Double-Gate Metal-Oxide-Semiconductor Field-Effect Transistor (DG-MOSFET) for label free electrical detection of biomolecules has been proposed. The conformal mapping technique has been used to derive the expressions for surface potential, lateral electric field, energy bands (i.e. conduction and valence band) and threshold voltage (Vth). Subsequently a full drain current model to analyze the sensitivity of the biosensor has been developed. The shift in the threshold voltage and drain current (after the biomolecules interaction with the gate underlap channel region of the MOS transistor) has been used as a sensing metric. All the characteristic trends have been verified through ATLAS (SILVACO) device simulation results.

  10. Voltage-gated sodium channels: therapeutic targets for pain.

    Science.gov (United States)

    Dib-Hajj, Sulayman D; Black, Joel A; Waxman, Stephen G

    2009-10-01

    To provide an overview of the role of voltage-gated sodium channels in pathophysiology of acquired and inherited pain states, and of recent developments that validate these channels as therapeutic targets for treating chronic pain. Neuropathic and inflammatory pain conditions are major medical needs worldwide with only partial or low efficacy treatment options currently available. An important role of voltage-gated sodium channels in many different pain states has been established in animal models and, empirically, in humans, where sodium channel blockers partially ameliorate pain. Animal studies have causally linked changes in sodium channel expression and modulation that alter channel gating properties or current density in nociceptor neurons to different pain states. Biophysical and pharmacological studies have identified the sodium channel isoforms Na(v)1.3, Na(v)1.7, Na(v)1.8, and Na(v)1.9 as particularly important in the pathophysiology of different pain syndromes. Recently, gain-of-function mutations in SCN9A, the gene which encodes Na(v)1.7, have been linked to two human-inherited pain syndromes, inherited erythromelalgia and paroxysmal extreme pain disorder, while loss-of-function mutations in SCN9A have been linked to complete insensitivity to pain. Studies on firing properties of sensory neurons of dorsal root ganglia demonstrate that the effects of gain-of-function mutations in Na(v)1.7 on the excitability of these neurons depend on the presence of Na(v)1.8, which suggests a similar physiological interaction of these two channels in humans carrying the Na(v)1.7 pain mutation. These studies suggest that isoform-specific blockers of these channels or targeting of their modulators may provide novel approaches to treatment of pain.

  11. Neurotoxins and their binding areas on voltage-gated sodium channels

    Directory of Open Access Journals (Sweden)

    Marijke eStevens

    2011-11-01

    Full Text Available Voltage-gated Sodium Channels (VGSCs are large transmembrane proteins that conduct sodium ions across the membrane and by doing so they generate signals of communication between many kinds of tissues. They are responsible for the generation and propagation of action potentials in excitable cells, in close collaboration with other channels like potassium channels. Genetic defects in sodium channel genes therefore can cause a wide variety of diseases, generally called ‘channelopathies’.The first insights into the mechanism of action potentials and the involvement of sodium channels originated from Hodgkin and Huxley for which they were awarded the Nobel Prize in 1963. Until now, these concepts still form the basis for understanding the functioning of VGSCs. When VGSCs sense a sufficient change in membrane potential, they are activated and will generate a massive influx of sodium ions. Immediately after, channels will start inactivating and currents decrease. In the inactivated state channels stay refractory for any new stimulus and they must return to the closed state before being susceptible to any new depolarization. On the other hand, studies with neurotoxins like tetrodotoxin (TTX and saxitoxin (STX also contributed largely to our today’s understanding of the structure and function of ion channels and specifically of VGSCs. Moreover, neurotoxins acting on ion channels turned out to be valuable tools in the development of new drugs for the enormous range of diseases in which ion channels are involved. A recent example of a synthetic neurotoxin that made it to the market is ziconotide (Prialt®, Elan. The original peptide, -MVIIA, is derived from the cone snail Conus magus and now FDA/EMEA-approved for the management of severe chronic pain by blocking the N-type voltage-gated calcium channels in neurons.This review focuses on the current status of research on neurotoxins acting on VGSC, their contribution to further unravel the

  12. Inhibition of voltage-gated sodium channels by sumatriptan bioisosteres

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    Roberta eCarbonara

    2015-07-01

    Full Text Available Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7-1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously-expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogues, namely 20b, (R-31b, and (S-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R-31b and (S-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors.

  13. Inhibition of voltage-gated sodium channels by sumatriptan bioisosteres.

    Science.gov (United States)

    Carbonara, Roberta; Carocci, Alessia; Roussel, Julien; Crescenzo, Giuseppe; Buonavoglia, Canio; Franchini, Carlo; Lentini, Giovanni; Camerino, Diana Conte; Desaphy, Jean-François

    2015-01-01

    Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7-1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogs, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors.

  14. The ?2? Subunit and Absence Epilepsy: Beyond Calcium Channels?

    National Research Council Canada - National Science Library

    Roberta Celli; Ines Santolini; Michela Guiducci; Gilles van Luijtelaar; Pasquale Parisi; Pasquale Striano; Roberto Gradini; Giuseppe Battaglia; Richard T. Ngomba; Ferdinando Nicoletti

    2017-01-01

    .... Activation of T-type voltage-sensitive calcium channels (VSCCs) contributes to the pathological oscillatory activity of this network, and some of the first-line drugs used in the treatment of absence epilepsy inhibit T-type calcium channels. The ?2...

  15. Modelling modal gating of ion channels with hierarchical Markov models.

    Science.gov (United States)

    Siekmann, Ivo; Fackrell, Mark; Crampin, Edmund J; Taylor, Peter

    2016-08-01

    Many ion channels spontaneously switch between different levels of activity. Although this behaviour known as modal gating has been observed for a long time it is currently not well understood. Despite the fact that appropriately representing activity changes is essential for accurately capturing time course data from ion channels, systematic approaches for modelling modal gating are currently not available. In this paper, we develop a modular approach for building such a model in an iterative process. First, stochastic switching between modes and stochastic opening and closing within modes are represented in separate aggregated Markov models. Second, the continuous-time hierarchical Markov model, a new modelling framework proposed here, then enables us to combine these components so that in the integrated model both mode switching as well as the kinetics within modes are appropriately represented. A mathematical analysis reveals that the behaviour of the hierarchical Markov model naturally depends on the properties of its components. We also demonstrate how a hierarchical Markov model can be parametrized using experimental data and show that it provides a better representation than a previous model of the same dataset. Because evidence is increasing that modal gating reflects underlying molecular properties of the channel protein, it is likely that biophysical processes are better captured by our new approach than in earlier models.

  16. 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.

  17. Analyses of Short Channel Effects of Single-Gate and Double-Gate Graphene Nanoribbon Field Effect Transistors

    Directory of Open Access Journals (Sweden)

    Hojjatollah Sarvari

    2016-01-01

    Full Text Available Short channel effects of single-gate and double-gate graphene nanoribbon field effect transistors (GNRFETs are studied based on the atomistic pz orbital model for the Hamiltonian of graphene nanoribbon using the nonequilibrium Green’s function formalism. A tight-binding Hamiltonian with an atomistic pz orbital basis set is used to describe the atomistic details in the channel of the GNRFETs. We have investigated the vital short channel effect parameters such as Ion and Ioff, the threshold voltage, the subthreshold swing, and the drain induced barrier lowering versus the channel length and oxide thickness of the GNRFETs in detail. The gate capacitance and the transconductance of both devices are also computed in order to calculate the intrinsic cut-off frequency and switching delay of GNRFETs. Furthermore, the effects of doping of the channel on the threshold voltage and the frequency response of the double-gate GNRFET are discussed. We have shown that the single-gate GNRFET suffers more from short channel effects if compared with those of the double-gate structure; however, both devices have nearly the same cut-off frequency in the range of terahertz. This work provides a collection of data comparing different features of short channel effects of the single gate with those of the double gate GNRFETs. The results give a very good insight into the devices and are very useful for their digital applications.

  18. Analytical models of calcium binding in a calcium channel

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jinn-Liang [Department of Applied Mathematics, National Hsinchu University of Education, Hsinchu 300, Taiwan (China); Eisenberg, Bob [Department of Molecular Biophysics and Physiology, Rush University, Chicago, Illinois 60612 (United States)

    2014-08-21

    The anomalous mole fraction effect of L-type calcium channels is analyzed using a Fermi like distribution with the experimental data of Almers and McCleskey [J. Physiol. 353, 585 (1984)] and the atomic resolution model of Lipkind and Fozzard [Biochemistry 40, 6786 (2001)] of the selectivity filter of the channel. Much of the analysis is algebraic, independent of differential equations. The Fermi distribution is derived from the configuration entropy of ions and water molecules with different sizes, different valences, and interstitial voids between particles. It allows us to calculate potentials and distances (between the binding ion and the oxygen ions of the glutamate side chains) directly from the experimental data using algebraic formulas. The spatial resolution of these results is comparable with those of molecular models, but of course the accuracy is no better than that implied by the experimental data. The glutamate side chains in our model are flexible enough to accommodate different types of binding ions in different bath conditions. The binding curves of Na{sup +} and Ca{sup 2+} for [CaCl{sub 2}] ranging from 10{sup −8} to 10{sup −2} M with a fixed 32 mM background [NaCl] are shown to agree with published Monte Carlo simulations. The Poisson-Fermi differential equation—that includes both steric and correlation effects—is then used to obtain the spatial profiles of energy, concentration, and dielectric coefficient from the solvent region to the filter. The energy profiles of ions are shown to depend sensitively on the steric energy that is not taken into account in the classical rate theory. We improve the rate theory by introducing a steric energy that lumps the effects of excluded volumes of all ions and water molecules and empty spaces between particles created by Lennard-Jones type and electrostatic forces. We show that the energy landscape varies significantly with bath concentrations. The energy landscape is not constant.

  19. The NH2terminus regulates voltage-dependent gating of CALHM ion channels.

    Science.gov (United States)

    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. STIM and calcium channel complexes in cancer.

    Science.gov (United States)

    Jardin, Isaac; Rosado, Juan A

    2016-06-01

    The ion Ca(2+) is a ubiquitous second messenger that mediates a variety of cellular functions. Dysfunction of the mechanisms involved in Ca(2+) homeostasis underlies a number of pathological processes, including cancer. Store-operated Ca(2+) entry (SOCE) is a major mechanism for Ca(2+) entry modulated by the intracellular Ca(2+) stores. The Ca(2+)-selective store-operated current (ICRAC) is mediated by the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and the store-operated Ca(2+) (SOC) channel Orai1, while other non-selective cation currents (ISOC) involves the participation of members of the canonical transient receptor potential (TRPC) channel family, including TRPC1. Distinct isoforms of the key components of SOCE have been described in mammalian cells, STIM1 and 2, Orai1-3 and TRPC1-7. In cancer cells, SOCE has been reported to play an important role in cell cycle progression and proliferation, migration, metastasis and evasion of apoptosis. Changes in the expression of the key elements of SOCE and Ca(2+) homeostasis remodeling have been account to play important roles in the phenotypic changes observed in transformed cells. Despite there are differences in the expression level of the molecular components of SOCE, as well as in the relevance of the STIM, Orai and TRPC isoforms in SOCE and tumorigenesis among cancer cell types, there is a body of evidence supporting an important role for SOCE underlying the phenotypic modifications of cancer cells that propose STIM and the SOC channels as suitable candidate targets for future prognostic or therapeutic strategies. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Modulation of L-type calcium channels by sodium ions.

    OpenAIRE

    Balke, C W; Wier, W G

    1992-01-01

    It is universally believed that the removal of external sodium ions is without effect on calcium current. We now report that in enzymatically isolated guinea pig ventricular cells, the replacement of external sodium ions with certain other cations causes a 3- to 6-fold increase in peak L-type calcium current. The increase in current is reversibly blocked by L-type calcium-channel antagonists, not mediated by changes in internal calcium, and is inhibited by intracellular 5'-adenylyl imidodipho...

  2. 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

  3. 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).…

  4. Identification of cyclic nucleotide gated channels using regular expressions

    KAUST Repository

    Zelman, Alice K.

    2013-09-03

    Cyclic nucleotide-gated channels (CNGCs) are nonselective cation channels found in plants, animals, and some bacteria. They have a six-transmembrane/one- pore structure, a cytosolic cyclic nucleotide-binding domain, and a cytosolic calmodulin-binding domain. Despite their functional similarities, the plant CNGC family members appear to have different conserved amino acid motifs within corresponding functional domains than animal and bacterial CNGCs do. Here we describe the development and application of methods employing plant CNGC-specific sequence motifs as diagnostic tools to identify novel candidate channels in different plants. These methods are used to evaluate the validity of annotations of putative orthologs of CNGCs from plant genomes. The methods detail how to employ regular expressions of conserved amino acids in functional domains of annotated CNGCs and together with Web tools such as PHI-BLAST and ScanProsite to identify novel candidate CNGCs in species including Physcomitrella patens. © Springer Science+Business Media New York 2013.

  5. The gating cycle of a K+ channel at atomic resolution

    Energy Technology Data Exchange (ETDEWEB)

    Cuello, Luis G. [Center for Membrane Protein Research, Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, United States; Cortes, D. Marien [Center for Membrane Protein Research, Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, United States; Perozo, Eduardo [Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, United States

    2017-11-22

    C-type inactivation in potassium channels helps fine-tune long-term channel activity through conformational changes at the selectivity filter. Here, through the use of cross-linked constitutively open constructs, we determined the structures of KcsA’s mutants that stabilize the selectivity filter in its conductive (E71A, at 2.25 Å) and deep C-type inactivated (Y82A at 2.4 Å) conformations. These structural snapshots represent KcsA’s transient open-conductive (O/O) and the stable open deep C-type inactivated states (O/I), respectively. The present structures provide an unprecedented view of the selectivity filter backbone in its collapsed deep C-type inactivated conformation, highlighting the close interactions with structural waters and the local allosteric interactions that couple activation and inactivation gating. Together with the structures associated with the closed-inactivated state (C/I) and in the well-known closed conductive state (C/O), this work recapitulates, at atomic resolution, the key conformational changes of a potassium channel pore domain as it progresses along its gating cycle.

  6. The crystal structure of a voltage-gated sodium channel.

    Science.gov (United States)

    Payandeh, Jian; Scheuer, Todd; Zheng, Ning; Catterall, William A

    2011-07-10

    Voltage-gated sodium (Na(V)) channels initiate electrical signalling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity and drug block is unknown. Here we report the crystal structure of a voltage-gated Na(+) channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage sensors at 2.7 Å resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures indicate that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ∼4.6 Å wide, and water filled, with four acidic side chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na(+) selectivity through partial dehydration via direct interaction with glutamate side chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. This structure provides the template for understanding electrical signalling in excitable cells and the actions of drugs used for pain, epilepsy and cardiac arrhythmia at the atomic level.

  7. Oxidative gating of water channels (aquaporins) in corn roots.

    Science.gov (United States)

    Ye, Qing; Steudle, Ernst

    2006-04-01

    An oxidative gating of water channels (aquaporins: AQPs) was observed in roots of corn seedlings as already found for the green alga Chara corallina. In the presence of 35 mM hydrogen peroxide (H2O2)--a precursor of hydroxyl radicals (*OH)--half times of water flow (as measured with the aid of pressure probes) increased at the level of both entire roots and individual cortical cells by factors of three and nine, respectively. This indicated decreases in the hydrostatic hydraulic conductivity of roots (Lp(hr)) and of cells (Lp(h)) by the same factors. Unlike other stresses, the plant hormone abscisic acid (ABA) had no ameliorative effect either on root LP(hr) or on cell Lp(h) when AQPs were inhibited by oxidative stress. Closure of AQPs reduced the permeability of acetone by factors of two in roots and 1.5 in cells. This indicated that AQPs were not ideally selective for water but allowed the passage of the organic solute acetone. In the presence of H2O2, channel closure caused anomalous (negative) osmosis at both the root and the cell level. This was interpreted by the fact that in the case of the rapidly permeating solute acetone, channel closure caused the solute to move faster than the water and the reflection coefficient (sigma s) reversed its sign. When H2O2 was removed from the medium, the effects were reversible, again at both the root and the cell level. The results provide evidence of oxidative gating of AQPs, which leads on to inhibition of water uptake by the roots. Possible mechanisms of the oxidative gating of AQPs induced by H2O2 (*OH) are discussed.

  8. Connexin domains relevant to the chemical gating of gap junction channels

    Directory of Open Access Journals (Sweden)

    C. Peracchia

    1997-05-01

    Full Text Available Most cells exchange ions and small metabolites via gap junction channels. These channels are made of two hemichannels (connexons, each formed by the radial arrangement of six connexin (Cx proteins. Connexins span the bilayer four times (M1-M4 and have both amino- and carboxy-termini (NT, CT at the cytoplasmic side of the membrane, forming two extracellular loops (E1, E2 and one inner (IL loop. The channels are regulated by gates that close with cytosolic acidification (e.g., CO2 treatment or increased calcium concentration, possibly via calmodulin activation. Although gap junction regulation is still unclear, connexin domains involved in gating are being defined. We have recently focused on the CO2 gating sensitivity of Cx32, Cx38 and various mutants and chimeras expressed in Xenopus oocytes and studied by double voltage clamp. Cx32 is weakly sensitive to CO2, whereas Cx38 is highly sensitive. A Cx32 chimera containing the second half of the inner loop (IL2 of Cx38 was as sensitive to CO2 as Cx38, indicating that this domain plays an important role. Deletion of CT by 84% did not affect CO2 sensitivity, but replacement of 5 arginines (R with sparagines (N at the beginning of CT (C1 greatly enhanced the CO2 sensitivity of Cx32. This suggests that whereas most of CT is irrelevant, positive charges of C1 maintain the CO2 sensitivity of Cx32 low. As a hypothesis we have proposed a model that involves charge interaction between negative residues of the beginning of IL1 and positive residues of either C1 or IL2. Open and closed channels would result from IL1-C1 and IL1-IL2 interactions, respectively

  9. Calcium channels in the brain as targets for the calcium-channel modulators used in the treatment of neurological disorders

    NARCIS (Netherlands)

    Peters, Thies; WILFFERT, B; VANHOUTTE, PM; VANZWIETEN, PA

    1991-01-01

    Recent investigations of calcium channels in brain cells by voltage-clamp techniques have revealed that, in spite of electrophysiological similarities, the pharmacological properties of these channels differ considerably from channels in peripheral tissues, e.g., heart and smooth muscle. Therefore,

  10. Use of voltage clamp fluorimetry in understanding potassium channel gating: a review of Shaker fluorescence data.

    Science.gov (United States)

    Horne, A J; Fedida, D

    2009-06-01

    Voltage clamp fluorimetry (VCF) utilizes fluorescent probes that covalently bind to cysteine residues introduced into proteins and emit light as a function of their environment. Measurement of this emitted light during membrane depolarization reveals changes in the emission level as the environment of the labelled residue changes. This allows for the correlation of channel gating events with movement of specific protein moieties, at nanosecond time resolution. Since the pioneering use of this technique to investigate Shaker potassium channel activation movements, VCF has become an invaluable technique used to understand ion channel gating. This review summarizes the theory and some of the data on the application of the VCF technique. Although its usage has expanded beyond voltage-gated potassium channels and VCF is now used in a number of other voltage- and ligand-gated channels, we will focus on studies conducted in Shaker potassium channels, and what they have told us about channel activation and inactivation gating.

  11. The sialic acid component of the beta1 subunit modulates voltage-gated sodium channel function.

    Science.gov (United States)

    Johnson, Daniel; Montpetit, Marty L; Stocker, Patrick J; Bennett, Eric S

    2004-10-22

    Voltage-gated sodium channels (Nav) are responsible for initiation and propagation of nerve, skeletal muscle, and cardiac action potentials. Nav are composed of a pore-forming alpha subunit and often one to several modulating beta subunits. Previous work showed that terminal sialic acid residues attached to alpha subunits affect channel gating. Here we show that the fully sialylated beta1 subunit induces a uniform, hyperpolarizing shift in steady state and kinetic gating of the cardiac and two neuronal alpha subunit isoforms. Under conditions of reduced sialylation, the beta1-induced gating effect was eliminated. Consistent with this, mutation of beta1 N-glycosylation sites abolished all effects of beta1 on channel gating. Data also suggest an interaction between the cis effect of alpha sialic acids and the trans effect of beta1 sialic acids on channel gating. Thus, beta1 sialic acids had no effect gating on the of the heavily glycosylated skeletal muscle alpha subunit. However, when glycosylation of the skeletal muscle alpha subunit was reduced through chimeragenesis such that alpha sialic acids did not impact gating, beta1 sialic acids caused a significant hyperpolarizing shift in channel gating. Together, the data indicate that beta1 N-linked sialic acids can modulate Nav gating through an apparent saturating electrostatic mechanism. A model is proposed in which a spectrum of differentially sialylated Nav can directly modulate channel gating, thereby impacting cardiac, skeletal muscle, and neuronal excitability.

  12. 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.

  13. Scientific evidence contradicts findings and assumptions of Canadian Safety Panel 6: microwaves act through voltage-gated calcium channel activation to induce biological impacts at non-thermal levels, supporting a paradigm shift for microwave/lower frequency electromagnetic field action.

    Science.gov (United States)

    Pall, Martin L

    2015-01-01

    This review considers a paradigm shift on microwave electromagnetic field (EMF) action from only thermal effects to action via voltage-gated calcium channel (VGCC) activation. Microwave/lower frequency EMFs were shown in two dozen studies to act via VGCC activation because all effects studied were blocked by calcium channel blockers. This mode of action was further supported by hundreds of studies showing microwave changes in calcium fluxes and intracellular calcium [Ca2+]i signaling. The biophysical properties of VGCCs/similar channels make them particularly sensitive to low intensity, non-thermal EMF exposures. Non-thermal studies have shown that in most cases pulsed fields are more active than are non-pulsed fields and that exposures within certain intensity windows have much large biological effects than do either lower or higher intensity exposures; these are both consistent with a VGCC role but inconsistent with only a heating/thermal role. Downstream effects of VGCC activation include calcium signaling, elevated nitric oxide (NO), NO signaling, peroxynitrite, free radical formation, and oxidative stress. Downstream effects explain repeatedly reported biological responses to non-thermal exposures: oxidative stress; single and double strand breaks in cellular DNA; cancer; male and female infertility; lowered melatonin/sleep disruption; cardiac changes including tachycardia, arrhythmia, and sudden cardiac death; diverse neuropsychiatric effects including depression; and therapeutic effects. Non-VGCC non-thermal mechanisms may occur, but none have been shown to have effects in mammals. Biologically relevant safety standards can be developed through studies of cell lines/cell cultures with high levels of different VGCCs, measuring their responses to different EMF exposures. The 2014 Canadian Report by a panel of experts only recognizes thermal effects regarding safety standards for non-ionizing radiation exposures. Its position is therefore contradicted by each

  14. Understanding alternative splicing of Cav1.2 calcium channels for a new approach towards individualized medicine

    Science.gov (United States)

    Liao, Ping; Soong, Tuck Wah

    2010-01-01

    Calcium channel blockers (CCBs) are widely used to treat cardiovascular diseases such as hypertension, angina pectoris, hypertrophic cardiomyopathy, and supraventricular tachycardia. CCBs selectively inhibit the inward flow of calcium ions through voltage-gated calcium channels, particularly Cav1.2, that are expressed in the cardiovascular system. Changes to the molecular structure of Cav1.2 channels could affect sensitivity of the channels to blockade by CCBs. Recently, extensive alternative splicing was found in Cav1.2 channels that generated wide phenotypic variations. Cardiac and smooth muscles express slightly different, but functionally important Cav1.2 splice variants. Alternative splicing could also modulate the gating properties of the channels and giving rise to different responses to inhibition by CCBs. Importantly, alternative splicing of Cav1.2 channels may play an important role to influence the outcome of many cardiovascular disorders. Therefore, the understanding of how alternative splicing impacts Cav1.2 channels pharmacology in various diseases and different organs may provide the possibility for individualized therapy with minimal side effects. PMID:23554629

  15. Voltage-gated calcium flux mediatesEscherichia colimechanosensation.

    Science.gov (United States)

    Bruni, Giancarlo N; Weekley, R Andrew; Dodd, Benjamin J T; Kralj, Joel M

    2017-08-29

    Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli , including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.

  16. Calcium binding and voltage gating in Cx46 hemichannels.

    Science.gov (United States)

    Pinto, Bernardo I; Pupo, Amaury; García, Isaac E; Mena-Ulecia, Karel; Martínez, Agustín D; Latorre, Ramón; Gonzalez, Carlos

    2017-11-20

    The opening of connexin (Cx) hemichannels in the membrane is tightly regulated by calcium (Ca 2+ ) and membrane voltage. Electrophysiological and atomic force microscopy experiments indicate that Ca 2+ stabilizes the hemichannel closed state. However, structural data show that Ca 2+ binding induces an electrostatic seal preventing ion transport without significant structural rearrangements. In agreement with the closed-state stabilization hypothesis, we found that the apparent Ca 2+ sensitivity is increased as the voltage is made more negative. Moreover, the voltage and Ca 2+ dependence of the channel kinetics indicate that the voltage sensor movement and Ca 2+ binding are allosterically coupled. An allosteric kinetic model in which the Ca 2+ decreases the energy necessary to deactivate the voltage sensor reproduces the effects of Ca 2+ and voltage in Cx46 hemichannels. In agreement with the model and suggesting a conformational change that narrows the pore, Ca 2+ inhibits the water flux through Cx hemichannels. We conclude that Ca 2+ and voltage act allosterically to stabilize the closed conformation of Cx46 hemichannels.

  17. An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells.

    Science.gov (United States)

    Koch, Peter; Herzig, Stefan; Matthes, Jan

    Pore-forming subunits of voltage gated calcium channels (VGCC) are large membrane proteins (260kDa) containing 24 transmembrane domains. Despite transfection with viral promoter driven vectors, biochemical analysis of VGCC is often hampered by rather low expression levels in heterologous systems rendering VGCC challenging targets. Especially in immunofluorescent detection, calcium channels are demanding proteins. We provide an expert step-by-step protocol with adapted conditions for handling procedures (tsA-201 cell culture, transient transfection, incubation time and temperature at 28°C or 37°C and immunostaining) to address the L-type calcium-channel pore Cav1.2 in an immunofluorescent approach. We performed immunocytochemical analysis of Cav1.2 expression at single-cell level in combination with detection of different markers for cellular organelles. We show confluency levels and shapes of tsA-201 cells at different time points during an experiment. Our experiments reveal sufficient levels of Cav1.2 protein and a correct Cav1.2 expression pattern in polygonal shaped cells already 12h after transfection. A sequence of elaborated protocol modifications allows subcellular localization analysis of Cav1.2 in an immunocytochemical approach. We provide a protocol that may be used to achieve insights into physiological and pathophysiological processes involving voltage gated calcium channels. Our protocol may be used for expression analysis of other challenging proteins and efficient overexpression may be exploited in related biochemical techniques requiring immunolabels. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. A Leucine Zipper Motif Essential for Gating of Hyperpolarization-activated Channels*

    Science.gov (United States)

    Wemhöner, Konstantin; Silbernagel, Nicole; Marzian, Stefanie; Netter, Michael F.; Rinné, Susanne; Stansfeld, Phillip J.; Decher, Niels

    2012-01-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are pacemakers in cardiac myocytes and neurons. Although their membrane topology closely resembles that of voltage-gated K+ channels, the mechanism of their unique gating behavior in response to hyperpolarization is still poorly understood. We have identified a highly conserved leucine zipper motif in the S5 segment of HCN family members. In order to study the role of this motif for channel function, the leucine residues of the zipper were individually mutated to alanine, arginine, or glutamine residues. Leucine zipper mutants traffic to the plasma membrane, but the channels lose their sensitivity to open upon hyperpolarization. Thus, our data indicate that the leucine zipper is an important molecular determinant for hyperpolarization-activated channel gating. Residues of the leucine zipper interact with the adjacent S6 segment of the channel. This interaction is essential for voltage-dependent gating of the channel. The lower part of the leucine zipper, at the intracellular mouth of the channel, is important for stabilizing the closed state. Mutations at these sites increase current amplitudes or result in channels with deficient closing and increased min-Po. Our data are further supported by homology models of the open and closed state of the HCN2 channel pore. Thus, we conclude that the leucine zipper of HCN channels is a major determinant for hyperpolarization-activated channel gating. PMID:23048023

  19. 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 diseas...

  20. A leucine zipper motif essential for gating of hyperpolarization-activated channels.

    Science.gov (United States)

    Wemhöner, Konstantin; Silbernagel, Nicole; Marzian, Stefanie; Netter, Michael F; Rinné, Susanne; Stansfeld, Phillip J; Decher, Niels

    2012-11-23

    It is poorly understood how hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) function. We have identified a leucine zipper in the S5 segment of HCNs, regulating hyperpolarization-activated and instantaneous current components. The leucine zipper is essential for HCN channel gating. The identification and functional characterization of the leucine zipper is an important step toward the understanding of HCN channel function. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are pacemakers in cardiac myocytes and neurons. Although their membrane topology closely resembles that of voltage-gated K(+) channels, the mechanism of their unique gating behavior in response to hyperpolarization is still poorly understood. We have identified a highly conserved leucine zipper motif in the S5 segment of HCN family members. In order to study the role of this motif for channel function, the leucine residues of the zipper were individually mutated to alanine, arginine, or glutamine residues. Leucine zipper mutants traffic to the plasma membrane, but the channels lose their sensitivity to open upon hyperpolarization. Thus, our data indicate that the leucine zipper is an important molecular determinant for hyperpolarization-activated channel gating. Residues of the leucine zipper interact with the adjacent S6 segment of the channel. This interaction is essential for voltage-dependent gating of the channel. The lower part of the leucine zipper, at the intracellular mouth of the channel, is important for stabilizing the closed state. Mutations at these sites increase current amplitudes or result in channels with deficient closing and increased min-P(o). Our data are further supported by homology models of the open and closed state of the HCN2 channel pore. Thus, we conclude that the leucine zipper of HCN channels is a major determinant for hyperpolarization-activated channel gating.

  1. Shellfish Toxins Targeting Voltage-Gated Sodium Channels

    Directory of Open Access Journals (Sweden)

    Fan Zhang

    2013-11-01

    Full Text Available Voltage-gated sodium channels (VGSCs play a central role in the generation and propagation of action potentials in excitable neurons and other cells and are targeted by commonly used local anesthetics, antiarrhythmics, and anticonvulsants. They are also common targets of neurotoxins including shellfish toxins. Shellfish toxins are a variety of toxic secondary metabolites produced by prokaryotic cyanobacteria and eukaryotic dinoflagellates in both marine and fresh water systems, which can accumulate in marine animals via the food chain. Consumption of shellfish toxin-contaminated seafood may result in potentially fatal human shellfish poisoning. This article provides an overview of the structure, bioactivity, and pharmacology of shellfish toxins that act on VGSCs, along with a brief discussion on their pharmaceutical potential for pain management.

  2. Maximum Caliber Analysis of Ion-Channel Gating

    Science.gov (United States)

    Campbell, Roy

    2011-10-01

    The principle of maximum caliber, MaxCal, is a generalization to nonequilibrium statistical mechanics of the principle of maximum entropy, MaxEnt. E. T. Jaynes introduced the MaxEnt approach to equilibrium statistical mechanics in 1957 and its MaxCal generalization in 1980. MaxCal has recently been used to derive dynamical laws of transport, analyze single particle two-state dynamics, and study few state models of non-equilibrium processes. We use MaxCal to analyze hidden Markov models of ion-channel gating and make logical inferences concerning the underlying dynamics. MaxCal is used to determine model parameters; test the adequacy of a model; and predict unmeasured quantities from the trajectory probability distribution. Results will be given for a MaxCal analysis of inositol trisphosphate receptor patch clamp data.

  3. Biophysical Adaptations of Prokaryotic Voltage-Gated Sodium Channels.

    Science.gov (United States)

    Vien, T N; DeCaen, P G

    2016-01-01

    This chapter describes the adaptive features found in voltage-gated sodium channels (NaVs) of prokaryotes and eukaryotes. These two families are distinct, having diverged early in evolutionary history but maintain a surprising degree of convergence in function. While prokaryotic NaVs are required for growth and motility, eukaryotic NaVs selectively conduct fast electrical currents for short- and long-range signaling across cell membranes in mammalian organs. Current interest in prokaryotic NaVs is stoked by their resolved high-resolution structures and functional features which are reminiscent of eukaryotic NaVs. In this chapter, comparisons between eukaryotic and prokaryotic NaVs are made to highlight the shared and unique aspects of ion selectivity, voltage sensitivity, and pharmacology. Examples of prokaryotic and eukaryotic NaV convergent evolution will be discussed within the context of their structural features. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Reporting sodium channel activity using calcium flux: pharmacological promiscuity of cardiac Nav1.5.

    Science.gov (United States)

    Zhang, Hongkang; Zou, Beiyan; Du, Fang; Xu, Kaiping; Li, Min

    2015-02-01

    Voltage-gated sodium (Nav) channels are essential for membrane excitability and represent therapeutic targets for treating human diseases. Recent reports suggest that these channels, e.g., Nav1.3 and Nav1.5, are inhibited by multiple structurally distinctive small molecule drugs. These studies give reason to wonder whether these drugs collectively target a single site or multiple sites in manifesting such pharmacological promiscuity. We thus investigate the pharmacological profile of Nav1.5 through systemic analysis of its sensitivity to diverse compound collections. Here, we report a dual-color fluorescent method that exploits a customized Nav1.5 [calcium permeable Nav channel, subtype 5 (SoCal5)] with engineered-enhanced calcium permeability. SoCal5 retains wild-type (WT) Nav1.5 pharmacological profiles. WT SoCal5 and SoCal5 with the local anesthetics binding site mutated (F1760A) could be expressed in separate cells, each with a different-colored genetically encoded calcium sensor, which allows a simultaneous report of compound activity and site dependence. The pharmacological profile of SoCal5 reveals a hit rate (>50% inhibition) of around 13% at 10 μM, comparable to that of hERG. The channel activity is susceptible to blockage by known drugs and structurally diverse compounds. The broad inhibition profile is highly dependent on the F1760 residue in the inner cavity, which is a residue conserved among all nine subtypes of Nav channels. Both promiscuity and dependence on F1760 seen in Nav1.5 were replicated in Nav1.4. Our evidence of a broad inhibition profile of Nav channels suggests a need to consider off-target effects on Nav channels. The site-dependent promiscuity forms a foundation to better understand Nav channels and compound interactions. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  5. Voltage-Gated Ion Channels in Cancer Cell Proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Rao, Vidhya R.; Perez-Neut, Mathew [Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago 2160 S. 1st Ave, Maywood, IL 60153 (United States); Kaja, Simon [Department of Ophthalmology and Vision Research Center, School of Medicine, University of Missouri-Kansas City, 2411 Holmes St., Kansas City, MO 64108 (United States); Gentile, Saverio, E-mail: sagentile@luc.edu [Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago 2160 S. 1st Ave, Maywood, IL 60153 (United States)

    2015-05-22

    Changes of the electrical charges across the surface cell membrane are absolutely necessary to maintain cellular homeostasis in physiological as well as in pathological conditions. The opening of ion channels alter the charge distribution across the surface membrane as they allow the diffusion of ions such as K{sup +}, Ca{sup ++}, Cl{sup −}, Na{sup +}. Traditionally, voltage-gated ion channels (VGIC) are known to play fundamental roles in controlling rapid bioelectrical signaling including action potential and/or contraction. However, several investigations have revealed that these classes of proteins can also contribute significantly to cell mitotic biochemical signaling, cell cycle progression, as well as cell volume regulation. All these functions are critically important for cancer cell proliferation. Interestingly, a variety of distinct VGICs are expressed in different cancer cell types, including metastasis but not in the tissues from which these tumors were generated. Given the increasing evidence suggesting that VGIC play a major role in cancer cell biology, in this review we discuss the role of distinct VGIC in cancer cell proliferation and possible therapeutic potential of VIGC pharmacological manipulation.

  6. Calcium-activated SK channels control firing regularity by modulating sodium channel availability in midbrain dopamine neurons.

    Science.gov (United States)

    Iyer, Rajeshwari; Ungless, Mark A; Faisal, Aldo A

    2017-07-12

    Dopamine neurons in the substantia nigra pars compacta and ventral tegmental area regulate behaviours such as reward-related learning, and motor control. Dysfunction of these neurons is implicated in Schizophrenia, addiction to drugs, and Parkinson's disease. While some dopamine neurons fire single spikes at regular intervals, others fire irregular single spikes interspersed with bursts. Pharmacological inhibition of calcium-activated potassium (SK) channels increases the variability in their firing pattern, sometimes also increasing the number of spikes fired in bursts, indicating that SK channels play an important role in maintaining dopamine neuron firing regularity and burst firing. However, the exact mechanisms underlying these effects are still unclear. Here, we develop a biophysical model of a dopamine neuron incorporating ion channel stochasticity that enabled the analysis of availability of ion channels in multiple states during spiking. We find that decreased firing regularity is primarily due to a significant decrease in the AHP that in turn resulted in a reduction in the fraction of available voltage-gated sodium channels due to insufficient recovery from inactivation. Our model further predicts that inhibition of SK channels results in a depolarisation of action potential threshold along with an increase in its variability.

  7. ALTERNATIVE EQUATIONS FOR DYNAMIC BEHAVIOR OF IONIC CHANNEL ACTIVATION AND INACTIVATION GATES

    Directory of Open Access Journals (Sweden)

    Mahmut ÖZER

    2003-03-01

    Full Text Available In this paper, alternative equations for dynamics of ionic channel activation and inactivation gates are proposed based on the path probability method. Dynamic behavior of a voltage-gated ionic channel is modeled by the conventional Hodgkin-Huxley (H-H mathematical formalism. In that model, conductance of the channel is defined in terms of activation and inactivation gates. Dynamics of the activation and inactivation gates is modeled by first-order differential equations dependent on the gate variable and the membrane potential. In the new approach proposed in this study, dynamic behavior of activation and inactivation gates is modeled by a firstorder differential equation dependent on internal energy and membrane potential by using the path probability method which is widely used in statistical physics. The new model doesn't require the time constant and steadystate values which are used explicitly in the H-H model. The numerical results show validity of the proposed method.

  8. Differential effect of brief electrical stimulation on voltage-gated potassium channels.

    Science.gov (United States)

    Cameron, Morven A; Al Abed, Amr; Buskila, Yossi; Dokos, Socrates; Lovell, Nigel H; Morley, John W

    2017-05-01

    Electrical stimulation of neuronal tissue is a promising strategy to treat a variety of neurological disorders. The mechanism of neuronal activation by external electrical stimulation is governed by voltage-gated ion channels. This stimulus, typically brief in nature, leads to membrane potential depolarization, which increases ion flow across the membrane by increasing the open probability of these voltage-gated channels. In spiking neurons, it is activation of voltage-gated sodium channels (Na V channels) that leads to action potential generation. However, several other types of voltage-gated channels are expressed that also respond to electrical stimulation. In this study, we examine the response of voltage-gated potassium channels (K V channels) to brief electrical stimulation by whole cell patch-clamp electrophysiology and computational modeling. We show that nonspiking amacrine neurons of the retina exhibit a large variety of responses to stimulation, driven by different K V -channel subtypes. Computational modeling reveals substantial differences in the response of specific K V -channel subtypes that is dependent on channel kinetics. This suggests that the expression levels of different K V -channel subtypes in retinal neurons are a crucial predictor of the response that can be obtained. These data expand our knowledge of the mechanisms of neuronal activation and suggest that K V -channel expression is an important determinant of the sensitivity of neurons to electrical stimulation. NEW & NOTEWORTHY This paper describes the response of various voltage-gated potassium channels (K V channels) to brief electrical stimulation, such as is applied during prosthetic electrical stimulation. We show that the pattern of response greatly varies between K V channel subtypes depending on activation and inactivation kinetics of each channel. Our data suggest that problems encountered when artificially stimulating neurons such as cessation in firing at high frequencies, or

  9. [Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

    Science.gov (United States)

    Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

    2012-07-01

    To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

  10. Alternative Splicing at C Terminus of CaV1.4 Calcium Channel Modulates Calcium-dependent Inactivation, Activation Potential, and Current Density

    Science.gov (United States)

    Tan, Gregory Ming Yeong; Yu, Dejie; Wang, Juejin; Soong, Tuck Wah

    2012-01-01

    The CaV1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type CaV1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native CaV1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the CaV1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the C terminus. The CaV1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ∼10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length CaV1.4. Naturally occurring alternative splice variants may in part contribute to the properties of the native CaV1.4 channels. PMID:22069316

  11. T-type calcium channels in synaptic plasticity.

    Science.gov (United States)

    Leresche, Nathalie; Lambert, Régis C

    2017-03-04

    The role of T-type calcium currents is rarely considered in the extensive literature covering the mechanisms of long-term synaptic plasticity. This situation reflects the lack of suitable T-type channel antagonists that till recently has hampered investigations of the functional roles of these channels. However, with the development of new pharmacological and genetic tools, a clear involvement of T-type channels in synaptic plasticity is starting to emerge. Here, we review a number of studies showing that T-type channels participate to numerous homo- and hetero-synaptic plasticity mechanisms that involve different molecular partners and both pre- and post-synaptic modifications. The existence of T-channel dependent and independent plasticity at the same synapse strongly suggests a subcellular localization of these channels and their partners that allows specific interactions. Moreover, we illustrate the functional importance of T-channel dependent synaptic plasticity in neocortex and thalamus.

  12. Structure of the voltage-gated K+ channel Eag1 reveals an alternative voltage sensing mechanism

    Science.gov (United States)

    Whicher, Jonathan R.; MacKinnon, Roderick

    2017-01-01

    Voltage-gated potassium channels (Kv) are gated by the movement of the transmembrane voltage sensor, which is coupled, through the helical S4–S5 linker, to the potassium pore. We determined the single-particle cryo-EM structure of mammalian Kv10.1 or Eag1, bound to the channel inhibitor calmodulin, at 3.78Å resolution. Unlike previous Kv structures, the S4–S5 linker of Eag1 is a 5-residue loop and the transmembrane segments are not domain swapped, suggesting an alternative mechanism of voltage-dependent gating. Additionally, the structure and position of the S4–S5 linker allows calmodulin to bind to the intracellular domains and close the potassium pore independent of voltage sensor position. The structure reveals an alternative gating mechanism for Kv channels and provides a template to further understand the gating properties of Eag1 and related channels. PMID:27516594

  13. Computer simulation of ion channel gating: the M(2) channel of influenza A virus in a lipid bilayer

    Science.gov (United States)

    Schweighofer, K. J.; Pohorille, A.

    2000-01-01

    The transmembrane fragment of the influenza virus M(2) protein forms a homotetrameric channel that transports protons. In this paper, we use molecular dynamics simulations to help elucidate the mechanism of channel gating by four histidines that occlude the channel lumen in the closed state. We test two competing hypotheses. In the "shuttle" mechanism, the delta nitrogen atom on the extracellular side of one histidine is protonated by the incoming proton, and, subsequently, the proton on the epsilon nitrogen atom is released on the opposite side. In the "water-wire" mechanism, the gate opens because of electrostatic repulsion between four simultaneously biprotonated histidines. This allows for proton transport along the water wire that penetrates the gate. For each system, composed of the channel embedded in a hydrated phospholipid bilayer, a 1.3-ns trajectory was obtained. It is found that the states involved in the shuttle mechanism, which contain either single-protonated histidines or a mixture of single-protonated histidines plus one biprotonated residue, are stable during the simulations. Furthermore, the orientations and dynamics of water molecules near the gate are conducive to proton transfer. In contrast, the fully biprotonated state is not stable. Additional simulations show that if only two histidines are biprotonated, the channel deforms but the gate remains closed. These results support the shuttle mechanism but not the gate-opening mechanism of proton gating in M(2).

  14. Regulation of CaV2 calcium channels by G protein coupled receptors

    Science.gov (United States)

    Zamponi, Gerald W.; Currie, Kevin P.M.

    2012-01-01

    Voltage gated calcium channels (Ca2+ channels) are key mediators of depolarization induced calcium influx into excitable cells, and thereby play pivotal roles in a wide array of physiological responses. This review focuses on the inhibition of CaV2 (N- and P/Q-type) Ca2+-channels by G protein coupled receptors (GPCRs), which exerts important autocrine/paracrine control over synaptic transmission and neuroendocrine secretion. Voltage-dependent inhibition is the most widespread mechanism, and involves direct binding of the G protein βγ dimer (Gβγ) to the α1 subunit of CaV2 channels. GPCRs can also recruit several other distinct mechanisms including phosphorylation, lipid signaling pathways, and channel trafficking that result in voltage-independent inhibition. Current knowledge of Gβγ-mediated inhibition is reviewed, including the molecular interactions involved, determinants of voltage-dependence, and crosstalk with other cell signaling pathways. A summary of recent developments in understanding the voltage-independent mechanisms prominent in sympathetic and sensory neurons is also included. PMID:23063655

  15. Diltiazem and verapamil preferentially block inactivated cardiac calcium channels.

    Science.gov (United States)

    Kanaya, S; Arlock, P; Katzung, B G; Hondeghem, L M

    1983-02-01

    Diltiazem has been proposed to act by blocking calcium channels of cardiac and smooth muscle since it has pharmacological [12-14] and clinical [10] effects that resemble those of verapamil, an agent that has been shown to block these channels [3]. However, block of the slow inward current by diltiazem has not been directly demonstrated. In fact, it has been suggested that diltiazem has an entirely different mechanism of action [7]. We therefore studied the blocking effects of diltiazem and verapamil on cardiac calcium channels by measuring the slow inward current in voltage-clamped ferret myocardium. Both drugs blocked the slow inward current in a use-dependent fashion, i.e. the block was enhanced by increased frequency of activating clamps and by more positive holding potentials. However, we found that short single activating clamps resulted in minimal block, whereas prolonging the clamp step progressively enhanced the blockade. Thus, a single long clamp caused as much blockade as a train of shorter pulses. These results demonstrate that diltiazem and verapamil block the slow inward current by binding to calcium channels in a state-dependent fashion, i.e. inactivated channels have a high affinity for the drugs, while rested and open channels have a lower affinity.

  16. Macroscopic kinetics of pentameric ligand gated ion channels: comparisons between two prokaryotic channels and one eukaryotic channel.

    Science.gov (United States)

    Laha, Kurt T; Ghosh, Borna; Czajkowski, Cynthia

    2013-01-01

    Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs). Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC) in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1β2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively) were relatively fast with time constants of 24.9 ± 5.1 ms and 1.2 ± 0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3 ± 0.3 s and deactivated even slower with a time constant of 4.6 ± 1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover mechanisms underlying p

  17. Na+ channel Nav1.9: in search of a gating mechanism.

    Science.gov (United States)

    Delmas, Patrick; Coste, Bertrand

    2003-02-01

    Voltage-gated Na(+) channels play key roles in generating and propagating action potentials. Their gating is believed to rely exclusively on changes in membrane potential. However, recent data from Blum, Kafitz and Konnerth provide direct evidence that the opening of Na(v)1.9, a member of the voltage-gated Na(+) channel family, is mediated by ligand binding rather than by voltage. This is arguably one of the most influential ideas brought to us in the history of the Na(+) channel field.

  18. TRPV5: an ingeniously controlled calcium channel.

    NARCIS (Netherlands)

    Groot, T. de; Bindels, R.J.M.; Hoenderop, J.G.J.

    2008-01-01

    Body Ca(2+) homeostasis is tightly controlled and slight disturbances in renal Ca(2+) reabsorption can lead to excessive urine Ca(2+) excretion and promote kidney stone formation. The epithelial Ca(2+) channel TRPV5 constitutes the rate-limiting step of active Ca(2+) reabsorption in the kidney.

  19. [Pain and analgesia : Mutations of voltage-gated sodium channels].

    Science.gov (United States)

    Eberhardt, M J; Leffler, A

    2017-02-01

    Voltage-gated sodium channels (Navs) are crucial for the generation and propagation of action potentials in all excitable cells, and therefore for the function of sensory neurons as well. Preclinical research over the past 20 years identified three Nav-isoforms in sensory neurons, namely Nav1.7, Nav1.8 and Nav1.9. A specific role for the function of nociceptive neurons was postulated for each. Whereas no selective sodium channel inhibitors have been established in the clinic so far, the relevance of all three isoforms regarding the pain sensitivity in humans is currently undergoing a remarkable verification through the translation of preclinical data into clinically manifest pictures. For the last ten years, Nav1.7 has been the main focus of clinical interest, as a large number of hereditary mutants were identified. The so-called "gain-of-function" mutations of Nav1.7 cause the pain syndromes hereditary erythromelalgia and paroxysmal extreme pain disorder. In addition, several Nav1.7 mutants were shown to be associated with small-fiber neuropathies. On the contrary, "loss-of-function" Nav1.7 mutants lead to a congenital insensitivity to pain. Recently, several gain-of-function mutations in Nav1.8 and Nav1.9 have been identified in patients suffering from painful peripheral neuropathies. However, another gain-of-function Nav1.9 mutation is associated with congenital insensitivity to pain. This review offers an overview of published work on painful Nav mutations with clinical relevance, and proposes possible consequences for the therapy of different pain symptoms resulting from these findings.

  20. Crystal structure of the channelrhodopsin light-gated cation channel

    Science.gov (United States)

    Kato, Hideaki E.; Zhang, Feng; Yizhar, Ofer; Ramakrishnan, Charu; Nishizawa, Tomohiro; Hirata, Kunio; Ito, Jumpei; Aita, Yusuke; Tsukazaki, Tomoya; Hayashi, Shigehiko; Hegemann, Peter; Maturana, Andrés D.; Ishitani, Ryuichiro; Deisseroth, Karl; Nureki, Osamu

    2014-01-01

    Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties. PMID:22266941

  1. Voltage-gated channel mechanosensitivity: Fact or Friction?

    Directory of Open Access Journals (Sweden)

    Catherine E Morris

    2011-05-01

    Full Text Available The heart is a continually active pulsatile fluid pump that generates forces by precisely timed and spaced engagement of contractile nano-machinery. This pump largely generates its own control signals, the most crucial of which involve precisely timed and spaced transmembrane ion fluxes. The timely opening and closing of the diverse voltage-gated channel (VGC sub-types is indispensible for pulsatile pumping with the appropriate rhythm. VGCs are large membrane proteins with four voltagesensors around a central ion-selective pore that opens and closes under the influence of membrane voltage (Fig 1. The operation of VGCs is tuned, in a secondary fashion, by the mechanical state of the bilayers in which they are embedded{18},{36},{44}. I focus here on the possibility that in the highly mechanically active environment of the myocardium and its vasculature, VGCs feel and respond to changing bilayer structures. Do they collectively transduce mechanical signals and tune rhythmicity? I suggest that tools now available could help answer this question. Some might wish to first peruse a section at the end of this essay summarizing a few fundamentals about VGC kinetics and about the modulation of their kinetics by mechanical factors; see Basic operation of a VGC.... That section is preceded by a list of abbreviations used in the essay and by lists of situations that might be expected to reversibly or irreversibly alter VGC-bearing membranes in the heart.

  2. Calcium channel blockers and cancer risk using the UK CPRD

    NARCIS (Netherlands)

    Grimaldi-Bensouda, Lamiae; De Groot, Mark; Reynolds, Robert; Klungel, Olaf; Rossignol, Michel

    2014-01-01

    Background: This study was part of the Pharmacoepidemiological Research on Outcomes (PROTECT) project which aims at monitoring of the benefit-risk of medicines in Europe. Few epidemiological studies have investigated the association between calcium channel blockers (CCB) and cancer, and have

  3. [Obtaining antibodies to 1,4-dihydropyridine calcium channel blockers].

    Science.gov (United States)

    Burkin, A A; Murkin, M A

    2008-01-01

    Immunization of rabbits with amlodipine conjugated with horseradish peroxidase resulted in raising polyclonal antibodies that allowed group determination of 1,4-dihydropyridine calcium channel blockers in aqueous solutions by ELISA with a sensitivity of 0.1 to 1.0 ng/ml for amlodipine, felodipine, nifedipine, and isradipine.

  4. Evaluation of nitrendipine -a new calcium channel blocker ...

    African Journals Online (AJOL)

    Nitrendipine (Baypress; Bayer-Miles), a new calcium channel blocker, was administered to 38 hypertensive patients in an oral dose of 20 mg once or twice daily. Both systolic and diastolic blood pressures were reduced to a clinically relevant extent within 2 hours of taking the medication. There was no loss of effect during ...

  5. Calcium-induced voltage gating in single conical nanopores.

    Science.gov (United States)

    Siwy, Zuzanna S; Powell, Matthew R; Petrov, Alexander; Kalman, Eric; Trautmann, Christina; Eisenberg, Robert S

    2006-08-01

    We examine time signals of ion current through single conically shaped nanopores in the presence of sub-millimolar concentrations of calcium ions. We show that calcium induces voltage-dependent ion current fluctuations in time in addition to the previously reported negative incremental resistance (Nano Lett. 2006, 6, 473-477). These current fluctuations occur on the millisecond time scale at voltages at which the effect of negative incremental resistance was observed. We explain the fluctuations as results of transient binding of calcium ions to carboxyl groups on the pore walls that cause transient changes in electric potential inside a conical nanopore. We support this explanation by recordings of ion current in the presence of manganese ions that bind to carboxyl groups 3 orders of magnitude more tightly than calcium ions. The system of a single conical nanopore with calcium ions is compared to a semiconductor device of a unijunction transistor in electronic circuits. A unijunction transistor also exhibits negative incremental resistance and current instabilities.

  6. 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...

  7. Effects of Calcium Ion, Calpains, and Calcium Channel Blockers on Retinitis Pigmentosa

    Directory of Open Access Journals (Sweden)

    Mitsuru Nakazawa

    2011-01-01

    Full Text Available Recent advances in molecular genetic studies have revealed many of the causative genes of retinitis pigmentosa (RP. These achievements have provided clues to the mechanisms of photoreceptor degeneration in RP. Apoptosis is known to be a final common pathway in RP and, therefore, a possible therapeutic target for photoreceptor rescue. However, apoptosis is not a single molecular cascade, but consists of many different reactions such as caspase-dependent and caspase-independent pathways commonly leading to DNA fractionation and cell death. The intracellular concentration of calcium ions is also known to increase in apoptosis. These findings suggest that calpains, one of the calcium-dependent proteinases, play some roles in the process of photoreceptor apoptosis and that calcium channel antagonists may potentially inhibit photoreceptor apoptosis. Herein, the effects of calpains and calcium channel antagonists on photoreceptor degeneration are reviewed.

  8. Comparison of short-circuit characteristics of trench gate and planar gate U-shaped channel SOI-LIGBTs

    Science.gov (United States)

    Zhang, Long; Zhu, Jing; Sun, Weifeng; Zhao, Minna; Huang, Xuequan; Chen, Jiajun; Shi, Longxing; Chen, Jian; Ding, Desheng

    2017-09-01

    Comparison of short-circuit (SC) characteristics of 500 V rated trench gate U-shaped channel (TGU) SOI-LIGBT and planar gate U-shaped channel (PGU) SOI-LIGBT is made for the first time in this paper. The on-state carrier profile of the TGU structure is reshaped by the dual trenches (a gate trench G1 and a hole barrier trench G2), which leads to a different conduction behavior from that of the PGU structure. The TGU structure exhibits a higher latchup immunity but a severer self-heating effect. At current density (JC) lattice temperature rise caused by the high current density at the emitter side in the TGU structure, the PGU exhibits a better JC-tSC trade-off at JC > 640 A/cm2. Comparison of layouts and fabrication processes are also made between the two types of devices.

  9. D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry.

    Science.gov (United States)

    Kisilevsky, Alexandra E; Mulligan, Sean J; Altier, Christophe; Iftinca, Mircea C; Varela, Diego; Tai, Chao; Chen, Lina; Hameed, Shahid; Hamid, Jawed; Macvicar, Brian A; Zamponi, Gerald W

    2008-05-22

    Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.

  10. Calcium channel-dependent molecular maturation of photoreceptor synapses.

    Directory of Open Access Journals (Sweden)

    Nawal Zabouri

    Full Text Available Several studies have shown the importance of calcium channels in the development and/or maturation of synapses. The Ca(V1.4(α(1F knockout mouse is a unique model to study the role of calcium channels in photoreceptor synapse formation. It features abnormal ribbon synapses and aberrant cone morphology. We investigated the expression and targeting of several key elements of ribbon synapses and analyzed the cone morphology in the Ca(V1.4(α(1F knockout retina. Our data demonstrate that most abnormalities occur after eye opening. Indeed, scaffolding proteins such as Bassoon and RIM2 are properly targeted at first, but their expression and localization are not maintained in adulthood. This indicates that either calcium or the Ca(V1.4 channel, or both are necessary for the maintenance of their normal expression and distribution in photoreceptors. Other proteins, such as Veli3 and PSD-95, also display abnormal expression in rods prior to eye opening. Conversely, vesicle related proteins appear normal. Our data demonstrate that the Ca(V1.4 channel is important for maintaining scaffolding proteins in the ribbon synapse but less vital for proteins related to vesicular release. This study also confirms that in adult retinae, cones show developmental features such as sprouting and synaptogenesis. Overall we present evidence that in the absence of the Ca(V1.4 channel, photoreceptor synapses remain immature and are unable to stabilize.

  11. Activation and inhibition of TMEM16A calcium-activated chloride channels.

    Directory of Open Access Journals (Sweden)

    Yu-Li Ni

    Full Text Available Calcium-activated chloride channels (CaCC encoded by family members of transmembrane proteins of unknown function 16 (TMEM16 have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca(2+-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1 by Ca(2+, Sr(2+, and Ba(2+, and discovered that Mg(2+ competes with Ca(2+ in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore-as revealed by the permeability ratios of these anions-appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA. On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1.

  12. Inhibitory effect of the recombinant Phoneutria nigriventer Tx1 toxin on voltage-gated sodium channels.

    Science.gov (United States)

    Silva, Anita O; Peigneur, Steve; Diniz, Marcelo R V; Tytgat, Jan; Beirão, Paulo S L

    2012-12-01

    Phoneutria nigriventer toxin Tx1 (PnTx1, also referred to in the literature as Tx1) exerts inhibitory effect on neuronal (Na(V)1.2) sodium channels in a way dependent on the holding potential, and competes with μ-conotoxins but not with tetrodotoxin for their binding sites. In the present study we investigated the electrophysiological properties of the recombinant toxin (rPnTx1), which has the complete amino acid sequence of the natural toxin with 3 additional residues: AM on the N-terminal and G on the C-terminal. At the concentration of 1.5 μM, the recombinant toxin inhibits Na(+) currents of dorsal root ganglia neurons (38.4 ± 6.1% inhibition at -80 mV holding potential) and tetrodotoxin-resistant Na(+) currents (26.2 ± 4.9% at the same holding potential). At -50 mV holding potential the inhibition of the total current reached 71.3 ± 2.3% with 1.5 μM rPnTx1. The selectivity of rPnTx1 was investigated on ten different isoforms of voltage-gated sodium channels expressed in Xenopus oocytes. The order of potency for rPnTx1 was: rNa(V)1.2 > rNa(V)1.7 ≈ rNa(V)1.4 ≥ rNa(V)1.3 > mNa(V)1.6 ≥ hNa(V)1.8. No effect was seen on hNa(V)1.5 and on the arthropods isoforms (DmNa(V)1, BGNa(V)1.1a and VdNa(V)1). The IC(50) for Na(V)1.2 was 33.7 ± 2.9 nM with a maximum inhibition of 83.3 ± 1.9%. The toxin did not alter the voltage-dependence of channel gating and was effective on Na(V)1.2 channels devoid of inactivation. It was ineffective on neuronal calcium channels. We conclude that rPnTx1 has a promising selectivity, and that it may be a valuable model to achieve pharmacological activities of interest for the treatment of channelopathies and neuropathic pain. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  13. PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1

    National Research Council Canada - National Science Library

    Herrmann, Solveig; Ninkovic, Milena; Kohl, Tobias; Pardo, Luis A

    2013-01-01

    .... In the case of the voltage-gated potassium channel KV10.1, this is determinant not only for its physiological function in brain, but also for its pathophysiology in tumors and possible use as a therapeutic target...

  14. Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels.

    Science.gov (United States)

    Guo, Kai; Wang, Xianming; Gao, Guofeng; Huang, Congxin; Elmslie, Keith S; Peterson, Blaise Z

    2010-11-01

    We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149-1159, 2010). The short 17 amino acid extracellular NH(2)-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate Ca(V)1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with Ca(V)1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on Ca(V)1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca(2+) dynamics in the heart.

  15. 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.

  16. Multiple mechanisms underlying rectification in retinal cyclic nucleotide-gated (CNGA1) channels.

    Science.gov (United States)

    Arcangeletti, Manuel; Marchesi, Arin; Mazzolini, Monica; Torre, Vincent

    2013-11-01

    In cyclic nucleotide-gated (CNGA1) channels, in the presence of symmetrical ionic conditions, current-voltage (I-V) relationship depends, in a complex way, on the radius of permeating ion. It has been suggested that both the pore and S4 helix contribute to the observed rectification. In the present manuscript, using tail and gating current measurements from homotetrameric CNGA1 channels expressed in Xenopus oocytes, we clarify and quantify the role of the pore and of the S4 helix. We show that in symmetrical Rb(+) and Cs(+) single-channel current rectification dominates macroscopic currents while voltage-dependent gating becomes larger in symmetrical ethylammonium and dimethylammonium, where the open probability strongly depends on voltage. Isochronal tail currents analysis in dimethylammonium shows that at least two voltage-dependent transitions underlie the observed rectification. Only the first voltage-dependent transition is sensible to mutation of charge residues in the S4 helix. Moreover, analysis of tail and gating currents indicates that the number of elementary charges per channel moving across the membrane is less than 2, when they are about 12 in K(+) channels. These results indicate the existence of distinct mechanisms underlying rectification in CNG channels. A restricted motion of the S4 helix together with an inefficient coupling to the channel gate render CNGA1 channels poorly sensitive to voltage in the presence of physiological Na(+) and K(+).

  17. Multiple mechanisms underlying rectification in retinal cyclic nucleotide-gated (CNGA1) channels

    Science.gov (United States)

    Arcangeletti, Manuel; Marchesi, Arin; Mazzolini, Monica; Torre, Vincent

    2013-01-01

    In cyclic nucleotide-gated (CNGA1) channels, in the presence of symmetrical ionic conditions, current–voltage (I-V) relationship depends, in a complex way, on the radius of permeating ion. It has been suggested that both the pore and S4 helix contribute to the observed rectification. In the present manuscript, using tail and gating current measurements from homotetrameric CNGA1 channels expressed in Xenopus oocytes, we clarify and quantify the role of the pore and of the S4 helix. We show that in symmetrical Rb+ and Cs+ single-channel current rectification dominates macroscopic currents while voltage-dependent gating becomes larger in symmetrical ethylammonium and dimethylammonium, where the open probability strongly depends on voltage. Isochronal tail currents analysis in dimethylammonium shows that at least two voltage-dependent transitions underlie the observed rectification. Only the first voltage-dependent transition is sensible to mutation of charge residues in the S4 helix. Moreover, analysis of tail and gating currents indicates that the number of elementary charges per channel moving across the membrane is less than 2, when they are about 12 in K+ channels. These results indicate the existence of distinct mechanisms underlying rectification in CNG channels. A restricted motion of the S4 helix together with an inefficient coupling to the channel gate render CNGA1 channels poorly sensitive to voltage in the presence of physiological Na+ and K+. PMID:24400150

  18. Optimal inverter logic gate using 10-nm double gate-all-around (DGAA transistor with asymmetric channel width

    Directory of Open Access Journals (Sweden)

    Myunghwan Ryu

    2016-01-01

    Full Text Available We investigate the electrical characteristics of a double-gate-all-around (DGAA transistor with an asymmetric channel width using three-dimensional device simulation. The DGAA structure creates a silicon nanotube field-effect transistor (NTFET with a core-shell gate architecture, which can solve the problem of loss of gate controllability of the channel and provides improved short-channel behavior. The channel width asymmetry is analyzed on both sides of the terminals of the transistors, i.e., source and drain. In addition, we consider both n-type and p-type DGAA FETs, which are essential to forming a unit logic cell, the inverter. Simulation results reveal that, according to the carrier types, the location of the asymmetry has a different effect on the electrical properties of the devices. Thus, we propose the N/P DGAA FET structure with an asymmetric channel width to form the optimal inverter. Various electrical metrics are analyzed to investigate the benefits of the optimal inverter structure over the conventional inverter structure. Simulation results show that 27% delay and 15% leakage power improvement are enabled in the optimum structure.

  19. 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.

  20. More gating charges are needed to open a Shaker K+ channel than are needed to open an rBIIA Na+ channel.

    Science.gov (United States)

    Gamal El-Din, Tamer M; Grögler, Dominik; Lehmann, Claudia; Heldstab, Hansjakob; Greeff, Nikolaus G

    2008-08-01

    This study presents what is, to our knowledge, a novel technique by means of which the ratio of the single gating charges of voltage-gated rat brain IIA (rBIIA) sodium and Shaker potassium ion channels was estimated. In the experiment, multiple tandems of enhanced green fluorescent protein were constructed and inserted into the C-terminals of Na(+) and K(+) ion channels. cRNA of Na(+) and K(+) ion channels was injected and expressed in Xenopus laevis oocytes. The two electrode voltage-clamp technique allowed us to determine the total gating charge of sodium and potassium ion channels, while a relative measure of the amount of expressed channels could be established on the basis of the quantification of the fluorescence intensity of membrane-bound channels marked by enhanced green fluorescent proteins. As a result, gating charge and fluorescence intensity were found to be positively correlated. A relative comparison of the single gating charges of voltage-gated sodium and potassium ion channels could thus be established: the ratio of the single gating charges of the Shaker potassium channel and the rBIIA sodium channel was found to be 2.5 +/- 0.4. Assuming the single channel gating charge of the Shaker K(+) channel to be approximately 13 elementary charges (well supported by other studies), this leads to approximately six elementary charges for the rBIIA sodium channel, which includes a fraction of gating charge that is missed during inactivation.

  1. Mechanism of magnesium activation of calcium-activated potassium channels.

    Science.gov (United States)

    Shi, Jingyi; Krishnamoorthy, Gayathri; Yang, Yanwu; Hu, Lei; Chaturvedi, Neha; Harilal, Dina; Qin, Jun; Cui, Jianmin

    2002-08-22

    Large-conductance (BK type) Ca(2+)-dependent K(+) channels are essential for modulating muscle contraction and neuronal activities such as synaptic transmission and hearing. BK channels are activated by membrane depolarization and intracellular Ca(2+) and Mg(2+) (refs 6-10). The energy provided by voltage, Ca(2+) and Mg(2+) binding are additive in activating the channel, suggesting that these signals open the activation gate through independent pathways. Here we report a molecular investigation of a Mg(2+)-dependent activation mechanism. Using a combined site-directed mutagenesis and structural analysis, we demonstrate that a structurally new Mg(2+)-binding site in the RCK/Rossman fold domain -- an intracellular structural motif that immediately follows the activation gate S6 helix -- is responsible for Mg(2+)-dependent activation. Mutations that impair or abolish Mg(2+) sensitivity do not affect Ca(2+) sensitivity, and vice versa. These results indicate distinct structural pathways for Mg(2+)- and Ca(2+)-dependent activation and suggest a possible mechanism for the coupling between Mg(2+) binding and channel opening.

  2. Alternative splicing modulates diltiazem sensitivity of cardiac and vascular smooth muscle Cav1.2 calcium channels

    Science.gov (United States)

    Zhang, Heng Yu; Liao, Ping; Wang, Jue Jin; Yu, De Jie; Soong, Tuck Wah

    2010-01-01

    Background and purpose: As a calcium channel blocker, diltiazem acts mainly on the voltage-gated calcium channels, Cav1.2, for its beneficial effects in cardiovascular diseases such as hypertension, angina and/or supraventricular arrhythmias. However, the effects of diltiazem on different isoforms of Cav1.2 channels expressed in heart and vascular smooth muscles remain to be investigated. Here, we characterized the effects of diltiazem on the splice variants of Cav1.2 channels, predominant in cardiac and vascular smooth muscles. Experimental approach: Cardiac and smooth muscle isoforms of Cav1.2 channels were expressed in human embryonic kidney cells and their electrophysiological properties were characterized using whole-cell patch-clamp techniques. Key results: Under closed-channel and use-dependent block (0.03 Hz), cardiac splice variant Cav1.2CM was less sensitive to diltiazem than two major smooth muscle splice variants, Cav1.2SM and Cav1.2b. Cav1.2CM has a more positive half-inactivation potential than the smooth muscle channels, and diltiazem shifted it less to negative potential. Additionally, the current decay was slower in Cav1.2CM channels. When we modified alternatively spliced exons of cardiac Cav1.2CM channels into smooth muscle exons, we found that all three loci contribute to the different diltiazem sensitivity between cardiac and smooth muscle splice isoforms. Conclusions and implications: Alternative splicing of Cav1.2 channels modifies diltiazem sensitivity in the heart and blood vessels. Gating properties altered by diltiazem are different in the three channels. PMID:20649567

  3. Effects of Anandamide and Noxious Heat on Intracellular Calcium Concentration in Nociceptive DRG Neurons of Rats

    National Research Council Canada - National Science Library

    Tilo Fischbach; Wolfgang Greffrath; Hermann Nawrath; Rolf-Detlef Treede

    2007-01-01

    ...+-free extracellular solution. Neither exclusion of voltage-gated sodium channels nor additional blockade of voltage-gated calcium channels of the L-, N-, and/or T-type, significantly reduced the anandamide...

  4. First direct electron microscopic visualization of a tight spatial coupling between GABAA-receptors and voltage-sensitive calcium channels

    DEFF Research Database (Denmark)

    Hansen, G H; Belhage, B; Schousboe, A

    1992-01-01

    Using cerebellar granule neurons in culture it was demonstrated that exposure of the cells to the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) leads to an increase in the number of voltage-gated calcium channels as revealed by quantitative preembedding indirect imm...... of THIP-treated cultures. This suggests that primarily low affinity GABAA-receptors are closely associated with Ca2+ channels and this may be important for the ability of these receptors to mediate an inhibitory action on transmitter release even under extreme depolarizing conditions....

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    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......-gated K(+) channels and blockers of hyperpolarization-activated cyclic nucleotide-gated channels that were found to reduce neuronal activity were also found to be effective in neuropathic and inflammatory pain states. The isoforms of these channels present on nociceptive axons have limited specificity....... The rationale for considering axonal voltage-gated ion channels as targets for pain treatment comes from the accumulating evidence that chronic pain states are associated with a dysregulation of these channels that could alter their specificity and make them more susceptible to pharmacological modulation...

  6. 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.

  7. Gating Modulation of the Tumor-Related Kv10.1 Channel by Mibefradil.

    Science.gov (United States)

    Gómez-Lagunas, Froylán; Carrillo, Elisa; Pardo, Luis A; Stühmer, Walter

    2017-08-01

    Several reports credit mibefradil with tumor suppressing properties arising from its known inhibition of Ca2+ currents. Given that mibefradil (Mb) is also known to inhibit K+ channels, we decided to study the interaction between this organic compound and the tumor-related Kv10.1 channel. Here we report that Mb modulates the gating of Kv10.1. Mb induces an apparent inactivation from both open and early closed states where the channels dwell at hyperpolarized potentials. Additionally, Mb accelerates the kinetics of current activation, in a manner that depends on initial conditions. Our observations suggest that Mb binds to the voltage sensor domain of Kv10.1 channels, thereby modifying the gating of the channels in a way that in some, but not all, aspects opposes to the gating effects exerted by divalent cations. J. Cell. Physiol. 232: 2019-2032, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Structural models of TREK channels and their gating mechanism

    National Research Council Canada - National Science Library

    Milac, Adina; Anishkin, Andriy; Fatakia, Sarosh N; Chow, Carson C; Sukharev, Sergei; Guy, H. Robert

    2011-01-01

    Mechanosensitive TREK channels belong to the family of K2P channels, a family of widely distributed, well modulated channels that uniquely have two similar or identical subunits, each with two TM1-P-TM2 motifs...

  9. Localization of calcium channels in Paramecium caudatum.

    Science.gov (United States)

    Dunlap, K

    1977-01-01

    1. Electrical recordings from Paramecium caudatum were made after removal of the cilia with chloral hydrate and during ciliary regrowth to study the electrical properties of that portion of the surface membrane enclosing the ciliary axoneme. 2. Removal of the somatic cilia (a 50% reduction in membrane surface area) results in an almost complete elimination of the regenerative Ca response, all-or-none Ba2+ spike, and delayed rectification. 3. A twofold increase in input resistance resulted from the 50% reduction in membrane surface area. 4. The electrical properties remained unchanged, despite prolonged exposure to the chloral hydrate, until the cilia were mechanically removed. 5. Restoration of the Ca response accompanied ciliary regrowth, so that complete excitability returns when the cilia regain their original lengths. 6. It is concluded that the voltage-sensitive Ca channels are localized to that portion of surface membrane surrounding the cilia. 7. Measurements of membrane constants before and after deciliation and estimations of the cable constants of a single cilium suggest that the cilia of Paramecium may be fully isopotential along their length and with the major cell compartment. Images Plate 1 Plate 2 PMID:915829

  10. Effects of stochastic channel gating and distribution on the cardiac action potential.

    Science.gov (United States)

    Lemay, Mathieu; de Lange, Enno; Kucera, Jan P

    2011-07-21

    Ion channels exhibit stochastic conformational changes determining their gating behavior. In addition, the process of protein turnover leads to a natural variability of the number of membrane and gap junctional channels. Nevertheless, in computational models, these two aspects are scarcely considered and their impacts are largely unknown. We investigated the effects of stochastic current fluctuations and channel distributions on action potential duration (APD), intercellular conduction delays (ICDs) and conduction blocks using a modified ventricular cell model (Rudy et al.) with Markovian formulations of the principal ion currents (to simulate their stochastic open-close gating behavior) and with channel counts drawn from Poisson distributions (to simulate their natural variability). In single cells, APD variability (coefficient of variation: 1.6% at BCL=1000ms) was essentially caused by stochastic channel gating of I(Ks), persistent I(Na) and I(Ca,L). In cell strands, ICD variability induced by stochastic channel gating and Poissonian channel distributions was low under normal conditions. Nonetheless, at low intercellular coupling levels, Poissonian gap junctional channel distribution resulted in a large ICD variability (coefficient of variation >20%), highly heterogeneous conduction patterns and conduction blocks. Therefore, the stochastic behavior of current fluctuations and channel distributions can contribute to the heterogeneity of conduction patterns and to conduction block, as observed previously in experiments in cardiac tissue with altered intercellular coupling. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. The involvement of the Mid1/Cch1/Yvc1 calcium channels in Aspergillus fumigatus virulence.

    Directory of Open Access Journals (Sweden)

    Patrícia Alves de Castro

    Full Text Available Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Calcium homeostasis and signaling is essential for numerous biological processes and also influences A. fumigatus pathogenicity. The presented study characterized the function of the A. fumigatus homologues of three Saccharomyces cerevisiae calcium channels, voltage-gated Cch1, stretch-activated Mid1 and vacuolar Yvc1. The A. fumigatus calcium channels cchA, midA and yvcA were regulated at transcriptional level by increased calcium levels. The YvcA::GFP fusion protein localized to the vacuoles. Both ΔcchA and ΔmidA mutant strains showed reduced radial growth rate in nutrient-poor minimal media. Interestingly, this growth defect in the ΔcchA strain was rescued by the exogenous addition of CaCl2. The ΔcchA, ΔmidA, and ΔcchA ΔmidA strains were also sensitive to the oxidative stress inducer, paraquat. Restriction of external Ca(2+ through the addition of the Ca(2+-chelator EGTA impacted upon the growth of the ΔcchA and ΔmidA strains. All the A. fumigatus ΔcchA, ΔmidA, and ΔyvcA strains demonstrated attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Infection with the parental strain resulted in a 100% mortality rate at 15 days post-infection, while the mortality rate of the ΔcchA, ΔmidA, and ΔyvcA strains after 15 days post-infection was only 25%. Collectively, this investigation strongly indicates that CchA, MidA, and YvcA play a role in A. fumigatus calcium homeostasis and virulence.

  12. Macroscopic kinetics of pentameric ligand gated ion channels: comparisons between two prokaryotic channels and one eukaryotic channel.

    Directory of Open Access Journals (Sweden)

    Kurt T Laha

    Full Text Available Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs. Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC and Gloeobacter violaceus (GLIC in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1β2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively were relatively fast with time constants of 24.9 ± 5.1 ms and 1.2 ± 0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3 ± 0.3 s and deactivated even slower with a time constant of 4.6 ± 1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover

  13. The KCNQ1 channel - remarkable flexibility in gating allows for functional versatility.

    Science.gov (United States)

    Liin, Sara I; Barro-Soria, Rene; Larsson, H Peter

    2015-06-15

    The KCNQ1 channel (also called Kv7.1 or KvLQT1) belongs to the superfamily of voltage-gated K(+) (Kv) channels. KCNQ1 shares several general features with other Kv channels but also displays a fascinating flexibility in terms of the mechanism of channel gating, which allows KCNQ1 to play different physiological roles in different tissues. This flexibility allows KCNQ1 channels to function as voltage-independent channels in epithelial tissues, whereas KCNQ1 function as voltage-activated channels with very slow kinetics in cardiac tissues. This flexibility is in part provided by the association of KCNQ1 with different accessory KCNE β-subunits and different modulators, but also seems like an integral part of KCNQ1 itself. The aim of this review is to describe the main mechanisms underlying KCNQ1 flexibility. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  14. The KCNQ1 channel – remarkable flexibility in gating allows for functional versatility

    Science.gov (United States)

    Liin, Sara I; Barro-Soria, Rene; Larsson, H Peter

    2015-01-01

    The KCNQ1 channel (also called Kv7.1 or KvLQT1) belongs to the superfamily of voltage-gated K+ (Kv) channels. KCNQ1 shares several general features with other Kv channels but also displays a fascinating flexibility in terms of the mechanism of channel gating, which allows KCNQ1 to play different physiological roles in different tissues. This flexibility allows KCNQ1 channels to function as voltage-independent channels in epithelial tissues, whereas KCNQ1 function as voltage-activated channels with very slow kinetics in cardiac tissues. This flexibility is in part provided by the association of KCNQ1 with different accessory KCNE β-subunits and different modulators, but also seems like an integral part of KCNQ1 itself. The aim of this review is to describe the main mechanisms underlying KCNQ1 flexibility. PMID:25653179

  15. Structures and functions of calcium channel beta subunits.

    Science.gov (United States)

    Birnbaumer, L; Qin, N; Olcese, R; Tareilus, E; Platano, D; Costantin, J; Stefani, E

    1998-08-01

    Calcium channel beta subunits have profound effects on how alpha1 subunits perform. In this article we summarize our present knowledge of the primary structures of beta subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with alpha1 subunits, the effects of beta subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by beta subunits, such as long-lasting prepulse facilitation of alpha1C (absent in alpha1E) and inhibition by G protein betagamma dimer of alpha1E, absent in alpha1C. One beta subunit, a brain beta2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of alpha1 in oocytes requires a beta subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for "alpha1 alone" channels seen in cells with limited beta subunit expression. In one model, beta dissociates from the mature alpha1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of beta would then have subunit composition alpha1beta. In the other model, the "chaperoning" beta remains associated with the mature channel and "alpha1 alone" channels would in fact be alpha1beta channels. Upon co-expression of high levels of beta the regulated channels would have composition [alpha1beta]beta.

  16. Artificial modulation of the gating behavior of a K+ channel in a KvAP-DNA chimera.

    Science.gov (United States)

    Wang, Andrew; Zocchi, Giovanni

    2011-04-19

    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.

  17. 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...... using the fluorescent calcium chelator fura-2. The types of calcium channels present at the synaptic terminal were determined by the inhibitory action of calcium channel blockers on potassium-induced [3H]GABA release in the same cell preparation. L-, N-, P-, Q- and R-/T-type voltage dependent calcium...

  18. Phosphodiesterase 3 and 5 and cyclic nucleotide-gated ion channel expression in rat trigeminovascular system

    DEFF Research Database (Denmark)

    Kruse, Lars S; Sandholdt, Nicolai T H; Gammeltoft, Steen

    2006-01-01

    may be associated with mutations in ion channels. The aim of the present study was to describe the expression of phosphodiesterase 3 (PDE3) and 5 (PDE5) and cyclic nucleotide-gated ion channels (CNG) in cerebral arteries, meninges, and the trigeminal ganglion. mRNA for PDE and CNG was determined...

  19. STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate

    Science.gov (United States)

    Yamashita, Megumi; Yeung, Priscilla S.-W.; Ing, Christopher E.; McNally, Beth A.; Pomès, Régis; Prakriya, Murali

    2017-02-01

    Store-operated Ca2+ release-activated Ca2+ (CRAC) channels constitute a major pathway for Ca2+ influx and mediate many essential signalling functions in animal cells, yet how they open remains elusive. Here, we investigate the gating mechanism of the human CRAC channel Orai1 by its activator, stromal interacting molecule 1 (STIM1). We find that two rings of pore-lining residues, V102 and F99, work together to form a hydrophobic gate. Mutations of these residues to polar amino acids produce channels with leaky gates that conduct ions in the resting state. STIM1-mediated channel activation occurs through rotation of the pore helix, which displaces the F99 residues away from the pore axis to increase pore hydration, allowing ions to flow through the V102-F99 hydrophobic band. Pore helix rotation by STIM1 also explains the dynamic coupling between CRAC channel gating and ion selectivity. This hydrophobic gating mechanism has implications for CRAC channel function, pharmacology and disease-causing mutations.

  20. 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.

  1. A peptide-gated ion channel from the freshwater polyp Hydra

    DEFF Research Database (Denmark)

    Golubovic, Andjelko; Kuhn, Anne; Williamson, Michael

    2007-01-01

    regarded as a curiosity, and it was not known whether peptide-gated ionotropic receptors are also present in other animal groups. Nervous systems first evolved in cnidarians, which extensively use neuropeptides. Here we report cloning from the freshwater cnidarian Hydra of a novel ion channel (Hydra sodium...... channel, HyNaC) that is directly gated by the neuropeptides Hydra-RFamides I and II and is related to FaNaC. The cells expressing HyNaC localize to the base of the tentacles, adjacent to the neurons producing the Hydra-RFamides, suggesting that the peptides are the natural ligands for this channel. Our...

  2. Precise analytical model for short channel Cylindrical Gate (CylG) Gate-All-Around (GAA) MOSFET

    Science.gov (United States)

    Sharma, Dheeraj; Vishvakarma, Santosh Kumar

    2013-08-01

    A compact analytical model is presented for device electrostatics of nanoscale Cylindrical Gate (CylG) Gate-All-Around (GAA) MOSFET, using isomorphic polynomial function for potential distribution. The model is based on solutions of 3D Laplace and Poisson's equations for subthreshold and strong inversion region respectively. In this paper, the short-channel effects are precisely accounted for by introducing z dependent characteristic length and the developed electrostatics is tested against analysis of crossover point for device under test. Further, the modeled subthreshold slope for lightly doped CylG GAA MOSFET has been improved by introducing z dependent characteristic length and the position of minimum center potential in the channel is obtained by virtual cathode position. A new model is proposed for threshold voltage, based on shifting of inversion charge from center line to silicon insulator interface.

  3. Monod-Wyman-Changeux Analysis of Ligand-Gated Ion Channel Mutants.

    Science.gov (United States)

    Einav, Tal; Phillips, Rob

    2017-04-20

    We present a framework for computing the gating properties of ligand-gated ion channel mutants using the Monod-Wyman-Changeux (MWC) model of allostery. We derive simple analytic formulas for key functional properties such as the leakiness, dynamic range, half-maximal effective concentration ([EC50]), and effective Hill coefficient, and explore the full spectrum of phenotypes that are accessible through mutations. Specifically, we consider mutations in the channel pore of nicotinic acetylcholine receptor (nAChR) and the ligand binding domain of a cyclic nucleotide-gated (CNG) ion channel, demonstrating how each mutation can be characterized as only affecting a subset of the biophysical parameters. In addition, we show how the unifying perspective offered by the MWC model allows us, perhaps surprisingly, to collapse the plethora of dose-response data from different classes of ion channels into a universal family of curves.

  4. 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

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

    Science.gov (United States)

    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.

  6. Inducible and titratable silencing of Caenorhabditis elegans neurons in vivo with histamine-gated chloride channels

    Science.gov (United States)

    Pokala, Navin; Liu, Qiang; Gordus, Andrew; Bargmann, Cornelia I.

    2014-01-01

    Recent progress in neuroscience has been facilitated by tools for neuronal activation and inactivation that are orthogonal to endogenous signaling systems. We describe here a chemical-genetic approach for inducible silencing of Caenorhabditis elegans neurons in intact animals, using the histamine-gated chloride channel HisCl1 from Drosophila and exogenous histamine. Administering histamine to freely moving C. elegans that express HisCl1 transgenes in neurons leads to rapid and potent inhibition of neural activity within minutes, as assessed by behavior, functional calcium imaging, and electrophysiology of neurons expressing HisCl1. C. elegans does not use histamine as an endogenous neurotransmitter, and exogenous histamine has little apparent effect on wild-type C. elegans behavior. HisCl1-histamine silencing of sensory neurons, interneurons, and motor neurons leads to behavioral effects matching their known functions. In addition, the HisCl1-histamine system can be used to titrate the level of neural activity, revealing quantitative relationships between neural activity and behavioral output. We use these methods to dissect escape circuits, define interneurons that regulate locomotion speed (AVA, AIB) and escape-related omega turns (AIB), and demonstrate graded control of reversal length by AVA interneurons and DA/VA motor neurons. The histamine-HisCl1 system is effective, robust, compatible with standard behavioral assays, and easily combined with optogenetic tools, properties that should make it a useful addition to C. elegans neurotechnology. PMID:24550306

  7. FM dyes enter via a store-operated calcium channel and modify calcium signaling of cultured astrocytes

    Science.gov (United States)

    Li, Dongdong; Hérault, Karine; Oheim, Martin; Ropert, Nicole

    2009-01-01

    The amphiphilic fluorescent styryl pyridinium dyes FM1-43 and FM4-64 are used to probe activity-dependent synaptic vesicle cycling in neurons. Cultured astrocytes can internalize FM1-43 and FM4-64 inside vesicles but their uptake is insensitive to the elevation of cytosolic calcium (Ca2+) concentration and the underlying mechanism remains unclear. Here we used total internal reflection fluorescence microscopy and pharmacological tools to study the mechanisms of FM4-64 uptake into cultured astrocytes from mouse neocortex. Our data show that: (i) endocytosis is not a major route for FM4-64 uptake into astrocytes; (ii) FM4-64 enters astrocytes through an aqueous pore and strongly affects Ca2+ homeostasis; (iii) partitioning of FM4-64 into the outer leaflet of the plasma membrane results in a facilitation of store-operated Ca2+ entry (SOCE) channel gating; (iv) FM4-64 permeates and competes with Ca2+ for entry through a SOCE channel; (v) intracellular FM4-64 mobilizes Ca2+ from the endoplasmic reticulum stores, conveying a positive feedback to activate SOCE and to sustain dye uptake into astrocytes. Our study demonstrates that FM dyes are not markers of cycling vesicles in astrocytes and calls for a careful interpretation of FM fluorescence. PMID:20007370

  8. Voltage sensitive calcium channels (VSCC) in cultured neuronal hybrid cells

    Energy Technology Data Exchange (ETDEWEB)

    Richard, C.L.; U' Prichard, D.C.; Noronha-Blob, L.

    1986-03-01

    Calcium entry via VSCC has been identified in selected, neuronal clonal cell lines using /sup 45/Ca uptake and the fluorescent calcium indicator, quin 2. VSCC in NG108-15 hybrid cells, differentiated with dibutyryl cyclic AMP (1 mM, 4 days) have been further characterized. Depolarization (50 mM K/sup +/, dp) resulted in a rapid (15 sec) influx of Ca/sup 2 +/. Intracellular calcium concentrations were elevated approx. 3 fold from 223 +- 68 nM to 666 +- 74 nM. Dp-sensitive calcium entry was voltage dependent, independent of Na/sup +/, stimulated (40%) by the agonist Bay K 8644 (1..mu..M) and blocked by divalent cations (..mu..M range) and organic calcium channel antagonists (nM range) Bay K 8644, in the absence of KCl, failed to stimulate Ca/sup 2 +/ influx. Tetrodotoxin (TTX) and tetraethylammonium had no effect on VSCC activity. Blockage of VSCC by nimodipine was reversed by increasing Ca/sup 2 +/ ions. IC/sub 50/ values were right shifted from 6.5 nM (1mM/sup 0/Ca/sup 2 +/) to 840 nM (10 mM Ca/sup 2 +/). Ca/sup 2 +/ entry was also stimulated by veratridine (VE), in a Na/sup +//sub 0/-sensitive manner. VE-induced Ca/sup 2 +/ entry was voltage-independent, TTX-sensitive, and was only 25% of dp-sensitive Ca/sup 2 +/ entry. These results together indicate that VSCC in neuronal cells offer a useful system for studying ion channel regulation.

  9. Physiology and Regulation of Calcium Channels in Stomatal Guard Cells

    Energy Technology Data Exchange (ETDEWEB)

    Schroeder, Julian I.

    2007-05-02

    Stomatal pores in the epidermis of leaves regulate the diffusion of CO2 into leaves for photosynthetic carbon fixation and control water loss of plants during drought periods. Guard cells sense CO2, water status, light and other environmental conditions to regulate stomatal apertures for optimization of CO2 intake and plant growth under drought stress. The cytosolic second messenger calcium contributes to stomatal movements by transducing signals and regulating ion channels in guard cells. Studies suggest that both plasma membrane Ca2+ influx channels and vacuolar/organellar Ca2+ release channels contribute to ABA-induced Ca2+ elevations in guard cells. Recent research in the P.I.'s laboratory has led to identification of a novel major cation-selective Ca2+-permeable influx channel (Ica) in the plasma membrane of Arabidopsis guard cells. These advances will allow detailed characterization of Ica plasma membrane Ca2+ influx channels in guard cells. The long term goal of this research project is to gain a first detailed characterization of these novel plasma membrane Ca2+-permeable channel currents in Arabidopsis guard cells. The proposed research will investigate the hypothesis that Ica represents an important Ca2+ influx pathway for ABA and CO2 signal transduction in Arabidopsis guard cells. These studies will lead to elucidation of key signal transduction mechanisms by which plants balance CO2 influx into leaves and transpirational water loss and may contribute to future strategies for manipulating gas exchange for improved growth of crop plants and for biomass production.

  10. Consequences of depleted SERCA2-gated calcium stores in the skin.

    Science.gov (United States)

    Müller, Eliane J; Caldelari, Reto; Kolly, Carine; Williamson, Lina; Baumann, Dominique; Richard, Gabriele; Jensen, Pamela; Girling, Peter; Delprincipe, Franco; Wyder, Marianne; Balmer, Vreni; Suter, Maja M

    2006-04-01

    Sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) pumps belong to the family of Ca2+-ATPases responsible for the maintenance of calcium in the endoplasmic reticulum. In epidermal keratinocytes, SERCA2-controlled calcium stores are involved in cell cycle exit and onset of terminal differentiation. Hence, their dysfunction was thought to provoke impaired keratinocyte cohesion and hampered terminal differentiation. Here, we assessed cultured keratinocytes and skin biopsies from a canine family with an inherited skin blistering disorder. Cells from lesional and phenotypically normal areas of one of these dogs revealed affected calcium homeostasis due to depleted SERCA2-gated stores. In phenotypically normal patient cells, this defect compromised upregulation of p21(WAF1) and delayed the exit from the cell cycle. Despite this abnormality it failed to impede the terminal differentiation process in the long term but instead coincided with enhanced apoptosis and appearance of chronic wounds, suggestive of secondary mutations. Collectively, these findings provide the first survey on phenotypic consequences of depleted SERCA-gated stores for epidermal homeostasis that explain how depleted SERCA2 calcium stores provoke focal lesions rather than generalized dermatoses, a phenotype highly reminiscent of the human genodermatosis Darier disease.

  11. Orai and TRPC channel characterization in FcεRI-mediated calcium signaling and mediator secretion in human mast cells.

    Science.gov (United States)

    Wajdner, Hannah E; Farrington, Jasmine; Barnard, Claire; Peachell, Peter T; Schnackenberg, Christine G; Marino, Joseph P; Xu, Xiaoping; Affleck, Karen; Begg, Malcolm; Seward, Elizabeth P

    2017-03-01

    Inappropriate activation of mast cells via the FcεRI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcεRI-initiated mediator release. However, until now the role of TRPC calcium channels in FcεRI-mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to FcεRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FcεRI-mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FcεRI-mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1-regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1-KK684-685EE - TRPC1 gating mutant) failed to alter FcεRI-mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that FcεRI-mediated calcium influx through Orai is necessary for histamine and TNFα release but is differentially involved in the generation of cytokines and eicosanoids. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  12. L-type calcium channels refine the neural population code of sound level.

    Science.gov (United States)

    Grimsley, Calum Alex; Green, David Brian; Sivaramakrishnan, Shobhana

    2016-12-01

    The coding of sound level by ensembles of neurons improves the accuracy with which listeners identify how loud a sound is. In the auditory system, the rate at which neurons fire in response to changes in sound level is shaped by local networks. Voltage-gated conductances alter local output by regulating neuronal firing, but their role in modulating responses to sound level is unclear. We tested the effects of L-type calcium channels (CaL: CaV1.1-1.4) on sound-level coding in the central nucleus of the inferior colliculus (ICC) in the auditory midbrain. We characterized the contribution of CaL to the total calcium current in brain slices and then examined its effects on rate-level functions (RLFs) in vivo using single-unit recordings in awake mice. CaL is a high-threshold current and comprises ∼50% of the total calcium current in ICC neurons. In vivo, CaL activates at sound levels that evoke high firing rates. In RLFs that increase monotonically with sound level, CaL boosts spike rates at high sound levels and increases the maximum firing rate achieved. In different populations of RLFs that change nonmonotonically with sound level, CaL either suppresses or enhances firing at sound levels that evoke maximum firing. CaL multiplies the gain of monotonic RLFs with dynamic range and divides the gain of nonmonotonic RLFs with the width of the RLF. These results suggest that a single broad class of calcium channels activates enhancing and suppressing local circuits to regulate the sensitivity of neuronal populations to sound level. Copyright © 2016 the American Physiological Society.

  13. Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

    DEFF Research Database (Denmark)

    Timmermann, D B; Lund, T M; Belhage, B

    2001-01-01

    using the fluorescent calcium chelator fura-2. The types of calcium channels present at the synaptic terminal were determined by the inhibitory action of calcium channel blockers on potassium-induced [3H]GABA release in the same cell preparation. L-, N-, P-, Q- and R-/T-type voltage dependent calcium...... 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...... in cytosolic calcium concentration. The results of this investigation demonstrate that pharmacologically distinct types of voltage dependent calcium channels are differentially localized in cell bodies, neurites and nerve terminals of mouse cortical neurons but that the Q-type calcium channel appears...

  14. Magnesium: Effect on ocular health as a calcium channel antagonist

    Directory of Open Access Journals (Sweden)

    Şafak Korkmaz

    2013-06-01

    Full Text Available Magnesium is the physiologic calcium channel blocker,involving in many different metabolic processes by maintainingcell membrane function, modulating smooth musclecontraction and influencing enzymatic activities. Magnesiumhas been shown to increase blood flow to tissuesby modifying endothelial function via endothelin-1 (ET-1and nitric Oxide (NO pathways. Magnesium also exhibitsneuroprotective role by blocking N-methyl-D-aspartate(NMDA receptor related calcium influx and by inhibitingthe release of glutamate, hence protects the cell againstoxidative stress and apoptosis. Both increase in bloodflow and its neuroprotective effect make magnesium agood candidate for glaucoma studies. Magnesium hasbeen shown to decrease oxidative stress and apoptosisin retinal tissue and to have retinal ganglion cell sparingeffect. A series of studies has been conducted aboutmagnesium could decrease insulin resistance in diabeticpatients, ease glycemia control and prevent diabetic retinopathy.Magnesium is found to be critically important inmaintaining normal ionic homeostasis of lens. Magnesiumdeficiency has been shown to cause increased lenticularoxidative stress and ionic imbalance in the lens so triggercataractogenesis. J Clin Exp Invest 2013; 4 (2: 244-251Key words: Magnesium, calcium channel blockage,glaucoma, neuroprotection, diabetic retinopathy, cataract

  15. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra.

    Science.gov (United States)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D; Williamson, Michael; Kalbacher, Hubert; Grimmelikhuijzen, Cornelis J P; Holstein, Thomas W; Gründer, Stefan

    2010-04-16

    Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na(+) channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore properties, like a low Na(+) selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new subunit is closely related to HyNaC2 and -3 and co-localizes with HyNaC2 and -3 to the base of the tentacles. Coexpression in Xenopus oocytes of HyNaC5 with HyNaC2 and -3 largely increases current amplitude after peptide stimulation and affinity of the channel to Hydra-RFamides I and II. Moreover, the HyNaC2/3/5 channel has altered pore properties and amiloride affinity, more similarly to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, -3, and -5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission.

  16. Domain and interdomain energetics underlying gating in Shaker-type Kv channels.

    Science.gov (United States)

    Peyser, Alexander; Gillespie, Dirk; Roth, Roland; Nonner, Wolfgang

    2014-10-21

    To understand gating events with a time-base many orders-of-magnitude slower than that of atomic motion in voltage-gated ion channels such as the Shaker-type KV channels, a multiscale physical model is constructed from the experimentally well-characterized voltage-sensor (VS) domains coupled to a hydrophobic gate. The four VS domains are described by a continuum electrostatic model under voltage-clamp conditions, the control of ion flow by the gate domain is described by a vapor-lock mechanism, and the simple coupling principle is informed by known experimental results and trial-and-error. The configurational energy computed for each element is used to produce a total Hamiltonian that is a function of applied voltage, VS positions, and gate radius. We compute statistical-mechanical expectation values of macroscopic laboratory observables. This approach stands in contrast with molecular-dynamic models which are challenged by increasing scale, and kinetic models which assume a probability distribution rather than derive it from the underlying physics. This generic model predicts well the Shaker charge/voltage and conductance/voltage relations; the tight constraints underlying these results allow us to quantitatively assess the underlying physical mechanisms. The total electrical work picked up by the VS domains is an order-of-magnitude larger than the work required to actuate the gate itself, suggesting an energetic basis for the evolutionary flexibility of the voltage-gating mechanism. The cooperative slide-and-interlock behavior of the VS domains described by the VS-gate coupling relation leads to the experimentally observed bistable gating. This engineering approach should prove useful in the investigation of various elements underlying gating characteristics and degraded behavior due to mutation.

  17. Evidence for a role of GABA- and glutamate-gated chloride channels in olfactory memory.

    Science.gov (United States)

    Boumghar, Katia; Couret-Fauvel, Thomas; Garcia, Mikael; Armengaud, Catherine

    2012-11-01

    In the honeybee, we investigated the role of transmissions mediated by GABA-gated chloride channels and glutamate-gated chloride channels (GluCls) of the mushroom bodies (MBs) on olfactory learning using a single-trial olfactory conditioning paradigm. The GABAergic antagonist picrotoxin (PTX) or the GluCl antagonist L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) was injected alone or in combination into the α-lobes of MBs. PTX impaired early long-term olfactory memory when injected before conditioning or before testing. L-trans-PDC alone induced no significant effect on learning and memory but induced a less specific response to the conditioned odor. When injected before PTX, L-trans-PDC was able to modulate PTX effects. These results emphasize the role of MB GABA-gated chloride channels in consolidation processes and strongly support that GluCls are involved in the perception of the conditioned stimulus.

  18. Fast sodium channel gating supports localized and efficient axonal action potential initiation.

    Science.gov (United States)

    Schmidt-Hieber, Christoph; Bischofberger, Josef

    2010-07-28

    Action potentials (APs) are initiated in the proximal axon of most neurons. In myelinated axons, a 50-times higher sodium channel density in the initial segment compared to the soma may account for this phenomenon. However, little is known about sodium channel density and gating in proximal unmyelinated axons. To study the mechanisms underlying AP initiation in unmyelinated hippocampal mossy fibers of adult mice, we recorded sodium currents in axonal and somatic membrane patches. We demonstrate that sodium channel density in the proximal axon is approximately 5 times higher than in the soma. Furthermore, sodium channel activation and inactivation are approximately 2 times faster. Modeling revealed that the fast activation localized the initiation site to the proximal axon even upon strong synaptic stimulation, while fast inactivation contributed to energy-efficient membrane charging during APs. Thus, sodium channel gating and density in unmyelinated mossy fiber axons appear to be specialized for robust AP initiation and propagation with minimal current flow.

  19. 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

  20. 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.

  1. Oscillatory Reaction Induced Periodic C-Quadruplex DNA Gating of Artificial Ion Channels.

    Science.gov (United States)

    Wang, Jian; Fang, Ruochen; Hou, Jue; Zhang, Huacheng; Tian, Ye; Wang, Huanting; Jiang, Lei

    2017-03-28

    Many biological ion channels controlled by biochemical reactions have autonomous and periodic gating functions, which play important roles in continuous mass transport and signal transmission in living systems. Inspired by these functional biological ion channel systems, here we report an artificial self-oscillating nanochannel system that can autonomously and periodically control its gating process under constant conditions. The system is constructed by integrating a chemical oscillator, consisting of BrO3(-), Fe(CN)6(4-), H(+), and SO3(2-), into a synthetic proton-sensitive nanochannel modified with C-quadruplex (C4) DNA motors. The chemical oscillator, containing H(+)-producing and H(+)-consuming reactions, can cyclically drive conformational changes of the C4-DNA motors on the channel wall between random coil and folded i-motif structures, thus leading to autonomous gating of the nanochannel between open and closed states. The autonomous gating processes are confirmed by periodic high-low ionic current oscillations of the channel monitored under constant reaction conditions. The utilization of a chemical oscillator integrated with DNA molecules represents a method to directly convert chemical energy of oscillating reactions to kinetic energy of conformational changes of the artificial nanochannels and even to achieve diverse autonomous gating functions in artificial nanofluidic devices.

  2. Energetic role of the paddle motif in voltage gating of Shaker K(+) channels.

    Science.gov (United States)

    Xu, Yanping; Ramu, Yajamana; Shin, Hyeon-Gyu; Yamakaze, Jayden; Lu, Zhe

    2013-05-01

    Voltage-gated ion channels underlie rapid electric signaling in excitable cells. Electrophysiological studies have established that the N-terminal half of the fourth transmembrane segment ((NT)S4) of these channels is the primary voltage sensor, whereas crystallographic studies have shown that (NT)S4 is not located within a proteinaceous pore. Rather, (NT)S4 and the C-terminal half of S3 ((CT)S3 or S3b) form a helix-turn-helix motif, termed the voltage-sensor paddle. This unexpected structural finding raises two fundamental questions: does the paddle motif also exist in voltage-gated channels in a biological membrane, and, if so, what is its function in voltage gating? Here, we provide evidence that the paddle motif exists in the open state of Drosophila Shaker voltage-gated K(+) channels expressed in Xenopus oocytes and that (CT)S3 acts as an extracellular hydrophobic 'stabilizer' for (NT)S4, thus biasing the gating chemical equilibrium toward the open state.

  3. Support for calcium channel gene defects in autism spectrum disorders

    Directory of Open Access Journals (Sweden)

    Lu Ake Tzu-Hui

    2012-12-01

    Full Text Available Abstract Background Alternation of synaptic homeostasis is a biological process whose disruption might predispose children to autism spectrum disorders (ASD. Calcium channel genes (CCG contribute to modulating neuronal function and evidence implicating CCG in ASD has been accumulating. We conducted a targeted association analysis of CCG using existing genome-wide association study (GWAS data and imputation methods in a combined sample of parent/affected child trios from two ASD family collections to explore this hypothesis. Methods A total of 2,176 single-nucleotide polymorphisms (SNP (703 genotyped and 1,473 imputed covering the genes that encode the α1 subunit proteins of 10 calcium channels were tested for association with ASD in a combined sample of 2,781 parent/affected child trios from 543 multiplex Caucasian ASD families from the Autism Genetics Resource Exchange (AGRE and 1,651 multiplex and simplex Caucasian ASD families from the Autism Genome Project (AGP. SNP imputation using IMPUTE2 and a combined reference panel from the HapMap3 and the 1,000 Genomes Project increased coverage density of the CCG. Family-based association was tested using the FBAT software which controls for population stratification and accounts for the non-independence of siblings within multiplex families. The level of significance for association was set at 2.3E-05, providing a Bonferroni correction for this targeted 10-gene panel. Results Four SNPs in three CCGs were associated with ASD. One, rs10848653, is located in CACNA1C, a gene in which rare de novo mutations are responsible for Timothy syndrome, a Mendelian disorder that features ASD. Two others, rs198538 and rs198545, located in CACN1G, and a fourth, rs5750860, located in CACNA1I, are in CCGs that encode T-type calcium channels, genes with previous ASD associations. Conclusions These associations support a role for common CCG SNPs in ASD.

  4. In vivo impact of presynaptic calcium channel dysfunction on motor axons in episodic ataxia type 2.

    Science.gov (United States)

    Tomlinson, Susan E; Tan, S Veronica; Burke, David; Labrum, Robyn W; Haworth, Andrea; Gibbons, Vaneesha S; Sweeney, Mary G; Griggs, Robert C; Kullmann, Dimitri M; Bostock, Hugh; Hanna, Michael G

    2016-02-01

    Ion channel dysfunction causes a range of neurological disorders by altering transmembrane ion fluxes, neuronal or muscle excitability, and neurotransmitter release. Genetic neuronal channelopathies affecting peripheral axons provide a unique opportunity to examine the impact of dysfunction of a single channel subtype in detail in vivo. Episodic ataxia type 2 is caused by mutations in CACNA1A, which encodes the pore-forming subunit of the neuronal voltage-gated calcium channel Cav2.1. In peripheral motor axons, this channel is highly expressed at the presynaptic neuromuscular junction where it contributes to action potential-evoked neurotransmitter release, but it is not expressed mid-axon or thought to contribute to action potential generation. Eight patients from five families with genetically confirmed episodic ataxia type 2 underwent neurophysiological assessment to determine whether axonal excitability was normal and, if not, whether changes could be explained by Cav2.1 dysfunction. New mutations in the CACNA1A gene were identified in two families. Nerve conduction studies were normal, but increased jitter in single-fibre EMG studies indicated unstable neuromuscular transmission in two patients. Excitability properties of median motor axons were compared with those in 30 age-matched healthy control subjects. All patients had similar excitability abnormalities, including a high electrical threshold and increased responses to hyperpolarizing (P ataxia type 2 thus has unexpected effects on axon excitability, which may reflect an indirect effect of abnormal calcium current fluxes during development. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.

  5. Graded-channel junctionless dual-gate MOSFETs for radiation tolerance

    Science.gov (United States)

    Wang, Ying; Shan, Chan; Liu, Chao-ming; Li, Xing-ji; Yang, Jian-qun; Tang, Yan; Bao, Meng-tian; Cao, Fei

    2017-12-01

    In this work, we investigate the bipolar gain and collected charge under heavy ion irradiation in graded-channel junctionless dual-gate MOSFETs. The transient response of the graded-channel device is compared with that of the conventional uniform-channel device. We present the different doping levels on the channel around the source and show that the bipolar gain and collected charge of the graded-channel device are lower than those of the uniform-channel device, owing to the lower doping level around the source in the channel, which induces lower floating body effects. We consider the effect of changing the doping level in a graded-channel device.

  6. Coulomb blockade in a Si channel gated by an Al single-electron transistor

    OpenAIRE

    Sun, L.; Brown, K. R.; Kane, B. E.

    2007-01-01

    We incorporate an Al-AlO_x-Al single-electron transistor as the gate of a narrow (~100 nm) metal-oxide-semiconductor field-effect transistor (MOSFET). Near the MOSFET channel conductance threshold, we observe oscillations in the conductance associated with Coulomb blockade in the channel, revealing the formation of a Si single-electron transistor. Abrupt steps present in sweeps of the Al transistor conductance versus gate voltage are correlated with single-electron charging events in the Si t...

  7. Interaction of H2S with Calcium Permeable Channels and Transporters

    Directory of Open Access Journals (Sweden)

    Weihua Zhang

    2015-01-01

    Full Text Available A growing amount of evidence has suggested that hydrogen sulfide (H2S, as a gasotransmitter, is involved in intensive physiological and pathological processes. More and more research groups have found that H2S mediates diverse cellular biological functions related to regulating intracellular calcium concentration. These groups have demonstrated the reciprocal interaction between H2S and calcium ion channels and transporters, such as L-type calcium channels (LTCC, T-type calcium channels (TTCC, sodium/calcium exchangers (NCX, transient receptor potential (TRP channels, β-adrenergic receptors, and N-methyl-D-aspartate receptors (NMDAR in different cells. However, the understanding of the molecular targets and mechanisms is incomplete. Recently, some research groups demonstrated that H2S modulates the activity of calcium ion channels through protein S-sulfhydration and polysulfide reactions. In this review, we elucidate that H2S controls intracellular calcium homeostasis and the underlying mechanisms.

  8. 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...... and discuss how they contribute to the establishment and maintenance of the specific localization of Kv channels in neurons....

  9. PIP2 regulation of KCNQ channels: biophysical and molecular mechanisms for lipid modulation of voltage-dependent gating

    Directory of Open Access Journals (Sweden)

    Mark Alan Zaydman

    2014-05-01

    Full Text Available Voltage-gated potassium (Kv channels contain voltage-sensing (VSD and pore-gate (PGD structural domains. During voltage-dependent gating, conformational changes in the two domains are coupled giving rise to voltage-dependent opening of the channel. In addition to membrane voltage, KCNQ (Kv7 channel opening requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2. Recent studies suggest that PIP2 serves as a cofactor to mediate VSD-PGD coupling in KCNQ1 channels. In this review, we put these findings in the context of the current understanding of voltage-dependent gating, lipid modulation of Kv channel activation, and PIP2-regulation of KCNQ channels. We suggest that lipid-mediated coupling of functional domains is a common mechanism among KCNQ channels that may be applicable to other Kv channels and membrane proteins.

  10. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra

    DEFF Research Database (Denmark)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D

    2010-01-01

    Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated...... by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na(+) channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore...... properties, like a low Na(+) selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new...

  11. Voltage-gated sodium channels in pain states: role in pathophysiology and targets for treatment.

    Science.gov (United States)

    Dib-Hajj, Sulayman D; Binshtok, Alexander M; Cummins, Theodore R; Jarvis, Michael F; Samad, Tarek; Zimmermann, Katharina

    2009-04-01

    Pain is a major unmet medical need which has been causally linked to changes in sodium channel expression, modulation, or mutations that alter channel gating properties or current density in nociceptor neurons. Voltage-gated sodium channels activate (open) then rapidly inactivate in response to a depolarization of the plasma membrane of excitable cells allowing the transient flow of sodium ions thus generating an inward current which underlies the generation and conduction of action potentials (AP) in these cells. Activation and inactivation, as well as other gating properties, of sodium channel isoforms have different kinetics and voltage-dependent properties, so that the ensemble of channels that are present determine the electrogenic properties of specific neurons. Biophysical and pharmacological studies have identified the peripheral-specific sodium channels Na(v)1.7, Na(v)1.8 and Na(v)1.9 as particularly important in the pathophysiology of different pain syndromes, and isoform-specific blockers of these channels or targeting their modulators hold the promise of a future effective therapy for treatment of pain.

  12. The S4---S5 linker - gearbox of TRP channel gating.

    Science.gov (United States)

    Hofmann, Laura; Wang, Hongmei; Zheng, Wang; Philipp, Stephan E; Hidalgo, Patricia; Cavalié, Adolfo; Chen, Xing-Zhen; Beck, Andreas; Flockerzi, Veit

    2017-11-01

    Transient receptor potential (TRP) channels are cation channels which participate in a wide variety of physiological processes in organisms ranging from fungi to humans. They fulfill roles in body homeostasis, are sensors for noxious chemicals and temperature in the mammalian somatosensory system and are activated by light stimulated phospholipase C activity in Drosophila or by hypertonicity in yeast. The transmembrane topology of TRP channels is similar to that of voltage-gated cation channels. TRP proteins assemble as tetramers with each subunit containing six transmembrane helices (S1-S6) and intracellular N- and C-termini. Here we focus on the emerging functions of the cytosolic S4-S5 linker on TRP channel gating. Most of this knowledge comes from pathogenic mutations within the S4-S5 linker that alter TRP channel activities. This knowledge has stimulated forward genetic approaches to identify additional residues around this region which are essential for channel gating and is supported, in part, by recent structures obtained for TRPV1, TRPV2, TRPV6, TRPA1, and TRPP2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Compartmentalized beta subunit distribution determines characteristics and ethanol sensitivity of somatic, dendritic, and terminal large-conductance calcium-activated potassium channels in the rat central nervous system.

    Science.gov (United States)

    Wynne, P M; Puig, S I; Martin, G E; Treistman, S N

    2009-06-01

    Neurons are highly differentiated and polarized cells, whose various functions depend upon the compartmentalization of ion channels. The rat hypothalamic-neurohypophysial system (HNS), in which cell bodies and dendrites reside in the hypothalamus, physically separated from their nerve terminals in the neurohypophysis, provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins. Using electrophysiological and immunohistochemical techniques, we characterized the large-conductance calcium-activated potassium (BK) channel in each of the three primary compartments (soma, dendrite, and terminal) of HNS neurons. We found that dendritic BK channels, in common with somatic channels but in contrast to nerve terminal channels, are insensitive to iberiotoxin. Furthermore, analysis of dendritic BK channel gating kinetics indicates that they, like somatic channels, have fast activation kinetics, in contrast to the slow gating of terminal channels. Dendritic and somatic channels are also more sensitive to calcium and have a greater conductance than terminal channels. Finally, although terminal BK channels are highly potentiated by ethanol, somatic and dendritic channels are insensitive to the drug. The biophysical and pharmacological properties of somatic and dendritic versus nerve terminal channels are consistent with the characteristics of exogenously expressed alphabeta1 versus alphabeta4 channels, respectively. Therefore, one possible explanation for our findings is a selective distribution of auxiliary beta1 subunits to the somatic and dendritic compartments and beta4 to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate beta1 or beta4 channel clusters in the membrane of somatic and dendritic or nerve terminal compartments, respectively.

  14. A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis

    Science.gov (United States)

    Guatimosim, C; Romano-Silva, M A; Cruz, J S; Beirão, P S L; Kalapothakis, E; Moraes-Santos, T; Cordeiro, M N; Diniz, C R; Gomez, M V; Prado, M A M

    1997-01-01

    The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. Using ω-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by ω-agatoxin IVA, an antagonist of P/Q calcium channels. ω-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. It is concluded that Tx3-3 potently inhibits ω-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes. PMID:9351520

  15. Reciprocal Interaction of Dendrite Geometry and Nuclear Calcium-VEGFD Signaling Gates Memory Consolidation and Extinction.

    Science.gov (United States)

    Hemstedt, Thekla J; Bengtson, C Peter; Ramírez, Omar; Oliveira, Ana M M; Bading, Hilmar

    2017-07-19

    Nuclear calcium is an important signaling end point in synaptic excitation-transcription coupling that is critical for long-term neuroadaptations. Here, we show that nuclear calcium acting via a target gene, VEGFD, is required for hippocampus-dependent fear memory consolidation and extinction in mice. Nuclear calcium-VEGFD signaling upholds the structural integrity and complexity of the dendritic arbor of CA1 neurons that renders those cells permissive for the efficient generation of synaptic input-evoked nuclear calcium transients driving the expression of plasticity-related genes. Therefore, the gating of memory functions rests on the reciprocally reinforcing maintenance of an intact dendrite geometry and a functional synapse-to-nucleus communication axis. In psychiatric and neurodegenerative disorders, therapeutic application of VEGFD may help to stabilize dendritic structures and network connectivity, which may prevent cognitive decline and could boost the efficacy of extinction-based exposure therapies. SIGNIFICANCE STATEMENT This study uncovers a reciprocal relationship between dendrite geometry, the ability to generate nuclear calcium transients in response to synaptic inputs, and the subsequent induction of expression of plasticity-related and dendritic structure-preserving genes. Insufficient nuclear calcium signaling in CA1 hippocampal neurons and, consequently, reduced expression of the nuclear calcium target gene VEGFD, a dendrite maintenance factor, leads to reduced-complexity basal dendrites of CA1 neurons, which severely compromises the animals' consolidation of both memory and extinction memory. The structure-protective function of VEGFD may prove beneficial in psychiatric disorders as well as neurodegenerative and aging-related conditions that are associated with loss of neuronal structures, dysfunctional excitation-transcription coupling, and cognitive decline. Copyright © 2017 the authors 0270-6474/17/376946-10$15.00/0.

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

    Science.gov (United States)

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

    2017-09-18

    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.

  17. Evidence for functional diversity between the voltage-gated proton channel Hv1 and its closest related protein HVRP1.

    Directory of Open Access Journals (Sweden)

    Iris H Kim

    Full Text Available The Hv1 channel and voltage-sensitive phosphatases share with voltage-gated sodium, potassium, and calcium channels the ability to detect changes in membrane potential through voltage-sensing domains (VSDs. However, they lack the pore domain typical of these other channels. NaV, KV, and CaV proteins can be found in neurons and muscles, where they play important roles in electrical excitability. In contrast, VSD-containing proteins lacking a pore domain are found in non-excitable cells and are not involved in neuronal signaling. Here, we report the identification of HVRP1, a protein related to the Hv1 channel (from which the name Hv1 Related Protein 1 is derived, which we find to be expressed primarily in the central nervous system, and particularly in the cerebellum. Within the cerebellar tissue, HVRP1 is specifically expressed in granule neurons, as determined by in situ hybridization and immunohistochemistry. Analysis of subcellular distribution via electron microscopy and immunogold labeling reveals that the protein localizes on the post-synaptic side of contacts between glutamatergic mossy fibers and the granule cells. We also find that, despite the similarities in amino acid sequence and structural organization between Hv1 and HVRP1, the two proteins have distinct functional properties. The high conservation of HVRP1 in vertebrates and its cellular and subcellular localizations suggest an important function in the nervous system.

  18. L- and T-type voltage-gated Ca2+ channels in human granulosa cells: functional characterization and cholinergic regulation.

    Science.gov (United States)

    Platano, Daniela; Magli, M Cristina; Ferraretti, Anna Pia; Gianaroli, Luca; Aicardi, Giorgio

    2005-04-01

    Using the whole-cell configuration of the patch-clamp technique, we have characterized two types of ionic currents through voltage-dependent Ca2+ channels in human granulosa cells. One is long-lasting, activates at approximately -20 mV, reaches the peak at approximately +20 mV, has an inactivation time constant of 132.5 +/- 5.6 msec at 20 mV, and is sensitive to dihydropyridines. The other is transient, activates at approximately -40 mV, peaks at approximately -10 mV, has an inactivation time constant of 38.8 +/- 1.8 msec at -10 mV, displays a voltage-dependent inactivation, and is sensitive to 100 microm Ni2+, but not to dihydropyridines. Biophysical and pharmacological properties of these currents indicate that they are gated through L- and T-type calcium channels, respectively. The cholinergic receptor agonist carbachol (50 microm) reduces the amplitude of the currents through both L-type (-34.7 +/- 6.4%; n = 10) and T-type (-52.6 +/- 7.4%; n = 8) channels, suggesting a possible role of these channels in the cholinergic regulation of human ovarian functions.

  19. Potentiation of Opioid-Induced Analgesia by L-Type Calcium Channel Blockers: Need for Clinical Trial in Cancer Pain

    Directory of Open Access Journals (Sweden)

    S Basu Ray

    2008-01-01

    Full Text Available Previous reports indicate that the analgesic effect of opioids is due to both closure of specific voltage-gated calcium channels (N- and P/Q-types and opening of G protein-coupled inwardly rectifying potassium channels (GIRKs in neurons concerned with transmission of pain. However, administration of opioids leads to unacceptable levels of side effects, particularly at high doses. Thus, current research is directed towards simultaneously targeting other voltage-gated calcium channels (VGCCs like the L-type VGCCs or even other cell signaling mechanisms, which would aug-ment opioid-mediated analgesic effect without a concurrent increase in the side effects. Unfortunately, the results of these studies are often conflicting considering the different experimental paradigms (variable drug selection and their doses and also the specific pain test used for studying analgesia adopted by researchers. The present review focuses on some of the interesting findings regarding the analgesic effect of Opioids + L-VGCC blockers and suggests that time has come for a clinical trial of this combination of drugs in the treatment of cancer pain.

  20. Imaging ligand-gated ion channels with quantum dots

    Science.gov (United States)

    Tomlinson, I. D.; Orndorff, Rebecca L.; Gussin, Hélène; Mason, John N.; Blakely, Randy D.; Pepperberg, David R.; Rosenthal, Sandra J.

    2007-02-01

    In this paper we report two different methodologies for labeling ligand-gated receptors. The first of these builds upon our earlier work with serotonin conjugated quantum dots and our studies with pegilated quantum dots to reduce non specific binding. In this approach a pegilated derivative of muscimol was synthesized and attached via an amide linkage to quantum dots coated in an amphiphillic polymer derivative of poly acrylamide. These conjugates were used to image the GABA C receptor in oocytes. An alternative approach was used to image tissue sections to study nicotinic acetylcholine receptors in the neuro muscular junction with biotinylated Bungerotoxin and streptavidin coated quantum dots.

  1. N Channel JFET Based Digital Logic Gate Structure

    Science.gov (United States)

    Krasowski, Michael J (Inventor)

    2013-01-01

    An apparatus is provided that includes a first field effect transistor with a source tied to zero volts and a drain tied to voltage drain drain (Vdd) through a first resistor. The apparatus also includes a first node configured to tie a second resistor to a third resistor and connect to an input of a gate of the first field effect transistor in order for the first field effect transistor to receive a signal. The apparatus also includes a second field effect transistor configured as a unity gain buffer having a drain tied to Vdd and an uncommitted source.

  2. Annotation of functional impact of voltage?gated sodium channel mutations

    OpenAIRE

    Hinard, Valérie; Britan, Aurore; Schaeffer, Mathieu; Zahn, Monique; Thomet, Urs; Rougier, Jean-Sébastien; Bairoch, Amos Marc; Abriel, Hugues; Gaudet, Pascale

    2017-01-01

    Abstract Voltage?gated sodium channels are pore?forming transmembrane proteins that selectively allow sodium ions to flow across the plasma membrane according to the electro?chemical gradient thus mediating the rising phase of action potentials in excitable cells and playing key roles in physiological processes such as neurotransmission, skeletal muscle contraction, heart rhythm, and pain sensation. Genetic variations in the nine human genes encoding these channels are known to cause a large ...

  3. Cortactin Controls Surface Expression of the Voltage-gated Potassium Channel KV10.1

    OpenAIRE

    Herrmann, Solveig; Ninkovic, Milena; Kohl, Tobias; Lörinczi, Éva; Pardo, Luis A.

    2012-01-01

    KV10.1 is a voltage-gated potassium channel aberrantly expressed in many cases of cancer, and participates in cancer initiation and tumor progression. Its action as an oncoprotein can be inhibited by a functional monoclonal antibody, indicating a role for channels located at the plasma membrane, accessible to the antibody. Cortactin is an actin-interacting protein implicated in cytoskeletal architecture and often amplified in several types of cancer. In this study, we describe a physical and ...

  4. PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1

    OpenAIRE

    Herrmann, Solveig; Ninkovic, Milena; Kohl, Tobias; Pardo, Luis A.

    2013-01-01

    Although crucial for their correct function, the mechanisms controlling surface expression of ion channels are poorly understood. In the case of the voltage-gated potassium channel KV10.1, this is determinant not only for its physiological function in brain, but also for its pathophysiology in tumors and possible use as a therapeutic target. The Golgi resident protein PIST binds several membrane proteins, thereby modulating their expression. Here we describe a PDZ domain-mediated interaction ...

  5. PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1

    OpenAIRE

    Solveig eHerrmann; Milena eNinkovic; Tobias eKohl; Pardo, Luis A.

    2013-01-01

    Although crucial for their correct function, the mechanisms controlling surface expression of ion channels are poorly understood. In the case of the voltage-gated potassium channel KV10.1,this is determinant not only for its physiological function in brain, but also for its pathophysiology in tumors and possible use as a therapeutic target. The Golgi resident protein PIST binds several membrane proteins, thereby modulating their expression. Here we describe a PDZ domain-mediated interaction o...

  6. Structural basis of slow activation gating in the cardiac IKs channel complex

    DEFF Research Database (Denmark)

    Strutz-Seebohm, Nathalie; Pusch, Michael; Wolf, Steffen

    2011-01-01

    Accessory ß-subunits of the KCNE gene family modulate the function of various cation channel a-subunits by the formation of heteromultimers. Among the most dramatic changes of biophysical properties of a voltage-gated channel by KCNEs are the effects of KCNE1 on KCNQ1 channels. KCNQ1 and KCNE1...... are believed to form nativeI(Ks) channels. Here, we characterize molecular determinants of KCNE1 interaction with KCNQ1 channels by scanning mutagenesis, double mutant cycle analysis, and molecular dynamics simulations. Our findings suggest that KCNE1 binds to the outer face of the KCNQ1 channel pore domain...... of the voltage sensor domain S4 of KCNQ1 in a putative pre-open channel state. Formation of this state may induce slow activation gating, the pivotal characteristic of native cardiac I(Ks) channels. This new KCNQ1-KCNE1 model may become useful for dynamic modeling of disease-associated mutant I(Ks) channels....

  7. Noradrenaline upregulates T-type calcium channels in rat pinealocytes.

    Science.gov (United States)

    Yu, Haijie; Seo, Jong Bae; Jung, Seung-Ryoung; Koh, Duk-Su; Hille, Bertil

    2015-02-15

    The mammalian pineal gland is a neuroendocrine organ that responds to circadian and seasonal rhythms. Its major function is to secrete melatonin as a hormonal night signal in response to nocturnal delivery of noradrenaline from sympathetic neurons. Culturing rat pinealocytes in noradrenaline for 24 h induced a low-voltage activated transient Ca(2+) current whose pharmacology and kinetics corresponded to a CaV3.1 T-type channel. The upregulation of the T-type Ca(2+) current is initiated by β-adrenergic receptors, cyclic AMP and cyclic AMP-dependent protein kinase. Messenger RNA for CaV3.1 T-type channels is significantly elevated by noradrenaline at 8 h and 24 h. The noradrenaline-induced T-type channel mediated an increased Ca(2+) entry and supported modest transient electrical responses to depolarizing stimuli, revealing the potential for circadian regulation of pinealocyte electrical excitability and Ca(2+) signalling. Our basic hypothesis is that mammalian pinealocytes have cycling electrical excitability and Ca(2+) signalling that may contribute to the circadian rhythm of pineal melatonin secretion. This study asked whether the functional expression of voltage-gated Ca(2+) channels (CaV channels) in rat pinealocytes is changed by culturing them in noradrenaline (NA) as a surrogate for the night signal. Channel activity was assayed as ionic currents under patch clamp and as optical signals from a Ca(2+)-sensitive dye. Channel mRNAs were assayed by quantitative polymerase chain reaction. Cultured without NA, pinealocytes showed only non-inactivating L-type dihydropyridine-sensitive Ca(2+) current. After 24 h in NA, additional low-voltage activated transient Ca(2+) current developed whose pharmacology and kinetics corresponded to a T-type CaV3.1 channel. This change was initiated by β-adrenergic receptors, cyclic AMP and protein kinase A as revealed by pharmacological experiments. mRNA for CaV3.1 T-type channels became significantly elevated, but mRNA for

  8. A Secondary Gate As a Mechanism for Inhibition of the M2 Proton Channel by Amantadine

    Science.gov (United States)

    Yi, Myunggi; Cross, Timothy A.; Zhou, Huan-Xiang

    2013-01-01

    The mechanism of inhibition of the influenza A virus M2 proton channel by the antiviral drug amantadine has been under intense investigation. The importance of a mechanistic understanding is heightened by the prevalence of amantadine-resistant mutations. To gain mechanistic insight at the molecular level, we carried out extensive molecular dynamics simulations of the tetrameric M2 proton channel in both apo and amantadine-bound forms in a lipid bilayer. The simulation of the apo form revealed that Val27 from the four M2 subunits can form a secondary gate near the channel entrance and break the water wire in the channel pore. This gate arises from physical occlusion and the elimination of hydrogen-bonding partners for water molecules. In the presence of amantadine, the secondary gate formed by Val27 and the drug molecule lying just below form an extended blockage, which breaks the water wire throughout the simulation. The location and orientation of amantadine inside of the channel pore as found in our simulation are supported by a host of experimental observations. Our study suggests a novel role for Val27 in the inhibition of the M2 proton channel by amantadine. PMID:18476738

  9. 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

  10. Structure of the inactivating gate from the Shaker voltage gated K{sup +} channel analyzed by NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Schott, M.K.; Antz, C. [Institute of Physiology, University of Tuebingen (Germany)]|[Department of Biophysics, Max-Planck-Institute for Medical Research, Heidelberg (Germany); Frank, R. [Center of Molecular Biology, Heidelberg (Germany); Ruppersberg, J.P. [Institute of Physiology, University of Tuebingen (Germany); Kalbitzer, H.R. [Department of Biophysics, University of Regensburg (Germany)]|[Department of Biophysics, Max-Planck-Institute for Medical Research, Heidelberg (Germany)

    1998-04-01

    Rapid inactivation of voltage-gated K{sup +} (K{sub V}) channels is mediated by an N-terminal domain (inactivating ball domain) which blocks the open channel from the cytoplasmic side. Inactivating ball domains of various K{sub V} channels are also biologically active when synthesized separately and added as a peptide to the solution. Synthetic inactivating ball domains from different K{sub V} channels with hardly any sequence homology mediate quite similar effects even on unrelated K{sub V} channel subtypes whose inactivation domain has been deleted. The solution structure of the inactivating ball peptide from Shaker (Sh-P22) was analyzed with NMR spectroscopy. The NMR data indicate a non-random structure in an aqueous environment. However, while other inactivating ball peptides showed well-defined three-dimensional structures under these conditions, Sh-P22 does not have a unique, compactly folded structure in solution. (orig.) With 3 figs., 2 tabs., 34 refs.

  11. Functional voltage-gated Ca2+ channels in muscle fibers of the platyhelminth Dugesia tigrina.

    Science.gov (United States)

    Cobbett, Peter; Day, Timothy A

    2003-03-01

    The presence and function of voltage-gated Ca(2+) channels were examined in individual muscle fibers freshly dispersed from the triclad turbellarian Dugesia tigrina. Individual muscle fibers contracted in response to elevated extracellular K(+) in a concentration-dependent fashion. These depolarization-induced contractions were blocked by extracellular Co(2+) (2.5 mM), suggesting that they were dependent on depolarization-induced Ca(2+) influx across the sarcolemma. A voltage-gated inward current was apparent in whole cell recordings when the outward K(+) current was abolished by replacement of intracellular K(+) by Cs(+). This inward current was amplified with increasing concentration (gated Ca(2+) channels. Further, and supporting the hypothesis that the inward current was mediated by these Ca(2+) channels, the Ba(2+) current was blocked by extracellular Co(2+) (2.5 mM) but not by tetrodotoxin (5 microM). Action potentials were generated by the muscle fibers in the presence of, but not in the absence of, extracellular Ba(2+) (5 mM). These data are the first clear demonstration of a voltage-gated Ca(2+) channel current in platyhelminth muscle, and they demonstrate a role for Ca(2+) influx in depolarization-induced contractions of muscle in these organisms.

  12. External Barium Affects the Gating of KCNQ1 Potassium Channels and Produces a Pore Block via Two Discrete Sites

    Science.gov (United States)

    Gibor, Gilad; Yakubovich, Daniel; Peretz, Asher; Attali, Bernard

    2004-01-01

    The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba2+ binding kinetics and the concentration and voltage dependence of Ba2+ steady-state block. Our results indicate that extracellular Ba2+ exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba2+ site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba2+ site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba2+ on channel gating in low external K+ solutions. Ba2+ binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K+ attenuates Ba2+ inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K+ channels, KCNQ1 channels display significant structural and functional uniqueness. PMID:15226366

  13. 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

  14. Involvement of Potassium Channels and Calcium-Independent Mechanisms in Hydrogen Sulfide-Induced Relaxation of Rat Mesenteric Small Arteries.

    Science.gov (United States)

    Hedegaard, Elise R; Gouliaev, Anja; Winther, Anna K; Arcanjo, Daniel D R; Aalling, Mathilde; Renaltan, Nirthika S; Wood, Mark E; Whiteman, Matthew; Skovgaard, Nini; Simonsen, Ulf

    2016-01-01

    Endogenous hydrogen sulfide (H2S) is involved in the regulation of vascular tone. We hypothesized that the lowering of calcium and opening of potassium (K) channels as well as calcium-independent mechanisms are involved in H2S-induced relaxation in rat mesenteric small arteries. Amperometric recordings revealed that free [H2S] after addition to closed tubes of sodium hydrosulfide (NaHS), Na2S, and GYY4137 [P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid] were, respectively, 14%, 17%, and 1% of added amount. The compounds caused equipotent relaxations in isometric myographs, but based on the measured free [H2S], GYY4137 caused more relaxation in relation to released free H2S than NaHS and Na2S in rat mesenteric small arteries. Simultaneous measurements of [H2S] and tension showed that 15 µM of free H2S caused 61% relaxation in superior mesenteric arteries. Simultaneous measurements of smooth muscle calcium and tension revealed that NaHS lowered calcium and caused relaxation of NE-contracted arteries, while high extracellular potassium reduced NaHS relaxation without corresponding calcium changes. In NE-contracted arteries, NaHS (1 mM) lowered the phosphorylation of myosin light chain, while phosphorylation of myosin phosphatase target subunit 1 remained unchanged. Protein kinase A and G, inhibitors of guanylate cyclase, failed to reduce NaHS relaxation, whereas blockers of voltage-gated KV7 channels inhibited NaHS relaxation, and blockers of mitochondrial complex I and III abolished NaHS relaxation. Our findings suggest that low micromolar concentrations of free H2S open K channels followed by lowering of smooth muscle calcium, and by another mechanism involving mitochondrial complex I and III leads to uncoupling of force, and hence vasodilation. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  15. The Role of Intracellular Linkers in Gating and Desensitization of Human Pentameric Ligand-Gated Ion Channels

    Science.gov (United States)

    Papke, David

    2014-01-01

    It has recently been proposed that post-translational modification of not only the M3–M4 linker but also the M1–M2 linker of pentameric ligand-gated ion channels modulates function in vivo. To estimate the involvement of the M1–M2 linker in gating and desensitization, we engineered a series of mutations to this linker of the human adult-muscle acetylcholine receptor (AChR), the α3β4 AChR and the homomeric α1 glycine receptor (GlyR). All tested M1–M2 linker mutations had little effect on the kinetics of deactivation or desensitization compared with the effects of mutations to the M2 α-helix or the extracellular M2–M3 linker. However, when the effects of mutations were assessed with 50 Hz trains of ∼1 ms pulses of saturating neurotransmitter, some mutations led to much more, and others to much less, peak-current depression than observed for the wild-type channels, suggesting that these mutations could affect the fidelity of fast synaptic transmission. Nevertheless, no mutation to this linker could mimic the irreversible loss of responsiveness reported to result from the oxidation of the M1–M2 linker cysteines of the α3 AChR subunit. We also replaced the M3–M4 linker of the α1 GlyR with much shorter peptides and found that none of these extensive changes affects channel deactivation strongly or reduces the marked variability in desensitization kinetics that characterizes the wild-type channel. However, we found that these large mutations to the M3–M4 linker can have pronounced effects on desensitization kinetics, supporting the notion that its post-translational modification could indeed modulate α1 GlyR behavior. PMID:24849357

  16. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

    Science.gov (United States)

    Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.

    2014-04-01

    Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

  17. Glucocorticoids specifically enhance L-type calcium current amplitude and affect calcium channel subunit expression in the mouse hippocampus.

    Science.gov (United States)

    Chameau, Pascal; Qin, Yongjun; Spijker, Sabine; Smit, August Benjamin; Smit, Guus; Joëls, Marian

    2007-01-01

    Previous studies have shown that corticosterone enhances whole cell calcium currents in CA1 pyramidal neurons, through a pathway involving binding of glucocorticoid receptor homodimers to the DNA. We examined whether glucocorticoids show selectivity for L- over N-type of calcium currents. Moreover, we addressed the putative gene targets that eventually lead to the enhanced calcium currents. Electrophysiological recordings were performed in nucleated patches that allow excellent voltage control. Calcium currents in these patches almost exclusively involve N- and L-type channels. We found that L- but not N-type calcium currents were largely enhanced after treatment with a high dose of corticosterone sufficient to activate glucocorticoid receptors. Voltage dependency and kinetic properties of the currents were unaffected by the hormone. Nonstationary noise analysis suggests that the increased current is not caused by a larger unitary conductance, but rather to a doubling of the number of functional channels. Quantitative real-time PCR revealed that transcripts of the Ca(v)1 subunits encoding for the N- or L-type calcium channels are not upregulated in the mouse CA1 area; instead, a strong, direct, and consistent upregulation of the beta4 subunit was observed. This indicates that the corticosteroid-induced increase in number of L-type calcium channels is not caused by a simple transcriptional regulation of the pore-forming subunit of the channels.

  18. Anomalous DIBL Effect in Fully Depleted SOI MOSFETs Using Nanoscale Gate-Recessed Channel Process

    Directory of Open Access Journals (Sweden)

    Avi Karsenty

    2015-01-01

    Full Text Available Nanoscale Gate-Recessed Channel (GRC Fully Depleted- (FD- SOI MOSFET device with a silicon channel thickness (tSi as low as 2.2 nm was first tested at room temperature for functionality check and then tested at low temperature (77 K for I-V characterizations. In spite of its FD-SOI nanoscale thickness and long channel feature, the device has surprisingly exhibited a Drain-Induced Barrier Lowering (DIBL effect at RT. However, this effect was suppressed at 77 K. If the apparition of such anomalous effect can be explained by a parasitic short channel transistor located at the edges of the channel, its suppression is explained by the decrease of the potential barrier between the drain and the channel when lowering the temperature.

  19. Estimation of charge effects of ultrafine channel utilizing junctionless transistor with nanodot-type floating gate

    Science.gov (United States)

    Ban, Takahiko; Migita, Shinji; Uenuma, Mutsunori; Okamoto, Naofumi; Ishikawa, Yasuaki; Uraoka, Yukiharu; Yamashita, Ichiro; Yamamoto, Shin-ichi

    2017-03-01

    Metal nanoparticles (NPs) embedded in junctionless field-effect transistors (JL-FETs) with a channel length of about sub-10-nm are fabricated and demonstrated. The anisotropic wet etching of a silicon-on-insulator (SOI) substrate was utilized to form V-grooves and define a nanometer-scale channel. Metal NPs are selectively placed onto the bottom of a V-groove using a bio nano process (BNP). A JL-FET is applied to a floating gate memory and used to study the impacts of charges close to the short channel. Low-voltage operation and memory behavior of broad threshold voltage appear. It is estimated by simulation that positive and negative charges equivalent to approximately 10 electrons are accumulated in one NP. It is expected that the JL-FETs can overcome the scaling limitations of floating gate memories.

  20. Selective calcium ion detection with functionalized ZnO nanorods-extended gate MOSFET.

    Science.gov (United States)

    Asif, M H; Nur, O; Willander, M; Danielsson, B

    2009-07-15

    Zinc oxide nanorod-extended gate field effect transistor (MOSFET) is demonstrated for the detection of calcium (Ca(2+)) ions. ZnO nanorods were grown on the surface of a silver wire to produce an electrochemical nanosensor for selectively detecting Ca(2+). The electrochemical response from the interaction between the ZnO nanorods and Ca(2+) in an aqueous solution is coupled directly to the gate of a field effect transistor (MOSFET). The induced voltage change on the gate results in a measureable current response. In order to adapt the sensors for Ca(2+) ions measurements in biological fluids with sufficient selectivity and stability, a plastic membrane coating containing ionophores was applied on the nanorods. The sensor exhibited a linear response within the range of interest from 1 microM to 1 mM. This work demonstrates a simple technique for sensitive detection of Ca(2+) ions by efficient transfer of the chemical response directly to a standard electronic component producing a low impedance signal.

  1. The isolated voltage sensing domain of the Shaker potassium channel forms a voltage-gated cation channel.

    Science.gov (United States)

    Zhao, Juan; Blunck, Rikard

    2016-10-06

    Domains in macromolecular complexes are often considered structurally and functionally conserved while energetically coupled to each other. In the modular voltage-gated ion channels the central ion-conducting pore is surrounded by four voltage sensing domains (VSDs). Here, the energetic coupling is mediated by interactions between the S4-S5 linker, covalently linking the domains, and the proximal C-terminus. In order to characterize the intrinsic gating of the voltage sensing domain in the absence of the pore domain, the Shaker Kv channel was truncated after the fourth transmembrane helix S4 (Shaker-iVSD). Shaker-iVSD showed significantly altered gating kinetics and formed a cation-selective ion channel with a strong preference for protons. Ion conduction in Shaker-iVSD developed despite identical primary sequence, indicating an allosteric influence of the pore domain. Shaker-iVSD also displays pronounced 'relaxation'. Closing of the pore correlates with entry into relaxation suggesting that the two processes are energetically related.

  2. The Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels: from Biophysics to Pharmacology of a Unique Family of Ion Channels.

    Science.gov (United States)

    Sartiani, Laura; Mannaioni, Guido; Masi, Alessio; Novella Romanelli, Maria; Cerbai, Elisabetta

    2017-10-01

    Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are important members of the voltage-gated pore loop channels family. They show unique features: they open at hyperpolarizing potential, carry a mixed Na/K current, and are regulated by cyclic nucleotides. Four different isoforms have been cloned (HCN1-4) that can assemble to form homo- or heterotetramers, characterized by different biophysical properties. These proteins are widely distributed throughout the body and involved in different physiologic processes, the most important being the generation of spontaneous electrical activity in the heart and the regulation of synaptic transmission in the brain. Their role in heart rate, neuronal pacemaking, dendritic integration, learning and memory, and visual and pain perceptions has been extensively studied; these channels have been found also in some peripheral tissues, where their functions still need to be fully elucidated. Genetic defects and altered expression of HCN channels are linked to several pathologies, which makes these proteins attractive targets for translational research; at the moment only one drug (ivabradine), which specifically blocks the hyperpolarization-activated current, is clinically available. This review discusses current knowledge about HCN channels, starting from their biophysical properties, origin, and developmental features, to (patho)physiologic role in different tissues and pharmacological modulation, ending with their present and future relevance as drug targets. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Boosting of synaptic potentials and spine Ca transients by the peptide toxin SNX-482 requires alpha-1E-encoded voltage-gated Ca channels.

    Directory of Open Access Journals (Sweden)

    Andrew J Giessel

    Full Text Available The majority of glutamatergic synapses formed onto principal neurons of the mammalian central nervous system are associated with dendritic spines. Spines are tiny protuberances that house the proteins that mediate the response of the postsynaptic cell to the presynaptic release of glutamate. Postsynaptic signals are regulated by an ion channel signaling cascade that is active in individual dendritic spines and involves voltage-gated calcium (Ca channels, small conductance (SK-type Ca-activated potassium channels, and NMDA-type glutamate receptors. Pharmacological studies using the toxin SNX-482 indicated that the voltage-gated Ca channels that signal within spines to open SK channels belong to the class Ca(V2.3, which is encoded by the Alpha-1E pore-forming subunit. In order to specifically test this conclusion, we examined the effects of SNX-482 on synaptic signals in acute hippocampal slices from knock-out mice lacking the Alpha-1E gene. We find that in these mice, application of SNX-482 has no effect on glutamate-uncaging evoked synaptic potentials and Ca influx, indicating that that SNX-482 indeed acts via the Alpha-1E-encoded Ca(V2.3 channel.

  4. Plasma membrane calcium channels in cancer: Alterations and consequences for cell proliferation and migration.

    Science.gov (United States)

    Déliot, Nadine; Constantin, Bruno

    2015-10-01

    The study of calcium channels in molecular mechanisms of cancer transformation is still a novel area of research. Several studies, mostly conducted on cancer cell lines, however support the idea that a diversity of plasma membrane channels participates in the remodeling of Ca2+ homeostasis, which regulates various cancer hallmarks such as uncontrolled multiplication and increase in migration and invasion abilities. However few is still understood concerning the intracellular signaling cascades mobilized by calcium influx participating to cancer cell behavior. This review intends to gather some of these pathways dependent on plasma membrane calcium channels and described in prostate, breast and lung cancer cell lines. In these cancer cell types, the calcium channels involved in calcium signaling pathways promoting cancer behaviors are mostly non-voltage activated calcium channels and belong to the TRP superfamily (TRPC, TPRPV and TRPM families) and the Orai family. TRP and Orai channels are part of many signaling cascades involving the activation of transmembrane receptors by extracellular ligand from the tumor environment. TRPV can sense changes in the physical and chemical environment of cancer cells and TRPM7 are stretch activated and sensitive to cholesterol. Changes in activation and or expression of plasma-membrane calcium channels affect calcium-dependent signaling processes relevant to tumorigenesis. The studies cited in this review suggest that an increase in plasma membrane calcium channel expression and/or activity sustain an elevated calcium entry (constitutive or under the control of extracellular signals) promoting higher cell proliferation and migration in most cases. A variety of non-voltage-operated calcium channels display change expression and/or activity in a same cancer type and cooperate to the same process relevant to cancer cell behavior, or can be involved in a different sequence of events during the tumorigenesis. This article is part of a

  5. T-Type Calcium Channels Are Required to Maintain Viability of Neural Progenitor Cells.

    Science.gov (United States)

    Kim, Ji-Woon; Oh, Hyun Ah; Lee, Sung Hoon; Kim, Ki Chan; Eun, Pyung Hwa; Ko, Mee Jung; Gonzales, Edson Luck T; Seung, Hana; Kim, Seonmin; Bahn, Geon Ho; Shin, Chan Young

    2018-02-21

    T-type calcium channels are low voltage-activated calcium channels that evoke small and transient calcium currents. Recently, T-type calcium channels have been implicated in neurodevelopmental disorders such as autism spectrum disorder and neural tube defects. However, their function during embryonic development is largely unknown. Here, we investigated the function and expression of T-type calcium channels in embryonic neural progenitor cells (NPCs). First, we compared the expression of T-type calcium channel subtypes (CaV3.1, 3.2, and 3.3) in NPCs and differentiated neural cells (neurons and astrocytes). We detected all subtypes in neurons but not in astrocytes. In NPCs, CaV3.1 was the dominant subtype, whereas CaV3.2 was weakly expressed, and CaV3.3 was not detected. Next, we determined CaV3.1 expression levels in the cortex during early brain development. Expression levels of CaV3.1 in the embryonic period were transiently decreased during the perinatal period and increased at postnatal day 11. We then pharmacologically blocked T-type calcium channels to determine the effects in neuronal cells. The blockade of T-type calcium channels reduced cell viability, and induced apoptotic cell death in NPCs but not in differentiated astrocytes. Furthermore, blocking T-type calcium channels rapidly reduced AKT-phosphorylation (Ser473) and GSK3β-phosphorylation (Ser9). Our results suggest that T-type calcium channels play essential roles in maintaining NPC viability, and T-type calcium channel blockers are toxic to embryonic neural cells, and may potentially be responsible for neurodevelopmental disorders.

  6. Ziconotide: neuronal calcium channel blocker for treating severe chronic pain.

    Science.gov (United States)

    Miljanich, G P

    2004-12-01

    Ziconotide (PRIALT) is a neuroactive peptide in the final stages of clinical development as a novel non-opioid treatment for severe chronic pain. It is the synthetic equivalent of omega-MVIIA, a component of the venom of the marine snail, Conus magus. The mechanism of action underlying ziconotide's therapeutic profile derives from its potent and selective blockade of neuronal N-type voltage-sensitive calcium channels (N-VSCCs). Direct blockade of N-VSCCs inhibits the activity of a subset of neurons, including pain-sensing primary nociceptors. This mechanism of action distinguishes ziconotide from all other analgesics, including opioid analgesics. In fact, ziconotide is potently anti-nociceptive in animal models of pain in which morphine exhibits poor anti-nociceptive activity. Moreover, in contrast to opiates, tolerance to ziconotide is not observed. Clinical studies of ziconotide in more than 2,000 patients reveal important correlations to ziconotide's non-clinical pharmacology. For example, ziconotide provides significant pain relief to severe chronic pain sufferers who have failed to obtain relief from opiate therapy and no evidence of tolerance to ziconotide is seen in these patients. Contingent on regulatory approval, ziconotide will be the first in a new class of neurological drugs: the N-type calcium channel blockers, or NCCBs. Its novel mechanism of action as a non-opioid analgesic suggests ziconotide has the potential to play a valuable role in treatment regimens for severe chronic pain. If approved for clinical use, ziconotide will further validate the neuroactive venom peptides as a source of new and useful medicines.

  7. The α2δ subunit and absence epilepsy: Beyond calcium channels?

    National Research Council Canada - National Science Library

    Celli, R; Santolini, I; Guiducci, M; Luijtelaar, E.L.J.M. van; Parisi, P; Striano, P; Gradini, R; Battaglia, G; Ngomba, R.T; Nicoletti, F

    2017-01-01

    .... Activation of T-type voltage-sensitive calcium channels (VSCCs) contributes to the pathological oscillatory activity of this network, and some of the first-line drugs used in the treatment of absence epilepsy inhibit T-type calcium channels. The α2δ...

  8. Role of L-type calcium-channel modulation in nonconvulsive epilepsy in rats

    NARCIS (Netherlands)

    Luijtelaar, E.L.J.M. van; Ates, N.; Coenen, A.M.L.

    1995-01-01

    Old male Wistar rats spontaneously showing hundreds of spike-wave discharges daily were used to investigate the role of calcium ions in nonconvulsive epilepsy. The effects of the L-type calcium channel blocker nimodipine and the L-type channel opener BAY K 8644 on number and duration of these

  9. Nonlinearity of a Voltage-Gated Potassium Channel Revealed by the Mechanical Susceptibility

    Science.gov (United States)

    Ariyaratne, Amila; Zocchi, Giovanni

    2013-01-01

    The voltage-gated potassium channel from Aeropyrum pernix operates by coupling the voltage-driven motion of a charged group of amino acids to the opening and closing of the pore. In this experiment, we drive this charged group with an ac field and observe the effect on the gating. The measurements for different frequencies and amplitudes of the forcing reveal an essential nonlinearity in the mechanical behavior of the molecule. Within a continuum-mechanics description, we extract the effective dissipation parameter γ for this conformational motion and find γ≈0.2g/s, similar to recent nanorheology measurements on the conformational motion of an enzyme.

  10. 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.

  11. Evaluating state dependence and subtype selectivity of calcium channel modulators in automated electrophysiology assays.

    Science.gov (United States)

    Kuryshev, Yuri A; Brown, Arthur M; Duzic, Emir; Kirsch, Glenn E

    2014-03-01

    Voltage-gated Ca2+ channels play essential roles in control of neurosecretion and muscle contraction. The pharmacological significance of Cav channels stem from their identification as the molecular targets of calcium blockers used in the treatment of cardiovascular diseases, such as hypertension, angina, and arrhythmia, and neurologic diseases, such as pain and seizure. It has been proposed that state-dependent Cav inhibitors, that is, those that preferentially bind to channels in open or inactivated states, may improve the therapeutic window over relatively state-independent Cav inhibitors. High-throughput fluorescent-based functional assays have been useful in screening chemical libraries to identify Cav inhibitors. However, hit confirmation, mechanism of action, and subtype selectivity are better suited to automated patch clamp assays that have sufficient capacity to handle the volume of compounds identified during screening, even of modest sized libraries (≤500,000 compounds), and the flexible voltage control that allows evaluation of state-dependent drug blocks. IonWorks Barracuda (IWB), the newest generation of IonWorks instruments, provides the opportunity to accelerate the Cav drug discovery studies in an automated patch clamp platform in 384-well format capable of medium throughput screening and profiling studies. We have validated hCav1.2, hCav2.1, hCav2.2, and hCav3.2 channels assays on the IWB platform (population patch clamp mode) and demonstrated that the biophysical characteristics of the channels (activation, inactivation, and steady-state inactivation) obtained with the IWB system are consistent with known subtype-specific characteristics. Using standard reference compounds (nifedipine, BAY K8644, verapamil, mibefradil, and pimozide), we demonstrated subtype-selective and state- and use-dependent characteristics of drug-channel interactions. Here we describe the design and validation of novel robust high-throughput Cav channel assays on the IWB

  12. L-type calcium channel targeting and local signalling in cardiac myocytes.

    Science.gov (United States)

    Shaw, Robin M; Colecraft, Henry M

    2013-05-01

    In the heart, Ca(2+) influx via Ca(V)1.2 L-type calcium channels (LTCCs) is a multi-functional signal that triggers muscle contraction, controls action potential duration, and regulates gene expression. The use of LTCC Ca(2+) as a multi-dimensional signalling molecule in the heart is complicated by several aspects of cardiac physiology. Cytosolic Ca(2+) continuously cycles between ~100 nM and ~1 μM with each heartbeat due to Ca(2+) linked signalling from LTCCs to ryanodine receptors. This rapid cycling raises the question as to how cardiac myocytes distinguish the Ca(2+) fluxes originating through L-type channels that are dedicated to contraction from Ca(2+) fluxes originating from other L-type channels that are used for non-contraction-related signalling. In general, disparate Ca(2+) sources in cardiac myocytes such as current through differently localized LTCCs as well as from IP3 receptors can signal selectively to Ca(2+)-dependent effectors in local microdomains that can be impervious to the cytoplasmic Ca(2+) transients that drive contraction. A particular challenge for diversified signalling via cardiac LTCCs is that they are voltage-gated and, therefore, open and presumably flood their microdomains with Ca(2+) with each action potential. Thus spatial localization of Cav1.2 channels to different types of microdomains of the ventricular cardiomyocyte membrane as well as the existence of particular macromolecular complexes in each Cav1.2 microdomain are important to effect different types of Cav1.2 signalling. In this review we examine aspects of Cav1.2 structure, targeting and signalling in two specialized membrane microdomains--transverse tubules and caveolae.

  13. Sensory neuron voltage-gated sodium channels as analgesic drug targets.

    Science.gov (United States)

    Momin, Aliakmal; Wood, John N

    2008-08-01

    Voltage-gated sodium channels are crucial determinants of neuronal excitability and signalling; some specific channel subtypes have been implicated in a number of chronic pain conditions. Human genetic studies show gain-of-function or loss-of-function mutations in Na(V)1.7 lead to an enhancement or lack of pain, respectively, whilst transgenic mouse and knockdown studies have implicated Na(V)1.3, Na(V)1.8 and Na(V)1.9 in peripheral pain pathways. The development of subtype-specific sodium channel blockers, though clearly desirable, has been technically challenging. Recent advances exploiting both natural products and small molecule selective channel blockers have demonstrated that this approach to pain control is feasible. These observations provide a rationale for the development of new analgesics without the side effect profile of broad spectrum sodium channel blockers.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Voltage-gated sodium channels contribute to action potentials and spontaneous contractility in isolated human lymphatic vessels.

    Science.gov (United States)

    Telinius, Niklas; Majgaard, Jens; Kim, Sukhan; Katballe, Niels; Pahle, Einar; Nielsen, Jørn; Hjortdal, Vibeke; Aalkjaer, Christian; Boedtkjer, Donna Briggs

    2015-07-15

    Voltage-gated sodium channels (VGSC) play a key role for initiating action potentials (AP) in excitable cells. VGSC in human lymphatic vessels have not been investigated. In the present study, we report the electrical activity and APs of small human lymphatic collecting vessels, as well as mRNA expression and function of VGSC in small and large human lymphatic vessels. The VGSC blocker TTX inhibited spontaneous contractions in six of 10 spontaneously active vessels, whereas ranolazine, which has a narrower VGSC blocking profile, had no influence on spontaneous activity. TTX did not affect noradrenaline-induced contractions. The VGSC opener veratridine induced contractions in a concentration-dependent manner (0.1-30 μm) eliciting a stable tonic contraction and membrane depolarization to -18 ± 0.6 mV. Veratridine-induced depolarizations and contractions were reversed ∼80% by TTX, and were dependent on Ca(2+) influx via L-type calcium channels and the sodium-calcium exchanger in reverse mode. Molecular analysis determined NaV 1.3 to be the predominantly expressed VGSC isoform. Electrophysiology of mesenteric lymphatics determined the resting membrane potential to be -45 ± 1.7 mV. Spontaneous APs were preceded by a slow depolarization of 5.3 ± 0.6 mV after which a spike was elicited that almost completely repolarized before immediately depolarizing again to plateau. Vessels transiently hyperpolarized prior to returning to the resting membrane potential. TTX application blocked APs. We have shown that VGSC are necessary for initiating and maintaining APs and spontaneous contractions in human lymphatic vessels and our data suggest the main contribution from comes NaV 1.3. We have also shown that activation of these channels augments the contractile activity of the vessels. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  16. Hyperpolarization-activated cyclic nucleotide-gated channels may contribute to regional anesthetic effects of lidocaine.

    Science.gov (United States)

    Zhou, Cheng; Ke, Bowen; Zhao, Yi; Liang, Peng; Liao, Daqing; Li, Tao; Liu, Jin; Chen, Xiangdong

    2015-03-01

    Local anesthetics (e.g., lidocaine) have been found to inhibit hyperpolarization-activated cyclic nucleotide-gated (HCN) channels besides sodium channels. However, the exact role of HCN channels in regional anesthesia in vivo is still elusive. Sciatic nerve block and intrathecal anesthesia were performed using lidocaine in wild-type and HCN1 channel knockout (HCN1) mice. EC50 of lidocaine and durations of 1% lidocaine were determined. In electrophysiologic recordings, effects of lidocaine on HCN channel currents, voltage-gated sodium channel currents, and neural membrane properties were recorded on dorsal root ganglia neurons. In both sciatic nerve block and intrathecal anesthesia, EC50 of lidocaine for tactile sensory blockade (2 g von Frey fiber) was significantly increased in HCN1 mice, whereas EC50 of lidocaine for pinprick blockade was unaffected. Durations of 1% lidocaine were significantly shorter in HCN1 mice for both sciatic nerve block and intrathecal anesthesia (n = 10). ZD7288 (HCN blocker) could significantly prolong durations of 1% lidocaine including pinprick blockade in sciatic nerve block (n = 10). Forskolin (raising cyclic adenosine monophosphate to enhance HCN2) could significantly shorten duration of pinprick blockade of 1% lidocaine in sciatic nerve block (n = 10). In electrophysiologic recordings, lidocaine could nonselectively inhibit HCN channel and sodium channel currents both in large and in small dorsal root ganglia neurons (n = 5 to 6). Meanwhile, lidocaine caused neural membrane hyperpolarization and increased input resistance of dorsal root ganglia neurons but not in large dorsal root ganglia neurons from HCN1 mice (n = 5-7). These data indicate that HCN channels may contribute to regional anesthetic effects of lidocaine. By inhibiting HCN channels, lidocaine could alter membrane properties of neurons.

  17. Menthol pain relief through cumulative inactivation of voltage-gated sodium channels.

    Science.gov (United States)

    Gaudioso, Christelle; Hao, Jizhe; Martin-Eauclaire, Marie-France; Gabriac, Mélanie; Delmas, Patrick

    2012-02-01

    Menthol is a natural compound of plant origin known to produce cool sensation via the activation of the TRPM8 channel. It is also frequently part of topical analgesic drugs available in a pharmacy, although its mechanism of action is still unknown. Compelling evidence indicates that voltage-gated Na(+) channels are critical for experiencing pain sensation. We tested the hypothesis that menthol may block voltage-gated Na(+) channels in dorsal root ganglion (DRG) neurons. By use of a patch clamp, we evaluated the effects of menthol application on tetrodotoxin (TTX)-resistant Nav1.8 and Nav1.9 channel subtypes in DRG neurons, and on TTX-sensitive Na(+) channels in immortalized DRG neuron-derived F11 cells. The results indicate that menthol inhibits Na(+) channels in a concentration-, voltage-, and frequency-dependent manner. Menthol promoted fast and slow inactivation states, causing use-dependent depression of Na(+) channel activity. In current clamp recordings, menthol inhibited firing at high-frequency stimulation with minimal effects on normal neuronal activity. We found that low concentrations of menthol cause analgesia in mice, relieving pain produced by a Na(+) channel-targeting toxin. We conclude that menthol is a state-selective blocker of Nav1.8, Nav1.9, and TTX-sensitive Na(+) channels, indicating a role for Na(+) channel blockade in the efficacy of menthol as topical analgesic compound. Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Yazdi, Samira; Stein, Matthias; Elinder, Fredrik; Andersson, Magnus; Lindahl, Erik

    2016-01-01

    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.

  19. 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.

  20. Evolutionary and structural perspectives of plant cyclic nucleotide-gated cation channels

    KAUST Repository

    Zelman, Alice K.

    2012-05-29

    Ligand-gated cation channels are a frequent component of signaling cascades in eukaryotes. Eukaryotes contain numerous diverse gene families encoding ion channels, some of which are shared and some of which are unique to particular kingdoms. Among the many different types are cyclic nucleotide-gated channels (CNGCs). CNGCs are cation channels with varying degrees of ion conduction selectivity. They are implicated in numerous signaling pathways and permit diffusion of divalent and monovalent cations, including Ca2+ and K+. CNGCs are present in both plant and animal cells, typically in the plasma membrane; recent studies have also documented their presence in prokaryotes. All eukaryote CNGC polypeptides have a cyclic nucleotide-binding domain and a calmodulin binding domain as well as a six transmembrane/one pore tertiary structure. This review summarizes existing knowledge about the functional domains present in these cation-conducting channels, and considers the evidence indicating that plant and animal CNGCs evolved separately. Additionally, an amino acid motif that is only found in the phosphate binding cassette and hinge regions of plant CNGCs, and is present in all experimentally confirmed CNGCs but no other channels was identified. This CNGC-specific amino acid motif provides an additional diagnostic tool to identify plant CNGCs, and can increase confidence in the annotation of open reading frames in newly sequenced genomes as putative CNGCs. Conversely, the absence of the motif in some plant sequences currently identified as probable CNGCs may suggest that they are misannotated or protein fragments. 2012 Zelman, Dawe, Gehring and Berkowitz.

  1. 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-gated

  2. Histochemical localisation of FMRFamide-gated Na+ channels in Helisoma trivolvis and Helix aspersa neurones.

    Science.gov (United States)

    Davey, F; Harris, S J; Cottrell, G A

    2001-11-01

    FMRFamide-gated Na+ channels of molluscan neurones belong to the ENa/Deg family of channels which have diverse functions. FMRFamide (Phe-Met-Arg-Phe-NH2) Na+ channels were detected electrophysiologically in specified neurones of Helix (Helix aspersa) and Helisoma (Helisoma trivolvis), and clones (FaNaCs) subsequently identified. We have now made a study to determine the distribution of mRNA for the clones HaFaNaC (Helix) and HtFaNaC (Helisoma) in the nervous systems of these species using standard in situ hybridization techniques. Immunohistochemical experiments were also made using an HtFaNaC antibody to detect the channel protein in Helisoma neurones. Many neurones in the central ganglia, including those which exhibit the FMRFamide Na+ current, stained for FaNaC-mRNA, suggesting a much wider distribution of the channel than was indicated by the earlier work. An immunoreactive response to the channel antibody was also observed in some Helisoma neurones, such as the giant dopamine neurone of the left pedal ganglion, also shown to possess HtFaNaC-mRNA and to exhibit the FMRFamide Na+ current. Taken together, these experiments suggest that the clones HaFaNaC and HtFaNaC are major, if not the only, subunits of the FMRFamide-gated Na+ channel detected electrophysiologically in the identified neurones of these species. However, fewer neurones in Helisoma reacted with the HtFaNaC-antibody than those which exhibited message for the channel. This discrepancy may be due to a difference in sensitivity of the two techniques, or because not all of the channel mRNA is normally expressed as a membrane protein.

  3. Involvement of voltage-gated sodium channels blockade in the analgesic effects of orphenadrine.

    Science.gov (United States)

    Desaphy, Jean-François; Dipalma, Antonella; De Bellis, Michela; Costanza, Teresa; Gaudioso, Christelle; Delmas, Patrick; George, Alfred L; Camerino, Diana Conte

    2009-04-01

    Orphenadrine is a drug acting on multiple targets, including muscarinic, histaminic, and NMDA receptors. It is used in the treatment of Parkinson's disease and in musculoskeletal disorders. It is also used as an analgesic, although its mechanism of action is still unknown. Both physiological and pharmacological results have demonstrated a critical role for voltage-gated sodium channels in many types of chronic pain syndromes. We tested the hypothesis that orphenadrine may block voltage-gated sodium channels. By using patch-clamp experiments, we evaluated the effects of the drug on whole-cell sodium currents in HEK293 cells expressing the skeletal muscle (Nav1.4), cardiac (Nav1.5) and neuronal (Nav1.1 and Nav1.7) subtypes of human sodium channels, as well as on whole-cell tetrodotoxin (TTX)-resistant sodium currents likely conducted by Nav1.8 and Nav1.9 channel subtypes in primary culture of rat DRG sensory neurons. The results indicate that orphenadrine inhibits sodium channels in a concentration-, voltage- and frequency-dependent manner. By using site-directed mutagenesis, we further show that orphenadrine binds to the same receptor as the local anesthetics. Orphenadrine affinities for resting and inactivated sodium channels were higher compared to those of known sodium channels blockers, such as mexiletine and flecainide. Low, clinically relevant orphenadrine concentration produces a significant block of Nav1.7, Nav1.8, and Nav1.9 channels, which are critical for experiencing pain sensations, indicating a role for sodium channel blockade in the clinical efficacy of orphenadrine as analgesic compound. On the other hand, block of Nav1.1 and Nav1.5 may contribute to the proconvulsive and proarrhythmic adverse reactions, especially observed during overdose.

  4. Dynamic PIP2 interactions with voltage sensor elements contribute to KCNQ2 channel gating

    Science.gov (United States)

    Zhang, Qiansen; Zhou, Pingzheng; Chen, Zhuxi; Li, Min; Jiang, Hualiang; Gao, Zhaobing; Yang, Huaiyu

    2013-01-01

    The S4 segment and the S4–S5 linker of voltage-gated potassium (Kv) channels are crucial for voltage sensing. Previous studies on the Shaker and Kv1.2 channels have shown that phosphatidylinositol-4,5-bisphosphate (PIP2) exerts opposing effects on Kv channels, up-regulating the current amplitude, while decreasing the voltage sensitivity. Interactions between PIP2 and the S4 segment or the S4–S5 linker in the closed state have been highlighted to explain the effects of PIP2 on voltage sensitivity. Here, we show that PIP2 preferentially interacts with the S4–S5 linker in the open-state KCNQ2 (Kv7.2) channel, whereas it contacts the S2–S3 loop in the closed state. These interactions are different from the PIP2–Shaker and PIP2–Kv1.2 interactions. Consistently, PIP2 exerts different effects on KCNQ2 relative to the Shaker and Kv1.2 channels; PIP2 up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. Disruption of the interaction of PIP2 with the S4–S5 linker by a single mutation decreases the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop does not alter voltage sensitivity. These results provide insight into the mechanism of PIP2 action on KCNQ channels. In the closed state, PIP2 is anchored at the S2–S3 loop; upon channel activation, PIP2 interacts with the S4–S5 linker and is involved in channel gating. PMID:24277843

  5. Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone.

    Science.gov (United States)

    Buckingham, Steven D; Higashino, Yoshiaki; Sattelle, David B

    2009-11-01

    The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D(f)) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D(f) motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.

  6. The influence of environmental calcium concentrations on calcium flux, compensatory drinking and epithelial calcium channel expression in a freshwater cartilaginous fish.

    Science.gov (United States)

    Allen, Peter J; Weihrauch, Dirk; Grandmaison, Vanessa; Dasiewicz, Patricia; Peake, Stephan J; Anderson, W Gary

    2011-03-15

    Calcium metabolism and mRNA levels of the epithelial calcium channel (ECaC) were examined in a freshwater cartilaginous fish, the lake sturgeon Acipenser fulvescens. Lake sturgeon were acclimated for ≥2 weeks to 0.1 (low), 0.4 (normal) or 3.3 (high) mmol l(-1) environmental calcium. Whole-body calcium flux was examined using (45)Ca as a radioactive marker. Net calcium flux was inward in all treatment groups; however, calcium influx was greatest in the low calcium environment and lowest in the high calcium environment, whereas efflux had the opposite relationship. A significant difference in the concentration of (45)Ca in the gastrointestinal tract (GIT) of fish in the low calcium environment led to the examination of drinking rate and calcium flux across the anterior-middle (mid) intestine. Drinking rate was not different between treatments; however, calcium influx across the mid-intestine in the low calcium treatment was significantly greater than that in both the normal and high calcium treatments. The lake sturgeon ECaC was 2831 bp in length, with a predicted protein sequence of 683 amino acids that shared a 66% identity with the closest sequenced ECaCs from the vertebrate phyla. ECaC mRNA levels were examined in the gills, kidney, pyloric caeca, mid-intestine and spiral intestine. Expression levels were highest in the gills, then the kidneys, and were orders of magnitude lower in the GIT. Contrary to existing models for calcium uptake in the teleost gill, ECaC expression was greatest in high calcium conditions and kidney ECaC expression was lowest in low calcium conditions, suggesting that cellular transport mechanisms for calcium may be distinctly different in these freshwater cartilaginous fishes.

  7. The voltage-gated sodium channel nav1.8 is expressed in human sperm.

    Directory of Open Access Journals (Sweden)

    Antonio Cejudo-Roman

    Full Text Available The role of Na(+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na(+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 μM, the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca(2+-containing or Ca(2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na(+, [Na(+]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na(+ channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

  8. The Voltage-Gated Sodium Channel Nav1.8 Is Expressed in Human Sperm

    Science.gov (United States)

    Cejudo-Roman, Antonio; Pinto, Francisco M.; Subirán, Nerea; Ravina, Cristina G.; Fernández-Sánchez, Manuel; Pérez-Hernández, Natalia; Pérez, Ricardo; Pacheco, Alberto; Irazusta, Jon; Candenas, Luz

    2013-01-01

    The role of Na+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 μM), the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca2+-containing or Ca2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na+, [Na+]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na+ channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function. PMID:24086692

  9. Signal transduction pathways in the pentameric ligand-gated ion channels.

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    David Mowrey

    Full Text Available The mechanisms of allosteric action within pentameric ligand-gated ion channels (pLGICs remain to be determined. Using crystallography, site-directed mutagenesis, and two-electrode voltage clamp measurements, we identified two functionally relevant sites in the extracellular (EC domain of the bacterial pLGIC from Gloeobacter violaceus (GLIC. One site is at the C-loop region, where the NQN mutation (D91N, E177Q, and D178N eliminated inter-subunit salt bridges in the open-channel GLIC structure and thereby shifted the channel activation to a higher agonist concentration. The other site is below the C-loop, where binding of the anesthetic ketamine inhibited GLIC currents in a concentration dependent manner. To understand how a perturbation signal in the EC domain, either resulting from the NQN mutation or ketamine binding, is transduced to the channel gate, we have used the Perturbation-based Markovian Transmission (PMT model to determine dynamic responses of the GLIC channel and signaling pathways upon initial perturbations in the EC domain of GLIC. Despite the existence of many possible routes for the initial perturbation signal to reach the channel gate, the PMT model in combination with Yen's algorithm revealed that perturbation signals with the highest probability flow travel either via the β1-β2 loop or through pre-TM1. The β1-β2 loop occurs in either intra- or inter-subunit pathways, while pre-TM1 occurs exclusively in inter-subunit pathways. Residues involved in both types of pathways are well supported by previous experimental data on nAChR. The direct coupling between pre-TM1 and TM2 of the adjacent subunit adds new insight into the allosteric signaling mechanism in pLGICs.

  10. Encephalitis due to antibodies to voltage gated potassium channel (VGKC with cerebellar involvement in a teenager

    Directory of Open Access Journals (Sweden)

    Megan M Langille

    2015-01-01

    Full Text Available Encephalitis due to antibodies to voltage gated potassium channel (VGKC typically presents with limbic encephalitis and medial temporal lobe involvement on neuroimaging. We describe a case of 13 year girl female with encephalitis due to antibodies to VGKC with signal changes in the cerebellar dentate nuclei bilaterally and clinical features that suggested predominant cerebellar involvement. These have never been reported previously in the literature. Our case expands the phenotypic spectrum of this rare condition.

  11. On the multiple roles of the voltage gated sodium channel β1 subunit in genetic diseases

    Directory of Open Access Journals (Sweden)

    Debora eBaroni

    2015-05-01

    Full Text Available Voltage-gated sodium channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are composed of a pore-forming α-subunit and associated β-subunits. The β1-subunit was the first accessory subunit to be cloned. It can be important for controlling cell excitability and modulating multiple aspects of sodium channel physiology. Mutations of β1 are implicated in a wide variety of inherited pathologies, including epilepsy and cardiac conduction diseases. This review summarizes β1-subunit related channelopathies pointing out the current knowledge concerning their genetic background and their underlying molecular mechanisms.

  12. Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster.

    Science.gov (United States)

    Remnant, Emily J; Williams, Adam; Lumb, Chris; Yang, Ying Ting; Chan, Janice; Duchêne, Sebastian; Daborn, Phillip J; Batterham, Philip; Perry, Trent

    2016-07-07

    Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets. Copyright © 2016 Remnant et al.

  13. 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.

  14. Gating machinery of InsP3R channels revealed by electron cryomicroscopy.

    Science.gov (United States)

    Fan, Guizhen; Baker, Matthew L; Wang, Zhao; Baker, Mariah R; Sinyagovskiy, Pavel A; Chiu, Wah; Ludtke, Steven J; Serysheva, Irina I

    2015-11-19

    Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are ubiquitous ion channels responsible for cytosolic Ca(2+) signalling and essential for a broad array of cellular processes ranging from contraction to secretion, and from proliferation to cell death. Despite decades of research on InsP3Rs, a mechanistic understanding of their structure-function relationship is lacking. Here we present the first, to our knowledge, near-atomic (4.7 Å) resolution electron cryomicroscopy structure of the tetrameric mammalian type 1 InsP3R channel in its apo-state. At this resolution, we are able to trace unambiguously ∼85% of the protein backbone, allowing us to identify the structural elements involved in gating and modulation of this 1.3-megadalton channel. Although the central Ca(2+)-conduction pathway is similar to other ion channels, including the closely related ryanodine receptor, the cytosolic carboxy termini are uniquely arranged in a left-handed α-helical bundle, directly interacting with the amino-terminal domains of adjacent subunits. This configuration suggests a molecular mechanism for allosteric regulation of channel gating by intracellular signals.

  15. Regulatory role of voltage-gated sodium channel β subunits in sensory neurons

    Directory of Open Access Journals (Sweden)

    Mohamed eChahine

    2011-11-01

    Full Text Available Voltage-gated Na+ channels are transmembrane-bound proteins incorporating aqueous conduction pores that are highly selective for Na+. The opening of these channels results in the rapid influx of Na+ ions that depolarize the cell and drive the rapid upstroke of nerve and muscle action potentials. While the concept of a Na+-selective ion channel had been formulated in the 1940s, it was not until the 1980s that the biochemical properties of the 260-kDa and 36-kDa auxiliary β subunits (β1, β2 were first described. Subsequent cloning and heterologous expression studies revealed that the  subunit forms the core of the channel and is responsible for both voltage-dependent gating and ionic selectivity. To date, ten isoforms of the Na+ channel α subunit have been identified that vary in their primary structures, tissue distribution, biophysical properties, and sensitivity to neurotoxins. Four β subunits (β1-β4 and two splice variants (β1A, β1B have been identified that modulate the subcellular distribution, cell surface expression, and functional properties of the α subunits. The purpose of this review is to provide a broad overview of β subunit expression and function in peripheral sensory neurons and examine their contributions to neuropathic pain.

  16. Finite element simulation of the gating mechanism of mechanosensitive ion channels

    Science.gov (United States)

    Bavi, Navid; Qin, Qinghua; Martinac, Boris

    2013-08-01

    In order to eliminate limitations of existing experimental or computational methods (such as patch-clamp technique or molecular dynamic analysis) a finite element (FE) model for multi length-scale and time-scale investigation on the gating mechanism of mechanosensitive (MS) ion channels has been established. Gating force value (from typical patch clamping values) needed to activate Prokaryotic MS ion channels was applied as tensional force to the FE model of the lipid bilayer. Making use of the FE results, we have discussed the effects of the geometrical and the material properties of the Escherichia coli MscL mechanosensitive ion channel opening in relation to the membrane's Young's modulus (which will vary depending on the cell type or cholesterol density in an artificial membrane surrounding the MscL ion channel). The FE model has shown that when the cell membrane stiffens the required channel activation force increases considerably. This is in agreement with experimental results taken from the literature. In addition, the present study quantifies the relationship between the membrane stress distribution around a `hole' for modeling purposes and the stress concentration in the place transmembrane proteins attached to the hole by applying an appropriate mesh refinement as well as well defining contact condition in these areas.

  17. Developmental expression of Kv1 voltage-gated potassium channels in the avian hypothalamus.

    Science.gov (United States)

    Doczi, Megan A; Vitzthum, Carl M; Forehand, Cynthia J

    2016-03-11

    Specialized hypothalamic neurons integrate the homeostatic balance between food intake and energy expenditure, processes that may become dysregulated during the development of diabetes, obesity, and other metabolic disorders. Shaker family voltage-gated potassium channels (Kv1) contribute to the maintenance of resting membrane potential, action potential characteristics, and neurotransmitter release in many populations of neurons, although hypothalamic Kv1 channel expression has been largely unexplored. Whole-cell patch clamp recordings from avian hypothalamic brain slices demonstrate a developmental shift in the electrophysiological properties of avian arcuate nucleus neurons, identifying an increase in outward ionic current that corresponds with action potential maturation. Additionally, RT-PCR experiments identified the early expression of Kv1.2, Kv1.3, and Kv1.5 mRNA in the embryonic avian hypothalamus, suggesting that these channels may underlie the electrophysiological changes observed in these neurons. Real-time quantitative PCR analysis on intact microdissections of embryonic hypothalamic tissue revealed a concomitant increase in Kv1.2 and Kv1.5 gene expression at key electrophysiological time points during development. This study is the first to demonstrate hypothalamic mRNA expression of Kv1 channels in developing avian embryos and may suggest a role for voltage-gated ion channel regulation in the physiological patterning of embryonic hypothalamic circuits governing energy homeostasis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Positive-bias gate-controlled metal-insulator transition in ultrathin VO2 channels with TiO2 gate dielectrics.

    Science.gov (United States)

    Yajima, Takeaki; Nishimura, Tomonori; Toriumi, Akira

    2015-12-14

    The next generation of electronics is likely to incorporate various functional materials, including those exhibiting ferroelectricity, ferromagnetism and metal-insulator transitions. Metal-insulator transitions can be controlled by electron doping, and so incorporating such a material in transistor channels will enable us to significantly modulate transistor current. However, such gate-controlled metal-insulator transitions have been challenging because of the limited number of electrons accumulated by gate dielectrics, or possible electrochemical reaction in ionic liquid gate. Here we achieve a positive-bias gate-controlled metal-insulator transition near the transition temperature. A significant number of electrons were accumulated via a high-permittivity TiO2 gate dielectric with subnanometre equivalent oxide thickness in the inverse-Schottky-gate geometry. An abrupt transition in the VO2 channel is further exploited, leading to a significant current modulation far beyond the capacitive coupling. This solid-state operation enables us to discuss the electrostatic mechanism as well as the collective nature of gate-controlled metal-insulator transitions, paving the pathway for developing functional field effect transistors.

  19. Divergent action of calcium channel blockers on ATP-binding cassette protein expression.

    Science.gov (United States)

    Hasegawa, Kazuhiro; Wakino, Shu; Kanda, Takeshi; Yoshioka, Kyoko; Tatematsu, Satoru; Homma, Koichiro; Takamatsu, Ichiro; Sugano, Naoki; Hayashi, Koichi

    2005-12-01

    Calcium channel blockers (CCBs) are widely used in clinical practice, and have been reported to be effective in preventing the progression of atherosclerosis. We examined whether various types of calcium channel blockers affected the expression of ATP binding cassette transporter A1 (ABCA1), a factor contributing to anti-atherogenesis. Undifferentiated monocytic cell line, THP-1 cells were maintained in RPMI 1640 medium and treated with different kinds of calcium channel blockers. Among the calcium channel blockers tested, aranidipine and efonidipine increased ABCA1 protein expression without an increase in ABCA1 mRNA expression, whereas other calcium channel blockers (eg, nifedipine, amlodipine, and nicardipine) or T-type calcium channel blockers (eg, mibefradil and nickel chloride) failed to upregulate ABCA1 expression. H89, a protein kinase A inhibitor inhibited the aranidipine-induced ABCA1 protein expression, whereas genistein (a tyrosine kinase inhibitor), or AG490 (a JAK-2 inhibitor) had no effects. Neither of these inhibitors suppressed the efonidipine-induced ABCA1 protein expression. Intracellular cAMP levels were elevated only by aranidipine, but not by efonidipine. In conclusion, aranidipine and efonidipine have the ability to induce ABCA1 protein by distinct mechanisms; protein kinase A is involved in the aranidipine-induced ABCA1 upregulation. This non-class effect of calcium channel blockers may potentially offer beneficial action in the treatment of hypertensive subjects with atherosclerosis.

  20. Effects of low-dose ionising radiation on pituitary adenoma: is there a role for L-type calcium channel?

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Marcella Araugio; Santos, Raquel Gouvea dos [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia]. E-mail: santosr@cdtn.br

    2005-10-15

    Pituitary adenomas constitute about 6-18% of brain tumours in adults. Activation of voltage gated calcium currents can account for growth hormone over secretion in some GH-secreting pituitary adenomas that produce an acromegaly appearance and increase mortality. Ca{sup 2+} ions, as mediators of intracellular signalling, are crucial for the development of apoptosis. However, the role of [Ca{sup 2+}] in the development of apoptosis is ambiguous. In this study, the effects of low-dose ionising gamma radiation ({sup 60} Co) on rat pituitary adenoma cells survival and proliferation and the role of calcium channels on the apoptosis radio-induced were evaluated. Doses as low as 3 Gy were found to inhibit GH3 cell proliferation. Even though there was a significant number of live cells,168 hours following irradiation, they were not able to proliferate. The results indicate that the blockade of extracellular calcium influx through these channels does not interfere in the radiation-induced apoptosis in GH3 cells. (author)

  1. Immunolocalization and expression of small-conductance calcium-activated potassium channels in human myometrium

    DEFF Research Database (Denmark)

    Rosenbaum, Sofia T; Svalø, Julie; Nielsen, Karsten

    2012-01-01

    Small-conductance calcium-activated potassium (SK3) channels have been detected in human myometrium and we have previously shown a functional role of SK channels in human myometrium in vitro. The aims of this study were to identify the precise localization of SK3 channels and to quantify SK3 mRNA...

  2. 5,6-EET potently inhibits T-type calcium channels

    DEFF Research Database (Denmark)

    Cazade, M.; Bidaud, I.; Hansen, Pernille B. Lærkegaard

    2014-01-01

    T-type calcium channels (T-channels) are important actors in neuronal pacemaking, in heart rhythm, and in the control of the vascular tone. T-channels are regulated by several endogenous lipids including the primary eicosanoid arachidonic acid (AA), which display an important role in vasodilation...

  3. Different calcium sources control somatic versus dendritic SK channel activation during action potentials.

    Science.gov (United States)

    Jones, Scott L; Stuart, Greg J

    2013-12-11

    Small-conductance calcium-activated potassium (SK) channels play an important role in regulating neuronal excitability. While SK channels at the soma have long been known to contribute to the medium afterhyperpolarization (mAHP), recent evidence indicates they also regulate NMDA receptor activation in dendritic spines. Here we investigate the activation of SK channels in spines and dendrites of rat cortical pyramidal neurons during action potentials (APs), and compare this to SK channel activation at the soma. Using confocal calcium imaging, we demonstrate that the inhibition of SK channels with apamin results in a location-dependent increase in calcium influx into dendrites and spines during backpropagating APs (average increase, ~40%). This effect was occluded by block of R-type voltage-dependent calcium channels (VDCCs), but not by inhibition of N- or P/Q-type VDCCs, or block of calcium release from intracellular stores. During these experiments, we noticed that the calcium indicator (Oregon Green BAPTA-1) blocked the mAHP. Subsequent experiments using low concentrations of EGTA (1 mm) produced the same result, suggesting that somatic SK channels are not tightly colocalized with their calcium source. Consistent with this idea, all known subtypes of VDCCs except R-type were calcium sources for the apamin-sensitive mAHP at the soma. We conclude that SK channels in spines and dendrites of cortical pyramidal neurons regulate calcium influx during backpropagating APs in a distance-dependent manner, and are tightly coupled to R-type VDCCs. In contrast, SK channels activated by APs at the soma of these neurons are weakly coupled to a variety of VDCCs.

  4. n-Alcohols Inhibit Voltage-Gated Na+ Channels Expressed in Xenopus Oocytes

    Science.gov (United States)

    Horishita, Takafumi; Harris, R. Adron

    2008-01-01

    Voltage-gated sodium channels are essential for the initiation and propagation of action potentials in excitable cells and are known as a target of local anesthetics. In addition, inhibition of sodium channels by volatile anesthetics has been proposed as a mechanism of general anesthesia. The n-alcohols produce anesthesia, and their potency increases with carbon number until a “cut-off” is reached. In this study, we examined effects of a range of n-alcohols on Nav1.2 subunits to determine the alcohol cut-off for this channel. We also studied the effect of a short-chain alcohol (ethanol) and a long-chain alcohol (octanol) on Nav1.2, Nav1.4, Nav1.6, and Nav1.8 subunits, and we investigated the effects of alcohol on channel kinetics. Ethanol and octanol inhibited sodium currents of all subunits, and the inhibition of the Nav1.2 channel by n-alcohols indicated a cut-off at nonanol. Ethanol and octanol produced open-channel block, which was more pronounced for Nav1.8 than for the other sodium channels. Inhibition of Nav1.2 was due to decreased activation and increased inactivation. These results suggest that sodium channels may have a hydrophobic binding site for n-alcohols and demonstrate the differences in the kinetic mechanisms of inhibition for n-alcohols and inhaled anesthetics. PMID:18434586

  5. Hyperpolarization-activated cyclic-nucleotide-gated channels potentially modulate axonal excitability at different thresholds.

    Science.gov (United States)

    Weerasinghe, Dinushi; Menon, Parvathi; Vucic, Steve

    2017-12-01

    Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels mediate differences in sensory and motor axonal excitability at different thresholds in animal models. Importantly, HCN channels are responsible for voltage-gated inward rectifying (Ih) currents activated during hyperpolarization. The Ih currents exert a crucial role in determining the resting membrane potential and have been implicated in a variety of neurological disorders, including neuropathic pain. In humans, differences in biophysical properties of motor and sensory axons at different thresholds remain to be elucidated and could provide crucial pathophysiological insights in peripheral neurological diseases. Consequently, the aim of this study was to characterize sensory and motor axonal function at different threshold. Median nerve motor and sensory axonal excitability studies were undertaken in 15 healthy subjects (45 studies in total). Tracking targets were set to 20, 40, and 60% of maximum for sensory and motor axons. Hyperpolarizing threshold electrotonus (TEh) at 90-100 ms was significantly increased in lower threshold sensory axons times (F = 11.195, P sensory axons. In conclusion, variation in the kinetics of HCN isoforms could account for the findings in motor and sensory axons. Importantly, assessing the function of HCN channels in sensory and motor axons of different thresholds may provide insights into the pathophysiological processes underlying peripheral neurological diseases in humans, particularly focusing on the role of HCN channels with the potential of identifying novel treatment targets.NEW & NOTEWORTHY Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which underlie inward rectifying currents (Ih), appear to mediate differences in sensory and motor axonal properties. Inward rectifying currents are increased in lower threshold motor and sensory axons, although different HCN channel isoforms appear to underlie these changes. While faster activating HCN

  6. The role of T-type calcium channel genes in absence seizures

    Directory of Open Access Journals (Sweden)

    Yucai eChen

    2014-05-01

    Full Text Available The thalamic relay neurons, reticular thalamic nucleus, and neocortical pyramidal cells form a circuit that sustains oscillatory burst firing, and is regarded as the underlying mechanism of absence seizures. T-type calcium channels play a key role in this circuit. Here we review the role of T-type calcium channel genes in the development of absence seizures, and emphasize gain or loss of function mutations, and other variations that alter both quantity and quality of transcripts, and methylation status of isoforms of T-type calcium channel proteins might be of equal importance in understanding the pathological mechanism of absence seizures.

  7. Calcium channels and their blockers in intraocular pressure and glaucoma.

    Science.gov (United States)

    Mayama, Chihiro

    2014-09-15

    Several factors besides high intraocular pressure assumed to be associated with the development and progression of glaucoma, and calcium channel blockers (CCBs) have been an anticipated option for glaucoma treatment by improving ocular perfusion and/or exerting neuroprotective effects on retinal ganglion cells with safety established in wide and long-term usage. Decrease in IOP has been reported after topical application of CCBs, however, the effect is much smaller and almost negligible after systemic application. Various CCBs have been reported to increase posterior ocular blood flow in vivo and to exert direct neuroprotection in neurons in vitro. Distribution of the drug at a pharmacologically active concentration in the posterior ocular tissues across the blood-brain barrier or blood-retina barrier, especially in the optic nerve head and retina where the ganglion cells mainly suffer from glaucomatous damage, is essential for clinical treatment of glaucoma. Improved visual functions such as sensitivity in the visual field test have been reported after administration of CCBs, but evidences from the randomized studies have been limited and effects of CCBs on blood flow and direct neuroprotection are hardly distinguished from each other. © 2013 Published by Elsevier B.V.

  8. Semi-quantitative model of the gating of KcsA ion channel. 2. Dynamic self-organization model of the gating

    Directory of Open Access Journals (Sweden)

    Boiteux C.

    2009-12-01

    Full Text Available The aim of this series of papers is to develop the semi-quantitative theory of the gating of KcsA channel. Methods. For this purpose available structural and electrophysiological data and the results of molecular dynamics simulations were used in the context of the concept of dynamical self-organization. In the second paper we describe the principles of dynamic self-organization and develop the theory of KcsA channel gating based on this concept. Conclusions. Present work is the first successful attempt of combining the structure and dynamics of real protein and the general concept of dynamic self-organization

  9. The role of protons in fast and slow gating of the Torpedo chloride channel ClC-0.

    Science.gov (United States)

    Zifarelli, Giovanni; Pusch, Michael

    2010-05-01

    Transmembrane proton transport is of fundamental importance for life. The list of H(+) transporting proteins has been recently expanded with the discovery that some members of the CLC gene family are stoichiometrically coupled Cl(-)/H(+) antiporters. Other CLC proteins are instead passive Cl(-) selective anion channels. The gating of these CLC channels is, however, strongly regulated by pH, likely reflecting the evolutionary relationship with CLC Cl(-)/H(+) antiporters. The role of protons in the gating of the model Torpedo channel ClC-0 is best understood. ClC-0 is a homodimer with separate pores in each subunit. Each protopore can be opened and closed independently from the other pore by a "fast gate". A common, slow gate acts on both pores simultaneously. The opening of the fast gate is controlled by a critical glutamate (E166), whose protonation state determines the fast gate's pH dependence. Extracellular protons likely can arrive directly at E166. In contrast, protonation of E166 from the inside has been proposed to be mediated by the dissociation of an intrapore water molecule. The OH(-) anion resulting from the water dissociation is stabilized in one of the anion binding sites of the channel, competing with intracellular Cl(-) ions. The pH dependence of the slow gate is less well understood. It has been shown that proton translocation drives irreversible gating transitions associated with the slow gate. However, the relationship of the fast gate's pH dependence on the proton translocation and the molecular basis of the slow gate remain to be discovered.

  10. Calcium channel blockers for inhibiting preterm labour and birth.

    Science.gov (United States)

    Flenady, Vicki; Wojcieszek, Aleena M; Papatsonis, Dimitri N M; Stock, Owen M; Murray, Linda; Jardine, Luke A; Carbonne, Bruno

    2014-06-05

    Preterm birth is a major contributor to perinatal mortality and morbidity, affecting around 9% of births in high-income countries and an estimated 13% of births in low- and middle-income countries. Tocolytics are drugs used to suppress uterine contractions for women in preterm labour. The most widely used tocolytic are the betamimetics, however, these are associated with a high frequency of unpleasant and sometimes severe maternal side effects. Calcium channel blockers (CCBs) (such as nifedipine) may have similar tocolytic efficacy with less side effects than betamimetics. Oxytocin receptor antagonists (ORAs) (e.g. atosiban) also have a low side-effect profile. To assess the effects on maternal, fetal and neonatal outcomes of CCBs, administered as a tocolytic agent, to women in preterm labour. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (12 November 2013). All published and unpublished randomised trials in which CCBs were used for tocolysis for women in labour between 20 and 36 completed weeks' gestation. Two review authors independently assessed trial eligibility, undertook quality assessment and data extraction. Results are presented using risk ratio (RR) for categorical data and mean difference (MD) for data measured on a continuous scale with the 95% confidence interval (CI). The number needed to treat to benefit (NNTB) and the number needed to treat to harm (NNTH) were calculated for categorical outcomes that were statistically significantly different. This update includes 26 additional trials involving 2511 women, giving a total of 38 included trials (3550 women). Thirty-five trials used nifedipine as the CCB and three trials used nicardipine. Blinding of intervention and outcome assessment was undertaken in only one of the trials (a placebo controlled trial). However, objective outcomes defined according to timing of birth and perinatal mortality were considered to have low risk of detection bias.Two small trials comparing CCBs

  11. A promoter in the coding region of the calcium channel gene CACNA1C generates the transcription factor CCAT.

    Directory of Open Access Journals (Sweden)

    Natalia Gomez-Ospina

    Full Text Available The C-terminus of the voltage-gated calcium channel Cav1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT, that regulates neurite extension and inhibits Cav1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of CACNA1C, the gene that encodes CaV1.2. Expression of CCAT is independent of Cav1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs, providing strong evidence that CCAT is not generated by cleavage of CaV1.2. Analysis of the transcriptional start sites in CACNA1C and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3' end of the CACNA1C gene. This study provides new insights into the regulation of CACNA1C, and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.

  12. Proton transfer unlocks inactivation in cyclic nucleotide-gated A1 channels.

    Science.gov (United States)

    Marchesi, Arin; Arcangeletti, Manuel; Mazzolini, Monica; Torre, Vincent

    2015-02-15

    Desensitization and inactivation provide a form of short-term memory controlling the firing patterns of excitable cells and adaptation in sensory systems. Unlike many of their cousin K(+) channels, cyclic nucleotide-gated (CNG) channels are thought not to desensitize or inactivate. Here we report that CNG channels do inactivate and that inactivation is controlled by extracellular protons. Titration of a glutamate residue within the selectivity filter destabilizes the pore architecture, which collapses towards a non-conductive, inactivated state in a process reminiscent of the usual C-type inactivation observed in many K(+) channels. These results indicate that inactivation in CNG channels represents a regulatory mechanism that has been neglected thus far, with possible implications in several physiological processes ranging from signal transduction to growth cone navigation. Ion channels control ionic fluxes across biological membranes by residing in any of three functionally distinct states: deactivated (closed), activated (open) or inactivated (closed). Unlike many of their cousin K(+) channels, cyclic nucleotide-gated (CNG) channels do not desensitize or inactivate. Using patch recording techniques, we show that when extracellular pH (pHo ) is decreased from 7.4 to 6 or lower, wild-type CNGA1 channels inactivate in a voltage-dependent manner. pHo titration experiments show that at pHo  < 7 the I-V relationships are outwardly rectifying and that inactivation is coupled to current rectification. Single-channel recordings indicate that a fast mechanism of proton blockage underlines current rectification while inactivation arises from conformational changes downstream from protonation. Furthermore, mutagenesis and ionic substitution experiments highlight the role of the selectivity filter in current decline, suggesting analogies with the C-type inactivation observed in K(+) channels. Analysis with Markovian models indicates that the non-independent binding of two

  13. Movements of native C505 during channel gating in CNGA1 channels.

    NARCIS (Netherlands)

    Nair, A.V.; Anselmi, C.; Mazzolini, M.

    2009-01-01

    We investigated conformational changes occurring in the C-linker and cyclic nucleotide-binding (CNB) domain of CNGA1 channels by analyzing the inhibition induced by thiol-specific reagents in mutant channels Q409C and A414C in the open and closed state. Cd(2+) (200 microM) inhibited irreversibly

  14. 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

  15. Low threshold T-type calcium channels as targets for novel epilepsy treatments.

    Science.gov (United States)

    Powell, Kim L; Cain, Stuart M; Snutch, Terrance P; O'Brien, Terence J

    2014-05-01

    Low voltage-activated T-type calcium channels were originally cloned in the 1990s and much research has since focused on identifying the physiological roles of these channels in health and disease states. T-type calcium channels are expressed widely throughout the brain and peripheral tissues, and thus have been proposed as therapeutic targets for a variety of diseases such as epilepsy, insomnia, pain, cancer and hypertension. This review discusses the literature concerning the role of T-type calcium channels in physiological and pathological processes related to epilepsy. T-type calcium channels have been implicated in pathology of both the genetic and acquired epilepsies and several anti-epileptic drugs (AEDs) in clinical use are known to suppress seizures via inhibition of T-type calcium channels. Despite the fact that more than 15 new AEDs have become clinically available over the past 20 years at least 30% of epilepsy patients still fail to achieve seizure control, and many patients experience unwanted side effects. Furthermore there are no treatments that prevent the development of epilepsy or mitigate the epileptic state once established. Therefore there is an urgent need for the development of new AEDs that are effective in patients with drug resistant epilepsy, are anti-epileptogenic and are better tolerated. We also review the mechanisms of action of the current AEDs with known effects on T-type calcium channels and discuss novel compounds that are being investigated as new treatments for epilepsy. © 2013 The British Pharmacological Society.

  16. The calcium channel β2 (CACNB2 subunit repertoire in teleosts

    Directory of Open Access Journals (Sweden)

    Mueller Rachel

    2008-04-01

    Full Text Available Abstract Background Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts. Results Cloning of two zebrafish β2 subunit genes (β2.1 and β2.2 indicated they are membrane-associated guanylate kinase (MAGUK-family genes. Zebrafish β2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but β2.2 is much more divergent in sequence than β2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both β2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single β2 subunit gene loci. Comparative analysis of the teleost and human β2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and

  17. Regulation of Voltage Gated K+ Channel KCNE1/KCNQ1 by the Janus Kinase JAK3

    Directory of Open Access Journals (Sweden)

    Jamshed Warsi

    2015-12-01

    Full Text Available Background/Aims: Janus kinase 3 (JAK3, a kinase mainly expressed in hematopoietic cells, has been shown to down-regulate the Na+/K+ ATPase and participate in the regulation of several ion channels and carriers. Channels expressed in thymus and regulating the abundance of T lymphocytes include the voltage gated K+ channel KCNE1/KCNQ1. The present study explored whether JAK3 contributes to the regulation of KCNE1/KCNQ1. Methods: cRNA encoding KCNE1/KCNQ1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active A568VJAK3, or inactive K851AJAK3. Voltage gated K+ channel activity was measured utilizing two electrode voltage clamp. Results: KCNE1/KCNQ1 activity was significantly increased by wild-type JAK3 and A568VJAK3, but not by K851AJAK3. The difference between oocytes expressing KCNE1/KCNQ1 alone and oocytes expressing KCNE1/KCNQ1 with A568VJAK3 was virtually abrogated by JAK3 inhibitor WHI-P154 (22 µM but not by inhibition of transcription with actinomycin D (50 nM. Inhibition of KCNE1/KCNQ1 protein insertion into the cell membrane by brefeldin A (5 µM resulted in a decline of the voltage gated current, which was similar in the absence and presence of A568VJAK3, suggesting that A568VJAK3 did not accelerate KCNE1/KCNQ1 protein retrieval from the cell membrane. Conclusion: JAK3 contributes to the regulation of membrane KCNE1/KCNQ1 activity, an effect sensitive to JAK3 inhibitor WHI-P154.

  18. Regulation of Voltage Gated K+ Channel KCNE1/KCNQ1 by the Janus Kinase JAK3.

    Science.gov (United States)

    Warsi, Jamshed; Abousaab, Abeer; Fezai, Myriam; Elvira, Bernat; Lang, Florian

    2015-01-01

    Janus kinase 3 (JAK3), a kinase mainly expressed in hematopoietic cells, has been shown to down-regulate the Na+/K+ ATPase and participate in the regulation of several ion channels and carriers. Channels expressed in thymus and regulating the abundance of T lymphocytes include the voltage gated K+ channel KCNE1/KCNQ1. The present study explored whether JAK3 contributes to the regulation of KCNE1/KCNQ1. cRNA encoding KCNE1/KCNQ1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active A568VJAK3, or inactive K851AJAK3. Voltage gated K+ channel activity was measured utilizing two electrode voltage clamp. KCNE1/KCNQ1 activity was significantly increased by wild-type JAK3 and A568VJAK3, but not by K851AJAK3. The difference between oocytes expressing KCNE1/KCNQ1 alone and oocytes expressing KCNE1/KCNQ1 with A568VJAK3 was virtually abrogated by JAK3 inhibitor WHI-P154 (22 µM) but not by inhibition of transcription with actinomycin D (50 nM). Inhibition of KCNE1/KCNQ1 protein insertion into the cell membrane by brefeldin A (5 µM) resulted in a decline of the voltage gated current, which was similar in the absence and presence of A568VJAK3, suggesting that A568VJAK3 did not accelerate KCNE1/KCNQ1 protein retrieval from the cell membrane. JAK3 contributes to the regulation of membrane KCNE1/KCNQ1 activity, an effect sensitive to JAK3 inhibitor WHI-P154. © 2015 The Author(s) Published by S. Karger AG, Basel.

  19. pH-dependent gating in a FocA formate channel.

    Science.gov (United States)

    Lü, Wei; Du, Juan; Wacker, Tobias; Gerbig-Smentek, Elke; Andrade, Susana L A; Einsle, Oliver

    2011-04-15

    The formate transporter FocA was described to switch its mode of operation from a passive export channel at high external pH to a secondary active formate/H(+) importer at low pH. The crystal structure of Salmonella typhimurium FocA at pH 4.0 shows that this switch involves a major rearrangement of the amino termini of individual protomers in the pentameric channel. The amino-terminal helices open or block transport in a concerted, cooperative action that indicates how FocA is gated in a pH-dependent way. Electrophysiological studies show that the protein acts as a specific formate channel at pH 7.0 and that it closes upon a shift of pH to 5.1.

  20. Low threshold T‐type calcium channels as targets for novel epilepsy treatments

    National Research Council Canada - National Science Library

    Powell, Kim L; Cain, Stuart M; Snutch, Terrance P; O'Brien, Terence J

    2014-01-01

    .... T ‐type calcium channels are expressed widely throughout the brain and peripheral tissues, and thus have been proposed as therapeutic targets for a variety of diseases such as epilepsy, insomnia, pain...

  1. A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome.

    NARCIS (Netherlands)

    Doyle, J.J.; Doyle, A.J.; Wilson, N.K.; Habashi, J.P.; Bedja, D.; Whitworth, R.E.; Lindsay, M.E.; Schoenhoff, F.; Myers, L.; Huso, N.; Bachir, S.; Squires, O.; Rusholme, B.; Ehsan, H.; Huso, D.; Thomas, C.J.; Caulfield, M.J.; Eyk, J.E. Van; Judge, D.P.; Dietz, H.C.; Loeys, B.L.

    2015-01-01

    Calcium channel blockers (CCBs) are prescribed to patients with Marfan syndrome for prophylaxis against aortic aneurysm progression, despite limited evidence for their efficacy and safety in the disorder. Unexpectedly, Marfan mice treated with CCBs show accelerated aneurysm expansion, rupture, and

  2. Ceramide modulates HERG potassium channel gating by translocation into lipid rafts

    Science.gov (United States)

    Ganapathi, Sindura B.; Fox, Todd E.; Elmslie, Keith S.

    2010-01-01

    Human ether-à-go-go-related gene (HERG) potassium channels play an important role in cardiac action potential repolarization, and HERG dysfunction can cause cardiac arrhythmias. However, recent evidence suggests a role for HERG in the proliferation and progression of multiple types of cancers, making it an attractive target for cancer therapy. Ceramide is an important second messenger of the sphingolipid family, which due to its proapoptotic properties has shown promising results in animal models as an anticancer agent. Yet the acute effects of ceramide on HERG potassium channels are not known. In the present study we examined the effects of cell-permeable C6-ceramide on HERG potassium channels stably expressed in HEK-293 cells. C6-ceramide (10 μM) reversibly inhibited HERG channel current (IHERG) by 36 ± 5%. Kinetically, ceramide induced a significant hyperpolarizing shift in the current-voltage relationship (ΔV1/2 = −8 ± 0.5 mV) and increased the deactivation rate (43 ± 3% for τfast and 51 ± 3% for τslow). Mechanistically, ceramide recruited HERG channels within caveolin-enriched lipid rafts. Cholesterol depletion and repletion experiments and mathematical modeling studies confirmed that inhibition and gating effects are mediated by separate mechanisms. The ceramide-induced hyperpolarizing gating shift (raft mediated) could offset the impact of inhibition (raft independent) during cardiac action potential repolarization, so together they may nullify any negative impact on cardiac rhythm. Our results provide new insights into the effects of C6-ceramide on HERG channels and suggest that C6-ceramide can be a promising therapeutic for cancers that overexpress HERG. PMID:20375276

  3. Sea anemone toxins affecting voltage-gated sodium channels--molecular and evolutionary features.

    Science.gov (United States)

    Moran, Yehu; Gordon, Dalia; Gurevitz, Michael

    2009-12-15

    The venom of sea anemones is rich in low molecular weight proteinaceous neurotoxins that vary greatly in structure, site of action, and phyletic (insect, crustacean or vertebrate) preference. This toxic versatility likely contributes to the ability of these sessile animals to inhabit marine environments co-habited by a variety of mobile predators. Among these toxins, those that show prominent activity at voltage-gated sodium channels and are critical in predation and defense, have been extensively studied for more than three decades. These studies initially focused on the discovery of new toxins, determination of their covalent and folded structures, understanding of their mechanisms of action on different sodium channels, and identification of the primary sites of interaction of the toxins with their channel receptors. The channel binding site for Type I and the structurally unrelated Type III sea anemone toxins was identified as neurotoxin receptor site 3, a site previously shown to be targeted by scorpion alpha-toxins. The bioactive surfaces of toxin representatives from these two sea anemone types have been characterized by mutagenesis. These analyses pointed to heterogeneity of receptor site 3 at various sodium channels. A turning point in evolutionary studies of sea anemone toxins was the recent release of the genome sequence of Nematostella vectensis, which enabled analysis of the genomic organization of the corresponding genes. This analysis demonstrated that Type I toxins in Nematostella and other species are encoded by gene families and suggested that these genes developed by concerted evolution. The current review provides a brief historical description of the discovery and characterization of sea anemone toxins that affect voltage-gated sodium channels and delineates recent advances in the study of their structure-activity relationship and evolution.

  4. Adenosine versus intravenous calcium channel antagonists for supraventricular tachycardia.

    Science.gov (United States)

    Alabed, Samer; Sabouni, Ammar; Providencia, Rui; Atallah, Edmond; Qintar, Mohammed; Chico, Timothy Ja

    2017-10-12

    People with supraventricular tachycardia (SVT) frequently are symptomatic and present to the emergency department for treatment. Although vagal manoeuvres may terminate SVT, they often fail, and subsequently adenosine or calcium channel antagonists (CCAs) are administered. Both are known to be effective, but both have a significant side effect profile. This is an update of a Cochrane review previously published in 2006. To review all randomised controlled trials (RCTs) that compare effects of adenosine versus CCAs in terminating SVT. We identified studies by searching CENTRAL, MEDLINE, Embase, and two trial registers in July 2017. We checked bibliographies of identified studies and applied no language restrictions. We planned to include all RCTs that compare adenosine versus a CCA for patients of any age presenting with SVT. We used standard methodological procedures as expected by Cochrane. Two review authors independently checked results of searches to identify relevant studies and resolved differences by discussion with a third review author. At least two review authors independently assessed each included study and extracted study data. We entered extracted data into Review Manager 5. Primary outcomes were rate of reversion to sinus rhythm and major adverse effects of adenosine and CCAs. Secondary outcomes were rate of recurrence, time to reversion, and minor adverse outcomes. We measured outcomes by calculating odds ratios (ORs) and assessed the quality of primary outcomes using the GRADE approach through the GRADEproGDT website. We identified two new studies for inclusion in the review update; the review now includes seven trials with 622 participants who presented to an emergency department with SVT. All included studies were RCTs, but only three described the randomisation process, and none had blinded participants, personnel, or outcome assessors to the intervention given. Moderate-quality evidence shows no differences in the number of people reverting to

  5. Calcium current-dependent and voltage-dependent inactivation of calcium channels in Helix aspersa

    Science.gov (United States)

    Brown, A. M.; Morimoto, K.; Tsuda, Y.; Wilson, D. L.

    1981-01-01

    -exponential inactivation process that persists in the presence of EGTAi is similar to that occurring when extracellular Ba ion carries current through the Ca channel. Steady-state inactivation also persists and is similar in the two cases. Therefore it is concluded that inactivation is voltage-dependent as well as Ca current-dependent. 8. Diffusion models that included reasonable values for the effect of binding on diffusion, even when combined with declining influxes, did not account for this `mixed' form of calcium- and voltage-dependent inactivation. A compartmental model in which the particular kinetic model of voltage-dependent inactivation was not critical described the Ca current-dependent inactivation. PMID:6275075

  6. Ryanodine receptor/calcium release channel PKA phosphorylation: A critical mediator of heart failure progression

    OpenAIRE

    Wehrens, Xander H. T.; Lehnart, Stephan E.; Reiken, Steven; Vest, John A.; Wronska, Anetta; Marks, Andrew R.

    2006-01-01

    Defective regulation of the cardiac ryanodine receptor (RyR2)/calcium release channel, required for excitation-contraction coupling in the heart, has been linked to cardiac arrhythmias and heart failure. For example, diastolic calcium “leak” via RyR2 channels in the sarcoplasmic reticulum has been identified as an important factor contributing to impaired contractility in heart failure and ventricular arrhythmias that cause sudden cardiac death. In patients with heart failure, chronic activat...

  7. Role of T-type calcium channels in myogenic tone of skeletal muscle resistance arteries

    DEFF Research Database (Denmark)

    VanBavel, Ed; Sorop, Oana; Andreasen, Ditte

    2002-01-01

    T-type calcium channels may be involved in the maintenance of myogenic tone. We tested their role in isolated rat cremaster arterioles obtained after CO(2) anesthesia and decapitation. Total RNA was analyzed by RT-PCR and Southern blotting for calcium channel expression. We observed expression......); K(+) -5.4 +/- 0.3 (n = 4); all log(IC(50)) P maintenance of myogenic tone in rat cremaster muscle arterioles....

  8. 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.

  9. Study of strained-Si p-channel MOSFETs with HfO2 gate dielectric

    Science.gov (United States)

    Pradhan, Diana; Das, Sanghamitra; Dash, Tara Prasanna

    2016-10-01

    In this work, the transconductance of strained-Si p-MOSFETs with high-K dielectric (HfO2) as gate oxide, has been presented through simulation using the TCAD tool Silvaco-ATLAS. The results have been compared with a SiO2/strained-Si p-MOSFET device. Peak transconductance enhancement factors of 2.97 and 2.73 has been obtained for strained-Si p-MOSFETs in comparison to bulk Si channel p-MOSFETs with SiO2 and high-K dielectric respectively. This behavior is in good agreement with the reported experimental results. The transconductance of the strained-Si device at low temperatures has also been simulated. As expected, the mobility and hence the transconductance increases at lower temperatures due to reduced phonon scattering. However, the enhancements with high-K gate dielectric is less as compared to that with SiO2.

  10. Nonlinearity of a Voltage-Gated Potassium Channel Revealed by the Mechanical Susceptibility

    Directory of Open Access Journals (Sweden)

    Amila Ariyaratne

    2013-02-01

    Full Text Available The voltage-gated potassium channel from Aeropyrum pernix operates by coupling the voltage-driven motion of a charged group of amino acids to the opening and closing of the pore. In this experiment, we drive this charged group with an ac field and observe the effect on the gating. The measurements for different frequencies and amplitudes of the forcing reveal an essential nonlinearity in the mechanical behavior of the molecule. Within a continuum-mechanics description, we extract the effective dissipation parameter γ for this conformational motion and find γ≈0.2  g/s, similar to recent nanorheology measurements on the conformational motion of an enzyme.

  11. Logic Gates Made of N-Channel JFETs and Epitaxial Resistors

    Science.gov (United States)

    Krasowski, Michael J.

    2008-01-01

    Prototype logic gates made of n-channel junction field-effect transistors (JFETs) and epitaxial resistors have been demonstrated, with a view toward eventual implementation of digital logic devices and systems in silicon carbide (SiC) integrated circuits (ICs). This development is intended to exploit the inherent ability of SiC electronic devices to function at temperatures from 300 to somewhat above 500 C and withstand large doses of ionizing radiation. SiC-based digital logic devices and systems could enable operation of sensors and robots in nuclear reactors, in jet engines, near hydrothermal vents, and in other environments that are so hot or radioactive as to cause conventional silicon electronic devices to fail. At present, current needs for digital processing at high temperatures exceed SiC integrated circuit production capabilities, which do not allow for highly integrated circuits. Only single to small number component production of depletion mode n-channel JFETs and epitaxial resistors on a single substrate is possible. As a consequence, the fine matching of components is impossible, resulting in rather large direct-current parameter distributions within a group of transistors typically spanning multiples of 5 to 10. Add to this the lack of p-channel devices to complement the n-channel FETs, the lack of precise dropping diodes, and the lack of enhancement mode devices at these elevated temperatures and the use of conventional direct coupled and buffered direct coupled logic gate design techniques is impossible. The presented logic gate design is tolerant of device parameter distributions and is not hampered by the lack of complementary devices or dropping diodes. In addition to n-channel JFETs, these gates include level-shifting and load resistors (see figure). Instead of relying on precise matching of parameters among individual JFETS, these designs rely on choosing the values of these resistors and of supply potentials so as to make the circuits perform

  12. Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms.

    Science.gov (United States)

    Wanke, Enzo; Zaharenko, André Junqueira; Redaelli, Elisa; Schiavon, Emanuele

    2009-12-15

    As voltage-gated Na(+) channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (except nematodes), they have become important targets for the toxins of venomous animals, from sea anemones to molluscs, scorpions, spiders and even fishes. During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of this channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Na(+) channels when exerting their different pharmacological effects, or at distinct receptor sites in different Na(v) channels subtypes whose particular properties lead to these pharmacological differences. The alpha-subunits of voltage-gated Na(+) channels (Na(v)1.x) have been divided into at least nine subtypes on the basis of amino acid sequences. Sea anemones have been extensively studied from the toxinological point of view for more than 40 years. There are about 40 sea anemone type 1 peptides known to be active on Na(v)1.x channels and all are 46-49 amino acid residues long, with three disulfide bonds and their molecular weights range between 3000 and 5000 Da. About 12 years ago a general model of Na(v)1.2-toxin interaction, developed for the alpha-scorpion toxins, was shown to fit also to action of sea anemone toxin such as ATX-II. According to this model these peptides are specifically acting on the type 3 site known to be between segments 3 and 4 in domain IV of the Na(+) channel protein. This region is indeed responsible for the normal Na(+) currents fast inactivation that is potently slowed by these toxins. This fundamental "gain-of-function" mechanism is responsible for the strong increase in the action potential duration. They constitute a class of tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins. As

  13. "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.

  14. Ginsenoside Rg3, a Gating Modifier of EAG Family K+ Channels.

    Science.gov (United States)

    Wu, Wei; Gardner, Alison; Sachse, Frank B; Sanguinetti, Michael C

    2016-10-01

    Ginsenoside 20(S)-Rg3 (Rg3) is a steroid glycoside that induces human ether-à-go-go-related gene type 1 (hERG1, Kv11.1) channels to activate at more negative potentials and to deactivate more slowly than normal. However, it is unknown whether this action is unique to hERG1 channels. Here we compare and contrast the mechanisms of actions of Rg3 on hERG1 with three other members of the ether-à-go-go (EAG) K(+) channel gene family, including EAG1 (Kv10.1), ERG3 (Kv11.3), and ELK1 (Kv12.1). All four channel types were heterologously expressed in Xenopus laevis oocytes, and K(+) currents were measured using the two-microelectrode voltage-clamp technique. At a maximally effective concentration, Rg3 shifted the half-point of voltage-dependent activation of currents by -14 mV for ERG1 (EC50 = 414 nM), -20 mV for ERG3 (EC50 = 374 nM), -28 mV for EAG1 (EC50 = 1.18 μM), and more than -100 mV for ELK1 (EC50 = 197 nM) channels. Rg3 also induced slowing of ERG1, ERG3, and ELK1 channel deactivation and accelerated the rate of EAG1 channel activation. A Markov model was developed to simulate gating and the effects of Rg3 on the voltage dependence of activation of hELK1 channels. Understanding the mechanism underlying the action of Rg3 may facilitate the development of more potent and selective EAG family channel activators as therapies for cardiovascular and neural disorders. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  15. Differential sensitivity of two insect GABA-gated chloride channels to dieldrin, fipronil and picrotoxinin.

    Science.gov (United States)

    Le Corronc, Hervé; Alix, Philippe; Hue, Bernard

    2002-04-01

    In the central nervous system of both vertebrates and invertebrates inhibitory neurotransmission is mainly achieved through activation of gamma-aminobutyric acid (GABA) receptors. Extensive studies have established the structural and pharmacological properties of vertebrate GABA receptors. Although the vast majority of insect GABA-sensitive responses share some properties with vertebrate GABAA receptors, peculiar pharmacological properties of these receptors led us to think that several GABA-gated chloride channels are present in insects. We describe here the pharmacological properties of two GABA receptor subtypes coupled to a chloride channel on dorsal unpaired median (DUM) neurones of the adult male cockroach. Long applications of GABA induce a large biphasic hyperpolarization, consisting of an initial transient hyperpolarization followed by a slow phase of hyperpolarization that is not quickly desensitized. With GABA, the transient hyperpolarization is sensitive to picrotoxinin, fipronil and dieldrin whereas the slow response is insensitive to these insecticides.When GABA is replaced by muscimol and cis-4-aminocrotonic acid (CACA) a biphasic hyperpolarization consisting of an initial transient hyperpolarization followed by a sustained phase is evoked which is blocked by picrotoxinin and fipronil. Exposure to dieldrin decreases only the early phase of the muscimol and CACA-induced biphasic response, suggesting that two GABA-gated chloride channel receptor subtypes are present in DUM neurones. This study describes, for the first time, a dieldrin resistant component different to the dieldrin- and picrotoxinin-resistant receptor found in several insect species.

  16. Actions of a hydrogen sulfide donor (NaHS) on transient sodium, persistent sodium, and voltage-gated calcium currents in neurons of the subfornical organ.

    Science.gov (United States)

    Kuksis, Markus; Ferguson, Alastair V

    2015-09-01

    Hydrogen sulfide (H2S) is an endogenously found gasotransmitter that has been implicated in a variety of beneficial physiological functions. This study was performed to investigate the cellular mechanisms underlying actions of H2S previously observed in subfornical organ (SFO), where H2S acts to regulate blood pressure through a depolarization of the membrane and an overall increase in the excitability of SFO neurons. We used whole cell patch-clamp electrophysiology in the voltage-clamp configuration to analyze the effect of 1 mM NaHS, an H2S donor, on voltage-gated potassium, sodium, and calcium currents. We observed no effect of NaHS on potassium currents; however, both voltage-gated sodium currents (persistent and transient) and the N-type calcium current had a depolarized activation curve and an enhanced peak-induced current in response to a series of voltage-step and ramp protocols run in the control and NaHS conditions. These effects were not responsible for the previously observed depolarization of the membrane potential, as depolarizing effects of H2S were still observed following block of these conductances with tetrodotoxin (5 μM) and ω-conotoxin-GVIA (100 nM). Our studies are the first to investigate the effect of H2S on a variety of voltage-gated conductances in a single brain area, and although they do not explain mechanisms underlying the depolarizing actions of H2S on SFO neurons, they provide evidence of potential mechanisms through which this gasotransmitter influences the excitability of neurons in this important brain area as a consequence of the modulation of multiple ion channels. Copyright © 2015 the American Physiological Society.

  17. Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons.

    Science.gov (United States)

    Dibattista, Michele; Amjad, Asma; Maurya, Devendra Kumar; Sagheddu, Claudia; Montani, Giorgia; Tirindelli, Roberto; Menini, Anna

    2012-07-01

    The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction.

  18. Channelrhodopsin unchained: structure and mechanism of a light-gated cation channel.

    Science.gov (United States)

    Lórenz-Fonfría, Víctor A; Heberle, Joachim

    2014-05-01

    The new and vibrant field of optogenetics was founded by the seminal discovery of channelrhodopsin, the first light-gated cation channel. Despite the numerous applications that have revolutionised neurophysiology, the functional mechanism is far from understood on the molecular level. An arsenal of biophysical techniques has been established in the last decades of research on microbial rhodopsins. However, application of these techniques is hampered by the duration and the complexity of the photoreaction of channelrhodopsin compared with other microbial rhodopsins. A particular interest in resolving the molecular mechanism lies in the structural changes that lead to channel opening and closure. Here, we review the current structural and mechanistic knowledge that has been accomplished by integrating the static structure provided by X-ray crystallography and electron microscopy with time-resolved spectroscopic and electrophysiological techniques. The dynamical reactions of the chromophore are effectively coupled to structural changes of the protein, as shown by ultrafast spectroscopy. The hierarchical sequence of structural changes in the protein backbone that spans the time range from 10(-12)s to 10(-3)s prepares the channel to open and, consequently, cations can pass. Proton transfer reactions that are associated with channel gating have been resolved. In particular, glutamate 253 and aspartic acid 156 were identified as proton acceptor and donor to the retinal Schiff base. The reprotonation of the latter is the critical determinant for channel closure. The proton pathway that eventually leads to proton pumping is also discussed. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Converging roles of ion channels, calcium, metabolic stress, and activity pattern of Substantia nigra dopaminergic neurons in health and Parkinson's disease.

    Science.gov (United States)

    Duda, Johanna; Pötschke, Christina; Liss, Birgit

    2016-10-01

    Dopamine-releasing neurons within the Substantia nigra (SN DA) are particularly vulnerable to degeneration compared to other dopaminergic neurons. The age-dependent, progressive loss of these neurons is a pathological hallmark of Parkinson's disease (PD), as the resulting loss of striatal dopamine causes its major movement-related symptoms. SN DA neurons release dopamine from their axonal terminals within the dorsal striatum, and also from their cell bodies and dendrites within the midbrain in a calcium- and activity-dependent manner. Their intrinsically generated and metabolically challenging activity is created and modulated by the orchestrated function of different ion channels and dopamine D2-autoreceptors. Here, we review increasing evidence that the mechanisms that control activity patterns and calcium homeostasis of SN DA neurons are not only crucial for their dopamine release within a physiological range but also modulate their mitochondrial and lysosomal activity, their metabolic stress levels, and their vulnerability to degeneration in PD. Indeed, impaired calcium homeostasis, lysosomal and mitochondrial dysfunction, and metabolic stress in SN DA neurons represent central converging trigger factors for idiopathic and familial PD. We summarize double-edged roles of ion channels, activity patterns, calcium homeostasis, and related feedback/feed-forward signaling mechanisms in SN DA neurons for maintaining and modulating their physiological function, but also for contributing to their vulnerability in PD-paradigms. We focus on the emerging roles of maintained neuronal activity and calcium homeostasis within a physiological bandwidth, and its modulation by PD-triggers, as well as on bidirectional functions of voltage-gated L-type calcium channels and metabolically gated ATP-sensitive potassium (K-ATP) channels, and their probable interplay in health and PD. We propose that SN DA neurons possess several feedback and feed-forward mechanisms to protect and adapt

  20. Calcium channel TRPV6 is involved in murine maternal-fetal calcium transport.

    Science.gov (United States)

    Suzuki, Yoshiro; Kovacs, Christopher S; Takanaga, Hitomi; Peng, Ji-Bin; Landowski, Christopher P; Hediger, Matthias A

    2008-08-01

    Maternal-fetal calcium (Ca(2+)) transport is crucial for fetal Ca(2+) homeostasis and bone mineralization. In this study, the physiological significance of the transient receptor potential, vanilloid 6 (TRPV6) Ca(2+) channel in maternal-fetal Ca(2+) transport was investigated using Trpv6 knockout mice. The Ca(2+) concentration in fetal blood and amniotic fluid was significantly lower in Trpv6 knockout fetuses than in wildtypes. The transport activity of radioactive Ca(2+) ((45)Ca) from mother to fetuses was 40% lower in Trpv6 knockout fetuses than in wildtypes. The ash weight was also lower in Trpv6 knockout fetuses compared with wildtype fetuses. TRPV6 mRNA and protein were mainly localized in intraplacental yolk sac and the visceral layer of extraplacental yolk sac, which are thought to be the places for maternal-fetal Ca(2+) transport in mice. These expression sites were co-localized with calbindin D(9K) in the yolk sac. In wildtype mice, placental TRPV6 mRNA increased 14-fold during the last 4 days of gestation, which coincides with fetal bone mineralization. These results provide the first in vivo evidence that TRPV6 is involved in maternal-fetal Ca(2+) transport. We propose that TRPV6 functions as a Ca(2+) entry pathway, which is critical for fetal Ca(2+) homeostasis.

  1. Signatures of protein structure in the cooperative gating of mechanosensitive ion channels

    CERN Document Server

    Kahraman, Osman; Haselwandter, Christoph A

    2016-01-01

    Membrane proteins deform the surrounding lipid bilayer, which can lead to membrane-mediated interactions between neighboring proteins. Using the mechanosensitive channel of large conductance (MscL) as a model system, we demonstrate how the observed differences in protein structure can affect membrane-mediated interactions and cooperativity among membrane proteins. We find that distinct oligomeric states of MscL lead to distinct gateway states for the clustering of MscL, and predict signatures of MscL structure and spatial organization in the cooperative gating of MscL. Our modeling approach establishes a quantitative relation between the observed shapes and cooperative function of membrane~proteins.

  2. 18F-FDG PET/CT findings in voltage-gated potassium channel limbic encephalitis.

    Science.gov (United States)

    Kamaleshwaran, Koramadai Karuppuswamy; Iyer, Rajesh Shankar; Antony, Joppy; Radhakrishnan, Edathuruthy Kalarickal; Shinto, Ajit

    2013-05-01

    Limbic encephalitis (LE) can be associated with cancer, viral infection, or be idiopathic. One such rare but treatable form is associated with voltage-gated potassium channel (VGKC) antibodies. Typical abnormalities are seen in FDG PET/CT. We report a 39-year-old female patient who presented with 3 months of progressive faciobrachial dystonic seizures and limbic encephalitis. Her serum and cerebrospinal fluid Lgi1 antibody titers were elevated. FDG PET/CT showed basal ganglial hypermetabolism and associated abnormalities. Serial MRI demonstrated atrophic changes predominantly involving the temporal lobes. She is on immunosuppressive therapy and shows clinical improvement with lowering of antibody titers.

  3. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting...... in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal......43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium....

  4. Effect of L- type Calcium Channel Blocker Nimodipine and T-type Calcium Channel Blocker Flunarizine on Motor Control in Mice

    Directory of Open Access Journals (Sweden)

    Swapnil Balkrishna Kaikade

    2015-05-01

    Full Text Available Objective: To study the effect of L-type of Calcium channel blocker nimodipine and T-type of calcium channel blocker funarizine on locomotor activity in mice without pretreatment by any other drug. Materials and method: The study was carried out following permission from the Institutional animal ethics committee. Healthy Swiss albino mice of either sex were selected by the strict inclusion and exclusion criteria and the grouping is done. Group A is control treated with normal saline, Group B and C received two titrated doses of nimodipine while Group D and E received two titrated doses of flunarizine. The animals were then observed for motor control on inclined plane and the Statistical analysis was done by using unpaired‘t’ test. Results: L-type calcium channel blocker nimodipine has dose dependent effect on motor control on inclined plane while the T- type calcium channel blocker flunarizine has no effect on motor control. Conclusion: Nimodipine has significant dose dependent depressant action on motor control on inclined plane while flunarizine has no effect on the above mentioned parameter.

  5. Meta-Analysis of Public Microarray Datasets Reveals Voltage-Gated Calcium Gene Signatures in Clinical Cancer Patients.

    Directory of Open Access Journals (Sweden)

    Chih-Yang Wang

    Full Text Available Voltage-gated calcium channels (VGCCs are well documented to play roles in cell proliferation, migration, and apoptosis; however, whether VGCCs regulate the onset and progression of cancer is still under investigation. The VGCC family consists of five members, which are L-type, N-type, T-type, R-type and P/Q type. To date, no holistic approach has been used to screen VGCC family genes in different types of cancer. We analyzed the transcript expression of VGCCs in clinical cancer tissue samples by accessing ONCOMINE (www.oncomine.org, a web-based microarray database, to perform a systematic analysis. Every member of the VGCCs was examined across 21 different types of cancer by comparing mRNA expression in cancer to that in normal tissue. A previous study showed that altered expression of mRNA in cancer tissue may play an oncogenic role and promote tumor development; therefore, in the present findings, we focus only on the overexpression of VGCCs in different types of cancer. This bioinformatics analysis revealed that different subtypes of VGCCs (CACNA1C, CACNA1D, CACNA1B, CACNA1G, and CACNA1I are implicated in the development and progression of diverse types of cancer and show dramatic up-regulation in breast cancer. CACNA1F only showed high expression in testis cancer, whereas CACNA1A, CACNA1C, and CACNA1D were highly expressed in most types of cancer. The current analysis revealed that specific VGCCs likely play essential roles in specific types of cancer. Collectively, we identified several VGCC targets and classified them according to different cancer subtypes for prospective studies on the underlying carcinogenic mechanisms. The present findings suggest that VGCCs are possible targets for prospective investigation in cancer treatment.

  6. Computer Simulation Studies of Ion Channel Gating: Characteristics of the M2 Channel of Influenza-A Virus in a Phospholipid Bilayer

    Science.gov (United States)

    Schweighofer, Karl J.; Pohorille, Andrew; DeVincenzi, D. (Technical Monitor)

    1999-01-01

    The 25 amino acids long, transmembrane fragment of the Influenza virus M2 protein forms a homotetrameric channel that transports protons across lipid bilayers. It has been postulated that high efficiency and selectivity of this process is due to gating by four histidine residues that occlude the channel lumen in the closed state. Two mechanisms of gating have been postulated. In one mechanism, the proton is "shuttled" through the gate by attaching to the delta nitrogen atom on the extracellular side of the imidazole ring, followed by the release of the proton attached to the epsilon nitrogen atom on the opposite side. In the second mechanism, the four histidines move away from each other due to electrostatic repulsion upon protonation, thus opening the gate sufficiently that a wire of water molecules can penetrate the gate. Then, protons are transported by "hopping" along the wire. In this paper, both mechanisms are evaluated in a series of molecular dynamics simulations by investigating stability of different protonation states of the channel that are involved in these mechanisms. For the shuttle mechanism, these are states with all epsilon protonated histidines, one biprotonated residue or one histidine protonated in the delta position. For the gate opening mechanism, this is the state in which all four histidines are biprotonated. In addition, a state with two biprotonated histidines is considered. For each system, composed of the protein channel embedded in phospholipid bilayer located between two water lamellae, a molecular dynamics trajectory of approximately 1.3 ns (after equilibration) was obtained. It is found that the states involved in the shuttle mechanism are stable during the simulations. Furthermore, the orientations and dynamics of water molecules near the gate are conducive to proton transfers involved in the shuttle. In contract, the fully biprotonated state, implicated in the gate opening mechanism, is not stable and the channel looses its

  7. Expanded alternative splice isoform profiling of the mouse Cav3.1/α1G T-type calcium channel

    Directory of Open Access Journals (Sweden)

    Ernst Wayne L

    2009-05-01

    Full Text Available Abstract Background Alternative splicing of low-voltage-activated T-type calcium channels contributes to the molecular and functional diversity mediating complex network oscillations in the normal brain. Transcript scanning of the human CACNA1G gene has revealed the presence of 11 regions within the coding sequence subjected to alternative splicing, some of which enhance T-type current. In mouse models of absence epilepsy, elevated T-type calcium currents without clear increases in channel expression are found in thalamic neurons that promote abnormal neuronal synchronization. To test whether enhanced T-type currents in these models reflect pathogenic alterations in channel splice isoforms, we determined the extent of alternative splicing of mouse Cacna1g transcripts and whether evidence of altered transcript splicing could be detected in mouse absence epilepsy models. Results Transcript scanning of the murine Cacna1g gene detected 12 regions encoding alternative splice isoforms of Cav3.1/α1G T-type calcium channels. Of the 12 splice sites, six displayed homology to the human CACNA1G splice sites, while six novel mouse-specific splicing events were identified, including one intron retention, three alternative acceptor sites, one alternative donor site, and one exon exclusion. In addition, two brain region-specific alternative splice patterns were observed in the cerebellum. Comparative analyses of brain regions from four monogenic absence epilepsy mouse models with altered thalamic T-type currents and wildtype controls failed to reveal differences in Cacna1g splicing patterns. Conclusion The determination of six novel alternative splice sites within the coding region of the mouse Cacna1g gene greatly expands the potential biophysical diversity of voltage-gated T-type channels in the mouse central nervous system. Although alternative splicing of Cav3.1/α1G channels does not explain the enhancement of T-type current identified in four mouse

  8. The mechano-gated channel inhibitor GsMTx4 reduces the exercise pressor reflex in decerebrate rats.

    Science.gov (United States)

    Copp, Steven W; Kim, Joyce S; Ruiz-Velasco, Victor; Kaufman, Marc P

    2016-02-01

    Mechanical and metabolic stimuli from contracting muscles evoke reflex increases in blood pressure, heart rate and sympathetic nerve activity. Little is known, however, about the nature of the mechano-gated channels on the thin fibre muscle afferents that contribute to evoke this reflex, termed the exercise pressor reflex. We determined the effect of GsMTx4, an inhibitor of mechano-gated Piezo channels, on the exercise pressor reflex evoked by intermittent contraction of the triceps surae muscles in decerebrated, unanaesthetized rats. GsMTx4 reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to intermittent contraction but did not reduce the pressor responses to femoral arterial injection of compounds that stimulate the metabolically-sensitive thin fibre muscle afferents. Expression levels of Piezo2 channels were greater than Piezo1 channels in rat dorsal root ganglia. Our findings suggest that mechanically-sensitive Piezo proteins contribute to the generation of the mechanical component of the exercise pressor reflex in rats. Mechanical and metabolic stimuli within contracting skeletal muscles evoke reflex autonomic and cardiovascular adjustments. In cats and rats, gadolinium has been used to investigate the role played by the mechanical component of this reflex, termed the exercise pressor reflex. Gadolinium, however, has poor selectivity for mechano-gated channels and exerts multiple off-target effects. We tested the hypothesis that GsMTX4, a more selective mechano-gated channel inhibitor than gadolinium and a particularly potent inhibitor of mechano-gated Piezo channels, reduced the exercise pressor reflex in decerebrate rats. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 5, GsMTx4: 12 ± 5 mmHg, P decerebrate rats and that the reduction was attributable, at least in part, to its effect on mechano-gated Piezo channels. © 2015 The Authors. The Journal of Physiology

  9. K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac.

    Science.gov (United States)

    Dong, Yin Yao; Pike, Ashley C W; Mackenzie, Alexandra; McClenaghan, Conor; Aryal, Prafulla; Dong, Liang; Quigley, Andrew; Grieben, Mariana; Goubin, Solenne; Mukhopadhyay, Shubhashish; Ruda, Gian Filippo; Clausen, Michael V; Cao, Lishuang; Brennan, Paul E; Burgess-Brown, Nicola A; Sansom, Mark S P; Tucker, Stephen J; Carpenter, Elisabeth P

    2015-03-13

    TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac. Copyright © 2015, American Association for the Advancement of Science.

  10. Characterization of P-channel power trench MOSFETs with polycrystalline silicon germanium gate electrode for faster switching

    Science.gov (United States)

    Dikshit, Rohit; Daggubati, Manmohan

    2012-02-01

    Electrical switching characteristics using polycrystalline silicon-germanium (poly-Si l-xGe x) gate for P-channel power trench MOSFETs was investigated. Switching time reduction of over 22% was observed when the boron-doped poly-Si gate was replaced with a similarly boron-doped poly-SiGe gate on the P-channel power MOSFETs. The fall time ( T f) on MOSFETs with poly-SiGe gate, was found to be ˜11 ns lesser than the poly-Si gate MOSFET which is ˜60% improvement in switching performance. However, all the switching improvement was observed during the fall times ( T f). The reason could be the higher series resistance in the switching test circuit masking any reduction in the rise times ( T r). Faster switching is achieved due to a lower gate resistance ( R g) offered by the poly-SiGe gate electrode as compared to poly-silicon (pSi) material. The pSi gate resistance was found to be 6.25 Ω compared to 3.75 Ω on the poly-SiGe gate measured on the same device. Lower gate resistance ( R g) also means less power is lost during switching thereby less heat is generated in the device. A very uniform boron doping profile was achieved with-in the pSiGe gate electrode, which is critical for uniform die turn on and better thermal response for the power trench MOSFET. pSiGe thin film optimization, properties and device characteristics are discussed in details in the following sections.

  11. 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.

  12. Analytical model for subthreshold current and subthreshold swing of short-channel double-material-gate MOSFETs with strained-silicon channel on silicon—germanium substrates

    Science.gov (United States)

    Tiwari, Pramod Kumar; Krishna Saramekala, Gopi; Dubey, Sarvesh; Mukhopadhyay, Anand Kumar

    2014-10-01

    The present work gives some insight into the subthreshold behaviour of short-channel double-material-gate strained-silicon on silicon—germanium MOSFETs in terms of subthreshold swing and off-current. The formulation of subthreshold current and, thereupon, the subthreshold swing have been done by exploiting the expression of potential distribution in the channel region of the device. The dependence of the subthreshold characteristics on the device parameters, such as Ge mole fraction, gate length ratio, work function of control gate metal and gate length, has been tested in detail. The analytical models have been validated by the numerical simulation results that were obtained from the device simulation software ATLAS™ by Silvaco Inc.

  13. The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties.

    NARCIS (Netherlands)

    Kovalevskaya, N.V.; Bokhovchuk, F.M.; Vuister, G.W.

    2012-01-01

    The epithelial Ca(2+) channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca(2+) reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal

  14. 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

  15. Inhibitory effect of calcium channel blockers on proliferation of human glioma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kunert-Radek, J.; Stepien, H.; Lyson, K.; Pawlikowski, M.; Radek, A.

    1989-01-01

    The effects of 2 specific calcium channel blockers, verapamil and nimodipine, on the proliferation of human glioma tumour cells were investigated in vitro. Tumour tissues for primary cell cultures were obtained bioptically from 3 patients with the histopathological diagnosis of glioblastoma. The (/sup 3/H)-thymidine incorporation into glioma tumour cells DNA was used as a sensitive index of the cell proliferation. It was found that varapamil (10/sup 4/-10/sup 5/M) and nimodipine (10/sup 4/-10/sup 6/M) significantly inhibited the (/sup 3/H)-thymidine uptake in a dose-related manner. The inhibitory effect of both calcium channel antagonists was reversed by stimultancous addition of calcium chloride (5x10/sup 3/M). These results indicate that verapamil and nimodipine may exert an antiproliferative effect on glioma cells growth acting through a blokade of specific voltage-dependent calcium channels.

  16. Sources of energy for gating by neurotransmitters in acetylcholine receptor channels.

    Science.gov (United States)

    Purohit, Prasad; Bruhova, Iva; Auerbach, Anthony

    2012-06-12

    Nicotinic acetylcholine receptors (AChRs) mediate signaling in the central and peripheral nervous systems. The AChR gating conformational change is powered by a low- to high-affinity change for neurotransmitters at two transmitter binding sites. We estimated (from single-channel currents) the components of energy for gating arising from binding site aromatic residues in the α-subunit. All mutations reduced the energy (TyrC1>TrpB≈TyrC2>TyrA), with TyrC1 providing ~40% of the total. Considered one at a time, the fractional energy contributions from the aromatic rings were TrpB ~35%, TyrC1 ~28%, TyrC2 ~28%, and TyrA ~10%. Together, TrpB, TyrC1, and TyrC2 comprise an "aromatic triad" that provides much of the total energy from the transmitter for gating. Analysis of mutant pairs suggests that the energy contributions from some residues are nearly independent. Mutations of TyrC1 cause particularly large energy reductions because they remove two favorable and approximately equal interactions between the aromatic ring and the quaternary amine of the agonist and between the hydroxyl and αLysβ7.

  17. Mg2+-dependent gating of bacterial MgtE channel underlies Mg2+ homeostasis

    Science.gov (United States)

    Hattori, Motoyuki; Iwase, Norihiko; Furuya, Noritaka; Tanaka, Yoshiki; Tsukazaki, Tomoya; Ishitani, Ryuichiro; Maguire, Michael E; Ito, Koichi; Maturana, Andres; Nureki, Osamu

    2009-01-01

    The MgtE family of Mg2+ transporters is ubiquitously distributed in all phylogenetic domains. Recent crystal structures of the full-length MgtE and of its cytosolic domain in the presence and absence of Mg2+ suggested a Mg2+-homeostasis mechanism, in which the MgtE cytosolic domain acts as a ‘Mg2+ sensor' to regulate the gating of the ion-conducting pore in response to the intracellular Mg2+ concentration. However, complementary functional analyses to confirm the proposed model have been lacking. Moreover, the limited resolution of the full-length structure precluded an unambiguous characterization of these regulatory divalent-cation-binding sites. Here, we showed that MgtE is a highly Mg2+-selective channel gated by Mg2+ and elucidated the Mg2+-dependent gating mechanism of MgtE, using X-ray crystallographic, genetic, biochemical, and electrophysiological analyses. These structural and functional results have clarified the control of Mg2+ homeostasis through cooperative Mg2+ binding to the MgtE cytosolic domain. PMID:19798051

  18. Nine-channel mid-power bipolar pulse generator based on a field programmable gate array

    Energy Technology Data Exchange (ETDEWEB)

    Haylock, Ben, E-mail: benjamin.haylock2@griffithuni.edu.au; Lenzini, Francesco; Kasture, Sachin; Fisher, Paul; Lobino, Mirko [Centre for Quantum Dynamics, Griffith University, Brisbane (Australia); Queensland Micro and Nanotechnology Centre, Griffith University, Brisbane (Australia); Streed, Erik W. [Centre for Quantum Dynamics, Griffith University, Brisbane (Australia); Institute for Glycomics, Griffith University, Gold Coast (Australia)

    2016-05-15

    Many channel arbitrary pulse sequence generation is required for the electro-optic reconfiguration of optical waveguide networks in Lithium Niobate. Here we describe a scalable solution to the requirement for mid-power bipolar parallel outputs, based on pulse patterns generated by an externally clocked field programmable gate array. Positive and negative pulses can be generated at repetition rates up to 80 MHz with pulse width adjustable in increments of 1.6 ns across nine independent outputs. Each channel can provide 1.5 W of RF power and can be synchronised with the operation of other components in an optical network such as light sources and detectors through an external clock with adjustable delay.

  19. Reconfigurable dual-channel all-optical logic gate in a silicon waveguide using polarization encoding.

    Science.gov (United States)

    Gao, Shiming; Wang, Xiaoyan; Xie, Yanqiao; Hu, Peiran; Yan, Qiang

    2015-04-01

    A reconfigurable dual-channel all-optical logic gate is proposed and experimentally demonstrated using four-wave mixing in a silicon waveguide for polarization encoding signals. Six logic functions, XNOR, AND, NOR, XOR, AB¯, and A¯B are implemented at two different wavelength channels by adjusting the polarization states of two 10 Gb/s non-return-to-zero polarization-shift keying (NRZ-PolSK) signals modulated by 10-bit on-off keying (OOK) sequences. The eye diagrams of the logic signals are clearly observed, and the logic functions are well demonstrated as the two incident NRZ-PolSK signals are both modulated by the OOK sequences, which originate from 2(31)-1 pseudo-random binary sequences.

  20. Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating.

    Science.gov (United States)

    Lórenz-Fonfría, Víctor A; Resler, Tom; Krause, Nils; Nack, Melanie; Gossing, Michael; Fischer von Mollard, Gabriele; Bamann, Christian; Bamberg, Ernst; Schlesinger, Ramona; Heberle, Joachim

    2013-04-02

    The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements. We have resolved the vibrational changes associated with the open states of the channel (P(2)(390) and P(3)(520)) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane α-helices with a t(1/2) = 60 μs, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t(1/2) = 10 μs), with the latter being reprotonated by aspartic acid 156 (t(1/2) = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. Previous conclusions on the involvement of glutamic acid 90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the nonconductive P(4)(480) state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions and the protein conformational changes to the gating of the cation channel.

  1. Molecular mechanisms underlying pimaric acid-induced modulation of voltage-gated K+channels.

    Science.gov (United States)

    Sakamoto, Kazuho; Suzuki, Yoshiaki; Yamamura, Hisao; Ohya, Susumu; Muraki, Katsuhiko; Imaizumi, Yuji

    2017-04-01

    Voltage-gated K + (K V ) channels, which control firing and shape of action potentials in excitable cells, are supposed to be potential therapeutic targets in many types of diseases. Pimaric acid (PiMA) is a unique opener of large conductance Ca 2+ -activated K + channel. Here, we report that PiMA modulates recombinant rodent K V channel activity. The enhancement was significant at low potentials (voltage-activation relationships (V 1/2 ) of rodent K V 1.1, 1.2, 1.3, 1.4, 1.6 and 2.1 channels (K V 1.1-K V 2.1) but K V 4.3 to lower potentials and prolonged their half-decay times of the deactivation (T 1/2D ). The amino acid sequence which is responsible for the difference in response to PiMA was examined between K V 1.1-K V 2.1 and K V 4.3 by site-directed mutagenesis of residues in S5 and S6 segments of Kv1.1. The point mutation of Phe 332 into Tyr mimics the effects of PiMA on V 1/2 and T 1/2D and also abolished the further change by addition of PiMA. The results indicate that PiMA enhances voltage sensitivity of K V 1.1-K V 2.1 channels and suggest that the lipophilic residues including Phe 332 in S5 of K V 1.1-K V 2.1 channels may be critical for the effects of PiMA, providing beneficial information for drug development of K V channel openers. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  2. Differential effects of organic calcium-channel blockers on diastolic SR calcium-handling in the frog heart.

    Science.gov (United States)

    Subramani, Sathya; Vijayanand, Caroline; Tharion, Elizabeth

    2002-11-01

    1. Gradual loss of sarcoplasmic reticular (SR) calcium during a rest-period is responsible for the rest-induced decay (RID) of force in mammalian myocardium. Effect of verapamil and diltiazem on a similar RID in the frog myocardium suggests a new mechanism of action of these drugs. 2. Strips of frog-ventricle were paced at 0.2 Hz and the rhythm was interrupted by varying rest-periods ranging from 10 to 180 s. In control conditions, the amplitude of the post-rest beat was significantly lower than that of the pre-rest beat for rest-periods more than 40 s (RID). 3. Verapamil and diltiazem (which are organic calcium-channel blockers (OCCB)) changed the pattern of RID in the control solution to a 'rest-induced potentiation' (RIP) in the same preparation while another OCCB nifedipine and the inorganic calcium-channel blocker cadmium did not alter the post-rest phenomenon. 4. We propose that verapamil and diltiazem produce an RIP due to either blockade of SR calcium-leak during rest or enhancement of SR calcium-uptake during rest.

  3. Aberrant Splicing Induced by Dysregulated Rbfox2 Produces Enhanced Function of CaV1.2 Calcium Channel and Vascular Myogenic Tone in Hypertension.

    Science.gov (United States)

    Zhou, Yingying; Fan, Jia; Zhu, Huayuan; Ji, Li; Fan, Wenyong; Kapoor, Isha; Wang, Yue; Wang, Yuan; Zhu, Guoqing; Wang, Juejin

    2017-12-01

    Calcium influx from activated voltage-gated calcium channel Ca V 1.2 in vascular smooth muscle cells is indispensable for maintaining myogenic tone and blood pressure. The function of Ca V 1.2 channel can be optimized by alternative splicing, one of post-transcriptional modification mechanisms. The splicing factor Rbfox2 is known to regulate the Ca V 1.2 pre-mRNA alternative splicing events during neuronal development. However, Rbfox2's roles in modulating the key function of vascular Ca V 1.2 channel and in the pathogenesis of hypertension remain elusive. Here, we report that the proportion of Ca V 1.2 channels with alternative exon 9* is increased by 10.3%, whereas that with alternative exon 33 is decreased by 10.5% in hypertensive arteries. Surprisingly, the expression level of Rbfox2 is increased ≈3-folds, presumably because of the upregulation of a dominant-negative isoform of Rbfox2. In vascular smooth muscle cells, we find that knockdown of Rbfox2 dynamically increases alternative exon 9*, whereas decreases exon 33 inclusion of Ca V 1.2 channels. By patch-clamp studies, we show that diminished Rbfox2-induced alternative splicing shifts the steady-state activation and inactivation curves of vascular Ca V 1.2 calcium channel to hyperpolarization, which makes the window current potential to more negative. Moreover, siRNA-mediated knockdown of Rbfox2 increases the pressure-induced vascular myogenic tone of rat mesenteric artery. Taken together, our data indicate that Rbfox2 modulates the functions of vascular Ca V 1.2 calcium channel by dynamically regulating the expressions of alternative exons 9* and 33, which in turn affects the vascular myogenic tone. Therefore, our work suggests a key role for Rbfox2 in hypertension, which provides a rational basis for designing antihypertensive therapies. © 2017 American Heart Association, Inc.

  4. Molecular cloning and characterization of novel glutamate-gated chloride channel subunits from Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Vanessa Dufour

    Full Text Available Cys-loop ligand-gated ion channels (LGICs mediate fast ionotropic neurotransmission. They are proven drug targets in nematodes and arthropods, but are poorly characterized in flatworms. In this study, we characterized the anion-selective, non-acetylcholine-gated Cys-loop LGICs from Schistosoma mansoni. Full-length cDNAs were obtained for SmGluCl-1 (Smp_096480, SmGluCl-2 (Smp_015630 and SmGluCl-3 (Smp_104890. A partial cDNA was retrieved for SmGluCl-4 (Smp_099500/Smp_176730. Phylogenetic analyses suggest that SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 belong to a novel clade of flatworm glutamate-gated chloride channels (GluCl that includes putative genes from trematodes and cestodes. The flatworm GluCl clade was distinct from the nematode-arthropod and mollusc GluCl clades, and from all GABA receptors. We found no evidence of GABA receptors in S. mansoni. SmGluCl-1, SmGluCl-2 and SmGluCl-3 subunits were characterized by two-electrode voltage clamp (TEVC in Xenopus oocytes, and shown to encode Cl⁻-permeable channels gated by glutamate. SmGluCl-2 and SmGluCl-3 produced functional homomers, while SmGluCl-1 formed heteromers with SmGluCl-2. Concentration-response relationships revealed that the sensitivity of SmGluCl receptors to L-glutamate is among the highest reported for GluCl receptors, with EC₅₀ values of 7-26 µM. Chloride selectivity was confirmed by current-voltage (I/V relationships. SmGluCl receptors are insensitive to 1 µM ivermectin (IVM, indicating that they do not belong to the highly IVM-sensitive GluClα subtype group. SmGluCl receptors are also insensitive to 10 µM meclonazepam, a schistosomicidal benzodiazepine. These results provide the first molecular evidence showing the contribution of GluCl receptors to L-glutamate signaling in S. mansoni, an unprecedented finding in parasitic flatworms. Further work is needed to elucidate the roles of GluCl receptors in schistosomes and to explore their potential as drug targets.

  5. Synthetic Ion Channels and DNA Logic Gates as Components of Molecular Robots.

    Science.gov (United States)

    Kawano, Ryuji

    2018-02-19

    A molecular robot is a next-generation biochemical machine that imitates the actions of microorganisms. It is made of biomaterials such as DNA, proteins, and lipids. Three prerequisites have been proposed for the construction of such a robot: sensors, intelligence, and actuators. This Minireview focuses on recent research on synthetic ion channels and DNA computing technologies, which are viewed as potential candidate components of molecular robots. Synthetic ion channels, which are embedded in artificial cell membranes (lipid bilayers), sense ambient ions or chemicals and import them. These artificial sensors are useful components for molecular robots with bodies consisting of a lipid bilayer because they enable the interface between the inside and outside of the molecular robot to function as gates. After the signal molecules arrive inside the molecular robot, they can operate DNA logic gates, which perform computations. These functions will be integrated into the intelligence and sensor sections of molecular robots. Soon, these molecular machines will be able to be assembled to operate as a mass microrobot and play an active role in environmental monitoring and in vivo diagnosis or therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Differential distribution of glutamate- and GABA-gated chloride channels in the housefly Musca domestica.

    Science.gov (United States)

    Kita, Tomo; Ozoe, Fumiyo; Azuma, Masaaki; Ozoe, Yoshihisa

    2013-09-01

    l-Glutamic acid (glutamate) mediates fast inhibitory neurotransmission by affecting glutamate-gated chloride channels (GluCls) in invertebrates. The molecular function and pharmacological properties of GluCls have been well studied, but not much is known about their physiological role and localization in the insect body. The distribution of GluCls in the housefly (Musca domestica L.) was thus compared with the distribution of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls). Quantitative PCR and ligand-binding experiments indicate that the GluCl and GABACl transcripts and proteins are predominantly expressed in the adult head. Intense GluCl immunostaining was detected in the lamina, leg motor neurons, and legs of adult houseflies. The GABACl (Rdl) immunostaining was more widely distributed, and was found in the medulla, lobula, lobula plate, mushroom body, antennal lobe, and ellipsoid body. The present findings suggest that GluCls have physiological roles in different tissues than GABACls. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Aging-associated changes in motor axon voltage-gated Na(+) channel function in mice.

    Science.gov (United States)

    Moldovan, Mihai; Rosberg, Mette Romer; Alvarez, Susana; Klein, Dennis; Martini, Rudolf; Krarup, Christian

    2016-03-01

    Accumulating myelin abnormalities and conduction slowing occur in peripheral nerves during aging. In mice deficient of myelin protein P0, severe peripheral nervous system myelin damage is associated with ectopic expression of Nav1.8 voltage-gated Na(+) channels on motor axons aggravating the functional impairment. The aim of the present study was to investigate the effect of regular aging on motor axon function with particular emphasis on Nav1.8. We compared tibial nerve conduction and excitability measures by threshold tracking in 12 months (mature) and 20 months (aged) wild-type (WT) mice. With aging, deviations during threshold electrotonus were attenuated and the resting current-threshold slope and early refractoriness were increased. Modeling indicated that, in addition to changes in passive membrane properties, motor fibers in aged WT mice were depolarized. An increased Nav1.8 isoform expression was found by immunohistochemistry. The depolarizing excitability features were absent in Nav1.8 null mice, and they were counteracted in WT mice by a Nav1.8 blocker. Our data suggest that alteration in voltage-gated Na(+) channel isoform expression contributes to changes in motor axon function during aging. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Phosphorylation of rat aquaporin-4 at Ser(111) is not required for channel gating.

    Science.gov (United States)

    Assentoft, Mette; Kaptan, Shreyas; Fenton, Robert A; Hua, Susan Z; de Groot, Bert L; MacAulay, Nanna

    2013-07-01

    Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4 is therefore of therapeutic interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser(111) has been reported to increase the water permeability of AQP4 expressed in an astrocytic cell line. This possibility was, however, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser(111) was indeed a site involved in phosphorylation-mediated gating of AQP4. The water permeability of AQP4-expressing Xenopus oocytes was not altered by a range of activators and inhibitors of PKG and PKA. Mutation of Ser(111) to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of AQP4. PKG activation had no effect on the water permeability of AQP4 in primary cultures of rat astrocytes. Molecular dynamics simulations of a phosphorylation of AQP4.Ser(111) recorded no phosphorylation-induced change in water permeability. A phospho-specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser(111) , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. Thus, our data indicate a lack of phosphorylation of Ser(111) and of phosphorylation-dependent gating of AQP4. Copyright © 2013 Wiley Periodicals, Inc.

  9. Interactions of divalent cations with single calcium channels from rat brain synaptosomes.

    Science.gov (United States)

    Nelson, M T

    1986-02-01

    Voltage-dependent calcium channels from a rat brain membrane preparation ("synaptosomes") were incorporated into planar lipid bilayers. The effects of calcium, barium, strontium, manganese, and cadmium ions on the amplitudes and kinetics of single channel currents were examined. The order of single channel conductances was gBa greater than gSr greater than gMn, which was the inverse of the order of the mean channel open times: TMn greater than TCa = TSr greater than TBa. In contrast, the identity of the charge carrier had little or no effect on the mean closed times of the channel. Manganese, in the absence of other permeant ions, can pass through single channels (gMn = 4 pS). However, when added to a solution that contained another type of permeant divalent cation, manganese reduced the single channel current in a voltage-dependent manner. Cadmium, a potent blocker of macroscopic "ensemble" calcium currents in many preparations, reduced the current through an open channel in a manner consistent with Cd ions both not being measurably permeant and interacting with a single site. The permeant ions competed with cadmium for this site with the following order: Mn greater than Sr = Ca greater than Ba. These results are consistent with the existence of no less than one divalent cation binding site in the channel that regulates ion permeation.

  10. Voltage-gated potassium channel (K(v) 1) autoantibodies in patients with chagasic gut dysmotility and distribution of K(v) 1 channels in human enteric neuromusculature (autoantibodies in GI dysmotility).

    Science.gov (United States)

    Hubball, A W; Lang, B; Souza, M A N; Curran, O D; Martin, J E; Knowles, C H

    2012-08-01

    Autoantibodies directed against specific neuronal antigens are found in a significant number of patients with gastrointestinal neuromuscular diseases (GINMDs) secondary to neoplasia. This study examined the presence of antineuronal antibodies in idiopathic GINMD and GINMD secondary to South American Trypanosomiasis. The GI distribution of voltage-gated potassium channels (VGKCs) was also investigated. Seventy-three patients were included in the study with diagnoses of primary achalasia, enteric dysmotility, chronic intestinal pseudo-obstruction, esophageal or colonic dysmotility secondary to Chagas' disease. Sera were screened for specific antibodies to glutamic acid decarboxylase, voltage-gated calcium channels (VGCCs; P/Q subtype), nicotinic acetylcholine receptors (nAChRs; α3 subtype), and voltage-gated potassium channels (VGKCs, K(V) 1 subtype) using validated immunoprecipitation assays. The distribution of six VGKC subunits (K(V) 1.1-1.6), including those known to be antigenic targets of anti-VGKC antibodies was immunohistochemically investigated in all main human GI tract regions. Three patients (14%) with chagasic GI dysmotility were found to have positive anti-VGKC antibody titers. No antibodies were detected in patients with idiopathic GINMD. The VGKCs were found in enteric neurons at every level of the gut in unique yet overlapping distributions. The VGKC expression in GI smooth muscle was found to be limited to the esophagus. A small proportion of patients with GI dysfunction secondary to Chagas' disease have antibodies against VGKCs. The presence of these channels in the human enteric nervous system may have pathological relevance to the growing number of GINMDs with which anti-VGKC antibodies have been associated. © 2012 Blackwell Publishing Ltd.

  11. Gate-Defined One-Dimensional Channel and Broken Symmetry States in MoS2 van der Waals Heterostructures

    Science.gov (United States)

    Pisoni, Riccardo; Lee, Yongjin; Overweg, Hiske; Eich, Marius; Simonet, Pauline; Watanabe, Kenji; Taniguchi, Takashi; Gorbachev, Roman; Ihn, Thomas; Ensslin, Klaus

    2017-08-01

    We have realized encapsulated trilayer MoS$_2$ devices with gated graphene contacts. In the bulk, we observe an electron mobility as high as 7000~cm$^{2}$/(V s) at a density of 3 $\\times$ 10$^{12}$~cm$^{-2}$ at a temperature of 1.9~K. Shubnikov--de Haas oscillations start at magnetic fields as low as 0.9~T. The observed 3-fold Landau level degeneracy can be understood based on the valley Zeeman effect. Negatively biased split gate electrodes allow us to form a channel that can be completely pinched off for sufficiently large gate voltages. The measured conductance displays plateau-like features.

  12. The HCN4 channel mutation D553N associated with bradycardia has a C-linker mediated gating defect.

    Science.gov (United States)

    Netter, Michael F; Zuzarte, Marylou; Schlichthörl, Günter; Klöcker, Nikolaj; Decher, Niels

    2012-01-01

    The D553N mutation located in the C-linker of the cardiac pacemaker channel HCN4 is thought to cause sino-atrial dysfunction via a pronounced dominant-negative trafficking defect. Since HCN4 mutations usually have a minor defect in channel gating, it was our aim to further characterize the disease causing mechanism of D553N. Fluorescence microscopy, FACS, TEVC and patch-clamp recordings were performed to characterize D553N. Surprisingly, we found that D553N channels reach the plasma membrane and have no apparent trafficking defect. Co-expression of D553N with HCN4 also revealed no dominant-negative effect on wild-type channels. Consistent with the normal cell surface expression of D553N, it was possible to extensively characterize D553N mutants in Xenopus oocytes and mammalian cells. D553N channels generate currents with reduced amplitude, while the kinetics of activation and deactivation are not altered. While the regulation of D553N by tyrosine kinases is normal, we observed a change in the cAMP regulation which however cannot account for the strong loss-of-function of the mutant. The pronounced current reduction and the regular surface expression indicate a major gating defect of the C-linker gate. We hypothesize that the D553N mutation stabilizes a previously reported salt bridge important for the gating of the channel. Copyright © 2012 S. Karger AG, Basel.

  13. Is there a role for voltage-gated Na+ channels in the aggressiveness of breast cancer?

    Directory of Open Access Journals (Sweden)

    P. Rhana

    Full Text Available Breast cancer is the most common cancer among women and its metastatic potential is responsible for numerous deaths. Thus, the need to find new targets for improving treatment, and even finding the cure, becomes increasingly greater. Ion channels are known to participate in several physiological functions, such as muscle contraction, cell volume regulation, immune response and cell proliferation. In breast cancer, different types of ion channels have been associated with tumorigenesis. Recently, voltage-gated Na+ channels (VGSC have been implicated in the processes that lead to increased tumor aggressiveness. To explain this relationship, different theories, associated with pH changes, gene expression and intracellular Ca2+, have been proposed in an attempt to better understand the role of these ion channels in breast cancer. However, these theories are having difficulty being accepted because most of the findings are contrary to the present scientific knowledge. Several studies have shown that VGSC are related to different types of cancer, making them a promising pharmacological target against this debilitating disease. Molecular biology and cell electrophysiology have been used to look for new forms of treatment aiming to reduce aggressiveness and the disease progress.

  14. [Study of fractal properties of single ionic channel gating mechanism by the fast Fourier transform].

    Science.gov (United States)

    Kazachenko, V N; Kochetkov, K V; Aslanidi, O V; Grinevich, A A

    2001-01-01

    Sets of the channel open times (tau o), closed times (tau c) and the full set of the channel open and closed times (tau o, tau c) in the activity of single Ca(2+)-activated K+ channels in cultured kidney cells Vero were analyzed using the fast Fourier transform. It was found that in the low-frequency range (about 0.01-10 Hz), power density can be described by the equation S(f) approximately f-alpha (as a rule, 0 < alpha < 1), and this part of the Fourier spectrum usually consists of narrow peaks at almost multiple frequencies. It was shown that the upper frequency boundary of this spectrum is determined by the kinetic parameters tau o [symbol: see text] tau c. The data obtained show that ion channel gating is a fractal process (correlated in time) and can be regarded as a random signal modulated by some periodical functions (sinuses). The data obtained by the Fourier method are in agreement with the earlier results obtained using the rescaled-range analysis.

  15. Mechanism of Ion Permeation in Mammalian Voltage-Gated Sodium Channels.

    Directory of Open Access Journals (Sweden)

    Somayeh Mahdavi

    Full Text Available Recent determination of the crystal structures of bacterial voltage-gated sodium (NaV channels have raised hopes that modeling of the mammalian counterparts could soon be achieved. However, there are substantial differences between the pore domains of the bacterial and mammalian NaV channels, which necessitates careful validation of mammalian homology models constructed from the bacterial NaV structures. Such a validated homology model for the NaV1.4 channel was constructed recently using the extensive mutagenesis data available for binding of μ-conotoxins. Here we use this NaV1.4 model to study the ion permeation mechanism in mammalian NaV channels. Linking of the DEKA residues in the selectivity filter with residues in the neighboring domains is found to be important for keeping the permeation pathway open. Molecular dynamics simulations and potential of mean force calculations reveal that there is a binding site for a Na+ ion just inside the DEKA locus, and 1-2 Na+ ions can occupy the vestibule near the EEDD ring. These sites are separated by a low free energy barrier, suggesting that inward conduction occurs when a Na+ ion in the vestibule goes over the free energy barrier and pushes the Na+ ion in the filter to the intracellular cavity, consistent with the classical knock-on mechanism. The NaV1.4 model also provides a good description of the observed Na+/K+ selectivity.

  16. Measurement of gating forces of mechanosensitive channels of large conductance in Escherichia coli

    Science.gov (United States)

    Pandzic, Elvis; Wiseman, Paul; Kilfoil, Maria

    2006-03-01

    In order to sense and respond to external mechanical stimuli, cells have evolved schemes to incorporate mechanosensors within their plasma membranes. Mechanosensitive channels of large conductance (MscL) are used by bacterial cells to respond quickly and effectively to hypo-osmotic shock: the opening of this channel permits cells to quickly release large amounts of osmolytes in order to quickly equalize unbalanced osmotic pressure across a membrane. In this study, we are investigating the physical mechanism of the MscL gating within the native environment of the Escherichia coli cells. We are using the green fluorescent protein (GFP) and derivative proteins (CFP, BFP) to label the C-termini of MscL subunits in order to observe the channels in live bacteria by fluorescence microscopy. Moreover, we label the opposite termini with a different chromophore system that constitutes an excellent fluorescence resonance energy transfer (FRET) pair with CFP. Channels are activated within the bacterial membrane by osmotic stress and interactions between differently labeled subunits are measured by fluorescence microscopy.

  17. 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

    . The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels...... to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate...... a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain....

  18. Effect of insecticidal fusion proteins containing spider toxins targeting sodium and calcium ion channels on pyrethroid-resistant strains of peach-potato aphid (Myzus persicae).

    Science.gov (United States)

    Yang, Sheng; Fitches, Elaine; Pyati, Prashant; Gatehouse, John A

    2015-07-01

    The recombinant fusion proteins Pl1a/GNA and Hv1a/GNA contain the spider venom peptides δ-amaurobitoxin-PI1a or ω-hexatoxin-Hv1a respectively, linked to snowdrop lectin (GNA). Pl1a targets receptor site 4 of insect voltage-gated sodium channels (NaCh), while Hv1a targets voltage-gated calcium channels. Insecticide-resistant strains of peach-potato aphid (Myzus persicae) contain mutations in NaCh. The pyrethroid-resistant kdr (794J) and super-kdr (UKO) strains contain mutations at residues L1014 and M918 in the channel α-subunit respectively, while the kdr + super-kdr strain (4824J), insensitive to pyrethroids, contains mutations at both L1014 and M918. Pl1a/GNA and Hv1a/GNA fusion proteins have estimated LC50 values of 0.35 and 0.19 mg mL(-1) when fed to wild-type M. persicae. For insecticide-resistant aphids, LC50 for the Pl1a/GNA fusion protein increased by 2-6-fold, correlating with pyrethroid resistance (wild type < kdr < super-kdr < kdr + super-kdr strains). In contrast, LC50 for the Hv1a/GNA fusion protein showed limited correlation with pyrethroid resistance. Mutations in the sodium channel in pyrethroid-resistant aphids also protect against a fusion protein containing a sodium-channel-specific toxin, in spite of differences in ligand-channel interactions, but do not confer resistance to a fusion protein targeting calcium channels. The use of fusion proteins with differing targets could play a role in managing pesticide resistance. © 2014 Society of Chemical Industry.

  19. /sup 3/H)Nitrendipine binding to calcium channels in bovine and rat pituitary

    Energy Technology Data Exchange (ETDEWEB)

    Titeler, M.; De Souza, E.B.; Kuhar, M.J.

    1985-06-01

    (/sup 3/H)Nitrendipine was used to label sites in homogenates of bovine anterior and neurointermediate lobes of the pituitary gland. The amount of specific binding in the anterior lobe was 1.82 +/- 0.30 pmol/g wet weight of tissue and the KD was 1.44 +/- 0.02 X 10(-10) M. Preliminary experiments indicated a similar amount of binding in bovine neurointermediate lobe. In competition studies nimodipine and nisoldipine (two potent voltage-sensitive calcium channel blockers) displayed IC50 values of 1.6 and 6.8 X 10(-10) M, respectively. Verapamil and the verapamil-like calcium channel blockers D-600 and tiapamil competed in a complex manner for the (/sup 3/H)nitrendipine specific binding to bovine anterior pituitary homogenates. Autoradiographical studies demonstrated specific (/sup 3/H)nitrendipine binding sites distributed approximately equally in the anterior and posterior lobes, but not in the intermediate lobe of the rat pituitary. In general the properties of (/sup 3/H)nitrendipine binding in the pituitary tissue resemble strongly the properties of (/sup 3/H)nitrendipine binding in the brain which is believed to be to voltage-sensitive calcium channels. These results provide support for the hypothesis that calcium channels are involved in pituitary hormone secretion and that drugs that interact with calcium channels may modulate the secretory process directly at the level of the pituitary.

  20. L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.

    Science.gov (United States)

    Jacquemet, Guillaume; Baghirov, Habib; Georgiadou, Maria; Sihto, Harri; Peuhu, Emilia; Cettour-Janet, Pierre; He, Tao; Perälä, Merja; Kronqvist, Pauliina; Joensuu, Heikki; Ivaska, Johanna

    2016-12-02

    Mounting in vitro, in vivo and clinical evidence suggest an important role for filopodia in driving cancer cell invasion. Using a high-throughput microscopic-based drug screen, we identify FDA-approved calcium channel blockers (CCBs) as potent inhibitors of filopodia formation in cancer cells. Unexpectedly, we discover that L-type calcium channels are functional and frequently expressed in cancer cells suggesting a previously unappreciated role for these channels during tumorigenesis. We further demonstrate that, at filopodia, L-type calcium channels are activated by integrin inside-out signalling, integrin activation and Src. Moreover, L-type calcium channels promote filopodia stability and maturation into talin-rich adhesions through the spatially restricted regulation of calcium entry and subsequent activation of the protease calpain-1. Altogether we uncover a novel and clinically relevant signalling pathway that regulates filopodia formation in cancer cells and propose that cycles of filopodia stabilization, followed by maturation into focal adhesions, directs cancer cell migration and invasion.

  1. 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.

  2. Characterization of an insect heterodimeric voltage-gated sodium channel with unique alternative splicing mode.

    Science.gov (United States)

    Jiang, Xuan-Zhao; Pei, Yu-Xia; Lei, Wei; Wang, Ke-Yi; Shang, Feng; Jiang, Hong-Bo; Wang, Jin-Jun

    2017-01-01

    Recent discovery of the heterodimeric voltage-gated sodium channels (Na v ) in two aphid species, Acyrthosiphon pisum and Myzus persicae, aroused interest in exploring whether this kind of channel is conserved for aphids. Herewith, we aim to provide evidence for the conservation of heterodimeric Na v s in aphids and investigate whether they have unique splicing patterns. We found that the only identifiable Na v from Toxoptera citricida consisted of two subunits, forming a heterodimeric Na v , which carried an atypical "DENS" ion selectivity filter and a conventional "MFM" inactivation gate, confirming the heterodimeric Na v s' conservation within aphids. These unique heterodimeric channels may form a new Na v subfamily, specific to aphids. A more ancient member of four-domain Na v homolog was well preserved in T. citricida, carrying a typical "DEEA" and "MFL" motif. The presence of "DENS" in mammalian Na x s and "DEKT" in a fungus Na v suggested that the heterodimeric Na v s may still preserve Na + permeability. Sequencing 46 clones from nymphs and adults exposed unique splicing patterns for this heterodimeric Na v from T. citricida, revealing 7 alternatively spliced exons, evidencing that exon 5 was no longer unique to Bombyx mori, and exon k/l was semi-mutually exclusive. Two previously undescribed optional exons and a SNP site seemingly unique to aphids were identified. In conclusion, the dimeric Na v s might form a new aphids-specific heterodimeric N a v subfamily. This dimeric Na v from T. citricida was characterized with distinguishable alternative splicing modes, exemplified by the discovery of two novel alternative exons and unique usage patterns of alternative exons. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Acetaldehyde - ethanol interactions on calcium-activated potassium (BK channels in pituitary tumor (GH3 cells

    Directory of Open Access Journals (Sweden)

    Astrid G. Handlechner

    2013-06-01

    Full Text Available Background: In the central nervous system ethanol (EtOH is metabolized to acetaldehyde (ACA primarily by the oxidative enzyme catalase. Evidence suggests that ACA is responsible for at least some of the effects on the brain that have been attributed to EtOH. Various types of ion channels which are involved in electrical signaling are targets of EtOH like maxi calcium-activated potassium (BK channels. BK channels exhibit various functions like action potential repolarization, blood pressure regulation, hormone secretion, or transmitter release. In most neuronal and neuroendocrine preparations at physiological intracellular calcium levels, EtOH increases BK channel activity. The simultaneous presence of ACA and EtOH reflects the physiological situation after drinking and may result in synergistic as well as antagonistic actions compared to a single application of either drug. The action of ACA on electrical activity has yet not been fully established.Methods: GH3 pituitary tumor cells were used for outside-out and inside-out patch-clamp recordings of BK activity in excised patches. Unitary current amplitude, open probability and channel mean open time of BK channels were measured. Results: Extracellular EtOH raised BK channel activity. In the presence of intracellular ACA this increment of BK activity was suppressed in a dose- as well as calcium-dependent manner. Mean channel open time was significantly reduced by internal ACA, whereas BK channel amplitudes were not affected. The EtOH counteracting effect of ACA was found to depend on succession of application. EtOH was prevented from activating BK channels by pre-exposure of membrane patches to ACA. In contrast BK activation by a hypotonic solution was not affected by internal ACA. Conclusions: Our data suggest an inhibitory impact of ACA on BK activation by EtOH. ACA appears to interact specifically with EtOH at BK channels since intracellular ACA had no effect when BK channels were activated by

  4. Oxidative Regulation of Large Conductance Calcium-Activated Potassium Channels

    Science.gov (United States)

    Tang, Xiang D.; Daggett, Heather; Hanner, Markus; Garcia, Maria L.; McManus, Owen B.; Brot, Nathan; Weissbach, Herbert; Heinemann, Stefan H.; Hoshi, Toshinori

    2001-01-01

    Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca2+-activated K+ channels (BKCa or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca2+, the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K+ channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism. PMID:11222629

  5. The medieval physician Avicenna used an herbal calcium channel blocker, Taxus baccata L.

    Science.gov (United States)

    Tekol, Yalcin

    2007-07-01

    Calcium channel blockers are drugs which are important for current medical therapy. The first examples of synthetic congeners of this class of drugs appear around at the beginning of the 1960s. Review of the current and historical literature shows that Avicenna (Ibn Sina) (980-1037) had used the herbal drug 'Zarnab' (Taxus baccata L.) as a cardiac remedy. The leaves of T. baccata contain an alkaloid mixture (taxines). It was recently demonstrated that this drug possessed calcium channel blocking activity. So, it is evident that Avicenna used a drug with calcium channel blocking activity much earlier than the arrival of synthetic drugs belonging to the same pharmacological group. Copyright 2007 John Wiley & Sons, Ltd.

  6. CaV2.2 Gates Calcium-Independent but Voltage-Dependent Secretion in Mammalian Sensory Neurons.

    Science.gov (United States)

    Chai, Zuying; Wang, Changhe; Huang, Rong; Wang, Yuan; Zhang, Xiaoyu; Wu, Qihui; Wang, Yeshi; Wu, Xi; Zheng, Lianghong; Zhang, Chen; Guo, Wei; Xiong, Wei; Ding, Jiuping; Zhu, Feipeng; Zhou, Zhuan

    2017-12-20

    Action potential induces membrane depolarization and triggers intracellular free Ca 2+ concentration (Ca 2+ )-dependent secretion (CDS) via Ca 2+ influx through voltage-gated Ca 2+ channels. We report a new type of somatic exocytosis triggered by the action potential per se-Ca 2+ -independent but voltage-dependent secretion (CiVDS)-in dorsal root ganglion neurons. Here we uncovered the molecular mechanism of CiVDS, comprising a voltage sensor, fusion machinery, and their linker. Specifically, the voltage-gated N-type Ca 2+ channel (Ca V 2.2) is the voltage sensor triggering CiVDS, the SNARE complex functions as the vesicle fusion machinery, the "synprint" of Ca V 2.2 serves as a linker between the voltage sensor and the fusion machinery, and ATP is a cargo of CiVDS vesicles. Thus, CiVDS releases ATP from the soma while CDS releases glutamate from presynaptic terminals, establishing the Ca V 2.2-SNARE "voltage-gating fusion pore" as a novel pathway co-existing with the canonical "Ca 2+ -gating fusion pore" pathway for neurotransmitter release following action potentials in primary sensory neurons. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Comparing coronary artery calcium and thoracic aorta calcium for prediction of all-cause mortality and cardiovascular events on low-dose non-gated computed tomography in a high-risk population of heavy smokers

    NARCIS (Netherlands)

    Jacobs, Peter C.; Prokop, Mathias; van der Graaf, Yolanda; Gondrie, Martijn J.; Janssen, Kristel J.; de Koning, Harry J.; Isgum, Ivana; van Klaveren, Rob J.; Oudkerk, Matthijs; van Ginneken, Bram; Mali, Willem P.

    Background: Coronary artery calcium (CAC) and thoracic aorta calcium (TAC) can be detected simultaneously on low-dose, non-gated computed tomography (CT) scans. CAC has been shown to predict cardiovascular (CVD) and coronary (CHD) events. A comparable association between TAC and CVD events has yet

  8. Materials characterization for process integration of multi-channel gate all around (GAA) devices

    Science.gov (United States)

    Muthinti, Gangadhara Raja; Loubet, Nicolas; Chao, Robin; de la Peña, Abraham A.; Li, Juntao; Guillorn, Michael A.; Yamashita, Tenko; Kanakasabapathy, Sivananda; Gaudiello, John; Cepler, Aron J.; Sendelbach, Matthew; Emans, Susan; Wolfling, Shay; Ger, Avron; Kandel, Daniel; Koret, Roy; Lee, Wei Ti; Gin, Peter; Matney, Kevin; Wormington, Matthew

    2017-03-01

    Multi-channel gate all around (GAA) semiconductor devices march closer to becoming a reality in production as their maturity in development continues. From this development, an understanding of what physical parameters affecting the device has emerged. The importance of material property characterization relative to that of other physical parameters has continued to increase for GAA architecture when compared to its relative importance in earlier architectures. Among these materials properties are the concentration of Ge in SiGe channels and the strain in these channels and related films. But because these properties can be altered by many different process steps, each one adding its own variation to these parameters, their characterization and control at multiple steps in the process flow is crucial. This paper investigates the characterization of strain and Ge concentration, and the relationships between these properties, in the PFET SiGe channel material at the earliest stages of processing for GAA devices. Grown on a bulk Si substrate, multiple pairs of thin SiGe/Si layers that eventually form the basis of the PFET channel are measured and characterized in this study. Multiple measurement techniques are used to measure the material properties. In-line X-Ray Photoelectron Spectroscopy (XPS) and Low Energy X-Ray Fluorescence (LE-XRF) are used to characterize Ge content, while in-line High Resolution X-Ray Diffraction (HRXRD) is used to characterize strain. Because both patterned and un-patterned structures were investigated, scatterometry (also called optical critical dimension, or OCD) is used to provide valuable geometrical metrology.

  9. Emerging roles of calcium-activated K channels and TRPV4 channels in lung oedema and pulmonary circulatory collapse.

    Science.gov (United States)

    Simonsen, U; Wandall-Frostholm, C; Oliván-Viguera, A; Köhler, R

    2017-01-01

    It has been suggested that the transient receptor potential cation (TRP) channel subfamily V (vanilloid) type 4 (TRPV4) and intermediate conductance calcium-activated potassium (KCa3.1) channels contribute to endothelium-dependent vasodilation. Here, we summarize very recent evidence for a synergistic interplay of TRPV4 and KCa3.1 channels in lung disease. Among the endothelial Ca2+ -permeable TRPs, TRPV4 is best characterized and produces arterial dilation by stimulating Ca2+ -dependent nitric oxide synthesis and endothelium-dependent hyperpolarization. Besides these roles, some TRP channels control endothelial/epithelial barrier functions and vascular integrity, while KCa3.1 channels provide the driving force required for Cl- and water transport in some cells and most secretory epithelia. The three conditions, increased pulmonary venous pressure caused by left heart disease, high inflation pressure and chemically induced lung injury, may lead to activation of TRPV4 channels followed by Ca2+ influx leading to activation of KCa3.1 channels in endothelial cells ultimately leading to acute lung injury. We find that a deficiency in KCa3.1 channels protects against TRPV4-induced pulmonary arterial relaxation, fluid extravasation, haemorrhage, pulmonary circulatory collapse and cardiac arrest in vivo. These data identify KCa3.1 channels as crucial molecular components in downstream TRPV4 signal transduction and as a potential target for the prevention of undesired fluid extravasation, vasodilatation and pulmonary circulatory collapse. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  10. Distribution of high-voltage-activated calcium channels in cultured gamma-aminobutyric acidergic neurons from mouse cerebral cortex.

    Science.gov (United States)

    Timmermann, Daniel B; Westenbroek, Ruth E; Schousboe, Arne; Catterall, William A

    2002-01-01

    The localization of voltage-gated calcium channel (VGCC) alpha(1) subunits in cultured GABAergic mouse cortical neurons was examined by immunocytochemical methods. Ca(v)1.2 and Ca(v)1.3 subunits of L-type VGCCs were found in cell bodies and dendrites of GABA-immunopositive neurons. Likewise, the Ca(v)2.3 subunit of R-type VGCCs was expressed in a somatodendritic pattern. Ca(v)2.2 subunits of N-type channels were found exclusively in small varicosities that were identified as presynaptic nerve terminals based on their expression of synaptic marker proteins. Two splice variants of the Ca(v)2.1 subunit of P/Q-type VGCCs showed widely differing expression patterns. The rbA isoform displayed a purely somatodendritic staining pattern, whereas the BI isoform was confined to axon-like fibers and nerve terminals. The nerve terminals of these cultured GABAergic neurons express Ca(v)2.2 either alone or in combination with Ca(v)2.1 (BI isoform) but never express Ca(v)2.1 alone. The functional association between VGCCs and the neurotransmitter release machinery was probed using the FM1-43 dye-labeling technique. N-type VGCCs were found to be tightly coupled to exocytosis in these cultured cortical neurons, and P-type VGCCs were also important in a fraction of the cells. The predominant role of N-type VGCCs in neurotransmitter release and the specific localization of the BI isoform of Ca(v)2.1 in the nerve terminals of these neurons distinguish them from previously studied central neurons. The complementary localization patterns observed for two different isoforms of the Ca(v)2.1 subunits provide direct evidence for alternative splicing as a means of generating functional diversity among neuronal calcium channels. Copyright 2002 Wiley-Liss, Inc.

  11. Biogenesis of the T1-S1 linker of voltage-gated K+ channels.

    Science.gov (United States)

    Tu, LiWei; Wang, Jing; Deutsch, Carol

    2007-07-10

    In the model derived from the crystal structure of Kv1.2, a six-transmembrane voltage-gated potassium channel, the linker between a cytosolic tetramerization domain, T1, and the first transmembrane segment, S1, is projected radially outward from the channel's central axis. This T1-S1 linker was modeled as two polyglycine helices to accommodate the residues between T1 and S1 [Long et al. (2005) Science 309, 897-903]; however, the structure of this linker is not known. Here, we investigate whether a compact secondary structure of the T1-S1 linker exists at an early stage of Kv channel biogenesis. We have used a mass-tagging accessibility assay to report the biogenesis of secondary structure for three consecutive regions of Kv1.3, a highly homologous isoform of Kv1.2. The three regions include the T1-S1 linker and its two flanking regions, alpha5 of the T1 domain and S1. Both alpha5 and S1 manifest compact structures (helical) inside the ribosomal exit tunnel, whereas the T1-S1 linker does not. Moreover, the location of the peptide in the tunnel influences compaction.

  12. Voltage-gated sodium channels confer excitability to human odontoblasts: possible role in tooth pain transmission.

    Science.gov (United States)

    Allard, Bruno; Magloire, Henry; Couble, Marie Lise; Maurin, Jean Christophe; Bleicher, Françoise

    2006-09-29

    Odontoblasts are responsible for the dentin formation. They are suspected to play a role in tooth pain transmission as sensor cells because of their close relationship with nerve, but this role has never been evidenced. We demonstrate here that human odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na(+) currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts express neuronal isoforms of alpha2 and beta2 subunits of sodium channels. Co-cultures of odontoblasts with trigeminal neurons indicate a clustering of alpha2 and beta2 sodium channel subunits and, at the sites of cell-cell contact, a co-localization of odontoblasts beta2 subunits with peripherin. In vivo, sodium channels are expressed in odontoblasts. Ankyrin(G) and beta2 co-localize, suggesting a link for signal transduction between axons and odontoblasts. Evidence for excitable properties of odontoblasts and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts may operate as sensor cells that initiate tooth pain transmission.

  13. Peptide-gated ion channels and the simple nervous system of Hydra.

    Science.gov (United States)

    Gründer, Stefan; Assmann, Marc

    2015-02-15

    Neurons either use electrical or chemical synapses to communicate with each other. Transmitters at chemical synapses are either small molecules or neuropeptides. After binding to their receptors, transmitters elicit postsynaptic potentials, which can either be fast and transient or slow and longer lasting, depending on the type of receptor. Fast transient potentials are mediated by ionotropic receptors and slow long-lasting potentials by metabotropic receptors. Transmitters and receptors are well studied for animals with a complex nervous system such as vertebrates and insects, but much less is known for animals with a simple nervous system like Cnidaria. As cnidarians arose early in animal evolution, nervous systems might have first evolved within this group and the study of neurotransmission in cnidarians might reveal an ancient mechanism of neuronal communication. The simple nervous system of the cnidarian Hydra extensively uses neuropeptides and, recently, we cloned and functionally characterized an ion channel that is directly activated by neuropeptides of the Hydra nervous system. These results demonstrate the existence of peptide-gated ion channels in Hydra, suggesting they mediate fast transmission in its nervous system. As related channels are also present in the genomes of the cnidarian Nematostella, of placozoans and of ctenophores, it should be considered that the early nervous systems of cnidarians and ctenophores have co-opted neuropeptides for fast transmission at chemical synapses. © 2015. Published by The Company of Biologists Ltd.

  14. Phylogenetic conservation of protein-lipid motifs in pentameric ligand-gated ion channels.

    Science.gov (United States)

    Barrantes, Francisco J

    2015-09-01

    Using the crosstalk between the nicotinic acetylcholine receptor (nAChR) and its lipid microenvironment as a paradigm, this short overview analyzes the occurrence of structural motifs which appear not only to be conserved within the nAChR family and contemporary eukaryotic members of the pentameric ligand-gated ion channel (pLGIC) superfamily, but also extend to prokaryotic homologues found in bacteria. The evolutionarily conserved design is manifested in: 1) the concentric three-ring architecture of the transmembrane region, 2) the occurrence in this region of distinct lipid consensus motifs in prokaryotic and eukaryotic pLGIC and 3) the key participation of the outer TM4 ring in conveying the influence of the lipid membrane environment to the middle TM1-TM3 ring and this, in turn, to the inner TM2 channel-lining ring, which determines the ion selectivity of the channel. The preservation of these constant structural-functional features throughout such a long phylogenetic span likely points to the successful gain-of-function conferred by their early acquisition. This article is part of a Special Issue entitled: Lipid-protein interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons.

    Science.gov (United States)

    Bao, Lan

    2015-09-30

    Voltage-gated sodium channels (Navs) comprise at least nine pore-forming α subunits. Of these, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied in primary sensory neurons located in the dorsal root ganglion and are mainly localized to the cytoplasm. A large pool of intracellular Navs raises the possibility that changes in Nav trafficking could alter channel function. The molecular mediators of Nav trafficking mainly consist of signals within the Navs themselves, interacting proteins and extracellular factors. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring, and it is also regulated by channel phosphorylation and ubiquitination in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons.

  16. Cortactin Controls Surface Expression of the Voltage-gated Potassium Channel KV10.1

    Science.gov (United States)

    Herrmann, Solveig; Ninkovic, Milena; Kohl, Tobias; Lörinczi, Éva; Pardo, Luis A.

    2012-01-01

    KV10.1 is a voltage-gated potassium channel aberrantly expressed in many cases of cancer, and participates in cancer initiation and tumor progression. Its action as an oncoprotein can be inhibited by a functional monoclonal antibody, indicating a role for channels located at the plasma membrane, accessible to the antibody. Cortactin is an actin-interacting protein implicated in cytoskeletal architecture and often amplified in several types of cancer. In this study, we describe a physical and functional interaction between cortactin and KV10.1. Binding of these two proteins occurs between the C terminus of KV10.1 and the proline-rich domain of cortactin, regions targeted by many post-translational modifications. This interaction is specific for KV10.1 and does not occur with KV10.2. Cortactin controls the abundance of KV10.1 at the plasma membrane and is required for functional expression of KV10.1 channels. PMID:23144454

  17. Zn2+Interaction with Alzheimer Amyloid β Protein Calcium Channels

    National Research Council Canada - National Science Library

    Nelson Arispe; Harvey B. Pollard; Eduardo Rojas

    1996-01-01

    The Alzheimer disease 40-residue amyloid β protein (Aβ P[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS...

  18. Transparent field-effect transistors based on AlN-gate dielectric and IGZO-channel semiconductor

    Energy Technology Data Exchange (ETDEWEB)

    Besleaga, C.; Stan, G.E.; Pintilie, I. [National Institute of Materials Physics, 405A Atomistilor, 077125 Magurele-Ilfov (Romania); Barquinha, P.; Fortunato, E. [CENIMAT/I3N, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, and CEMOP-UNINOVA, 2829-516 Caparica (Portugal); Martins, R., E-mail: rm@uninova.pt [CENIMAT/I3N, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, and CEMOP-UNINOVA, 2829-516 Caparica (Portugal)

    2016-08-30

    Highlights: • TFTs based on IGZO channel semiconductor and AlN gate dielectric were fabricated. • AlN films – a viable and cheap gate dielectric alternative for transparent TFTs. • Influence of gate dielectric layer thickness on TFTs electrical characteristics. • No degradation of AlN gate dielectric was observed during devices stress testing. - Abstract: The degradation of thin-film transistors (TFTs) caused by the self-heating effect constitutes a problem to be solved for the next generation of displays. Aluminum nitride (AlN) is a viable alternative for gate dielectric of TFTs due to its good thermal conductivity, matching coefficient of thermal expansion to indium–gallium–zinc-oxide, and excellent stability at high temperatures. Here, AlN thin films of different thicknesses were fabricated by a low temperature reactive radio-frequency magnetron sputtering process, using a low cost, metallic Al target. Their electrical properties have been thoroughly assessed. Furthermore, the 200 nm and 500 nm thick AlN layers have been integrated as gate-dielectric in transparent TFTs with indium–gallium–zinc-oxide as channel semiconductor. Our study emphasizes the potential of AlN thin films for transparent electronics, whilst the functionality of the fabricated field-effect transistors is explored and discussed.

  19. The Segregated Expression of Voltage-Gated Potassium and Sodium Channels in Neuronal Membranes: Functional Implications and Regulatory Mechanisms.

    Science.gov (United States)

    Duménieu, Maël; Oulé, Marie; Kreutz, Michael R; Lopez-Rojas, Jeffrey

    2017-01-01

    Neurons are highly polarized cells with apparent functional and morphological differences between dendrites and axon. A critical determinant for the molecular and functional identity of axonal and dendritic segments is the restricted expression of voltage-gated ion channels (VGCs). Several studies show an uneven distribution of ion channels and their differential regulation within dendrites and axons, which is a prerequisite for an appropriate integration of synaptic inputs and the generation of adequate action potential (AP) firing patterns. This review article will focus on the signaling pathways leading to segmented expression of voltage-gated potassium and sodium ion channels at the neuronal plasma membrane and the regulatory mechanisms ensuring segregated functions. We will also discuss the relevance of proper ion channel targeting for neuronal physiology and how alterations in polarized distribution contribute to neuronal pathology.

  20. Lidocaine reduces the transition to slow inactivation in Nav1.7 voltage-gated sodium channels

    Science.gov (United States)

    Sheets, Patrick L; Jarecki, Brian W; Cummins, Theodore R

    2011-01-01

    BACKGROUND AND PURPOSE The primary use of local anaesthetics is to prevent or relieve pain by reversibly preventing action potential propagation through the inhibition of voltage-gated sodium channels. The tetrodotoxin-sensitive voltage-gated sodium channel subtype Nav1.7, abundantly expressed in pain-sensing neurons, plays a crucial role in perception and transmission of painful stimuli and in inherited chronic pain syndromes. Understanding the interaction of lidocaine with Nav1.7 channels could provide valuable insight into the drug's action in alleviating pain in distinct patient populations. The aim of this study was to determine how lidocaine interacts with multiple inactivated conformations of Nav1.7 channels. EXPERIMENTAL APPROACH We investigated the interactions of lidocaine with wild-type Nav1.7 channels and a paroxysmal extreme pain disorder mutation (I1461T) that destabilizes fast inactivation. Whole cell patch clamp recordings were used to examine the activity of channels expressed in human embryonic kidney 293 cells. KEY RESULTS Depolarizing pulses that increased slow inactivation of Nav1.7 channels also reduced lidocaine inhibition. Lidocaine enhanced recovery of Nav1.7 channels from prolonged depolarizing pulses by decreasing slow inactivation. A paroxysmal extreme pain disorder mutation that destabilizes fast inactivation of Nav1.7 channels decreased lidocaine inhibition. CONCLUSIONS AND IMPLICATIONS Lidocaine decreased the transition of Nav1.7 channels to the slow inactivated state. The fast inactivation gate (domain III–IV linker) is important for potentiating the interaction of lidocaine with the Nav1.7 channel. PMID:21232038

  1. Cross-Sectional Channel Shape Dependence of Short-Channel Effects in Fin-Type Double-Gate Metal Oxide Semiconductor Field-Effect Transistors

    Science.gov (United States)

    Liu, Yongxun; Ishii, Kenichi; Masahara, Meishoku; Tsutsumi, Toshiyuki; Takashima, Hidenori; Yamauchi, Hiromi; Suzuki, Eiichi

    2004-04-01

    The dependence of short-channel effects (SCEs) on the cross-sectional channel shape of the fin-type double-gate metal oxide semiconductor field-effect transistors (MOSFETs) has been experimentally investigated from the viewpoint of fin fabrication. The three types of fin-type double-gate MOSFETs (FinFETs) with a rectangular-cross-section channel on a (110)-oriented silicon-on-insulator (SOI) wafer, and a triangular and trapezoidal channels on a (100)-oriented SOI wafer were fabricated using the same orientation-dependent wet etching process. The experimental results show that the SCEs in rectangular-cross-section silicon (Si)-fin channel devices are well suppressed compared with those in a triangular or a trapezoidal Si-fin channel device fabricated using a similar mask pattern, in the regimes of the gate length of less than 85 nm and Si fin height of larger than 65 nm. The presented experimental results are valuable for FinFET design and fabrication.

  2. Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance.

    Science.gov (United States)

    Lórenz-Fonfría, Víctor A; Bamann, Christian; Resler, Tom; Schlesinger, Ramona; Bamberg, Ernst; Heberle, Joachim

    2015-10-27

    The discovery of channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time-resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(-) and 1,648(+) cm(-1). These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance.

  3. Source-Coupled, N-Channel, JFET-Based Digital Logic Gate Structure Using Resistive Level Shifters

    Science.gov (United States)

    Krasowski, Michael J.

    2011-01-01

    A circuit topography is used to create usable, digital logic gates using N (negatively doped) channel junction field effect transistors (JFETs), load resistors, level shifting resistors, and supply rails whose values are based on the DC parametric distributions of these JFETs. This method has direct application to the current state-of-the-art in high-temperature (300 to 500 C and higher) silicon carbide (SiC) device production, and defines an adaptation to the logic gate described in U.S. Patent 7,688,117 in that, by removing the level shifter from the output of the gate structure described in the patent (and applying it to the input of the same gate), a source-coupled gate topography is created. This structure allows for the construction AND/OR (sum of products) arrays that use far fewer transistors and resistors than the same array as constructed from the gates described in the aforementioned patent. This plays a central role when large multiplexer constructs are necessary; for example, as in the construction of memory. This innovation moves the resistive level shifter from the output of the basic gate structure to the front as if the input is now configured as what would be the output of the preceding gate, wherein the output is the two level shifting resistors. The output of this innovation can now be realized as the lone follower transistor with its source node as the gate output. Additionally, one may leave intact the resistive level shifter on the new gate topography. A source-coupled to direct-coupled logic translator will be the result.

  4. The TRPM7 channel kinase regulates store-operated calcium entry.

    Science.gov (United States)

    Faouzi, Malika; Kilch, Tatiana; Horgen, F David; Fleig, Andrea; Penner, Reinhold

    2017-05-15

    Pharmacological and molecular inhibition of transient receptor potential melastatin 7 (TRPM7) reduces store-operated calcium entry (SOCE). Overexpression of TRPM7 in TRPM7-/- cells restores SOCE. TRPM7 is not a store-operated calcium channel. TRPM7 kinase rather than channel modulates SOCE. TRPM7 channel activity contributes to the maintenance of store Ca2+ levels at rest. The transient receptor potential melastatin 7 (TRPM7) is a protein that combines an ion channel with an intrinsic kinase domain, enabling it to modulate cellular functions either by conducting ions through the pore or by phosphorylating downstream proteins via its kinase domain. In the present study, we report store-operated calcium entry (SOCE) as a novel target of TRPM7 kinase activity. TRPM7-deficient chicken DT40 B lymphocytes exhibit a strongly impaired SOCE compared to wild-type cells as a result of reduced calcium release activated calcium currents, and independently of potassium channel regulation, membrane potential changes or changes in cell-cycle distribution. Pharmacological blockade of TRPM7 with NS8593 or waixenicin A in wild-type B lymphocytes results in a significant decrease in SOCE, confirming that TRPM7 activity is acutely linked to SOCE, without TRPM7 representing a store-operated channel itself. Using kinase-deficient mutants, we find that TRPM7 regulates SOCE through its kinase domain. Furthermore, Ca2+ influx through TRPM7 is essential for the maintenance of endoplasmic reticulum Ca2+ concentration in resting cells, and for the refilling of Ca2+ stores after a Ca2+ signalling event. We conclude that the channel kinase TRPM7 and SOCE are synergistic mechanisms regulating intracellular Ca2+ homeostasis. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

  5. Role of calcium influx through voltage-operated calcium channels and of calcium mobilization in the physiology of Schistosoma mansoni muscle contractions.

    Science.gov (United States)

    Mendonça-Silva, D L; Novozhilova, E; Cobbett, P J R; Silva, C L M; Noël, F; Totten, M I J; Maule, A G; Day, T A

    2006-07-01

    We tested the hypothesis that voltage-operated Ca2+ channels mediate an extracellular Ca2+ influx in muscle fibres from the human parasite Schistosoma mansoni and, along with Ca2+ mobilization from the sarcoplasmic reticulum, contribute to muscle contraction. Indeed, whole-cell voltage clamp revealed voltage-gated inward currents carried by divalent ions with a peak current elicited by steps to +20 mV (from a holding potential of -70 mV). Depolarization of the fibres by elevated extracellular K+ elicited contractions that were completely dependent on extracellular Ca2+ and inhibited by nicardipine (half inhibition at 4.1 microM). However these contractions were not very sensitive to other classical blockers of voltage-gated Ca2+ channels, indicating that the schistosome muscle channels have an atypical pharmacology when compared to their mammalian counterparts. Futhermore, the contraction induced by 5 mM caffeine was inhibited after depletion of the sarcoplasmic reticulum either with thapsigargin (10 microM) or ryanodine (10 microM). These data suggest that voltage-operated Ca2+ channels do contribute to S. mansoni contraction as does the mobilization of stored Ca2+, despite the small volume of sarcoplasmic reticulum in schistosome smooth muscles.

  6. Analytical drain current model for Gate and Channel Engineered RingFET (GCE-RingFET)

    Science.gov (United States)

    Kumar, Sachin; Kumari, Vandana; Singh, Sanjeev; Saxena, Manoj; Gupta, Mridula

    2017-11-01

    In this work, an analytical drain current model for Gate and Channel Engineered RingFET (GCE-RingFET) has been developed by solving 2D-Poisson equation in cylindrical coordinates. The authenticity of proposed model for GCE-RingFET architecture has been justified by comparing the analytical results with simulation results obtained using ATLAS 3D device simulation. Performance comparison of GCE-RingFET with the conventional RingFET device architectures has been performed. Various important performance metrics such as surface potential, transfer characteristic (Ids-Vgs), ION/IOFF ratio, Threshold voltage roll off, Sub-threshold Slope (SS), Drain Induced Barrier Lowering (DIBL), Trans-conductance Generation Efficiency (gm/Ids), have been investigated.

  7. 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.

  8. 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 amin...... acids into proteins. We also review insights into the function and pharmacology of voltage-gated ion channels that have emerged from unnatural amino acid mutagenesis approaches....

  9. In-depth study of the interaction, sensitivity and gating modulation by PUFAs on K+ channels; interaction and new targets

    Directory of Open Access Journals (Sweden)

    Cristina Moreno

    2016-11-01

    Full Text Available Voltage gated potassium channels (Kv are membrane proteins that allow selective flow of K+ ions in a voltage-dependent manner. These channels play an important role in several excitable cells as neurons, cardiomyocytes and vascular smooth muscle. Over the last 20 years, it has been shown that omega-3 polyunsaturated fatty acids (PUFAs enhance or decrease the activity of several cardiac Kv channels. PUFAs-dependent modulation of potassium ion channels has been reported to be cardioprotective. However, the precise cellular mechanism underlying the cardiovascular benefits remained unclear in part because new PUFAs targets and signaling pathways continue being discovered. In this review, we will focus on recent data available concerning the following aspects of the Kv channel modulation by PUFAs: i the exact residues involved in PUFAs-Kv channels interaction; ii the structural PUFAs determinants important for their effects on Kv channels; iii the mechanism of the gating modulation of KV channels and, finally, iv the PUFAs modulation of a few new targets present in smooth muscle cells, KCa1.1, K2P and KATP channels, involved in vascular relaxation.

  10. Optical modulation of neurotransmission using calcium photocurrents through the ion channel LiGluR

    Directory of Open Access Journals (Sweden)

    Mercè eIzquierdo-Serra

    2013-03-01

    Full Text Available A wide range of light-activated molecules (photoswitches and phototriggers have been used to the study of computational properties of an isolated neuron by acting pre and postsynaptically. However, new tools are being pursued to elicit a presynaptic calcium influx that triggers the release of neurotransmitters, most of them based in calcium-permeable Channelrhodopsin-2 mutants. Here we describe a method to control exocytosis of synaptic vesicles through the use of a light-gated glutamate receptor (LiGluR, which has recently been demonstrated that supports secretion by means of calcium influx in chromaffin cells. Expression of LiGluR in hippocampal neurons enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the wavelength of illumination. This method may be useful for the determination of the complex transfer function of individual synapses.

  11. Conformational rearrangements in the S6 domain and C-linker during gating in CNGA1 channels.

    NARCIS (Netherlands)

    Nair, A.V.; Nguyen, C.H.; Mazzolini, M.

    2009-01-01

    This work completes previous findings and, using cysteine scanning mutagenesis (CSM) and biochemical methods, provides detailed analysis of conformational changes of the S6 domain and C-linker during gating of CNGA1 channels. Specific residues between Phe375 and Val424 were mutated to a cysteine in

  12. Effect of Gating Modifier Toxins on Membrane Thickness: Implications for Toxin Effect on Gramicidin and Mechanosensitive Channels

    Directory of Open Access Journals (Sweden)

    Shin-Ho Chung

    2013-02-01

    Full Text Available Various gating modifier toxins partition into membranes and interfere with the gating mechanisms of biological ion channels. For example, GsMTx4 potentiates gramicidin and several bacterial mechanosensitive channels whose gating kinetics are sensitive to mechanical properties of the membrane, whereas binding of HpTx2 shifts the voltage-activity curve of the voltage-gated potassium channel Kv4.2 to the right. The detailed process by which the toxin partitions into membranes has been difficult to probe using molecular dynamics due to the limited time scale accessible. Here we develop a protocol that allows the spontaneous assembly of a polypeptide toxin into membranes in atomistic molecular dynamics simulations of tens of nanoseconds. The protocol is applied to GsMTx4 and HpTx2. Both toxins, released in water at the start of the simulation, spontaneously bind into the lipid bilayer within 50 ns, with their hydrophobic patch penetrated into the bilayer beyond the phosphate groups of the lipids. It is found that the bilayer is about 2 Å thinner upon the binding of a GsMTx4 monomer. Such a thinning effect of GsMTx4 on membranes may explain its potentiation effect on gramicidin and mechanosensitive channels.

  13. Carisbamate, a novel neuromodulator, inhibits voltage-gated sodium channels and action potential firing of rat hippocampal neurons.

    Science.gov (United States)

    Liu, Yi; Yohrling, George J; Wang, Yan; Hutchinson, Tasha L; Brenneman, Douglas E; Flores, Christopher M; Zhao, Boyu

    2009-01-01

    Carisbamate (RWJ-333369; (S)-2-O-carbamoyl-1-o-chlorophenyl-ethanol) is a novel investigational antiepileptic drug that exhibits a broad-spectrum of activity in a number of animal models of seizure and drug refractory epilepsy. In an effort to understand the molecular mechanism by which carisbamate produces its antiepileptic actions, we studied its effects on the function of voltage-gated, rat brain sodium and potassium channels and on the repetitive firing of action potentials in cultured rat hippocampal neurons. In whole-cell patch clamp recording, carisbamate resulted in a concentration-, voltage- and use-dependent inhibition of rat Nav1.2, with an IC(50) value of 68 microM at -67 mV. In rat hippocampal neurons, carisbamate similarly blocked voltage-gated sodium channels, with an IC(50) value of 89 microM at -67 mV, and inhibited repetitive firing of action potentials in a concentration-dependent manner (by 46% at 30 microM and 87% at 100 microM, respectively). Carisbamate had no effect on the steady-state membrane potential or voltage-gated potassium channels (K(v)) in these neurons. These inhibitory effects of carisbamate occurred at therapeutically relevant concentrations in vivo, raising the possibility that block of voltage-gated sodium channels by carisbamate contributes to its antiepileptic activity.

  14. Effect of gating modifier toxins on membrane thickness: implications for toxin effect on gramicidin and mechanosensitive channels.

    Science.gov (United States)

    Chen, Rong; Chung, Shin-Ho

    2013-02-22

    Various gating modifier toxins partition into membranes and interfere with the gating mechanisms of biological ion channels. For example, GsMTx4 potentiates gramicidin and several bacterial mechanosensitive channels whose gating kinetics are sensitive to mechanical properties of the membrane, whereas binding of HpTx2 shifts the voltage-activity curve of the voltage-gated potassium channel Kv4.2 to the right. The detailed process by which the toxin partitions into membranes has been difficult to probe using molecular dynamics due to the limited time scale accessible. Here we develop a protocol that allows the spontaneous assembly of a polypeptide toxin into membranes in atomistic molecular dynamics simulations of tens of nanoseconds. The protocol is applied to GsMTx4 and HpTx2. Both toxins, released in water at the start of the simulation, spontaneously bind into the lipid bilayer within 50 ns, with their hydrophobic patch penetrated into the bilayer beyond the phosphate groups of the lipids. It is found that the bilayer is about 2 Å thinner upon the binding of a GsMTx4 monomer. Such a thinning effect of GsMTx4 on membranes may explain its potentiation effect on gramicidin and mechanosensitive channels.

  15. A new RF trench-gate multi-channel laterally-diffused MOSFET on InGaAs

    Science.gov (United States)

    Payal, M.; Singh, Y.

    2017-09-01

    In this work, a new RF power trench-gate multi-channel laterally-diffused MOSFET (TGMC-LDMOS) on InGaAs is proposed. The gate-electrodes of the new structure are placed vertically in the trenches built in the drift layer. Each gate results in the formation of two channels in the p-body region of the device. The drain metal is also placed in a trench to take contact from the n+-InGaAs region located over the substrate. In a cell length of 5 {{μ }}{{m}}, the TGMC-LDMOS structure has seven channels, which conduct simultaneously to carry drain current in parallel. The formation of multi-channels in the proposed device increases the drive current ({I}{{D}}) leading to a large reduction in the specific on-resistance ({R}{{on-sp}}). Due to better control of gates on the drain current, the new structure exhibits substantially higher transconductance ({g}{{m}}) resulting in significant improvement in cut-off frequency ({f}{{T}}) and oscillation frequency ({f}\\max ). Using two-dimensional numerical simulations, a 55 V TGMC-LDMOS is demonstrated to achieve 7 times higher {I}{{D}}, 6.2 times lower {R}{{on-sp}}, 6.3 times higher peak {g}{{m}}, 2.6 times higher {f}{{T}}, and 2.5 times increase in {f}\\max in comparison to a conventional device for the identical cell length.

  16. Hyperpolarization-activated cyclic nucleotide-gated channels in peripheral diaphragmatic lymphatics.

    Science.gov (United States)

    Negrini, Daniela; Marcozzi, Cristiana; Solari, Eleonora; Bossi, Elena; Cinquetti, Raffaella; Reguzzoni, Marcella; Moriondo, Andrea

    2016-10-01

    Diaphragmatic lymphatic function is mainly sustained by pressure changes in the tissue and serosal cavities during cardiorespiratory cycles. The most peripheral diaphragmatic lymphatics are equipped with muscle cells (LMCs), which exhibit spontaneous contraction, whose molecular machinery is still undetermined. Hypothesizing that spontaneous contraction might involve hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in lymphatic LMCs, diaphragmatic specimens, including spontaneously contracting lymphatics, were excised from 33 anesthetized rats, moved to a perfusion chamber containing HEPES-Tyrode's solution, and treated with HCN channels inhibitors cesium chloride (CsCl), ivabradine, and ZD-7288. Compared with control, exposure to 10 mM CsCl reduced (-65%, n = 13, P < 0.01) the contraction frequency (FL) and increased end-diastolic diameter (DL-d, +7.3%, P < 0.01) without changes in end-systolic diameter (DL-s). Ivabradine (300 μM) abolished contraction and increased DL-d (-14%, n = 10, P < 0.01) or caused an incomplete inhibition of FL (n = 3, P < 0.01), leaving DL-d and DL-s unaltered. ZD-7288 (200 μM) completely (n = 12, P < 0.01) abolished FL, while DL-d decreased to 90.9 ± 2.7% of control. HCN gene expression and immunostaining confirmed the presence of HCN1-4 channel isoforms, likely arranged in different configurations, in LMCs. Hence, all together, data suggest that HCN channels might play an important role in affecting contraction frequency of LMCs. Copyright © 2016 the American Physiological Society.

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

    Directory of Open Access Journals (Sweden)

    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

  18. The pharmacology of voltage-gated sodium channels in sensory neurones.

    Science.gov (United States)

    Docherty, Reginald J; Farmer, Clare E

    2009-01-01

    Voltage-gated sodium channels (VGSCs) are vital for the normal functioning of most excitable cells. At least nine distinct functional subtypes of VGSCs are recognized, corresponding to nine genes for their pore-forming alpha-subunits. These have different developmental expression patterns, different tissue distributions in the adult and are differentially regulated at the cellular level by receptor-coupled cell signalling systems. Unsurprisingly, VGSC blockers are found to be useful as drugs in diverse clinical applications where excessive excitability of tissue leads to pathological dysfunction, e.g. epilepsy or cardiac tachyarrhythmias. The effects of most clinically useful VGSC blockers are use-dependent, i.e. their efficacy depends on channel activity. In addition, many natural toxins have been discovered that interact with VGSCs in complex ways and they have been used as experimental probes to study the structure and function of the channels and to better understand how drugs interact with the channels. Here we have attempted to summarize the properties of VGSCs in sensory neurones, discuss how they are regulated by cell signalling systems and we have considered briefly current concepts of their physiological function. We discuss in detail how drugs and toxins interact with archetypal VGSCs and where possible consider how they act on VGSCs in peripheral sensory neurones. Increasingly, drugs that block VGSCs are being used as systemic analgesic agents in chronic pain syndromes, but the full potential for VGSC blockers in this indication is yet to be realized and other applications in sensory dysfunction are also possible. Drugs targeting VGSC subtypes in sensory neurones are likely to provide novel systemic analgesics that are tissue-specific and perhaps even disease-specific, providing much-needed novel therapeutic approaches for the relief of chronic pain.

  19. Effect of propionyl-L-carnitine on L-type calcium channels in human heart sarcolemma

    Energy Technology Data Exchange (ETDEWEB)

    Bevilacqua, M.; Vago, T.; Norbiato, G. (Servizio di Endocrinologia, Milano, (Italy))

    1991-02-01

    Propionyl-L-carnitine (PC) protects perfused rat hearts against damage by ischemia-reperfusion. Activation of L-type calcium channel play a role on ischemia-reperfusion damage. Therefore, we studied the effect of PC on some properties of L-type calcium channels in an in vitro preparation from human myocardium sarcolemma (from patients with idiopathic dilated cardiomyopathy). Binding of the L-type calcium channel blockers isradipine ({sup 3}H)-PN 200-110 (PN) to plasma membrane preparations revealed a single population of binding sites (total number: Bmax = 213 +/- 34 fM/mg protein and affinity: Kd = 152 +/- 19 nM; n = 6). The characteristics of these binding sites were evaluated in the presence and in the absence of Ca{sup 2}{sup +} and of calcium blockers (D-888, a verapamillike drug, and diltiazem). Incubation in a Ca{sup 2}{sup +}-containing buffer increased the affinity of PN binding sites. Binding sites for PN were modulated by organic calcium channel blockers; in competition isotherms at 37{degree}C, D-888 (desmethoxyverapamil) decreased the PN binding, whereas diltiazem increased it. These results strongly suggest that the site labelled by PN is the voltage-operated calcium channel of the human myocardium. The addition of PC (1 mM) to plasma membranes labelled with PN at 37{degree}C decreased the affinity of the binding; this effect was counteracted by the addition of Ca{sup 2}{sup +} to the medium. This result was consistent with a competition between Ca{sup 2}{sup +} and PC. The effect of PC incubation at 4{degree}C was the opposite; at this temperature PC increased the affinity of the binding sites and the effect was obscured by Ca{sup 2}{sup +}.

  20. Identifying Calcium Channels and Porters in Plant Membranes

    Energy Technology Data Exchange (ETDEWEB)

    Sze, Heven

    1998-04-01

    The overall objectives of the proposal submitted in 6/90 was to understand how Ca was transported across plant membranes, and how these transport pathways were regulated. Ca participates in many cellular processes, including the transduction of hormonal and environmental signals, secretion, and protein folding. These processes depend on the coordination of passive Ca fluxes via channels and active Ca pumps; however these transport pathways are poorly understood in plants. We had, therefore, proposed to identify and characterize Ca transport proteins, such as the inositol-1 ,4,5-trisphosphate (IP3)-sensitive Ca channels and Ca pumps. We have had difficulties characterizing and cloning the IP3-sensitive Ca channel, but have made considerable progress on the biochemical characterization, and partial purification of a 120 kD Ca-pumping ATPase. We have begun to determine the structure of Ca pumps by molecular cloning and have already obtained a partial cDNA with features characteristic of Ca pumps.

  1. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex.

    Directory of Open Access Journals (Sweden)

    Weiping Zhang

    Full Text Available Calcium-activated chloride channels of the anoctamin (alias TMEM16 protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. G protein-gated inwardly rectifying potassium channel subunits 1 and 2 are down-regulated in rat dorsal root ganglion neurons and spinal cord after peripheral axotomy.

    Science.gov (United States)

    Lyu, Chuang; Mulder, Jan; Barde, Swapnali; Sahlholm, Kristoffer; Zeberg, Hugo; Nilsson, Johanna; Århem, Peter; Hökfelt, Tomas; Fried, Kaj; Shi, Tie-Jun Sten

    2015-07-22

    Increased nociceptive neuronal excitability underlies chronic pain conditions. Various ion channels, including sodium, calcium and potassium channels have pivotal roles in the control of neuronal excitability. The members of the family of G protein-gated inwardly rectifying potassium (GIRK) channels, GIRK1-4, have been implicated in modulating excitability. Here, we investigated the expression and distribution of GIRK1 and GIRK2 in normal and injured dorsal root ganglia (DRGs) and spinal cord of rats. We found that ~70% of the DRG neurons expressed GIRK1, while only <10% expressed GIRK2. The neurochemical profiles of GIRK1- and GIRK2-immunoreactive neurons were characterized using the neuronal markers calcitonin gene-related peptide, isolectin-B4 and neurofilament-200, and the calcium-binding proteins calbindin D28k, calretinin, parvalbumin and secretagogin. Both GIRK subunits were expressed in DRG neurons with nociceptive characteristics. However, while GIRK1 was widely expressed in several sensory neuronal subtypes, GIRK2 was detected mainly in a group of small C-fiber neurons. In the spinal dorsal horn, GIRK1- and -2-positive cell bodies and processes were mainly observed in lamina II, but also in superficial and deeper layers. Abundant GIRK1-, but not GIRK2-like immunoreactivity, was found in the ventral horn (laminae VI-X). Fourteen days after axotomy, GIRK1 and GIRK2 were down-regulated in DRG neurons at the mRNA and protein levels. Both after axotomy and rhizotomy there was a reduction of GIRK1- and -2-positive processes in the dorsal horn, suggesting a presynaptic localization of these potassium channels. Furthermore, nerve ligation caused accumulation of both subunits on both sides of the lesion, providing evidence for anterograde and retrograde fast axonal transport. Our data support the hypothesis that reduced GIRK function is associated with increased neuronal excitability and causes sensory disturbances in post-injury conditions, including neuropathic

  4. Mutations in voltage-gated potassium channel KCNC3 cause degenerative and developmental central nervous system phenotypes.

    Science.gov (United States)

    Waters, Michael F; Minassian, Natali A; Stevanin, Giovanni; Figueroa, Karla P; Bannister, John P A; Nolte, Dagmar; Mock, Allan F; Evidente, Virgilio Gerald H; Fee, Dominic B; Müller, Ulrich; Dürr, Alexandra; Brice, Alexis; Papazian, Diane M; Pulst, Stefan M

    2006-04-01

    Potassium channel mutations have been described in episodic neurological diseases. We report that K+ channel mutations cause disease phenotypes with neurodevelopmental and neurodegenerative features. In a Filipino adult-onset ataxia pedigree, the causative gene maps to 19q13, overlapping the SCA13 disease locus described in a French pedigree with childhood-onset ataxia and cognitive delay. This region contains KCNC3 (also known as Kv3.3), encoding a voltage-gated Shaw channel with enriched cerebellar expression. Sequencing revealed two missense mutations, both of which alter KCNC3 function in Xenopus laevis expression systems. KCNC3(R420H), located in the voltage-sensing domain, had no channel activity when expressed alone and had a dominant-negative effect when co-expressed with the wild-type channel. KCNC3(F448L) shifted the activation curve in the negative direction and slowed channel closing. Thus, KCNC3(R420H) and KCNC3(F448L) are expected to change the output characteristics of fast-spiking cerebellar neurons, in which KCNC channels confer capacity for high-frequency firing. Our results establish a role for KCNC3 in phenotypes ranging from developmental disorders to adult-onset neurodegeneration and suggest voltage-gated K+ channels as candidates for additional neurodegenerative diseases.

  5. Calcium channels are involved in EphB/ephrinB reverse signaling‑induced apoptosis in a rat chronic ocular hypertension model.

    Science.gov (United States)

    Dong, Lingdan; Cheng, Xianglin; Zhou, Long; Hu, Yanhong

    2018-02-01

    Erythropoietin-producing hepatocyte receptor B (EphB)/ephrinB reverse signaling has been revealed to be activated in chronic ocular hypertension (COH) by increasing the apoptosis of retinal ganglion cells (RGCs). However, the exact mechanism is not well understood. The present study investigated the involvement of Ca2+ channels in the apoptosis of RGCs induced by EphB/ephrinB reverse signaling in a rat CHO model, which was established by cauterizing 3 out of the 4 episcleral veins. The expression levels of four voltage‑gated Ca2+ channel subunits (Cav3.1‑3.3 and Cav1.2) were detected using immunofluorescence and western blot analysis. TUNEL staining was performed to assess RGC apoptosis following an injection with the T type Ca2+ channel blocker. Ca2+ channels, mainly the T type, were upregulated in COH rat retinas when compared with the sham group (P<0.01). Additionally, the Cav3.2 subunit of T type calcium channels was predominantly expressed in Müller cells and RGCs, such as ephrinB2. Furthermore, an intravitreal injection of the Ca2+ channel blocker Mibefradil (3 µM) reduced EphB2‑fragment crystallizable region‑induced RGC apoptosis in normal rats. Thus, the results suggest that Ca2+ channels in a COH model may be a pathway involved in ephrinB/EphB signaling‑induced RGC apoptosis.

  6. T-type Ca(2+) channels and Autoregulation of Local Blood Flow

    DEFF Research Database (Denmark)

    Jensen, Lars Jørn; Nielsen, Morten Schak; Salomonsson, Max

    2017-01-01

    L-type voltage gated Ca(2+) channels are considered to be the primary source of calcium influx during the myogenic response. However, many vascular beds also express T-type voltage gated Ca(2+) channels. Recent studies suggest that these channels may also play a role in autoregulation. At low...

  7. The mechano‐gated channel inhibitor GsMTx4 reduces the exercise pressor reflex in decerebrate rats

    Science.gov (United States)

    Kim, Joyce S.; Ruiz‐Velasco, Victor; Kaufman, Marc P.

    2016-01-01

    Key points Mechanical and metabolic stimuli from contracting muscles evoke reflex increases in blood pressure, heart rate and sympathetic nerve activity. Little is known, however, about the nature of the mechano‐gated channels on the thin fibre muscle afferents that contribute to evoke this reflex, termed the exercise pressor reflex.We determined the effect of GsMTx4, an inh