WorldWideScience

Sample records for voltage dependent channel

  1. 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.

  2. Pharmacology of the human cell voltage-dependent cation channel. Part II: inactivation and blocking

    DEFF Research Database (Denmark)

    Bennekou, Poul; Barksmann, Trine L.; Kristensen, Berit I.

    2004-01-01

    Human red cells; Nonselective voltage-dependent cation channel; NSVDC channel; Thiol group reagents......Human red cells; Nonselective voltage-dependent cation channel; NSVDC channel; Thiol group reagents...

  3. Conductance hysteresis in the voltage-dependent anion channel.

    Science.gov (United States)

    Rappaport, Shay M; Teijido, Oscar; Hoogerheide, David P; Rostovtseva, Tatiana K; Berezhkovskii, Alexander M; Bezrukov, Sergey M

    2015-09-01

    Hysteresis in the conductance of voltage-sensitive ion channels is observed when the transmembrane voltage is periodically varied with time. Although this phenomenon has been used in studies of gating of the voltage-dependent anion channel, VDAC, from the outer mitochondrial membrane for nearly four decades, full hysteresis curves have never been reported, because the focus was solely on the channel opening branches of the hysteresis loops. We studied the hysteretic response of a multichannel VDAC system to a triangular voltage ramp the frequency of which was varied over three orders of magnitude, from 0.5 mHz to 0.2 Hz. We found that in this wide frequency range the area encircled by the hysteresis curves changes by less than a factor of three, suggesting broad distribution of the characteristic times and strongly non-equilibrium behavior. At the same time, quasi-equilibrium two-state behavior is observed for hysteresis branches corresponding to VDAC opening. This enables calculation of the usual equilibrium gating parameters, gating charge and voltage of equipartitioning, which were found to be almost insensitive to the ramp frequency. To rationalize this peculiarity, we hypothesize that during voltage-induced closure and opening the system explores different regions of the complex free energy landscape, and, in the opening branch, follows quasi-equilibrium paths.

  4. Pharmacology of the human red cell voltage-dependent cation channel Part I. Activation by clotrimazole and analogues

    DEFF Research Database (Denmark)

    Barksmann, Trine Lyberth; Kristensen, Berit I.; Christophersen, Palle.

    2004-01-01

    Human red cells, Nonselective voltage dependent cation channel, NSVDC channel, Gárdos channel blockers, NSVDC channel activators......Human red cells, Nonselective voltage dependent cation channel, NSVDC channel, Gárdos channel blockers, NSVDC channel activators...

  5. [Role of voltage-dependent ion channels in epileptogenesis].

    Science.gov (United States)

    Ricard-Mousnier, B; Couraud, F

    1993-10-01

    The aim of this review is to gather information in favour of the involvement of voltage-dependent ion channels in epileptogenesis. Although, up to now, no study has shown that epilepsy is accompanied by a modification in the activity to these channels, the recently acquired knowledge of their physiology allows to presume would favor their involvement in epileptogenesis. The results from electrophysiological studies are as follows: a persistent sodium current increases neuronal excitability whereas potassium currents have an inhibitory role. In particular, calcium-dependent potassium current are involved in the post-hyperpolarization phases which follows PDS. Calcium currents are also involved in the genesis of the "bursting pacemaker" activity displayed by the neurons presumed to be inducers of the epileptic activity. Biochemical data has shown that as a consequence of epileptic activity, sodium and calcium channels are down regulated. This down-regulation could be a way to reduces neuronal hyperexcitability. Pharmacological data demonstrate the drugs which activate calcium channels or which inhibit potassium channels have a convusilvant effect. On the contrary, agents which block calcium or sodium channels or which properties. Among the latter ones, some antiepileptic drugs can be found. In summary situations which lead to increase in calcium and sodium currents and/or to an inhibition in potassium currents are potentially epileptogenic.

  6. A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion.

    Science.gov (United States)

    Grefen, Christopher; Karnik, Rucha; Larson, Emily; Lefoulon, Cécile; Wang, Yizhou; Waghmare, Sakharam; Zhang, Ben; Hills, Adrian; Blatt, Michael R

    2015-01-01

    Growth in plants depends on ion transport for osmotic solute uptake and secretory membrane trafficking to deliver material for wall remodelling and cell expansion. The coordination of these processes lies at the heart of the question, unresolved for more than a century, of how plants regulate cell volume and turgor. Here we report that the SNARE protein SYP121 (SYR1/PEN1), which mediates vesicle fusion at the Arabidopsis plasma membrane, binds the voltage sensor domains (VSDs) of K(+) channels to confer a voltage dependence on secretory traffic in parallel with K(+) uptake. VSD binding enhances secretion in vivo subject to voltage, and mutations affecting VSD conformation alter binding and secretion in parallel with channel gating, net K(+) concentration, osmotic content and growth. These results demonstrate a new and unexpected mechanism for secretory control, in which a subset of plant SNAREs commandeer K(+) channel VSDs to coordinate membrane trafficking with K(+) uptake for growth.

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

    Directory of Open Access Journals (Sweden)

    Rajeev Gupta

    2017-06-01

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

  8. G Protein-induced Trafficking of Voltage-dependent Calcium Channels

    National Research Council Canada - National Science Library

    Eugene Tombler; Nory Jun Cabanilla; Paul Carman; Natasha Permaul; John J. Hall; Ryan W. Richman; Jessica Lee; Jennifer Rodriguez; Dan P. Felsenfeld; Robert F. Hennigan; María A. Diversé-Pierluissi

    2006-01-01

    .... Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane...

  9. State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels

    OpenAIRE

    Miranda, Pablo; Contreras, Jorge E.; Plested, Andrew J. R.; Sigworth, Fred J.; Holmgren, Miguel; Giraldez, Teresa

    2013-01-01

    Large-conductance voltage- and calcium-dependent potassium channels (BK, “Big K+”) are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a “gating-ring” structure has been proposed to transduce Ca2+ binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductan...

  10. The human red cell voltage-dependent cation channel. Part III: Distribution homogeneity and pH dependence

    DEFF Research Database (Denmark)

    Bennekou, P.; Barksmann, T. L.; Christophersen, P.

    2006-01-01

    The homogeneity of the distribution of the non-selective voltage-dependent cation channel (the NSVDC channel) in the human erythrocyte, and the pH dependence was investigated. Activation of this channel caused a uniform cellular dehydration, which was characterized by the changes in the erythrocyte...

  11. Voltage-dependent Ca2+ channel and Na+ channel in frog taste cells.

    Science.gov (United States)

    Kashiwayanagi, M; Miyake, M; Kurihara, K

    1983-01-01

    Frog taste cells were hyperpolarized by injecting an inward current pulse, and regenerative anode-break potentials were observed at the termination of the current pulse. The results obtained are as follows. 1) The magnitude of the anode-break potentials increased with the extent of hyperpolarization of taste cells and reached a saturation level around -200 mV. 2) The magnitudes of the anode-break potentials observed in 80 different taste cells hyperpolarized to about -200 mV were distributed widely from cell to cell. The average magnitude was 39 mV. 3) The anode-break potentials were recorded after the lingual artery was perfused with artificial solutions containing various channel blockers. The results indicated that the anode-break potentials are composed of Na+ and Ca2+ components. 4) The slope of the current-voltage relation obtained with cells hyperpolarized to 100 mV was appreciably decreased above -50 mV by application of tetrodotoxin to the perfusing solution. Discussion was made on possible roles of the voltage-dependent Na+ and Ca2+ channels in the electrotonic spreading of the depolarization at the receptor membranes to the synaptic area and in releasing a chemical transmitter.

  12. Voltage-dependent metabolic regulation of Kv2.1 channels in pancreatic beta-cells.

    Science.gov (United States)

    Yoshida, Masashi; Nakata, Masanori; Yamato, Shiho; Dezaki, Katsuya; Sugawara, Hitoshi; Ishikawa, San-e; Kawakami, Masanobu; Yada, Toshihiko; Kakei, Masafumi

    2010-05-28

    Voltage-gated potassium channels (Kv channels) play a crucial role in formation of action potentials in response to glucose stimulation in pancreatic beta-ells. We previously reported that the Kv channel is regulated by glucose metabolism, particularly by MgATP. We examined whether the regulation of Kv channels is voltage-dependent and mechanistically related with phosphorylation of the channels. In rat pancreatic beta-cells, suppression of glucose metabolism with low glucose concentrations of 2.8mM or less or by metabolic inhibitors decreased the Kv2.1-channel activity at positive membrane potentials, while increased it at potentials negative to -10 mV, suggesting that modulation of Kv channels by glucose metabolism is voltage-dependent. Similarly, in HEK293 cells expressing the recombinant Kv2.1 channels, 0mM but not 10mM MgATP modulated the channel activity in a manner similar to that in beta-cells. Both steady-state activation and inactivation kinetics of the channel were shifted toward the negative potential in association with the voltage-dependent modulation of the channels by cytosolic dialysis of alkaline phosphatase in beta-cells. The modulation of Kv-channel current-voltage relations were also observed during and after glucose-stimulated electrical excitation. These results suggest that the cellular metabolism including MgATP production and/or channel phosphorylation/dephosphorylation underlie the physiological modulation of Kv2.1 channels during glucose-induced insulin secretion.

  13. The Mechanism of Voltage Dependent Gating of the NaChBac Prokaryotic Sodium Channel

    Science.gov (United States)

    Decaen, Paul G.

    Electrical signaling in cells depends on selective conductance of ions through membrane proteins called 'voltage gated ion channels'. These channels are characterized by their ability turn on and off the flow of ionic current by opening and closing their conductive pore in response to changes in membrane potential. The opening and closing of the pore is a mechanically linked to conformational movement of the positively charged fourth transmembrane segment (S4) in 'the voltage sensor' region. How the S4 moves in response to membrane potential is a controversial subject. In this thesis, we used the prokaryotic sodium channel NaChBac as our model sodium channel to study voltage dependent movement of the S4 in the voltage sensor. We use a disulfide-locking method where we introduced pairs of cysteines in the voltage sensor that crosslink and trap the S4 in its path after depolarization. We screened over one hundred mutations of the NaChBac channel in the whole cell patch clamp assay and demonstrated discrete and sequential voltage dependent ion pair interactions that occur in at least three states between the positively charged residues of the S4 segment and the acidic residues in the S1, S2 and S3 segments. In conjunction with structural modeling of the voltage sensor and our disulfide locking data, we propose that the S4 moves in and out of the plane of the membrane 8-13 A, forming distinct gating charge interactions with counter charges of the voltage sensor and adopts a 310 helix over a portion of its structure during activation. These findings are compatible with the sliding helix model and refine our understanding of the structural determinates of voltage sensor function in voltage gated ion channels.

  14. Voltage-dependent calcium channels from brain incorporated into planar lipid bilayers

    Science.gov (United States)

    Nelson, Mark T.; French, Robert J.; Krueger, Bruce K.

    1984-03-01

    Many important physiological processes, including neurotransmitter release and muscle contraction1-3, are regulated by the concentration of Ca2+ ions in the cell. Levels of cytoplasmic Ca2+ can be elevated by the entry of Ca2+ ions through voltage-dependent channels which are selective for Ca2+, Ba2+ and Sr2+ ions4-14. We have measured currents through single, voltage-dependent calcium channels from rat brain that have been incorporated into planar lipid bilayers. Channel gating was voltage-dependent: membrane depolarization increased the channel open times and decreased the closed times. The channels were selective for divalent cations over monovalent ions. The well-known calcium channel blockers, lanthanum and cadmium, produced a concentration-dependent reduction of the apparent single-channel conductance. Contrary to expectations14, the nature of the divalent cation carrying current through the channel affected not only the single-channel conductance, but also the channel open times, with mean open times being shortest for barium.

  15. Novel expression and regulation of voltage-dependent potassium channels in placentas from women with preeclampsia.

    Science.gov (United States)

    Mistry, Hiten D; McCallum, Laura A; Kurlak, Lesia O; Greenwood, Iain A; Broughton Pipkin, Fiona; Tribe, Rachel M

    2011-09-01

    Preeclampsia is associated with structural/functional alterations in placental and maternal vasculature. Voltage-dependant potassium channels encoded by KCNQ1-5 genes have been detected in several types of blood vessels where they promote vascular relaxation. Voltage-dependant potassium channel function can be modulated by KCNE1-5-encoded accessory proteins. The aim of this study was to determine whether KCNQ and KCNE genes are differentially expressed in placentas from women with preeclampsia compared with normotensive controls and to examine any differences in those who delivered preterm (voltage-dependant potassium channels are expressed and markedly modulated in placentas from preeclamptic women. Differential expression of isoforms may lead to altered cell proliferation. The correlation between KCNQ3 and KCNE5 expression is indicative of a novel channel complex and warrants further investigation.

  16. Endocytic regulation of voltage-dependent potassium channels in the heart.

    Science.gov (United States)

    Ishii, Kuniaki; Norota, Ikuo; Obara, Yutaro

    2012-01-01

    Understanding the regulation of cardiac ion channels is critical for the prevention of arrhythmia caused by abnormal excitability. Ion channels can be regulated by a change in function (qualitative) and a change in number (quantitative). Functional changes have been extensively investigated for many ion channels including cardiac voltage-dependent potassium channels. By contrast, the regulation of ion channel numbers has not been widely examined, particularly with respect to acute modulation of ion channels. This article briefly summarizes stimulus-induced endocytic regulation of major voltage-dependent potassium channels in the heart. The stimuli known to cause their endocytosis include receptor activation, drugs, and low extracellular [K(+)], following which the potassium channels undergo either clathrin-mediated or caveolin-mediated endocytosis. Receptor-mediated endocytic regulation has been demonstrated for Kv1.2, Kv1.5, KCNQ1 (Kv7.1), and Kv4.3, while drug-induced endocytosis has been demonstrated for Kv1.5 and hERG. Low [K(+)](o)-induced endocytosis might be unique for hERG channels, whose electrophysiological characteristics are known to be under strong influence of [K(+)](o). Although the precise mechanisms have not been elucidated, it is obvious that major cardiac voltage-dependent potassium channels are modulated by endocytosis, which leads to changes in cardiac excitability.

  17. 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...

  18. Voltage dependence of Hodgkin-Huxley rate functions for a multistage K+ channel voltage sensor within a membrane

    Science.gov (United States)

    Vaccaro, S. R.

    2014-11-01

    The activation of a K+channel sensor in two sequential stages during a voltage clamp may be described as the translocation of a Brownian particle in an energy landscape with two large barriers between states. A solution of the Smoluchowski equation for a square-well approximation to the potential function of the S4 voltage sensor satisfies a master equation and has two frequencies that may be determined from the forward and backward rate functions. When the higher-frequency terms have small amplitude, the solution reduces to the relaxation of a rate equation, where the derived two-state rate functions are dependent on the relative magnitude of the forward rates (α and γ ) and the backward rates (β and δ ) for each stage. In particular, the voltage dependence of the Hodgkin-Huxley rate functions for a K+channel may be derived by assuming that the rate functions of the first stage are large relative to those of the second stage—α ≫γ and β ≫δ . For a Shaker IR K+ channel, the first forward and backward transitions are rate limiting (α <γ and δ ≪β ), and for an activation process with either two or three stages, the derived two-state rate functions also have a voltage dependence that is of a similar form to that determined for the squid axon. The potential variation generated by the interaction between a two-stage K+ ion channel and a noninactivating Na+ ion channel is determined by the master equation for K+channel activation and the ionic current equation when the Na+channel activation time is small, and if β ≪δ and α ≪γ , the system may exhibit a small amplitude oscillation between spikes, or mixed-mode oscillation, in which the slow closed state modulates the K+ ion channel conductance in the membrane.

  19. 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.

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

    Science.gov (United States)

    Zaydman, Mark A; Cui, Jianmin

    2014-01-01

    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.

  1. G protein-induced trafficking of voltage-dependent calcium channels.

    Science.gov (United States)

    Tombler, Eugene; Cabanilla, Nory Jun; Carman, Paul; Permaul, Natasha; Hall, John J; Richman, Ryan W; Lee, Jessica; Rodriguez, Jennifer; Felsenfeld, Dan P; Hennigan, Robert F; Diversé-Pierluissi, María A

    2006-01-20

    Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.

  2. Voltage dependence of Hodgkin-Huxley rate functions for a multistage K^{+} channel voltage sensor within a membrane.

    Science.gov (United States)

    Vaccaro, S R

    2014-11-01

    The activation of a K^{+} channel sensor in two sequential stages during a voltage clamp may be described as the translocation of a Brownian particle in an energy landscape with two large barriers between states. A solution of the Smoluchowski equation for a square-well approximation to the potential function of the S4 voltage sensor satisfies a master equation and has two frequencies that may be determined from the forward and backward rate functions. When the higher-frequency terms have small amplitude, the solution reduces to the relaxation of a rate equation, where the derived two-state rate functions are dependent on the relative magnitude of the forward rates (α and γ) and the backward rates (β and δ) for each stage. In particular, the voltage dependence of the Hodgkin-Huxley rate functions for a K^{+} channel may be derived by assuming that the rate functions of the first stage are large relative to those of the second stage-α≫γ and β≫δ. For a Shaker IR K^{+} channel, the first forward and backward transitions are rate limiting (αchannel and a noninactivating Na^{+} ion channel is determined by the master equation for K^{+} channel activation and the ionic current equation when the Na^{+} channel activation time is small, and if β≪δ and α≪γ, the system may exhibit a small amplitude oscillation between spikes, or mixed-mode oscillation, in which the slow closed state modulates the K^{+} ion channel conductance in the membrane.

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

    Directory of Open Access Journals (Sweden)

    Xiaohui eSun

    2012-04-01

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

  4. Proper Voltage-Dependent Ion Channel Function in Dysferlin-Deficient Cardiomyocytes.

    Science.gov (United States)

    Rubi, Lena; Gawali, Vaibhavkumar S; Kubista, Helmut; Todt, Hannes; Hilber, Karlheinz; Koenig, Xaver

    2015-01-01

    Dysferlin plays a decisive role in calcium-dependent membrane repair in myocytes. Mutations in the encoding DYSF gene cause a number of myopathies, e.g. limb-girdle muscular dystrophy type 2B (LGMD2B). Besides skeletal muscle degenerative processes, dysferlin deficiency is also associated with cardiac complications. Thus, both LGMD2B patients and dysferlin-deficient mice develop a dilated cardiomyopathy. We and others have recently reported that dystrophin-deficient ventricular cardiomyocytes from mouse models of Duchenne muscular dystrophy show significant abnormalities in voltage-dependent ion channels, which may contribute to the pathophysiology in dystrophic cardiomyopathy. The aim of the present study was to investigate if dysferlin, like dystrophin, is a regulator of cardiac ion channels. By using the whole cell patch-clamp technique, we compared the properties of voltage-dependent calcium and sodium channels, as well as action potentials in ventricular cardiomyocytes isolated from the hearts of normal and dysferlin-deficient (dysf) mice. In contrast to dystrophin deficiency, the lack of dysferlin did not impair the ion channel properties and left action potential parameters unaltered. In connection with normal ECGs in dysf mice these results suggest that dysferlin deficiency does not perturb cardiac electrophysiology. Our study demonstrates that dysferlin does not regulate cardiac voltage-dependent ion channels, and implies that abnormalities in cardiac ion channels are not a universal characteristic of all muscular dystrophy types. © 2015 S. Karger AG, Basel.

  5. Proper Voltage-Dependent Ion Channel Function in Dysferlin-Deficient Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Lena Rubi

    2015-06-01

    Full Text Available Background/Aims: Dysferlin plays a decisive role in calcium-dependent membrane repair in myocytes. Mutations in the encoding DYSF gene cause a number of myopathies, e.g. limb-girdle muscular dystrophy type 2B (LGMD2B. Besides skeletal muscle degenerative processes, dysferlin deficiency is also associated with cardiac complications. Thus, both LGMD2B patients and dysferlin-deficient mice develop a dilated cardiomyopathy. We and others have recently reported that dystrophin-deficient ventricular cardiomyocytes from mouse models of Duchenne muscular dystrophy show significant abnormalities in voltage-dependent ion channels, which may contribute to the pathophysiology in dystrophic cardiomyopathy. The aim of the present study was to investigate if dysferlin, like dystrophin, is a regulator of cardiac ion channels. Methods and Results: By using the whole cell patch-clamp technique, we compared the properties of voltage-dependent calcium and sodium channels, as well as action potentials in ventricular cardiomyocytes isolated from the hearts of normal and dysferlin-deficient (dysf mice. In contrast to dystrophin deficiency, the lack of dysferlin did not impair the ion channel properties and left action potential parameters unaltered. In connection with normal ECGs in dysf mice these results suggest that dysferlin deficiency does not perturb cardiac electrophysiology. Conclusion: Our study demonstrates that dysferlin does not regulate cardiac voltage-dependent ion channels, and implies that abnormalities in cardiac ion channels are not a universal characteristic of all muscular dystrophy types.

  6. Purification and Characterization of Two Voltage-Dependent Anion Channel Isoforms from Plant Seeds1

    Science.gov (United States)

    Abrecht, Helge; Wattiez, Ruddy; Ruysschaert, Jean-Marie; Homblé, Fabrice

    2000-01-01

    Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 m urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates. PMID:11080295

  7. KCNQ1 Channels Voltage Dependence through a Voltage-dependent Binding of the S4-S5 Linker to the Pore Domain*

    OpenAIRE

    2010-01-01

    Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1–S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5–S6) is surrounded by four voltage sensor domains (S1–S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5L) and S6 C terminus (S6T). From these data, we hypothesiz...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-07-29

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

  9. Voltage dependence of rate functions for Na+ channel inactivation within a membrane

    CERN Document Server

    Vaccaro, Samuel R

    2015-01-01

    The inactivation of a Na+ channel occurs when the activation of the charged S4 segment of domain IV, with rate functions $\\alpha_{i}$ and $\\beta_{i}$, is followed by the binding of an intracellular hydrophobic motif which blocks conduction through the ion pore, with rate functions $\\gamma_{i}$ and $\\delta_{i}$. During a voltage clamp of the Na+ channel, the solution of the master equation for inactivation reduces to the relaxation of a rate equation when the binding of the inactivation motif is rate limiting ($\\alpha_{i} \\gg \\gamma_{i}$ and $\\beta_{i} \\gg \\delta_{i}$). The voltage dependence of the derived forward rate function for Na+ channel inactivation has an exponential dependence on the membrane potential for small depolarizations and approaches a constant value for larger depolarizations, whereas the voltage dependence of the backward rate function is exponential, and each rate has a similar form to the Hodgkin-Huxley empirical rate functions for Na+ channel inactivation in the squid axon.

  10. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels.

    Science.gov (United States)

    Yagoda, Nicholas; von Rechenberg, Moritz; Zaganjor, Elma; Bauer, Andras J; Yang, Wan Seok; Fridman, Daniel J; Wolpaw, Adam J; Smukste, Inese; Peltier, John M; Boniface, J Jay; Smith, Richard; Lessnick, Stephen L; Sahasrabudhe, Sudhir; Stockwell, Brent R

    2007-06-14

    Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.

  11. A comparative study of the action of tolperisone on seven different voltage dependent sodium channel isoforms.

    Science.gov (United States)

    Hofer, Doris; Lohberger, Birgit; Steinecker, Bibiane; Schmidt, Kurt; Quasthoff, Stefan; Schreibmayer, Wolfgang

    2006-05-24

    The specific, acute interaction of tolperisone, an agent used as a muscle relaxant and for the treatment of chronic pain conditions, with the Na(v1.2), Na(v1.3), Na(v1.4), Na(v1.5), Na(v1.6), Na(v1.7), and Na(v1.8) isoforms of voltage dependent sodium channels was investigated and compared to that of lidocaine. Voltage dependent sodium channels were expressed in the Xenopus laevis oocyte expression system and sodium currents were recorded with the two electrode voltage clamp technique. Cumulative dose response relations revealed marked differences in IC(50) values between the two drugs on identical isoforms, as well as between isoforms. A detailed kinetic analysis uncovered that tolperisone as well as lidocaine exhibited their blocking action not only via state dependent association/dissociation with voltage dependent sodium channels, but a considerable fraction of inhibition is tonic, i.e. permanent and basic in nature. Voltage dependent activation was affected to a minor extent only. A shift in steady-state inactivation to more negative potentials could be observed for most drug/isoform combinations. The contribution of this shift to overall block was, however, small at drug concentrations resulting in considerable overall block. Recovery from inactivation was affected notably by both drugs. Lidocaine application led to a pronounced prolongation of the time constant of the fast recovery process for the Na(v1.3), Na(v1.5), and Na(v1.7) isoforms, indicating common structural properties in the local anesthetic receptor site of these three proteins. Interestingly, this characteristic drug action was not observed for tolperisone.

  12. Voltage-dependent K channels in protoplasts of trap-lobe cells of Dionaea muscipula.

    Science.gov (United States)

    Iijima, T; Hagiwara, S

    1987-01-01

    The outward rectification of the K+ current in mesophyll cell protoplasts from trap-lobes of Dionaea muscipula was studied with the patch-clamp technique. The rectification had instantaneous and time-dependent components. Changes in [K+]i strongly affected the conductance voltage relation of the plasma membrane while changes in [K+]o had little effect on the relation. Thus, the outward rectification depends on the membrane voltage and the concentration of intracellular K+. Corresponding single-channel activities were observed both in the intact membrane (cell-attached recording) and in excised patches. The single-channel conductance was about 3.3 pS with symmetrical solutions containing 30 mM K+.

  13. Lavender Oil-Potent Anxiolytic Properties via Modulating Voltage Dependent Calcium Channels

    OpenAIRE

    2013-01-01

    Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs) as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, bioc...

  14. The Eag domain regulates the voltage-dependent inactivation of rat Eag1 K+ channels.

    Directory of Open Access Journals (Sweden)

    Ting-Feng Lin

    Full Text Available Eag (Kv10 and Erg (Kv11 belong to two distinct subfamilies of the ether-à-go-go K+ channel family (KCNH. While Erg channels are characterized by an inward-rectifying current-voltage relationship that results from a C-type inactivation, mammalian Eag channels display little or no voltage-dependent inactivation. Although the amino (N-terminal region such as the eag domain is not required for the C-type inactivation of Erg channels, an N-terminal deletion in mouse Eag1 has been shown to produce a voltage-dependent inactivation. To further discern the role of the eag domain in the inactivation of Eag1 channels, we generated N-terminal chimeras between rat Eag (rEag1 and human Erg (hERG1 channels that involved swapping the eag domain alone or the complete cytoplasmic N-terminal region. Functional analyses indicated that introduction of the homologous hERG1 eag domain led to both a fast phase and a slow phase of channel inactivation in the rEag1 chimeras. By contrast, the inactivation features were retained in the reverse hERG1 chimeras. Furthermore, an eag domain-lacking rEag1 deletion mutant also showed the fast phase of inactivation that was notably attenuated upon co-expression with the rEag1 eag domain fragment, but not with the hERG1 eag domain fragment. Additionally, we have identified a point mutation in the S4-S5 linker region of rEag1 that resulted in a similar inactivation phenotype. Biophysical analyses of these mutant constructs suggested that the inactivation gating of rEag1 was distinctly different from that of hERG1. Overall, our findings are consistent with the notion that the eag domain plays a critical role in regulating the inactivation gating of rEag1. We propose that the eag domain may destabilize or mask an inherent voltage-dependent inactivation of rEag1 K+ channels.

  15. Eugenol dilates rat cerebral arteries by inhibiting smooth muscle cell voltage-dependent calcium channels.

    Science.gov (United States)

    Peixoto-Neves, Dieniffer; Leal-Cardoso, Jose Henrique; Jaggar, Jonathan H

    2014-11-01

    Plants high in eugenol, a phenylpropanoid compound, are used as folk medicines to alleviate diseases including hypertension. Eugenol has been demonstrated to relax conduit and ear arteries and reduce systemic blood pressure, but mechanisms involved are unclear. Here, we studied eugenol regulation of resistance-size cerebral arteries that control regional brain blood pressure and flow and investigated mechanisms involved. We demonstrate that eugenol dilates arteries constricted by either pressure or membrane depolarization (60 mM K) in a concentration-dependent manner. Experiments performed using patch-clamp electrophysiology demonstrated that eugenol inhibited voltage-dependent calcium (Ca) currents, when using Ba as a charge carrier, in isolated cerebral artery smooth muscle cells. Eugenol inhibition of voltage-dependent Ca currents involved pore block, a hyperpolarizing shift (∼-10 mV) in voltage-dependent inactivation, an increase in the proportion of steady-state inactivating current, and acceleration of inactivation rate. In summary, our data indicate that eugenol dilates cerebral arteries by means of multimodal inhibition of voltage-dependent Ca channels.

  16. The effects of S4 segments on the voltage-dependence of inactivation for Cav3.1 calcium channels

    Institute of Scientific and Technical Information of China (English)

    LI JunYing

    2007-01-01

    T-type calcium channels exhibit fast voltage-dependent inactivation,for which the underlying structure-function relationship still remains unclear.To investigate the roles of S4 segments in voltage-dependent inactivation of T-type calcium channels,we created S4 replacement chimeras between Cav3.1 calcium channels(fast voltage-dependent inactivation)and Cav1.2 calcium channels(little oltage-dependent inactivation)by replacing S4s in Cav3.1 with the corresponding regions in Cav1.2.Wild type and chimeric channels were expressed in Xenopus oocytes and channel currents were recorded with two-electrode voltage-clamp.We showed that replacing S4 region in domain I shifted voltage-dependence for inactivation of Cav3.1 to the left,and the V0.5 inact and kinact value were significantly changed.However replacing S4s in domains Ⅱ-Ⅳ had no effects on the voltage-dependent inactivation properties.These results suggest that the roles of S4 segments in domains Ⅰ-Ⅳ are different,and S4 in domain I is likely to be involved in voltage-dependent Inactivation process.Its movement during membrane depolarization may trigger a conformational change in the inactivation gate.

  17. Selective modulation of cellular voltage dependent calcium channels by hyperbaric pressure - a suggested HPNS partial mechanism

    Directory of Open Access Journals (Sweden)

    Ben eAviner

    2014-05-01

    Full Text Available Professional deep sea divers experience motor and cognitive impairment, known as High Pressure Neurological Syndrome (HPNS, when exposed to pressures of 100 msw (1.1MPa and above, considered to be the result of synaptic transmission alteration. Previous studies have indicated modulation of presynaptic Ca2+ currents at high pressure. We directly measured for the first time pressure effects on the currents of voltage dependent Ca2+ channels (VDCCs expressed in Xenopus oocytes. Pressure selectivity augmented the current in CaV1.2 and depressed it in CaV3.2 channels. Pressure application also affected the channels' kinetics, such as ƮRise, ƮDecay. Pressure modulation of VDCCs seems to play an important role in generation of HPNS signs and symptoms.

  18. Investigation of channel width-dependent threshold voltage variation in a-InGaZnO thin-film transistors

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Kuan-Hsien; Chou, Wu-Ching [Department of Electrophysics, National Chiao Tung University, Hsinchu, Taiwan (China); Chang, Ting-Chang, E-mail: tcchang@mail.phys.nsysu.edu.tw [Department of Physics, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Advanced Optoelectronics Technology Center, National Cheng Kung University, Taiwan (China); Wu, Ming-Siou; Hung, Yi-Syuan; Sze, Simon M. [Department of Electronics Engineering, National Chiao Tung University, Hsinchu, Taiwan (China); Hung, Pei-Hua; Chu, Ann-Kuo [Department of Photonics, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Hsieh, Tien-Yu [Department of Physics, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Yeh, Bo-Liang [Advanced Display Technology Research Center, AU Optronics, No. 1, Li-Hsin Rd. 2, Hsinchu Science Park, Hsinchu 30078, Taiwan (China)

    2014-03-31

    This Letter investigates abnormal channel width-dependent threshold voltage variation in amorphous indium-gallium-zinc-oxide (a-IGZO) thin-film transistors. Unlike drain-induced source barrier lowering effect, threshold voltage increases with increasing drain voltage. Furthermore, the wider the channel, the larger the threshold voltage observed. Because of the surrounding oxide and other thermal insulating material and the low thermal conductivity of the IGZO layer, the self-heating effect will be pronounced in wider channel devices and those with a larger operating drain bias. To further clarify the physical mechanism, fast IV measurement is utilized to demonstrate the self-heating induced anomalous channel width-dependent threshold voltage variation.

  19. Stochastic Dynamics of Electrical Membrane with Voltage-Dependent Ion Channel Fluctuations

    CERN Document Server

    Qian, Hong; Qian, Min

    2014-01-01

    Brownian ratchet like stochastic theory for the electrochemical membrane system of Hodgkin-Huxley (HH) is developed. The system is characterized by a continuous variable $Q_m(t)$, representing mobile membrane charge density, and a discrete variable $K_t$ representing ion channel conformational dynamics. A Nernst-Planck-Nyquist-Johnson type equilibrium is obtained when multiple conducting ions have a common reversal potential. Detailed balance yields a previously unknown relation between the channel switching rates and membrane capacitance, bypassing Eyring-type explicit treatment of gating charge kinetics. From a molecular structural standpoint, membrane charge $Q_m$ is a more natural dynamic variable than potential $V_m$; our formalism treats $Q_m$-dependent conformational transition rates $\\lambda_{ij}$ as intrinsic parameters. Therefore in principle, $\\lambda_{ij}$ vs. $V_m$ is experimental protocol dependent,e.g., different from voltage or charge clamping measurements. For constant membrane capacitance pe...

  20. Cortisone dissociates voltage-dependent K+ channel from its beta subunit

    Science.gov (United States)

    Pan, Yaping; Weng, Jun; Kabaleeswaran, Venkataraman; Li, Huiguang; Cao, Yu; Bhosle, Rahul C.; Zhou, Ming

    2009-01-01

    The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and essential for cellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked to episodic ataxia and atrial fibrillation. All Kv1 channels assemble with beta subunits (Kvβ) and certain Kvβs, for example Kvβ1, have an N-terminal segment that closes a channel by the N-type inactivation mechanism. In principle dissociation of Kvβ1, although never reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases mammalian (rat) Kv1 channel activity by binding to Kvβ1. A crystal structure of the Kvβ-cortisone complex was solved to 1.82 Å resolution and revealed novel cortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kvβ. The new mode of channel modulation may be explored by native or synthetic ligands to fine tune cellular excitability. PMID:18806782

  1. KCNQ1 channels voltage dependence through a voltage-dependent binding of the S4-S5 linker to the pore domain.

    Science.gov (United States)

    Choveau, Frank S; Rodriguez, Nicolas; Abderemane Ali, Fayal; Labro, Alain J; Rose, Thierry; Dahimène, Shehrazade; Boudin, Hélène; Le Hénaff, Carole; Escande, Denis; Snyders, Dirk J; Charpentier, Flavien; Mérot, Jean; Baró, Isabelle; Loussouarn, Gildas

    2011-01-07

    Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5(L)) and S6 C terminus (S6(T)). From these data, we hypothesized that S4S5(L) behaves like a ligand specifically interacting with S6(T) and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5(L) and S6(T) of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5(L) peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5(L) peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6(T), consistent with S4S5(L) binding to S6(T). Val(254) in S4S5(L) is known to contact Leu(353) in S6(T) when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5(L) binding to S6(T). Our results suggest a mechanistic model in which S4S5(L) acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5(L) away from S6(T), allowing channel opening.

  2. Reversal of HCN channel voltage dependence via bridging of the S4-S5 linker and Post-S6.

    Science.gov (United States)

    Prole, David L; Yellen, Gary

    2006-09-01

    Voltage-gated ion channels possess charged domains that move in response to changes in transmembrane voltage. How this movement is transduced into gating of the channel pore is largely unknown. Here we show directly that two functionally important regions of the spHCN1 pacemaker channel, the S4-S5 linker and the C-linker, come into close proximity during gating. Cross-linking these regions with high-affinity metal bridges or disulfide bridges dramatically alters channel gating in the absence of cAMP; after modification the polarity of voltage dependence is reversed. Instead of being closed at positive voltage and activating with hyperpolarization, modified channels are closed at negative voltage and activate with depolarization. Mechanistically, this reversal of voltage dependence occurs as a result of selectively eliminating channel deactivation, while retaining an existing inactivation process. Bridging also alters channel activation by cAMP, showing that interaction of these two regions can also affect the efficacy of physiological ligands.

  3. Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.

    Directory of Open Access Journals (Sweden)

    Dong-Hai Liu

    Full Text Available Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

  4. Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.

    Science.gov (United States)

    Liu, Dong-Hai; Huang, Xu; Guo, Xin; Meng, Xiang-Min; Wu, Yi-Song; Lu, Hong-Li; Zhang, Chun-Mei; Kim, Young-chul; Xu, Wen-Xie

    2014-01-01

    Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV) was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV) to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

  5. Functional unit size of the neurotoxin receptors on the voltage-dependent sodium channel.

    Science.gov (United States)

    Angelides, K J; Nutter, T J; Elmer, L W; Kempner, E S

    1985-03-25

    Radiation inactivation was used in situ to determine the functional unit sizes of the neurotoxin receptors of the voltage-dependent sodium channel from rat brain. Frozen or lyophilized synaptosomes were irradiated with high energy electrons generated by a linear accelerator and assayed for [3H]saxitoxin, 125I-Leiurus quinquestriatus quinquestriatus (alpha-scorpion toxin), 125I-Centruroides suffusus suffusus (beta-scorpion toxin), and batrachotoxinin-A 20 alpha-[3H]benzoate binding activity. The functional unit size of the neurotoxin receptors determined in situ by target analysis are 220,000 for saxitoxin, 263,000 for alpha-scorpion toxin, and 45,000 for beta-scorpion toxin. Analysis of the inactivation curve for batrachotoxinin-A 20 alpha-benzoate binding to the channel yields two target sizes of Mr approximately 287,000 (50%) and approximately 51,000 (50%). The results are independent of the purity of the membrane preparation. Comparison of the radiation inactivation data with the protein composition of the rat brain sodium channel indicates that there are at least two functional components.

  6. Over Expression of Voltage Dependent Anion Channel 2 (VDAC2 in Muscles of Electrically Stunned Chickens

    Directory of Open Access Journals (Sweden)

    Norshahida Abu Samah, Azura Amid, and Faridah Yusof

    2011-12-01

    Full Text Available Water bath stunning is a common practice in commercial slaughterhouses. Such treatment is economic and in line with animal welfare practice. However, the conditions applied for the stunning process may vary from a slaughterhouse to another slaughterhouse. Such a loose regulation on the stunning procedure has opened up doors for food adulteration such as over dose stunning. In this study, a simple and reliable approach using proteomics have been developed to study the effect of different currents and voltages in stunning on the protein expression of the chickens. Protein profiles of the chickens were constructed in order to detect any differences in protein expression and modifications. The different voltage studied were 10 V, 40 V and 70 V while the values for current studied were 0.25 A, 0.5 A, and 0.75 A. After the proteomics analyses using 2D Platinum ImageMaster 6.0 and Matrix-assisted laser desorption ionization- time of flight (MALDI TOF spectrometry identification, Voltage dependent anion channel 2 (VDAC2 was identified to be over expressed in the muscle sample of over stunned chicken. The over expression of VDAC2 was confirmed at the transcriptional level of RNA expression. Real Time PCR showed that all over stunned samples contained higher mRNA expression level for VDAC2 genes. The mRNA level of VDAC2 was up-regulated by 59.87 fold change when normalized with housekeeping gene. In conclusion, VDAC2 could serve as potential biomarkers for identification of electrically stimulated chickens. The existence of these biomarkers will help to monitor the slaughtering and stunning process in the future. It will revolutionize the food authentication field and give a new breathe to the meat industry.ABSTRAK: Kaedah "waterbath stunning" merupakan amalan biasa di pusat-pusat penyembelihan. Kaedah ini adalah ekonomik dan selari dengan amalan kebajikan haiwan. Walaubagaimanapun, syarat-syarat yang digunakan untuk proses kejutan tersebut mungkin

  7. Regulation of mitochondrial function by voltage dependent anion channels in ethanol metabolism and the Warburg effect.

    Science.gov (United States)

    Lemasters, John J; Holmuhamedov, Ekhson L; Czerny, Christoph; Zhong, Zhi; Maldonado, Eduardo N

    2012-06-01

    Voltage dependent anion channels (VDAC) are highly conserved proteins that are responsible for permeability of the mitochondrial outer membrane to hydrophilic metabolites like ATP, ADP and respiratory substrates. Although previously assumed to remain open, VDAC closure is emerging as an important mechanism for regulation of global mitochondrial metabolism in apoptotic cells and also in cells that are not dying. During hepatic ethanol oxidation to acetaldehyde, VDAC closure suppresses exchange of mitochondrial metabolites, resulting in inhibition of ureagenesis. In vivo, VDAC closure after ethanol occurs coordinately with mitochondrial uncoupling. Since acetaldehyde passes through membranes independently of channels and transporters, VDAC closure and uncoupling together foster selective and more rapid oxidative metabolism of toxic acetaldehyde to nontoxic acetate by mitochondrial aldehyde dehydrogenase. In single reconstituted VDAC, tubulin decreases VDAC conductance, and in HepG2 hepatoma cells, free tubulin negatively modulates mitochondrial membrane potential, an effect enhanced by protein kinase A. Tubulin-dependent closure of VDAC in cancer cells contributes to suppression of mitochondrial metabolism and may underlie the Warburg phenomenon of aerobic glycolysis. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

  8. Origin of the voltage dependence of G-protein regulation of P/Q-type Ca2+ channels.

    Science.gov (United States)

    Zhang, Yun; Chen, Yu-Hang; Bangaru, Saroja D; He, Linling; Abele, Kathryn; Tanabe, Shihori; Kozasa, Tohru; Yang, Jian

    2008-12-24

    G-protein (Gbetagamma)-mediated voltage-dependent inhibition of N- and P/Q-type Ca(2+) channels contributes to presynaptic inhibition and short-term synaptic plasticity. The voltage dependence derives from the dissociation of Gbetagamma from the inhibited channels, but the underlying molecular and biophysical mechanisms remain largely unclear. In this study we investigated the role in this process of Ca(2+) channel beta subunit (Ca(v)beta) and a rigid alpha-helical structure between the alpha-interacting domain (AID), the primary Ca(v)beta docking site on the channel alpha(1) subunit, and the pore-lining IS6 segment. Gbetagamma inhibition of P/Q-type channels was reconstituted in giant inside-out membrane patches from Xenopus oocytes. Large populations of channels devoid of Ca(v)beta were produced by washing out a mutant Ca(v)beta with a reduced affinity for the AID. These beta-less channels were still inhibited by Gbetagamma, but without any voltage dependence, indicating that Ca(v)beta is indispensable for voltage-dependent Gbetagamma inhibition. A truncated Ca(v)beta containing only the AID-binding guanylate kinase (GK) domain could fully confer voltage dependence to Gbetagamma inhibition. Gbetagamma did not alter inactivation properties, and channels recovered from Gbetagamma inhibition exhibited the same activation property as un-inhibited channels, indicating that Gbetagamma does not dislodge Ca(v)beta from the inhibited channel. Furthermore, voltage-dependent Gbetagamma inhibition was abolished when the rigid alpha-helix between the AID and IS6 was disrupted by insertion of multiple glycines, which also eliminated Ca(v)beta regulation of channel gating, revealing a pivotal role of this rigid alpha-helix in both processes. These results suggest that depolarization-triggered movement of IS6, coupled to the subsequent conformational change of the Gbetagamma-binding pocket through a rigid alpha-helix induced partly by the Ca(v)beta GK domain, causes the

  9. A Doping Dependent Threshold Voltage Model of Uniformly Doped Short-Channel Symmetric Double-Gate (DG MOSFET’s

    Directory of Open Access Journals (Sweden)

    P.K. Tiwari

    2011-01-01

    Full Text Available The paper presents a doping dependent threshold voltage model for the short-channel double-gate (DG MOSFETs. The channel potential has been determined by solving the two-dimensional (2D Poisson’s equation using the parabolic potential approximation in the vertical direction of channel. Threshold voltage sensitivity on acceptor doping and device parameters is discussed in detail. The threshold voltage expression has been modified by incorporating the effects of band gap narrowing for highly doped DG MOSFETs. Quantum mechanical corrections have also been employed in the threshold voltage model. The theoretical results have been compared with the ATLASTM simulation results. The present model is found to be valid for acceptor doping variation from 1014 cm–3 to 5 × 1018cm–3.

  10. 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

  11. Effects of arsenic trioxide on voltage-dependent potassium channels and on cell proliferation of human multiple myeloma cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Jin; WANG Wei; WEI Qing-fang; FENG Tie-ming; TAN Li-jun; YANG Bao-feng

    2007-01-01

    @@ Arsenic trioxide (ATO) can induce cellular apoptosis and inhibit the activities of multiple myeloma (MM)cells in vitro,1 but how it works is not very clear. Recent studies showed that ATO worked on the voltagedependent potassium channel and L-type calcium channel in myocardial cells,2-5 but the effect of ATO on ion channels of tumor cells was rarely reported. As the potassium channel plays an important role in controlling cell proliferation,6 we studied the effects of ATO on the voltage-dependent potassium current (Ikv) of the voltage-dependent potassium channel in an MM cell line,and probed into the relationship between changes of the Ikv caused by ATO and cell proliferation.

  12. Pharmacological Investigation of Voltage-dependent Ca2+ Channels in Human Ejaculatory Sperm in vitro

    Institute of Scientific and Technical Information of China (English)

    LI Lu; LIU Jihong; LI Jiagui; YE Zhangqun

    2006-01-01

    The types of the voltage-dependent calcium channels (VDCCs) in human ejaculatory sperm and the effects of calcium channel blocker (CCB) on human sperm motility parameters in vitro were investigated. The human sperm motility parameters in vitro in response to the pharmacological agents nifedipine (NIF, inhibitor of L-type VDCC) and ω-conotoxin (GVIA, inhibitor of N-type VDCC) were compared and analyzed statistically. The results showed that NIF (1, 5, 10 μmol/L)could not only significantly affect human sperm's shape but also spermatozoa motility after incubated at least 10 min in vitro (P<0.001). GVIA (0.1, 0.5 and 1 μmol/L) could just only significantly affect human sperm's progressive motility (a %+b %) after incubated for 20 min in vitro (P<0.01), but they both could not significantly affect spermic abnormality rate. It is suggested that L-type VDCC, non L-type VDCCs and isoform of L-type VDCC exist in the cell membrane of human sperm solely or together, and they participate in the spermic physiological processes especially the spermic motility.

  13. Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1.

    Science.gov (United States)

    Elinder, Fredrik; Madeja, Michael; Zeberg, Hugo; Århem, Peter

    2016-10-18

    The transmembrane voltage needed to open different voltage-gated K (Kv) channels differs by up to 50 mV from each other. In this study we test the hypothesis that the channels' voltage dependences to a large extent are set by charged amino-acid residues of the extracellular linkers of the Kv channels, which electrostatically affect the charged amino-acid residues of the voltage sensor S4. Extracellular cations shift the conductance-versus-voltage curve, G(V), by interfering with these extracellular charges. We have explored these issues by analyzing the effects of the divalent strontium ion (Sr(2+)) on the voltage dependence of the G(V) curves of wild-type and chimeric Kv channels expressed in Xenopus oocytes, using the voltage-clamp technique. Out of seven Kv channels, Kv1.2 was found to be most sensitive to Sr(2+) (50 mM shifted G(V) by +21.7 mV), and Kv2.1 to be the least sensitive (+7.8 mV). Experiments on 25 chimeras, constructed from Kv1.2 and Kv2.1, showed that the large Sr(2+)-induced G(V) shift of Kv1.2 can be transferred to Kv2.1 by exchanging the extracellular linker between S3 and S4 (L3/4) in combination with either the extracellular linker between S5 and the pore (L5/P) or that between the pore and S6 (LP/6). The effects of the linker substitutions were nonadditive, suggesting specific structural interactions. The free energy of these interactions was ∼20 kJ/mol, suggesting involvement of hydrophobic interactions and/or hydrogen bonds. Using principles from double-layer theory we derived an approximate linear equation (relating the voltage shifts to altered ionic strength), which proved to well match experimental data, suggesting that Sr(2+) acts on these channels mainly by screening surface charges. Taken together, these results highlight the extracellular surface potential at the voltage sensor as an important determinant of the channels' voltage dependence, making the extracellular linkers essential targets for evolutionary selection.

  14. 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).…

  15. 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).…

  16. Lavender oil-potent anxiolytic properties via modulating voltage dependent calcium channels.

    Science.gov (United States)

    Schuwald, Anita M; Nöldner, Michael; Wilmes, Thomas; Klugbauer, Norbert; Leuner, Kristina; Müller, Walter E

    2013-01-01

    Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs) as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, biochemical, and electrophysiological experiments were investigated in physiological concentrations in the nanomolar range, which correlate to a single dosage of 80 mg/d in humans that was used in clinical trials. We show for the first time that lavender oil bears some similarities with the established anxiolytic pregabalin. Lavender oil inhibits VOCCs in synaptosomes, primary hippocampal neurons and stably overexpressing cell lines in the same range such as pregabalin. Interestingly, Silexan does not primarily bind to P/Q type calcium channels such as pregabalin and does not interact with the binding site of pregabalin, the α2δ subunit of VOCCs. Lavender oil reduces non-selectively the calcium influx through several different types of VOCCs such as the N-type, P/Q-type and T-type VOCCs. In the hippocampus, one brain region important for anxiety disorders, we show that inhibition by lavender oil is mainly mediated via N-type and P/Q-type VOCCs. Taken together, we provide a pharmacological and molecular rationale for the clinical use of the oral application of lavender oil in patients suffering from anxiety.

  17. Lavender oil-potent anxiolytic properties via modulating voltage dependent calcium channels.

    Directory of Open Access Journals (Sweden)

    Anita M Schuwald

    Full Text Available Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, biochemical, and electrophysiological experiments were investigated in physiological concentrations in the nanomolar range, which correlate to a single dosage of 80 mg/d in humans that was used in clinical trials. We show for the first time that lavender oil bears some similarities with the established anxiolytic pregabalin. Lavender oil inhibits VOCCs in synaptosomes, primary hippocampal neurons and stably overexpressing cell lines in the same range such as pregabalin. Interestingly, Silexan does not primarily bind to P/Q type calcium channels such as pregabalin and does not interact with the binding site of pregabalin, the α2δ subunit of VOCCs. Lavender oil reduces non-selectively the calcium influx through several different types of VOCCs such as the N-type, P/Q-type and T-type VOCCs. In the hippocampus, one brain region important for anxiety disorders, we show that inhibition by lavender oil is mainly mediated via N-type and P/Q-type VOCCs. Taken together, we provide a pharmacological and molecular rationale for the clinical use of the oral application of lavender oil in patients suffering from anxiety.

  18. Functional coupling between sodium-activated potassium channels and voltage-dependent persistent sodium currents in cricket Kenyon cells.

    Science.gov (United States)

    Takahashi, Izumi; Yoshino, Masami

    2015-10-01

    In this study, we examined the functional coupling between Na(+)-activated potassium (KNa) channels and Na(+) influx through voltage-dependent Na(+) channels in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. Single-channel activity of KNa channels was recorded with the cell-attached patch configuration. The open probability (Po) of KNa channels increased with increasing Na(+) concentration in a bath solution, whereas it decreased by the substitution of Na(+) with an equimolar concentration of Li(+). The Po of KNa channels was also found to be reduced by bath application of a high concentration of TTX (1 μM) and riluzole (100 μM), which inhibits both fast (INaf) and persistent (INaP) Na(+) currents, whereas it was unaffected by a low concentration of TTX (10 nM), which selectively blocks INaf. Bath application of Cd(2+) at a low concentration (50 μM), as an inhibitor of INaP, also decreased the Po of KNa channels. Conversely, bath application of the inorganic Ca(2+)-channel blockers Co(2+) and Ni(2+) at high concentrations (500 μM) had little effect on the Po of KNa channels, although Cd(2+) (500 μM) reduced the Po of KNa channels. Perforated whole cell clamp analysis further indicated the presence of sustained outward currents for which amplitude was dependent on the amount of Na(+) influx. Taken together, these results indicate that KNa channels could be activated by Na(+) influx passing through voltage-dependent persistent Na(+) channels. The functional significance of this coupling mechanism was discussed in relation to the membrane excitability of Kenyon cells and its possible role in the formation of long-term memory.

  19. Diverse roles for auxiliary subunits in phosphorylation-dependent regulation of mammalian brain voltage-gated potassium channels.

    Science.gov (United States)

    Vacher, Helene; Trimmer, James S

    2011-11-01

    Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse partly due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here, we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels.

  20. Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin

    DEFF Research Database (Denmark)

    Fuchs, W; Larsen, Erik Hviid; Lindemann, B

    1977-01-01

    ) was clamped to zero and step-changes of Na activity in the outer solution ((Na)(o)) were performed with a fast-flow chamber at constant ionic strength, while the short-circuit current was recorded.3. At pre-selected times after a step-change of (Na)(o) the current response (I) to a fast voltage staircase...... was recorded. This procedure was repeated after blocking the Na channels with amiloride to obtain the current-voltage curve of transmembrane and paracellular shunt pathways. The current-voltage curve of the Na channels was computed by subtracting the shunt current from the total current.4. The instantaneous I...... transport through open Na-selective channels of the outward facing membrane of the stratum granulosum cells can be described as an electrodiffusion process which as such does not saturate with increasing (Na)(o). However, when added to the outer border of the membrane Na causes a decrease of P(Na) within...

  1. Functional coupling between large-conductance potassium channels and Cav3.2 voltage-dependent calcium channels participates in prostate cancer cell growth

    Directory of Open Access Journals (Sweden)

    Florian Gackière

    2013-07-01

    It is strongly suspected that potassium (K+ channels are involved in various aspects of prostate cancer development, such as cell growth. However, the molecular nature of those K+ channels implicated in prostate cancer cell proliferation and the mechanisms through which they control proliferation are still unknown. This study uses pharmacological, biophysical and molecular approaches to show that the main voltage-dependent K+ current in prostate cancer LNCaP cells is carried by large-conductance BK channels. Indeed, most of the voltage-dependent current was inhibited by inhibitors of BK channels (paxillin and iberiotoxin and by siRNA targeting BK channels. In addition, we reveal that BK channels constitute the main K+ channel family involved in setting the resting membrane potential in LNCaP cells at around −40 mV. This consequently promotes a constitutive calcium entry through T-type Cav3.2 calcium channels. We demonstrate, using single-channel recording, confocal imaging and co-immunoprecipitation approaches, that both channels form macromolecular complexes. Finally, using flow cytometry cell cycle measurements, cell survival assays and Ki67 immunofluorescent staining, we show that both BK and Cav3.2 channels participate in the proliferation of prostate cancer cells.

  2. Correlation character of ionic current fluctuations: analysis of ion current through a voltage-dependent potassium single channel.

    Science.gov (United States)

    Tong-Han, Lan; Huang, Xi; Jia-Rui, Lin

    2005-10-03

    The gating of ion channels has widely been modeled by assuming the transition between open and closed states is a memoryless process. Nevertheless, the statistical analysis of an ionic current signal recorded from voltage dependence K(+) single channel is presented. Calculating the sample auto-correlation function of the ionic current based on the digitized signals, rather than the sequence of open and closed states duration time. The results provide evidence for the existence of memory. For different voltages, the ion channel current fluctuation has different correlation attributions. The correlations in data generated by simulation of two Markov models, on one hand, auto-correlation function of the ionic current shows a weaker memory, after a delayed period of time, the attribute of memory does not exist; on the other hand, the correlation depends on the number of states in the Markov model. For V(p)=-60 mV pipette potential, spectral analysis of ion channel current was conducted, the result indicates that the spectrum is not a flat spectrum, the data set from ionic current fluctuations shows considerable variability with a broad 1/f -like spectrum, alpha=1.261+/-0.24. Thus the ion current fluctuations give information about the kinetics of the channel protein, the results suggest the correlation character of ion channel protein nonlinear kinetics regardless of whether the channel is in open or closed state.

  3. Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Liu-Min Fan

    2009-01-01

    Free cytosolic Ca~(2+) ([Ca~(2+)]_(cyt)) is an ubiquitous second messenger in plant cell signaling, and [Ca~(2+)]_(cyt) elevation is associated with Ca~(2+)-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca~(2+) channels and their regulation remains limited in planta. A type of voltage-dependent Ca~(2+)-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba~(2+) and Ca~(2+), and their activities can be inhibited by micromolar Gd~(3+). The unitary conductance and the reversal potential of the channels depend on the Ca~(2+) or Ba~(2+) gradients across the plasma membrane. The inward whole-cell Ca~(2+) (Ba~(2+)) current, as well as the unitary current amplitude and NP. of the single Ca~(2+) channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NP_o of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.

  4. Selective serotonin reuptake inhibitor sertraline inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

    Indian Academy of Sciences (India)

    HAN SOL KIM; HONGLIANG LI; HYE WON KIM; SUNG EUN SHIN; IL-WHAN CHOI; AMY L FIRTH; HYOWEON BANG; YOUNG MIN BAE; WON SUN PARK

    2016-12-01

    We examined the effects of the selective serotonin reuptake inhibitor (SSRI) sertraline on voltage-dependent K+ (Kv)channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Sertralinedecreased the Kv channel current in a dose-dependent manner, with an IC50 value of 0.18 μM and a slope value (Hillcoefficient) of 0.61. Although the application of 1 μM sertraline did not affect the steady-state activation curves,sertraline caused a significant, negative shift in the inactivation curves. Pretreatment with another SSRI, paroxetine,had no significant effect on Kv currents and did not alter the inhibitory effects of sertraline on Kv currents. From theseresults, we concluded that sertraline dose-dependently inhibited Kv currents independently of serotonin reuptakeinhibition by shifting inactivation curves to a more negative potential.

  5. Selective serotonin reuptake inhibitor sertraline inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

    Science.gov (United States)

    Kim, Han Sol; Li, Hongliang; Kim, Hye Won; Shin, Sung Eun; Choi, Il-Whan; Firth, Amy L; Bang, Hyoweon; Bae, Young Min; Park, Won Sun

    2016-12-01

    We examined the effects of the selective serotonin reuptake inhibitor (SSRI) sertraline on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Sertraline decreased the Kv channel current in a dose-dependent manner, with an IC50 value of 0.18 mu M and a slope value (Hill coefficient) of 0.61. Although the application of 1 mu M sertraline did not affect the steady-state activation curves, sertraline caused a significant, negative shift in the inactivation curves. Pretreatment with another SSRI, paroxetine, had no significant effect on Kv currents and did not alter the inhibitory effects of sertraline on Kv currents. From these results, we concluded that sertraline dose-dependently inhibited Kv currents independently of serotonin reuptake inhibition by shifting inactivation curves to a more negative potential.

  6. Conducting and voltage-dependent behaviors of potassium ion channels reconstituted from diaphragm sarcoplasmic reticulum: comparison with the cardiac isoform.

    Science.gov (United States)

    Picher, M; Decrouy, A; Rousseau, E

    1996-02-21

    Sarcoplasmic reticulum (SR) K+ channels from canine diaphragm were studied upon fusion of longitudinal and junctional membrane vesicles into planar lipid bilayers (PLB). The large-conductance cation selective channel (gamma(max) = 250 pS; Km = 33 mM) displays long-lasting open events which are much more frequent at positive than at negative voltages. A major subconducting state about 45% of the fully-open state current amplitude was occasionally observed at all voltages. The voltage-dependence of the open probability displays a sigmoid relationship that was fitted by the Boltzmann equation and expressed in terms of thermodynamic parameters, namely the free energy (delta Gi) and the effective gating charge (Zs): delta Gi = 0.27 kcal/mol and Zs = -1.19 in 250 mM potassium gluconate (K-gluconate). Kinetic analyses also confirmed the voltage-dependent gating behavior of this channel, and indicate the implication of at least two open and three closed states. The diaphragm SR K+ channel shares several biophysical properties with the cardiac isoform: g = 180 pS, delta Gi = 0.75 kcal/mol, Zs = -1.45 in 150 mM K-gluconate, and a similar sigmoid P(o)/voltage relationship. Little is known about the regulation of the diaphragm and cardiac SR K+ channels. The conductance and gating of these channels were not influenced by physiological concentrations of Ca2+ (0.1 microM-1 mM) or Mg2+ (0.25-1 mM), as well as by cGMP (25-100 microM), lemakalim (1-100 microM), glyburide (up to 10 microM) or charybdotoxin (45-200 nM), added either to the cis or to the trans chamber. The apparent lack of biochemical or pharmacological modulation of these channels implies that they are not related to any of the well characterized surface membrane K+ channels. On the other hand, their voltage sensitivity strongly suggests that their activity could be modulated by putative changes in SR membrane potential that might occur during calcium fluxes.

  7. Molecular characterization and functional expression of the Apis mellifera voltage-dependent Ca2+ channels.

    Science.gov (United States)

    Cens, Thierry; Rousset, Matthieu; Collet, Claude; Charreton, Mercedes; Garnery, Lionel; Le Conte, Yves; Chahine, Mohamed; Sandoz, Jean-Christophe; Charnet, Pierre

    2015-03-01

    Voltage-gated Ca(2+) channels allow the influx of Ca(2+) ions from the extracellular space upon membrane depolarization and thus serve as a transducer between membrane potential and cellular events initiated by Ca(2+) transients. Most insects are predicted to possess three genes encoding Cavα, the main subunit of Ca(2+) channels, and several genes encoding the two auxiliary subunits, Cavβ and Cavα2δ; however very few of these genes have been cloned so far. Here, we cloned three full-length cDNAs encoding the three Cavα subunits (AmelCav1a, AmelCav2a and AmelCav3a), a cDNA encoding a novel variant of the Cavβ subunit (AmelCavβc), and three full-length cDNAs encoding three Cavα2δ subunits (AmelCavα2δ1 to 3) of the honeybee Apis mellifera. We identified several alternative or mutually exclusive exons in the sequence of the AmelCav2 and AmelCav3 genes. Moreover, we detected a stretch of glutamine residues in the C-terminus of the AmelCav1 subunit that is reminiscent of the motif found in the human Cav2.1 subunit of patients with Spinocerebellar Ataxia type 6. All these subunits contain structural domains that have been identified as functionally important in their mammalian homologues. For the first time, we could express three insect Cavα subunits in Xenopus oocytes and we show that AmelCav1a, 2a and 3a form Ca(2+) channels with distinctive properties. Notably, the co-expression of AmelCav1a or AmelCav2a with AmelCavβc and AmCavα2δ1 produces High Voltage-Activated Ca(2+) channels. On the other hand, expression of AmelCav3a alone leads to Low Voltage-Activated Ca(2+) channels.

  8. Voltage-gated lipid ion channels

    DEFF Research Database (Denmark)

    Blicher, Andreas; Heimburg, Thomas Rainer

    2013-01-01

    Synthetic lipid membranes can display channel-like ion conduction events even in the absence of proteins. We show here that these events are voltage-gated with a quadratic voltage dependence as expected from electrostatic theory of capacitors. To this end, we recorded channel traces and current...

  9. Lidocaine stabilizes the open state of CNS voltage-dependent sodium channels.

    Science.gov (United States)

    Castañeda-Castellanos, David R; Nikonorov, Igor; Kallen, Roland G; Recio-Pinto, E

    2002-03-28

    We have previously reported that the lidocaine action is different between CNS and muscle batrachotoxin-modified Na+ channels [Salazar et al., J. Gen. Physiol. 107 (1996) 743-754; Brain Res. 699 (1995) 305-314]. In this study we examined lidocaine action on CNS Na+ currents, to investigate the mechanism of lidocaine action on this channel isoform and to compare it with that proposed for muscle Na+ currents. Na+ currents were measured with the whole cell voltage clamp configuration in stably transfected cells expressing the brain alpha-subunit (type IIA) by itself (alpha-brain) or together with the brain beta(1)-subunit (alphabeta(1)-brain), or the cardiac alpha-subunit (hH1) (alpha-cardiac). Lidocaine (100 microM) produced comparable levels of Na+ current block at positive potentials and of hyperpolarizing shift of the steady-state inactivation curve in alpha-brain and alphabeta(1)-brain Na+ currents. Lidocaine accelerated the rates of activation and inactivation, produced an hyperpolarizing shift in the steady-state activation curve and increased the current magnitude at negative potentials in alpha-brain but not in alphabeta(1)-brain Na+ currents. The lidocaine action in alphabeta(1)-brain resembled that observed in alpha-cardiac Na+ currents. The lidocaine-induced increase in current magnitude at negative potentials and the hyperpolarizing shift in the steady-state activation curve of alpha-brain, are novel effects and suggest that lidocaine treatment does not always lead to current reduction/block when it interacts with Na+ channels. The data are explained by using a modified version of the model proposed by Vedantham and Cannon [J. Gen. Physiol., 113 (1999) 7-16] in which we postulate that the difference in lidocaine action between alpha-brain and alphabeta(1)-brain Na+ currents could be explained by differences in the lidocaine action on the open channel state.

  10. Ca2+ channel inhibitor NNC 55-0396 inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

    Science.gov (United States)

    Son, Youn Kyoung; Hong, Da Hye; Li, Hongliang; Kim, Dae-Joong; Na, Sung Hun; Park, Hongzoo; Jung, Won-Kyo; Choi, Il-Whan; Park, Won Sun

    2014-01-01

    We demonstrated the inhibitory effect of NNC 55-0396, a T-type Ca(2+) channel inhibitor, on voltage-dependent K(+) (K(V)) channels in freshly isolated rabbit coronary arterial smooth muscle cells. NNC 55-0396 decreased the amplitude of K(V) currents in a concentration-dependent manner, with an IC(50) of 0.080 μM and a Hill coefficient of 0.76.NNC 55-0396 did not affect steady-state activation and inactivation curves, indicating that the compound does not affect the voltage sensitivity of K(V) channel gating. Both the K(V) currents and the inhibitory effect of NNC 55-0396 on K(V) channels were not altered by depletion of extracellular Ca(2+) or intracellular ATP, suggesting that the inhibitory effect of NNC 55-0396 is independent of Ca(2+)-channel activity and phosphorylation-dependent signaling cascades. From these results, we concluded that NNC 55-0396 dosedependently inhibits K(V) currents, independently of Ca(2+)-channel activity and intracellular signaling cascades.

  11. Hysteresis in voltage-gated channels.

    Science.gov (United States)

    Villalba-Galea, Carlos A

    2016-09-30

    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.

  12. Neuroprotective activity of stiripentol with a possible involvement of voltage-dependent calcium and sodium channels.

    Science.gov (United States)

    Verleye, Marc; Buttigieg, Dorothée; Steinschneider, Rémy

    2016-02-01

    A growing body of data has shown that recurrent epileptic seizures may be caused by an excessive release of the excitatory neurotransmitter glutamate in the brain. Glutamatergic overstimulation results in massive neuronal influxes of calcium and sodium through N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainic acid glutamate subtype receptors and also through voltage-gated calcium and sodium channels. These persistent and abnormal sodium and calcium entry points have deleterious consequences (neurotoxicity) for neuronal function. The therapeutic value of an antiepileptic drug would include not only control of seizure activity but also protection of neuronal tissue. The present study examines the in vitro neuroprotective effects of stiripentol, an antiepileptic compound with γ-aminobutyric acidergic properties, on neuronal-astroglial cultures from rat cerebral cortex exposed to oxygen-glucose deprivation (OGD) or to glutamate (40 µM for 20 min), two in vitro models of brain injury. In addition, the affinity of stiripentol for the different glutamate receptor subtypes and the interaction with the cell influx of Na(+) and of Ca(2+) enhanced by veratridine and NMDA, respectively, are assessed. Stiripentol (10-100 µM) included in the culture medium during OGD or with glutamate significantly increased the number of surviving neurons relative to controls. Stiripentol displayed no binding affinity for different subtypes of glutamate receptors (IC50  >100 µM) but significantly blocked the entry of Na(+) and Ca(2+) activated by veratridine and NMDA, respectively. These results suggest that Na(+) and Ca(2+) channels could contribute to the neuroprotective properties of sitiripentol.

  13. Voltage-gated lipid ion channels

    DEFF Research Database (Denmark)

    Blicher, Andreas; Heimburg, Thomas Rainer

    2013-01-01

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

  14. Aspartic Acid Residue D3 Critically Determines Cx50 Gap Junction Channel Transjunctional Voltage-Dependent Gating and Unitary Conductance

    Science.gov (United States)

    Xin, Li; Nakagawa, So; Tsukihara, Tomitake; Bai, Donglin

    2012-01-01

    Previous studies have suggested that the aspartic acid residue (D) at the third position is critical in determining the voltage polarity of fast Vj-gating of Cx50 channels. To test whether another negatively charged residue (a glutamic acid residue, E) could fulfill the role of the D3 residue, we generated the mutant Cx50D3E. Vj-dependent gating properties of this mutant channel were characterized by double-patch-clamp recordings in N2A cells. Macroscopically, the D3E substitution reduced the residual conductance (Gmin) to near zero and outwardly shifted the half-inactivation voltage (V0), which is a result of both a reduced aggregate gating charge (z) and a reduced free-energy difference between the open and closed states. Single Cx50D3E gap junction channels showed reduced unitary conductance (γj) of the main open state, reduced open dwell time at ±40 mV, and absence of a long-lived substate. In contrast, a G8E substitution tested to compare the effects of the E residue at the third and eighth positions did not modify the Vj-dependent gating profile or γj. In summary, this study is the first that we know of to suggest that the D3 residue plays an essential role, in addition to serving as a negative-charge provider, as a critical determinant of the Vj-dependent gating sensitivity, open-closed stability, and unitary conductance of Cx50 gap junction channels. PMID:22404924

  15. The voltage-dependent K+ channels Kv1.3 and Kv1.5 in human cancer

    Science.gov (United States)

    Comes, Núria; Bielanska, Joanna; Vallejo-Gracia, Albert; Serrano-Albarrás, Antonio; Marruecos, Laura; Gómez, Diana; Soler, Concepció; Condom, Enric; Ramón y Cajal, Santiago; Hernández-Losa, Javier; Ferreres, Joan C.; Felipe, Antonio

    2013-01-01

    Voltage-dependent K+ channels (Kv) are involved in a number of physiological processes, including immunomodulation, cell volume regulation, apoptosis as well as differentiation. Some Kv channels participate in the proliferation and migration of normal and tumor cells, contributing to metastasis. Altered expression of Kv1.3 and Kv1.5 channels has been found in several types of tumors and cancer cells. In general, while the expression of Kv1.3 apparently exhibits no clear pattern, Kv1.5 is induced in many of the analyzed metastatic tissues. Interestingly, evidence indicates that Kv1.5 channel shows inversed correlation with malignancy in some gliomas and non-Hodgkin's lymphomas. However, Kv1.3 and Kv1.5 are similarly remodeled in some cancers. For instance, expression of Kv1.3 and Kv1.5 correlates with a certain grade of tumorigenicity in muscle sarcomas. Differential remodeling of Kv1.3 and Kv1.5 expression in human cancers may indicate their role in tumor growth and their importance as potential tumor markers. However, despite of this increasing body of information, which considers Kv1.3 and Kv1.5 as emerging tumoral markers, further research must be performed to reach any conclusion. In this review, we summarize what it has been lately documented about Kv1.3 and Kv1.5 channels in human cancer. PMID:24133455

  16. The voltage-dependent K(+) channels Kv1.3 and Kv1.5 in human cancer.

    Science.gov (United States)

    Comes, Núria; Bielanska, Joanna; Vallejo-Gracia, Albert; Serrano-Albarrás, Antonio; Marruecos, Laura; Gómez, Diana; Soler, Concepció; Condom, Enric; Ramón Y Cajal, Santiago; Hernández-Losa, Javier; Ferreres, Joan C; Felipe, Antonio

    2013-10-10

    Voltage-dependent K(+) channels (Kv) are involved in a number of physiological processes, including immunomodulation, cell volume regulation, apoptosis as well as differentiation. Some Kv channels participate in the proliferation and migration of normal and tumor cells, contributing to metastasis. Altered expression of Kv1.3 and Kv1.5 channels has been found in several types of tumors and cancer cells. In general, while the expression of Kv1.3 apparently exhibits no clear pattern, Kv1.5 is induced in many of the analyzed metastatic tissues. Interestingly, evidence indicates that Kv1.5 channel shows inversed correlation with malignancy in some gliomas and non-Hodgkin's lymphomas. However, Kv1.3 and Kv1.5 are similarly remodeled in some cancers. For instance, expression of Kv1.3 and Kv1.5 correlates with a certain grade of tumorigenicity in muscle sarcomas. Differential remodeling of Kv1.3 and Kv1.5 expression in human cancers may indicate their role in tumor growth and their importance as potential tumor markers. However, despite of this increasing body of information, which considers Kv1.3 and Kv1.5 as emerging tumoral markers, further research must be performed to reach any conclusion. In this review, we summarize what it has been lately documented about Kv1.3 and Kv1.5 channels in human cancer.

  17. Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D;

    2001-01-01

    .2 protein was demonstrated by immunochemical labeling of rat preglomerular vasculature and juxtamedullary efferent arterioles and vasa recta. Cortical efferent arterioles were not immunopositive. Recordings of intracellular calcium concentration with digital fluorescence imaging microscopy showed......The distribution of voltage-dependent calcium channels in kidney pre- and postglomerular resistance vessels was determined at the molecular and functional levels. Reverse transcription-polymerase chain reaction analysis of microdissected rat preglomerular vessels and cultured smooth muscle cells...... showed coexpression of mRNAs for T-type subunits (Ca(V)3.1, Ca(V)3.2) and for an L-type subunit (Ca(V)1.2). The same expression pattern was observed in juxtamedullary efferent arterioles and outer medullary vasa recta. No calcium channel messages were detected in cortical efferent arterioles. Ca(V)1...

  18. Characterization and functional analysis of voltage-dependent anion channel 1 (VDAC1) from orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Shi, Yan; Zhao, Zhe; Hong, Xiaoyou; Chen, Kunci; Zhu, Xinping

    2014-07-01

    The voltage-dependent anion channel (VDAC) is a highly conserved integral protein of mitochondria in different eukaryotic species. It forms a selective channel in the mitochondrial outer membrane that serves as the controlled pathway for small metabolites and ions. In this study, a VDAC gene, EcVDAC1, was isolated from orange-spotted grouper (Epinephelus coioides). The EcVDAC1 exhibits ubiquitous expression in various tissues of orange-spotted grouper and is upregulated in liver, gill, and spleen after stimulation with lipopolysaccharides (LPS). Subcellular localization analysis shows that the EcVDAC1 protein colocalized with the mitochondria. A caspase-3 assay demonstrates that overexpression of the EcVDAC1 induced apoptotic cell death in fathead minnow cells. The data presented in this study provide new information regarding the relationship between LPS and the EcVDAC1 gene, suggesting that the fish VDAC1 gene may play an important role in antibacterial immune response.

  19. Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D

    2001-01-01

    The distribution of voltage-dependent calcium channels in kidney pre- and postglomerular resistance vessels was determined at the molecular and functional levels. Reverse transcription-polymerase chain reaction analysis of microdissected rat preglomerular vessels and cultured smooth muscle cells...... showed coexpression of mRNAs for T-type subunits (Ca(V)3.1, Ca(V)3.2) and for an L-type subunit (Ca(V)1.2). The same expression pattern was observed in juxtamedullary efferent arterioles and outer medullary vasa recta. No calcium channel messages were detected in cortical efferent arterioles. Ca(V)1.......2 protein was demonstrated by immunochemical labeling of rat preglomerular vasculature and juxtamedullary efferent arterioles and vasa recta. Cortical efferent arterioles were not immunopositive. Recordings of intracellular calcium concentration with digital fluorescence imaging microscopy showed...

  20. Use-dependent block of the voltage-gated Na(+) channel by tetrodotoxin and saxitoxin: effect of pore mutations that change ionic selectivity.

    Science.gov (United States)

    Huang, Chien-Jung; Schild, Laurent; Moczydlowski, Edward G

    2012-10-01

    Voltage-gated Na(+) channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca(2+) permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca(2+) or Na(+) ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca(2+) permeability, suggesting that ion-toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.

  1. trans-Caryophyllene, a Natural Sesquiterpene, Causes Tracheal Smooth Muscle Relaxation through Blockade of Voltage-Dependent Ca2+ Channels

    Directory of Open Access Journals (Sweden)

    Jader Santos Cruz

    2012-10-01

    Full Text Available trans-Caryophyllene is a major component in the essential oils of various species of medicinal plants used in popular medicine in Brazil. It belongs to the chemical class of the sesquiterpenes and has been the subject of a number of studies. Here, we evaluated the effects of this compound in airway smooth muscle. The biological activities of trans-caryophyllene were examined in isolated bath organs to investigate the effect in basal tonus. Electromechanical and pharmacomechanical couplings were evaluated through the responses to K+ depolarization and exposure to acetylcholine (ACh, respectively. Isolated cells of rat tracheal smooth muscle were used to investigate trans-caryophyllene effects on voltage-dependent Ca2+ channels by using the whole-cell voltage-clamp configuration of the patch-clamp technique. trans-Caryophyllene showed more efficiency in the blockade of electromechanical excitation-contraction coupling while it has only minor inhibitory effect on pharmacomechanical coupling. Epithelium removal does not modify tracheal smooth muscle response elicited by trans-caryophyllene in the pharmacomechanical coupling. Under Ca2+-free conditions, pre-exposure to trans-caryophyllene did not reduce the contraction induced by ACh in isolated rat tracheal smooth muscle, regardless of the presence of intact epithelium. In the whole-cell configuration, trans-caryophyllene (3 mM, inhibited the inward Ba2+ current (IBa to approximately 50% of control levels. Altogether, our results demonstrate that trans-caryophyllene has anti-spasmodic activity on rat tracheal smooth muscle which could be explained, at least in part, by the voltage-dependent Ca2+ channels blockade.

  2. Multiphasic profiles for voltage-dependent K+ channels: Reanalysis of data of MacKinnon and coworkers

    CERN Document Server

    Nissen, Per

    2016-01-01

    In a study of the role that voltage-dependent K+ channels may have in the mechanosensation of living cells (Schmidt et al. Proc Soc Natl Acad Sci USA 109: 10352-10357. 2012), the data were as conventionally done fitted by a Boltzmann function. However, as also found for other data for ion channels, this interpretation must be rejected in favor of a multiphasic profile, a series of straight lines separated by discontinuous transitions, quite often in the form of noncontiguities (jumps). The data points in the present study are often very unevenly distributed around the curvilinear profiles. Thus, for 43 of the 75 profiles, the probability is less than 5% that the uneven distribution is due to chance, for 26 the probability is less than 1%, and for 12 the probability is less than 0.1%, giving a vanishingly low overall probability for all profiles. Especially at low voltages, the differences between the fits to curvilinear and multiphasic profiles may be huge. In the multiphasic profiles, adjacent lines are quit...

  3. Distribution of voltage-dependent and intracellular Ca2+ channels in submucosal neurons from rat distal colon.

    Science.gov (United States)

    Rehn, Matthias; Bader, Sandra; Bell, Anna; Diener, Martin

    2013-09-01

    We recently observed a bradykinin-induced increase in the cytosolic Ca2+ concentration in submucosal neurons of rat colon, an increase inhibited by blockers of voltage-dependent Ca2+ (Ca(v)) channels. As the types of Ca(v) channels used by this part of the enteric nervous system are unknown, the expression of various Ca(v) subunits has been investigated in whole-mount submucosal preparations by immunohistochemistry. Submucosal neurons, identified by a neuronal marker (microtubule-associated protein 2), are immunoreactive for Ca(v)1.2, Ca(v)1.3 and Ca(v)2.2, expression being confirmed by reverse transcription plus the polymerase chain reaction. These data agree with previous observations that the inhibition of L- and N-type Ca2+ currents strongly inhibits the response to bradykinin. However, whole-cell patch-clamp experiments have revealed that bradykinin does not enhance Ca2+ inward currents under voltage-clamp conditions. Consequently, bradykinin does not directly interact with Ca(v) channels. Instead, the kinin-induced Ca2+ influx is caused indirectly by the membrane depolarization evoked by this peptide. As intracellular Ca2+ channels on Ca(2+)-storing organelles can also contribute to Ca2+ signaling, their expression has been investigated by imaging experiments and immunohistochemistry. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) have been functionally demonstrated in submucosal neurons loaded with the Ca(2+)-sensitive fluorescent dye, fura-2. Histamine, a typical agonist coupled to the phospholipase C pathway, induces an increase in the fura-2 signal ratio, which is suppressed by 2-aminophenylborate, a blocker of IP3 receptors. The expression of IP3R1 has been confirmed by immunohistochemistry. In contrast, ryanodine, tested over a wide concentration range, evokes no increase in the cytosolic Ca2+ concentration nor is there immunohistochemical evidence for the expression of ryanodine receptors in these neurons. Thus, rat submucosal neurons are equipped

  4. Voltage regulation of connexin channel conductance.

    Science.gov (United States)

    Oh, Seunghoon; Bargiello, Thaddeus A

    2015-01-01

    Voltage is an important parameter that regulates the conductance of both intercellular and plasma membrane channels (undocked hemichannels) formed by the 21 members of the mammalian connexin gene family. Connexin channels display two forms of voltage-dependence, rectification of ionic currents and voltage-dependent gating. Ionic rectification results either from asymmetries in the distribution of fixed charges due to heterotypic pairing of different hemichannels, or by channel block, arising from differences in the concentrations of divalent cations on opposite sides of the junctional plaque. This rectification likely underpins the electrical rectification observed in some electrical synapses. Both intercellular and undocked hemichannels also display two distinct forms of voltage-dependent gating, termed Vj (fast)-gating and loop (slow)-gating. This review summarizes our current understanding of the molecular determinants and mechanisms underlying these conformational changes derived from experimental, molecular-genetic, structural, and computational approaches.

  5. Thiazolidinedione insulin sensitizers alter lipid bilayer properties and voltage-dependent sodium channel function: implications for drug discovery.

    Science.gov (United States)

    Rusinova, Radda; Herold, Karl F; Sanford, R Lea; Greathouse, Denise V; Hemmings, Hugh C; Andersen, Olaf S

    2011-08-01

    The thiazolidinediones (TZDs) are used in the treatment of diabetes mellitus type 2. Their canonical effects are mediated by activation of the peroxisome proliferator-activated receptor γ (PPARγ) transcription factor. In addition to effects mediated by gene activation, the TZDs cause acute, transcription-independent changes in various membrane transport processes, including glucose transport, and they alter the function of a diverse group of membrane proteins, including ion channels. The basis for these off-target effects is unknown, but the TZDs are hydrophobic/amphiphilic and adsorb to the bilayer-water interface, which will alter bilayer properties, meaning that the TZDs may alter membrane protein function by bilayer-mediated mechanisms. We therefore explored whether the TZDs alter lipid bilayer properties sufficiently to be sensed by bilayer-spanning proteins, using gramicidin A (gA) channels as probes. The TZDs altered bilayer elastic properties with potencies that did not correlate with their affinity for PPARγ. At concentrations where they altered gA channel function, they also altered the function of voltage-dependent sodium channels, producing a prepulse-dependent current inhibition and hyperpolarizing shift in the steady-state inactivation curve. The shifts in the inactivation curve produced by the TZDs and other amphiphiles can be superimposed by plotting them as a function of the changes in gA channel lifetimes. The TZDs' partition coefficients into lipid bilayers were measured using isothermal titration calorimetry. The most potent bilayer modifier, troglitazone, alters bilayer properties at clinically relevant free concentrations; the least potent bilayer modifiers, pioglitazone and rosiglitazone, do not. Unlike other TZDs tested, ciglitazone behaves like a hydrophobic anion and alters the gA monomer-dimer equilibrium by more than one mechanism. Our results provide a possible mechanism for some off-target effects of an important group of drugs, and

  6. Cloning, chromosomal localization, and functional expression of the alpha 1 subunit of the L-type voltage-dependent calcium channel from normal human heart

    NARCIS (Netherlands)

    Schultz, D; Mikala, G; Yatani, A; Engle, D B; Iles, D E; Segers, B; Sinke, R J; Weghuis, D O; Klöckner, U; Wakamori, M

    1993-01-01

    A unique structural variant of the cardiac L-type voltage-dependent calcium channel alpha 1 subunit cDNA was isolated from libraries derived from normal human heart mRNA. The deduced amino acid sequence shows significant homology to other calcium channel alpha 1 subunits. However, differences from t

  7. Inhibition of rat hippocampal excitability by the plant alkaloid 3-acetylaconitine mediated by interaction with voltage-dependent sodium channels.

    Science.gov (United States)

    Ameri, A

    1997-02-01

    The effects of the Aconitum alkaloid 3-acetylaconitine on neuronal activity were investigated in the slice preparation and on cultivated neurons of rat hippocampus by extracellular and patch-clamp recordings, respectively. 3-Acetylaconitine (0.01-1 microM) diminished the orthodromic and antidromic population spike in a concentration-dependent manner. The inhibitory action of the drug was preceded by a transiently enhanced excitability. The latency of onset of the inhibition was accelerated by increased stimulation frequency, whereas recovery during washout of the alkaloid was accelerated by decreased stimulation frequency. Moreover, the inhibitory effect of 3-acetylaconitine was evaluated in two different models of epileptiform activity induced either by blockade of GABA receptors by bicuculline (10 microM) or by a nominal Mg(2+)-free bathing medium. In accordance with the activity-dependent mode of action, this compound abolished the synaptically evoked population spikes in the presence of bicuculline or nominal Mg(2+)-free bathing medium, respectively. Whole-cell patch-clamp recordings revealed an interaction of 3-acetylaconitine with the voltage-dependent sodium channel. At a concentration of 1 microM, 3-acetylaconitine did not affect the peak amplitude of the sodium current, but shifted the current-voltage relationship in the hyperpolarized direction such that sodium currents were already activated at the resting potential.

  8. Cysteine mutagenesis in the voltage-dependent sodium channel structural insights and implications.

    Science.gov (United States)

    Tomaselli, G F

    1997-08-01

    The superfamily of ion channel proteins comprise multisubunit transmembrane glycoproteins that are the fundamental electrical signaling molecules in the heart and other excitable tissues. The large size and hydrophobicity of these proteins present a formidable obstacle to the generation of a crystal structure. In lieu of a high-resolution structure, complementary methods have been used to study the structure function relationships of these essential excitability proteins. Molecular cloning and biophysical analysis of heterologously expressed wild-type and mutant channel proteins have provided insights into the structural basis of the essential channel functions of permeation and gating. This powerful combination of techniques also provides dynamic structural information regarding channel proteins not likely to be forthcoming from a crystal structure. (Trends Cardiovasc Med 1997;7:211-218). © 1997, Elsevier Science Inc.

  9. Voltage-dependent anion channels (VDACs, porin) expressed in the plasma membrane regulate the differentiation and function of human osteoclasts.

    Science.gov (United States)

    Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki

    2013-01-01

    Fewer molecules have been identified on human than murine osteoclasts, the former differing from murine osteoclasts in many ways. We show that voltage-dependent anion channels (VDACs, porin) are expressed in the plasma membrane of human osteoclasts. A search for novel proteins expressed in the plasma membrane of human osteoclasts identified VDAC. Anti-VDAC antibodies inhibited human osteoclastogenesis in vitro. VDAC expression was detected in membranes by immunoelectron microscopy and immunocytochemical double staining. The VDAC protein functions as a Cl(-) channel. VDACs regulate bone resorption, which show using Osteologic™ plates. The epitope of the antibody lay within a 10-amino acid sequence in the VDAC. The findings suggest that the VDAC is, at least partly, a novel Cl(-) channel regulating the differentiation and function of human osteoclasts. VDACs may play a crucial role in acidifying the resorption lacunae between osteoclasts and bone. Inhibitors of VDACs could be used to treat diseases involving increased resorption, such as osteoporosis, rheumatoid arthritis, and Paget's disease. © 2012 International Federation for Cell Biology.

  10. Aconitum sp. alkaloids: The modulation of voltage-dependent Na+channels, toxicity and antinociceptive properties

    NARCIS (Netherlands)

    Friese, Jutta; Gleitz, Johannes; Gutser, Ulrike T.; Heubach, Jürgen F.; Matthiesen, Theo; Wilffert, Bob; Selve, Norma

    1997-01-01

    Alkaloids from Aconitum sp., used as analgesics in traditional Chinese medicine, were investigated to elucidate their antinociceptive and toxic properties considering: (1) binding to Na+channel epitope site 2, (2) alterations in synaptosoml Na+and Ca2+concentration ([Na+](i), [Ca2+](i)), (3) arrhyth

  11. Voltage dependence of cardiac excitation-contraction coupling: unitary Ca2+ current amplitude and open channel probability.

    Science.gov (United States)

    Altamirano, Julio; Bers, Donald M

    2007-09-14

    Excitation-contraction coupling in cardiac myocytes occurs by Ca2+-induced Ca2+ release, where L-type Ca2+ current evokes a larger sarcoplasmic reticulum (SR) Ca2+ release. The Ca2+-induced Ca2+ release amplification factor or gain (SR Ca2+ release/I(Ca)) is usually assessed by the V(m) dependence of current and Ca2+ transients. Gain rises at negative V(m), as does single channel I(Ca) (i(Ca)), which has led to the suggestion that the increases of i(Ca) amplitude enhances gain at more negative V(m). However, I(Ca) = NP(o) x i(Ca) (where NP(o) is the number of open channels), and NP(o) and i(Ca) both depend on V(m). To assess how i(Ca) and NP(o) separately influence Ca2+-induced Ca2+ release, we measured I(Ca) and junctional SR Ca2+ release in voltage-clamped rat ventricular myocytes using "Ca2+ spikes" (confocal microscopy). To vary i(Ca) alone, we changed [Ca2+](o) rapidly at constant test V(m) (0 mV) or abruptly repolarized from +120 mV to different V(m) (at constant [Ca2+](o)). To vary NP(o) alone, we altered Ca2+ channel availability by varying holding V(m) (at constant test V(m)). Reducing either i(Ca) or NP(o) alone increased excitation-contraction coupling gain. Thus, increasing i(Ca) does not increase gain at progressively negative test V(m). Such enhanced gain depends on lower NP(o) and reduced redundant Ca2+ channel openings (per junction) and a consequently smaller denominator in the gain equation. Furthermore, modest i(Ca) (at V(m) = 0 mV) may still effectively trigger SR Ca2+ release, whereas at positive V(m) (and smaller i(Ca)), high and well-synchronized channel openings are required for efficient excitation-contraction coupling. At very positive V(m), reduced i(Ca) must explain reduced SR Ca2+ release.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    cycle, including its direction, is reproduced by a model with 2×2 discrete states: the normal open/closed states and two different states of "gate tension". Rates of transitions between the two branches of the hysteresis curve are modeled with single-barrier kinetics by introducing a real......-valued "reaction coordinate" parametrizing the protein's conformational change between the two states of gate tension. The resulting scenario suggests a reanalysis of former experiments with NSVDC channels....

  13. Lack of negatively charged residues at the external mouth of Kir2.2 channels enable the voltage-dependent block by external Mg2+.

    Directory of Open Access Journals (Sweden)

    Junwei Li

    Full Text Available Kir channels display voltage-dependent block by cytosolic cations such as Mg2+ and polyamines that causes inward rectification. In fact, cations can regulate K channel activity from both the extracellular and intracellular sides. Previous studies have provided insight into the up-regulation of Kir channel activity by extracellular K+ concentration. In contrast, extracellular Mg2+ has been found to reduce the amplitude of the single-channel current at milimolar concentrations. However, little is known about the molecular mechanism of Kir channel blockade by external Mg2+ and the relationship between the Mg2+ blockade and activity potentiation by permeant K+ ions. In this study, we applied an interactive approach between theory and experiment. Electrophysiological recordings on Kir2.2 and its mutants were performed by heterologous expression in Xenopus laevis oocytes. Our results confirmed that extracellular Mg2+ could reduce heterologously expressed WT Kir2.2 currents in a voltage dependent manner. The kinetics of inhibition and recovery of Mg2+ exhibit a 3∼4s time constant. Molecular dynamics simulation results revealed a Mg2+ binding site located at the extracellular mouth of Kir2.2 that showed voltage-dependent Mg2+ binding. The mutants, G119D, Q126E and H128D, increased the number of permeant K+ ions and reduced the voltage-dependent blockade of Kir2.2 by extracellular Mg2+.

  14. Expression and localization of voltage dependent potassium channel Kv4.2 in epilepsy associated focal lesions

    NARCIS (Netherlands)

    Aronica, E.; Boer, K.; Doorn, K.J.; Zurolo, E.; Spliet, W.G.M.; van Rijen, P.C.; Baayen, J.C.; Gorter, J.A.; Jeromin, A.

    2009-01-01

    An increasing number of observations suggest an important role for voltage-gated potassium (Kv) channels in epilepsy. We studied the cell-specific distribution of Kv4.2, phosphorylated (p) Kv4.2 and the Kv4.2 interacting protein NCS-1 using immunocytochemistry in different epilepsy-associated focal

  15. Expression and localization of voltage dependent potassium channel Kv4.2 in epilepsy associated focal lesions

    NARCIS (Netherlands)

    Aronica, E.; Boer, K.; Doorn, K.J.; Zurolo, E.; Spliet, W.G.M.; van Rijen, P.C.; Baayen, J.C.; Gorter, J.A.; Jeromin, A.

    2009-01-01

    An increasing number of observations suggest an important role for voltage-gated potassium (Kv) channels in epilepsy. We studied the cell-specific distribution of Kv4.2, phosphorylated (p) Kv4.2 and the Kv4.2 interacting protein NCS-1 using immunocytochemistry in different epilepsy-associated focal

  16. Voltage-dependent regulation of CaV2.2 channels by Gq-coupled receptor is facilitated by membrane-localized β subunit.

    Science.gov (United States)

    Keum, Dongil; Baek, Christina; Kim, Dong-Il; Kweon, Hae-Jin; Suh, Byung-Chang

    2014-10-01

    G protein-coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca(2+), and phosphatidylinositol 4,5-bisphosphate (PIP2), to modulate N-type voltage-gated Ca(2+) (CaV2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which GqPCRs inhibit CaV2.2 channel current are not completely understood. Here, we report that the location of CaV β subunits is key to determining the voltage dependence of CaV2.2 channel modulation by GqPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M1 receptors, together with CaV N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for CaV2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of CaV2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of CaV2.2 current after M1 receptor activation was markedly reduced and delayed, whereas the delay between PIP2 hydrolysis and inhibition of CaV2.2 current was decreased. When the Gβγ-insensitive CaV2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M1 receptors act through Gβγ to inhibit CaV2.2 channels bearing membrane-localized CaV β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylation-negative mutant β2a(C3,4S), reduced the voltage dependence of M1 muscarinic inhibition of CaV2.2 channels, whereas it increased inhibition mediated by PIP2 depletion. Together, our results indicate that, with membrane-localized CaV β subunits, CaV2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP2-mediated voltage-independent regulation. Thus, the voltage dependence of

  17. Evidence for functional interaction of plasma membrane electron transport, voltage-dependent anion channel and volume-regulated anion channel in frog aorta

    Indian Academy of Sciences (India)

    Rashmi P Rao; J Prakasa Rao

    2010-12-01

    Frog aortic tissue exhibits plasma membrane electron transport (PMET) owing to its ability to reduce ferricyanide even in the presence of mitochondrial poisons, such as cyanide and azide. Exposure to hypotonic solution (108 mOsmol/kg H2O) enhanced the reduction of ferricyanide in excised aortic tissue of frog. Increment in ferricyanide reductase activity was also brought about by the presence of homocysteine (100 M dissolved in isotonic frog Ringer solution), a redox active compound and a potent modulator of PMET. Two plasma-membrane-bound channels, the volume regulated anion channel (VRAC) and the voltage-dependent anion channel (VDAC), are involved in the response to hypotonic stress. The presence of VRAC and VDAC antagonists–tamoxifen, glibenclamide, fluoxetine and verapamil, and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), respectively–inhibited this enhanced activity brought about by either hypotonic stress or homocysteine. The blockers do not affect the ferricyanide reductase activity under isotonic conditions. Taken together, these findings indicate a functional interaction of the three plasma membrane proteins, namely, ferricyanide reductase (PMET), VDAC and VRAC.

  18. Charged residues distribution modulates selectivity of the open state of human isoforms of the voltage dependent anion-selective channel.

    Science.gov (United States)

    Amodeo, Giuseppe Federico; Scorciapino, Mariano Andrea; Messina, Angela; De Pinto, Vito; Ceccarelli, Matteo

    2014-01-01

    Voltage Dependent Anion-selective Channels (VDACs) are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs) Molecular Dynamics (MD) simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10' to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities.

  19. Voltage-dependent ionic channels in differentiating neural precursor cells collected from adult mouse brains six hours post-mortem.

    Science.gov (United States)

    Bellardita, Carmelo; Bolzoni, Francesco; Sorosina, Melissa; Marfia, Giovanni; Carelli, Stephana; Gorio, Alfredo; Formenti, Alessandro

    2012-04-01

    A novel type of adult neural precursor cells (NPCs) has been isolated from the subventricular zone of the mouse 6 hr after animal death (T6-NPCs). This condition is supposed to select hypoxia-resistant cells of scientific and clinical interest. Ionic channels are ultimately the expression of the functional maturation of neurons, so the aim of this research was to characterize the pattern of the main voltage-dependent ionic channels in T6-NPCs differentiating to a neuronal phenotype, comparing it with NPCs isolated soon after death (T0-NPCs). T6- and T0-NPCs grow in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Differentiation was performed in small wells without the addition of growth factors, in the presence of adhesion molecules, fetal bovine serum, and leukemia inhibitory factor. Ionic currents, recorded by means of whole-cell patch-clamp, namely, I(Ca2+) HVA, both L- and non-L-type, I(K+) delayed rectifying, I(K+) inward rectifier, transient I(K+A) , and TTX-sensitive I(Na+) have been found, although Na(+) currents were found in only a small percentage of cells and after the fifth week of differentiation. No significant differences in current types, density, orcell capacitance were observed between T6-NPCs and T0-NPCs. The sequence in which the markers appear in new neural cells is not necessarily a fixed program, but the discrepancies in morphological, biochemical, and electrophysiological maturation of mouse NPCs to neurons, possibly different in vivo, suggest that the various steps of the differentiation are independently regulated. Therefore, in addition to morphological and biochemical data, functional tests should be considered for characterizing the maturation of neurons.

  20. The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy.

    Science.gov (United States)

    Smilansky, Angela; Dangoor, Liron; Nakdimon, Itay; Ben-Hail, Danya; Mizrachi, Dario; Shoshan-Barmatz, Varda

    2015-12-25

    The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼ 2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.

  1. Voltage-Dependent Anion Channel 1(VDAC1) Participates the Apoptosis of the Mitochondrial Dysfunction in Desminopathy

    Science.gov (United States)

    Mo, Yanqing; Gong, Qi; Jiang, Aihua; Zhao, Jing

    2016-01-01

    Desminopathies caused by the mutation in the gene coding for desmin are genetically protein aggregation myopathies. Mitochondrial dysfunction is one of pathological changes in the desminopathies at the earliest stage. The molecular mechanisms of mitochondria dysfunction in desminopathies remain exclusive. VDAC1 regulates mitochondrial uptake across the outer membrane and mitochondrial outer membrane permeabilization (MOMP). Relationships between desminopathies and Voltage-dependent anion channel 1 (VDAC1) remain unclear. Here we successfully constructed the desminopathy rat model, evaluated with conventional stains, containing hematoxylin and eosin (HE), Gomori Trichrome (MGT), (PAS), red oil (ORO), NADH-TR, SDH staining and immunohistochemistry. Immunofluorescence results showed that VDAC1 was accumulated in the desmin highly stained area of muscle fibers of desminopathy patients or desminopathy rat model compared to the normal ones. Meanwhile apoptosis related proteins bax and ATF2 were involved in desminopathy patients and desminopathy rat model, but not bcl-2, bcl-xl or HK2.VDAC1 and desmin are closely relevant in the tissue splices of deminopathies patients and rats with desminopathy at protein lever. Moreover, apoptotic proteins are also involved in the desminopathies, like bax, ATF2, but not bcl-2, bcl-xl or HK2. This pathological analysis presents the correlation between VDAC1 and desmin, and apoptosis related proteins are correlated in the desminopathy. Furthermore, we provide a rat model of desminopathy for the investigation of desmin related myopathy. PMID:27941998

  2. Frequency-dependent reliability of spike propagation is function of axonal voltage-gated sodium channels in cerebellar Purkinje cells.

    Science.gov (United States)

    Yang, Zhilai; Wang, Jin-Hui

    2013-12-01

    The spike propagation on nerve axons, like synaptic transmission, is essential to ensure neuronal communication. The secure propagation of sequential spikes toward axonal terminals has been challenged in the neurons with a high firing rate, such as cerebellar Purkinje cells. The shortfall of spike propagation makes some digital spikes disappearing at axonal terminals, such that the elucidation of the mechanisms underlying spike propagation reliability is crucial to find the strategy of preventing loss of neuronal codes. As the spike propagation failure is influenced by the membrane potentials, this process is likely caused by altering the functional status of voltage-gated sodium channels (VGSC). We examined this hypothesis in Purkinje cells by using pair-recordings at their somata and axonal blebs in cerebellar slices. The reliability of spike propagation was deteriorated by elevating spike frequency. The frequency-dependent reliability of spike propagation was attenuated by inactivating VGSCs and improved by removing their inactivation. Thus, the functional status of axonal VGSCs influences the reliability of spike propagation.

  3. Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells: regulation by free tubulin and erastin.

    Science.gov (United States)

    Maldonado, Eduardo N; Sheldon, Kely L; DeHart, David N; Patnaik, Jyoti; Manevich, Yefim; Townsend, Danyelle M; Bezrukov, Sergey M; Rostovtseva, Tatiana K; Lemasters, John J

    2013-04-26

    Respiratory substrates and adenine nucleotides cross the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC), comprising three isoforms--VDAC1, 2, and 3. We characterized the role of individual isoforms in mitochondrial metabolism by HepG2 human hepatoma cells using siRNA. With VDAC3 to the greatest extent, all VDAC isoforms contributed to the maintenance of mitochondrial membrane potential, but only VDAC3 knockdown decreased ATP, ADP, NAD(P)H, and mitochondrial redox state. Cells expressing predominantly VDAC3 were least sensitive to depolarization induced by increased free tubulin. In planar lipid bilayers, free tubulin inhibited VDAC1 and VDAC2 but not VDAC3. Erastin, a compound that interacts with VDAC, blocked and reversed mitochondrial depolarization after microtubule destabilizers in intact cells and antagonized tubulin-induced VDAC blockage in planar bilayers. In conclusion, free tubulin inhibits VDAC1/2 and limits mitochondrial metabolism in HepG2 cells, contributing to the Warburg phenomenon. Reversal of tubulin-VDAC interaction by erastin antagonizes Warburg metabolism and restores oxidative mitochondrial metabolism.

  4. Altered ischemic cerebral injury in mice lacking αIE subunit of the voltage-dependent Ca2+ channel

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective ①To set up a stable and reproducible focal cerebral infarct modelin mice; (②To examine theinvolvement of αIE subunit of voltage-dependent Ca2 + channel in cerebral ischemic injury. Methods Male C57BL/6J Jclmice 8 ~ 12w and F4 ~ F6αIE subunit of Ca2+ channel mutant mice were both used in this study. All animals were allowedto freely access to food and water before and after operation. Animals were anesthetized with pentobarbital sodium 60mg/kg,ip. Rectal temperature was continuously monitored before, during and after operation, and maintained at (36.6 +0.1 )°C by a autoregulating pad. To produce pilot models, the middle cerebral artery (MCA) was occluded either by sur-gical ligation or electrical coagulation and in some models the common carotid artery (CCA) was surgically ligated in tan-dem. In our latter work the MCA was cut off soon after it was ligated or coagulated in order to make sure that the bloodflow was occluded completely. The MCA was coagulated or ligated with a bipolar coagulator or microsurgery suture at thesite just superior to the rhinal fissure. Twenty~four hours after the operation, the mice were anesthetized and decapitated,then their brains were dissected from the skull and put into cold artificial brain spinal fluid as soon as possible. Lmm thickcoronal sections were cut by vibratome and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for30min. Every section was photographed positively and the whole infarction volume was calculated by summing up the in-farction volumes of all sections by NIH Image System. Infarction ratio ( % ) was also calculated by the following fommula:(contralateral volume-ipsilateral undamaged volume)/contralateral volume × 100% to eliminate the influence of edema.In brief, the mutant mice were produced with gene targeting technique. F4 ~ F6 mice were used in this experiment. Alloffsprings were genotyped by the polymerase chain reaction (PCR) and the genotypes remained umknown

  5. Effects of in vitro and in vivo lead exposure on voltage-dependent calcium channels in central neurons of Lymnaea stagnalis.

    Science.gov (United States)

    Audesirk, G

    1987-01-01

    Currents through calcium channels of members of an identified cluster of neurons (B cells) in the pond snail Lymnaea stagnalis were studied under voltage clamp. The normal physiological saline was modified to maximize the visibility of voltage-dependent calcium currents and minimize contamination by other currents. Barium was used as the charge carrier for the calcium channels. Depolarizing voltage steps induce an inward current, the magnitude of which varies with the barium concentration. In brains taken from animals not exposed in vivo to lead, in vitro addition of lead acetate to the recording medium (0.25 to 14 microM) inhibits the barium current by 59 +/- 14% (mean +/- s.d.), in a manner that is independent of the lead concentration. The magnitude of the residual current still varies with the barium concentration. The voltage dependence of the current appears to be unaffected by lead. In contrast to some other calcium-channel blockers, such as cobalt, the inhibition of barium currents by in vitro lead exposure is irreversible, at least in short-term experiments. Contrary to expectations based on these in vitro results, barium currents in B cells of animals exposed to 5 microM lead for 6 to 12 weeks in vivo were approximately twice as large as barium currents in B cells from unexposed controls, when both were recorded in lead-free saline. It is possible that chronic in vivo lead exposure causes an increase in the number of calcium channels in these neurons.

  6. Involvement of presynaptic voltage-dependent Kv3 channel in endothelin-1-induced inhibition of noradrenaline release from rat gastric sympathetic nerves.

    Science.gov (United States)

    Nakamura, Kumiko; Shimizu, Takahiro; Tanaka, Kenjiro; Taniuchi, Keisuke; Yokotani, Kunihiko

    2012-11-05

    We previously reported that two types of K(+) channels, the BK type Ca(2+)-activated K(+) channel coupled with phospholipase C (PLC) and the voltage-dependent K(+) channel (Kv channel), are, respectively, involved in the prostanoid TP receptor- and muscarinic M(2) receptor-mediated inhibition of noradrenaline (NA) release from rat gastric sympathetic nerves. In the present study, therefore, we examined whether these K(+) channels are involved in endothelin-1-induced inhibition of NA release, using an isolated, vascularly perfused rat stomach. The gastric sympathetic postganglionic nerves around the left gastric artery were electrically stimulated twice at 2.5 Hz for 1 min, and endothelin-1 was added during the second stimulation. Endothelin-1 (1, 2 and 10 nM) dose-dependently inhibited gastric NA release. Endothelin-1 (2 nM)-induced inhibition of NA release was neither attenuated by PLC inhibitors [U-73122 (3 μM) and ET-18-OCH(3) (3 μM)] nor by Ca(2+)-activated K(+) channel blockers [charybdotoxin (0.1 μM) (a blocker of BK type K(+) channel) and apamin (0.3 μM) (a blocker of SK type K(+) channel)]. The endothelin-1-induced inhibitory response was also not attenuated by α-dendrotoxin (0.1 μM) (a selective inhibitor of Kv1 channel), but abolished by 4-aminopyridine (20 μM) (a selectively inhibitory dose for Kv3 channel). These results suggest the involvement of a voltage-dependent Kv3 channel in the endothelin-1-induced inhibition of NA release from the gastric sympathetic nerves in rats.

  7. Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells.

    Science.gov (United States)

    Onetti, C G; Lara, J; García, E

    1996-05-01

    Effects of membrane potential, intracellular Ca2+ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K+ ions; the unitary conductance was 112 pS in symmetrical K+, and the K+ permeability (PK) was 1.3 x 10(-13) cm x s(-1). An inward rectification was observed when intracellular K+ was reduced. Using a quasi-physiological K+ gradient, a non-linear K+ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (Po) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K+ concentrations, the channel was activated from a threshold of about -60 mV, and the activation midpoint was -2 mV. Po decreased noticeably at 50 microM internal adenosine 5'-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 microM. Internal application of 5'-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased Po. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca2+ ions (0.1 to 10 microM) was required for the activation of this channel. The glucose--sensitive K+ channel of XO neurons could be considered as a subtype of ATP-sensitive K+ channel, contributing substantially to macroscopic outward current.

  8. "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.

  9. Neuroprotective effect of interleukin-6 regulation of voltage-gated Na+ channels of cortical neurons is time- and dose-dependent

    Directory of Open Access Journals (Sweden)

    Wei Xia

    2015-01-01

    Full Text Available Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour exposure of interleukin-6 on cortical neurons at various concentrations (0.1, 1, 5 and 10 ng/mL and the effects of 10 ng/mL interleukin-6 exposure to cortical neurons for various durations (2, 4, 8, 24 and 48 hours by studying voltage-gated Na + channels using a patch-clamp technique. Voltage-clamp recording results demonstrated that interleukin-6 suppressed Na + currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of cortical neurons, but did not change the action potential threshold. The regulation of voltage-gated Na + channels in rat cortical neurons by interleukin-6 is time- and dose-dependent.

  10. Neuroprotective effect of interleukin-6 regulation of voltage-gated Na(+) channels of cortical neurons is time- and dose-dependent.

    Science.gov (United States)

    Xia, Wei; Peng, Guo-Yi; Sheng, Jiang-Tao; Zhu, Fang-Fang; Guo, Jing-Fang; Chen, Wei-Qiang

    2015-04-01

    Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour exposure of interleukin-6 on cortical neurons at various concentrations (0.1, 1, 5 and 10 ng/mL) and the effects of 10 ng/mL interleukin-6 exposure to cortical neurons for various durations (2, 4, 8, 24 and 48 hours) by studying voltage-gated Na(+) channels using a patch-clamp technique. Voltage-clamp recording results demonstrated that interleukin-6 suppressed Na(+) currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of cortical neurons, but did not change the action potential threshold. The regulation of voltage-gated Na(+) channels in rat cortical neurons by interleukin-6 is time- and dose-dependent.

  11. Beta-scorpion toxin effects suggest electrostatic interactions in domain II of voltage-dependent sodium channels. : Electrostatic interactions between segments IIS2, IIS3 and IIS4 of Na+ channel.

    OpenAIRE

    Mantegazza, Massimo; Cestèle, Sandrine

    2005-01-01

    International audience; Beta-scorpion toxins specifically modulate the voltage dependence of sodium channel activation by acting through a voltage-sensor trapping model. We used mutagenesis, functional analysis and the action of beta-toxin as tools to investigate the existence and role in channel activation of molecular interactions between the charged residues of the S2, S3 and S4 segments in domain II of sodium channels. Mutating to arginine the acidic residues of the S2 and S3 transmembran...

  12. Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1.

    Science.gov (United States)

    Budelier, Melissa M; Cheng, Wayland W L; Bergdoll, Lucie; Chen, Zi-Wei; Janetka, James W; Abramson, Jeff; Krishnan, Kathiresan; Mydock-McGrane, Laurel; Covey, Douglas F; Whitelegge, Julian P; Evers, Alex S

    2017-06-02

    Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr(83) and Glu(73), respectively. When Glu(73) was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr(62) within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu(73) residue. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Reduced KCNQ4-encoded voltage-dependent potassium channel activity underlies impaired ß-adrenoceptor-mediated relaxation of renal arteries in hypertension

    DEFF Research Database (Denmark)

    Chadha, Preet S; Zunke, Friederike; Zhu, Hai-Lei;

    2012-01-01

    KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown...... strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to ß-adrenoceptor-mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein...... spontaneously hypertensive rats, which was associated with ˜60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to ß-adrenoceptor-mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired ß...

  14. A conserved threonine in the S1-S2 loop of KV7.2 and K V7.3 channels regulates voltage-dependent activation.

    Science.gov (United States)

    Füll, Yvonne; Seebohm, Guiscard; Lerche, Holger; Maljevic, Snezana

    2013-06-01

    The voltage-gated potassium channels KV7.2 and KV7.3 (KCNQ2/3 genes) play an important role in regulating neuronal excitability. More than 50 KCNQ2/3 mutations have been identified to cause an inherited form of epilepsy in newborns. For two of those (E119G and S122L) found in the S1-S2 region of KV7.2, we previously showed a decreased channel availability mainly at action potential subthreshold voltages caused by a slight depolarizing shift of the activation curve. Interestingly, recent studies revealed that a threonine residue within the S1-S2 loop, highly conserved among different classes of KV channels, is crucial for both their function and surface expression. To investigate the functional role of the homologous threonine residues in KV7.2 (T114) and KV7.3 (T144) channels, we replaced them with alanine and examined the electrophysiological properties using heterologous expression in CHO cells and whole cell patch clamping. Channels comprising mutant subunits yielded decreased potassium currents with slowed activation and accelerated deactivation kinetics. However, the most striking effect was a depolarizing shift in the voltage dependence of activation reaching +30 mV upon co-expression of both mutant subunits. Potential interactions of T114 within the channel were analyzed by creating a 3D homology model of KV7.2 in an open state suggesting that this residue plays a central role in the formation of a stable interface between the S1-S2 and the S5 segment helices. This could be the explanation why substitution of the conserved threonine in KV7.2 and KV7.3 channels destabilizes the open and favors the closed state of these channels.

  15. Neuroprotective effect of interleukin-6 regulation of voltage-gated Na+ channels of cortical neurons is time- and dose-dependent

    Institute of Scientific and Technical Information of China (English)

    Wei Xia; Guo-yi Peng; Jiang-tao Sheng; Fang-fang Zhu; Jing-fang Guo; Wei-qiang Chen

    2015-01-01

    Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour expo-sure of interleukin-6 on cortical neurons at various concentrations (0.1, 1, 5 and 10 ng/mL) and the effects of 10 ng/mL interleukin-6 exposure to cortical neurons for various durations (2, 4, 8, 24 and 48 hours) by studying voltage-gated Na+ channels using a patch-clamp technique. Volt-age-clamp recording results demonstrated that interleukin-6 suppressed Na+ currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of cortical neurons, but did not change the action potential threshold. The regulation of voltage-gated Na+channels in rat corti-cal neurons by interleukin-6 is time- and dose-dependent.

  16. Voltage-dependent gating of hyperpolarization-activated, cyclic nucleotide-gated pacemaker channels: molecular coupling between the S4-S5 and C-linkers.

    Science.gov (United States)

    Decher, Niels; Chen, Jun; Sanguinetti, Michael C

    2004-04-02

    Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have a transmembrane topology that is highly similar to voltage-gated K(+) channels, yet HCN channels open in response to membrane hyperpolarization instead of depolarization. The structural basis for the "inverted" voltage dependence of HCN gating and how voltage sensing by the S1-S4 domains is coupled to the opening of the intracellular gate formed by the S6 domain are unknown. Coupling could arise from interaction between specific residues or entire transmembrane domains. We previously reported that the mutation of specific residues in the S4-S5 linker of HCN2 (i.e. Tyr-331 and Arg-339) prevented normal channel closure presumably by disruption of a crucial interaction with the activation gate. Here we hypothesized that the C-linker, a carboxyl terminus segment that connects S6 to the cyclic nucleotide binding domain, interacts with specific residues of the S4-S5 linker to mediate coupling. The recently solved structure of the C-linker of HCN2 indicates that an alpha-helix (the A'-helix) is located near the end of each S6 domain, the presumed location of the activation gate. Ala-scanning mutagenesis of the end of S6 and the A'-helix identified five residues that were important for normal gating as mutations disrupted channel closure. However, partial deletion of the C-linker indicated that the presence of only two of these residues was required for normal coupling. Further mutation analyses suggested that a specific electrostatic interaction between Arg-339 of the S4-S5 linker and Asp-443 of the C-linker stabilizes the closed state and thus participates in the coupling of voltage sensing and activation gating in HCN channels.

  17. An autism-associated mutation in CaV1.3 channels has opposing effects on voltage- and Ca(2+)-dependent regulation.

    Science.gov (United States)

    Limpitikul, Worawan B; Dick, Ivy E; Ben-Johny, Manu; Yue, David T

    2016-06-03

    CaV1.3 channels are a major class of L-type Ca(2+) channels which contribute to the rhythmicity of the heart and brain. In the brain, these channels are vital for excitation-transcription coupling, synaptic plasticity, and neuronal firing. Moreover, disruption of CaV1.3 function has been associated with several neurological disorders. Here, we focus on the de novo missense mutation A760G which has been linked to autism spectrum disorder (ASD). To explore the role of this mutation in ASD pathogenesis, we examined the effects of A760G on CaV1.3 channel gating and regulation. Introduction of the mutation severely diminished the Ca(2+)-dependent inactivation (CDI) of CaV1.3 channels, an important feedback system required for Ca(2+) homeostasis. This reduction in CDI was observed in two major channel splice variants, though to different extents. Using an allosteric model of channel gating, we found that the underlying mechanism of CDI reduction is likely due to enhanced channel opening within the Ca(2+)-inactivated mode. Remarkably, the A760G mutation also caused an opposite increase in voltage-dependent inactivation (VDI), resulting in a multifaceted mechanism underlying ASD. When combined, these regulatory deficits appear to increase the intracellular Ca(2+) concentration, thus potentially disrupting neuronal development and synapse formation, ultimately leading to ASD.

  18. Coexpression of voltage-dependent calcium channels Cav1.2, 2.1a, and 2.1b in vascular myocytes

    DEFF Research Database (Denmark)

    Andreasen, Ditte; Friis, Ulla Glenert; Uhrenholt, Torben Rene

    2006-01-01

    , and blocking P-type currents (omega-agatoxin IVA 10 nmol/L) led to 20.2+/-3.0% inhibition, whereas 300 nmol/L of omega agatoxin IVA (blocking P/Q-type) inhibited 45.0+/-7.3%. In rat aortic smooth muscle cells (A7r5), blockade of L-type channels resulted in 28.5+/-6.1% inhibition, simultaneous blockade of L...... microscopy revealed expression of both channels in all of the smooth muscle cells. Whole-cell patch clamp on single preglomerular VSMCs from mice showed L-, P-, and Q-type currents. Blockade of the L-type currents by calciseptine (20 nmol/L) inhibited 35.6+/-3.9% of the voltage-dependent Ca2+ current......-type and P-type channels inhibited 58.0+/-11.8%, and simultaneous inhibition of L-, P-, and Q-type channels led to blockade (88.7+/-5.6%) of the Ca2+ current. We conclude that aortic and renal preglomerular smooth muscle cells express L-, P-, and Q-type voltage-dependent Ca2+ channels in the rat and mouse....

  19. Poly(ethylene glycol-cholesterol inhibits L-type Ca2+ channel currents and augments voltage-dependent inactivation in A7r5 cells.

    Directory of Open Access Journals (Sweden)

    Rikuo Ochi

    Full Text Available Cholesterol distributes at a high density in the membrane lipid raft and modulates ion channel currents. Poly(ethylene glycol cholesteryl ether (PEG-cholesterol is a nonionic amphipathic lipid consisting of lipophilic cholesterol and covalently bound hydrophilic PEG. PEG-cholesterol is used to formulate lipoplexes to transfect cultured cells, and liposomes for encapsulated drug delivery. PEG-cholesterol is dissolved in the external leaflet of the lipid bilayer, and expands it to flatten the caveolae and widen the gap between the two leaflets. We studied the effect of PEG-cholesterol on whole cell L-type Ca(2+ channel currents (I(Ca,L recorded from cultured A7r5 arterial smooth muscle cells. The pretreatment of cells with PEG-cholesterol decreased the density of ICa,L and augmented the voltage-dependent inactivation with acceleration of time course of inactivation and negative shift of steady-state inactivation curve. Methyl-β-cyclodextrin (MβCD is a cholesterol-binding oligosaccharide. The enrichment of cholesterol by the MβCD:cholesterol complex (cholesterol (MβCD caused inhibition of I(Ca,L but did not augment voltage-dependent inactivation. Incubation with MβCD increased I(Ca,L, slowed the time course of inactivation and shifted the inactivation curve to a positive direction. Additional pretreatment by a high concentration of MβCD of the cells initially pretreated with PEG-cholesterol, increased I(Ca,L to a greater level than the control, and removed the augmented voltage-dependent inactivation. Due to the enhancement of the voltage-dependent inactivation, PEG-cholesterol inhibited window I(Ca,L more strongly as compared with cholesterol (MβCD. Poly(ethylene glycol conferred to cholesterol the efficacy to induce sustained augmentation of voltage-dependent inactivation of I(Ca,L.

  20. Structural mapping of the voltage-dependent sodium channel. Distance between the tetrodotoxin and Centruroides suffusus suffusus II beta-scorpion toxin receptors.

    Science.gov (United States)

    Darbon, H; Angelides, K J

    1984-05-25

    A 7- dimethylaminocoumarin -4-acetate fluorescent derivative of toxin II from the venom of the scorpion Centruroides suffusus suffusus (Css II) has been prepared to study the structural, conformational, and cellular properties of the beta-neurotoxin receptor site on the voltage-dependent sodium channel. The derivative retains high affinity for its receptor site on the synaptosomal sodium channel with a KD of 7 nM and site capacity of 1.5 pmol/mg of synaptosomal protein. The fluorescent toxin is very environmentally sensitive and the fluorescence emission upon binding indicates that the Css II receptor is largely hydrophobic. Binding of tetrodotoxin or batrachotoxin does not alter the spectroscopic properties of bound Css II, whereas toxin V from Leiurus quinquestriatus effects a 10-nm blue shift to a more hydrophobic environment. This is the first direct indication of conformational coupling between these separate neurotoxin receptor sites. The distance between the tetrodotoxin and Css II scorpion toxin receptors on the sodium channel was measured by fluorescence resonance energy transfer. Efficiencies were measured by both donor quenching and acceptor-sensitized emission. The distance between these two neurotoxin sites is about 34 A. The implications of these receptor locations together with other known molecular distances are discussed in terms of a molecular structure of the voltage-dependent sodium channel.

  1. Flow- and voltage-dependent blocking effect of ethosuximide on the inward rectifier K⁺ (Kir2.1) channel.

    Science.gov (United States)

    Huang, Chiung-Wei; Kuo, Chung-Chin

    2015-08-01

    Absence seizures are manifestations of abnormal thalamocortical oscillations characterized by spike-and-wave complexes in EEG. Ethosuximide (ETX) is one of the principal medications against absence seizures. We investigate the effect of ETX on the Kir2.1 channel, a prototypical inward rectifier K(+) channel possibly playing an important role in the setting of neuronal membrane potential. We demonstrate that the outward currents of Kir2.1 channels are significantly inhibited by intracellular ETX. We further show that the movement of neutral molecule ETX in the Kir2.1 channel is accompanied by ∼1.2 K(+), giving rise to the vivid voltage dependence of ETX unbinding rate. Moreover, the apparent affinity (K d ) of ETX in the channels are decreased by single-point mutations involving M183, E224, and S165, and especially by double mutations involving T141/S165, which always also disrupt the flux-coupling feature of ETX block. Molecular dynamics simulation demonstrates narrowing of the pore at ∼D172 by binding of ETX to S165 or T141. ETX block of the Kir2.1 channels may cause a modest but critical depolarization of the relevant neurons, decreasing available T-type Ca(2+) channels and consequently lessening pathological thalamocortical burst discharges.

  2. Ropivacaine-Induced Contraction Is Attenuated by Both Endothelial Nitric Oxide and Voltage-Dependent Potassium Channels in Isolated Rat Aortae

    Directory of Open Access Journals (Sweden)

    Seong-Ho Ok

    2013-01-01

    Full Text Available This study investigated endothelium-derived vasodilators and potassium channels involved in the modulation of ropivacaine-induced contraction. In endothelium-intact rat aortae, ropivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: the nonspecific nitric oxide synthase (NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, the neuronal NOS inhibitor Nω-propyl-L-arginine hydrochloride, the inducible NOS inhibitor 1400W dihydrochloride, the nitric oxide-sensitive guanylyl cyclase (GC inhibitor ODQ, the NOS and GC inhibitor methylene blue, the phosphoinositide-3 kinase inhibitor wortmannin, the cytochrome p450 epoxygenase inhibitor fluconazole, the voltage-dependent potassium channel inhibitor 4-aminopyridine (4-AP, the calcium-activated potassium channel inhibitor tetraethylammonium (TEA, the inward-rectifying potassium channel inhibitor barium chloride, and the ATP-sensitive potassium channel inhibitor glibenclamide. The effect of ropivacaine on endothelial nitric oxide synthase (eNOS phosphorylation in human umbilical vein endothelial cells was examined by western blotting. Ropivacaine-induced contraction was weaker in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, and methylene blue enhanced ropivacaine-induced contraction, whereas wortmannin, Nω-propyl-L-arginine hydrochloride, 1400W dihydrochloride, and fluconazole had no effect. 4-AP and TEA enhanced ropivacaine-induced contraction; however, barium chloride and glibenclamide had no effect. eNOS phosphorylation was induced by ropivacaine. These results suggest that ropivacaine-induced contraction is attenuated primarily by both endothelial nitric oxide and voltage-dependent potassium channels.

  3. Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca(2+) channels.

    Science.gov (United States)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin; Korotchenko, Svetlana; Lievens, Patricia Marie-Jeanne; Berezin, Vladimir; Dityatev, Alexander

    2015-12-01

    Our previous studies revealed that L-type voltage-dependent Ca(2+) channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular domains of Cav1.2α1 to identify putative binding sites between the channel and hyaluronic acid or another class of polyanionic glycans, such as heparin/heparan sulfates. None of the tested peptides showed detectable interaction with hyaluronic acid, but two peptides derived from the first pore-forming domain of Cav1.2α1 subunit bound to heparin. At 25 °C the binding of the peptide P7 (MGKMHKTCYN) was at ~50 μM, and that of the peptide P8 (GHGRQCQNGTVCKPGWDGPKHG) was at ~21 μM. The Cav1.2α1 first pore forming segment that contained both peptides maintained a high affinity for heparin (~23 μM), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin-agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1.2 channels revealed that enzymatic digestion of highly sulfated heparan sulfates with heparinase 1 affects neither voltage-dependence of channel activation nor the level of steady state inactivation, but did speed up channel inactivation. Treatment of hippocampal cultures with heparinase 1 reduced the firing rate and led to appearance of long-lasting bursts in the same manner as treatment with the inhibitor of L-VDCC diltiazem. Thus, heparan sulfate proteoglycans may bind to and regulate L-VDCC inactivation and network activity.

  4. The episodic ataxia type 1 mutation I262T alters voltage-dependent gating and disrupts protein biosynthesis of human Kv1.1 potassium channels.

    Science.gov (United States)

    Chen, Szu-Han; Fu, Ssu-Ju; Huang, Jing-Jia; Tang, Chih-Yung

    2016-01-18

    Voltage-gated potassium (Kv) channels are essential for setting neuronal membrane excitability. Mutations in human Kv1.1 channels are linked to episodic ataxia type 1 (EA1). The EA1-associated mutation I262T was identified from a patient with atypical phenotypes. Although a previous report has characterized its suppression effect, several key questions regarding the impact of the I262T mutation on Kv1.1 as well as other members of the Kv1 subfamily remain unanswered. Herein we show that the dominant-negative effect of I262T on Kv1.1 current expression is not reversed by co-expression with Kvβ1.1 or Kvβ2 subunits. Biochemical examinations indicate that I262T displays enhanced protein degradation and impedes membrane trafficking of Kv1.1 wild-type subunits. I262T appears to be the first EA1 mutation directly associated with impaired protein stability. Further functional analyses demonstrate that I262T changes the voltage-dependent activation and Kvβ1.1-mediated inactivation, uncouples inactivation from activation gating, and decelerates the kinetics of cumulative inactivation of Kv1.1 channels. I262T also exerts similar dominant effects on the gating of Kv1.2 and Kv1.4 channels. Together our data suggest that I262T confers altered channel gating and reduced functional expression of Kv1 channels, which may account for some of the phenotypes of the EA1 patient.

  5. 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.

  6. The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K{sup +} channels in rabbit coronary arterial smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hongliang; Hong, Da Hye; Kim, Han Sol; Kim, Hye Won [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of); Jung, Won-Kyo [Department of Biomedical Engineering, Center for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University, Busan 608-737 (Korea, Republic of); Na, Sung Hun [Institute of Medical Sciences, Department of Obstetrics and Gynecology, Kangwon National University Hospital, School of Medicine, Kangwon National University, Chuncheon, 200-701 (Korea, Republic of); Jung, In Duk; Park, Yeong-Min [Department of Immunology, Lab of Dendritic Cell Differentiation and Regulation, College of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of); Choi, Il-Whan, E-mail: cihima@inje.ac.kr [Department of Microbiology, Inje University College of Medicine, Busan, 614-735 (Korea, Republic of); Park, Won Sun, E-mail: parkws@kangwon.ac.kr [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of)

    2015-06-15

    We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K{sup +} (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC{sub 50}) value of 0.81 μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77 ± 0.04 μM{sup −1} s{sup −1} and 2.55 ± 1.50 s{sup −1}, respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2 Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition. - Highlights: • We investigated the effects of CGS 9394B on Kv channels. • CGS 9394B inhibited Kv current in a state-, time-, and use-dependent manner. • Caution is required when using CGS 9394B in vascular function studies.

  7. Voltage Dependence of Supercapacitor Capacitance

    Directory of Open Access Journals (Sweden)

    Szewczyk Arkadiusz

    2016-09-01

    Full Text Available Electronic Double-Layer Capacitors (EDLC, called Supercapacitors (SC, are electronic devices that are capable to store a relatively high amount of energy in a small volume comparing to other types of capacitors. They are composed of an activated carbon layer and electrolyte solution. The charge is stored on electrodes, forming the Helmholtz layer, and in electrolyte. The capacitance of supercapacitor is voltage- dependent. We propose an experimental method, based on monitoring of charging and discharging a supercapacitor, which enables to evaluate the charge in an SC structure as well as the Capacitance-Voltage (C-V dependence. The measurement setup, method and experimental results of charging/discharging commercially available supercapacitors in various voltage and current conditions are presented. The total charge stored in an SC structure is proportional to the square of voltage at SC electrodes while the charge on electrodes increases linearly with the voltage on SC electrodes. The Helmholtz capacitance increases linearly with the voltage bias while a sublinear increase of total capacitance was found. The voltage on SC increases after the discharge of electrodes due to diffusion of charges from the electrolyte to the electrodes. We have found that the recovery voltage value is linearly proportional to the initial bias voltage value.

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

    Directory of Open Access Journals (Sweden)

    Samuel eGoodchild

    2012-06-01

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

  9. Altered calcium homeostasis in motor neurons following AMPA receptor but not voltage-dependent calcium channels' activation in a genetic model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Guatteo, Ezia; Carunchio, Irene; Pieri, Massimo; Albo, Federica; Canu, Nadia; Mercuri, Nicola B; Zona, Cristina

    2007-10-01

    Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disease characterized by a substantial loss of motor neurons in the spinal cord, brain stem and motor cortex. By combining electrophysiological recordings with imaging techniques, clearance/buffering capacity of cultured spinal cord motor neurons after a calcium accumulation has been analyzed in response to AMPA receptors' (AMPARs') activation and to depolarizing stimuli in a genetic mouse model of ALS (G93A). Our studies demonstrate that the amplitude of the calcium signal in response to AMPARs' or voltage-dependent calcium channels' activation is not significantly different in controls and G93A motor neurons. On the contrary, in G93A motor neurons, the [Ca(2+)](i) recovery to basal level is significantly slower compared to control neurons following AMPARs but not voltage-dependent calcium channels' activation. This difference was not observed in G93A cultured cortical neurons. This observation is the first to indicate a specific alteration of the calcium clearance linked to AMPA receptors' activation in G93A motor neurons and the involvement of AMPA receptor regulatory proteins controlling both AMPA receptor functionality and the sequence of events connected to them.

  10. 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.

  11. Reduced KCNQ4-encoded voltage-dependent potassium channel activity underlies impaired β-adrenoceptor-mediated relaxation of renal arteries in hypertension.

    Science.gov (United States)

    Chadha, Preet S; Zunke, Friederike; Zhu, Hai-Lei; Davis, Alison J; Jepps, Thomas A; Olesen, Søren P; Cole, William C; Moffatt, James D; Greenwood, Iain A

    2012-04-01

    KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to β-adrenoceptor-mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein expression of KCNQ1, KCNQ3, KCNQ4, KCNQ5, and Kv7.1, Kv7.4, and Kv7.5 in rat renal artery. Isoproterenol produced concentration-dependent relaxation of precontracted renal arteries and increased Kv7 channel currents in isolated smooth muscle cells. Application of the Kv7 blocker linopirdine attenuated isoproterenol-induced relaxation and current. Isoproterenol-induced relaxations were also reduced in arteries incubated with small interference RNAs targeted to KCNQ4 that produced a ≈60% decrease in Kv7.4 protein level. Relaxation to isoproterenol and the Kv7 activator S-1 were abolished in arteries from spontaneously hypertensive rats, which was associated with ≈60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to β-adrenoceptor-mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired β-adrenoceptor pathway in spontaneously hypertensive rats. These findings may provide a novel pathogenic link between arterial dysfunction and hypertension.

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

    Science.gov (United States)

    Guidelli, Rolando; Becucci, Lucia

    2016-04-01

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

  13. Potentiation of prolactin secretion following lactotrope escape from dopamine action. II. Phosphorylation of the alpha(1) subunit of L-type, voltage-dependent calcium channels.

    Science.gov (United States)

    Hernández, M E; del Mar Hernández, M; Díaz-Muñoz, M; Clapp, C; de la Escalera, G M

    1999-07-01

    Modulation of Ca(2+) channels has been shown to alter cellular functions. It can play an important role in the amplification of signals in the endocrine system, including the pleiotropically regulated pituitary lactotropes. Prolactin (PRL) secretion is tonically inhibited by dopamine (DA), the escape from which triggers acute episodes of hormone secretion. The magnitude of these episodes is explained by a potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). While the mechanisms of this potentiation are not fully understood, it is known to be mimicked by the dihydropyridine, L-type Ca(2+) channel agonist Bay K 8644 and blocked by nifedipine and methoxyverapamil. The potentiation is also blocked by inhibitors of cyclic AMP-dependent protein kinase and protein kinase C. We recently described that the escape from tonic actions of DA results in increased macroscopic Ca(2+) currents in GH(4)C(1) lactotropic clonal cells transfected with a cDNA encoding the long form of the human D(2)-DA receptor. Here we show that the withdrawal from DA potentiates the PRL-releasing action of TRH in GH(4)C(1)/D(2)-DAR cells to the same extent as in enriched lactotropes in primary culture. In both experimental models a low density of dihydropyridine receptors was shown by (+)-[(3)H]PN200-110 binding. Photoaffinity labelling with the dihydropyridine [(3)H]azidopine revealed a protein consistent with the alpha(1) subunit of L-type Ca(2+) channels of 165-170 kDa. In both experimental models, the facilitation of TRH action triggered by the escape from DA was correlated with an enhanced rate of phosphorylation of this putative alpha(1) subunit, the nature of which was further supported by immunoprecipitation with selective antibodies directed against the alpha(1C) and alpha(1D) subunit of voltage-gated calcium channels. We propose that PKA- and PKC-dependent phosphorylation of the alpha(1) subunit of high voltage activated, L-type Ca(2

  14. α-Synuclein Shows High Affinity Interaction with Voltage-dependent Anion Channel, Suggesting Mechanisms of Mitochondrial Regulation and Toxicity in Parkinson Disease.

    Science.gov (United States)

    Rostovtseva, Tatiana K; Gurnev, Philip A; Protchenko, Olga; Hoogerheide, David P; Yap, Thai Leong; Philpott, Caroline C; Lee, Jennifer C; Bezrukov, Sergey M

    2015-07-24

    Participation of the small, intrinsically disordered protein α-synuclein (α-syn) in Parkinson disease (PD) pathogenesis has been well documented. Although recent research demonstrates the involvement of α-syn in mitochondrial dysfunction in neurodegeneration and suggests direct interaction of α-syn with mitochondria, the molecular mechanism(s) of α-syn toxicity and its effect on neuronal mitochondria remain vague. Here we report that at nanomolar concentrations, α-syn reversibly blocks the voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane that controls most of the metabolite fluxes in and out of the mitochondria. Detailed analysis of the blockage kinetics of VDAC reconstituted into planar lipid membranes suggests that α-syn is able to translocate through the channel and thus target complexes of the mitochondrial respiratory chain in the inner mitochondrial membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. The functional interactions between VDAC and α-syn, revealed by the present study, point toward the long sought after physiological and pathophysiological roles for monomeric α-syn in PD and in other α-synucleinopathies.

  15. S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK channels

    Directory of Open Access Journals (Sweden)

    Michael J Shipston

    2014-08-01

    Full Text Available Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK channels are important determinants of their (pathophysiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs and acyl thioesterases. (APTs. S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signalling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease.

  16. The non-selective voltage-activated cation channel in the human red blood cell membrane: reconciliation between two conflicting reports and further characterisation

    DEFF Research Database (Denmark)

    Kaestner, Lars; Christophersen, Palle; Bernhardt, Ingolf;

    2000-01-01

    Erythrocyte; Patch-clamp; Non-specific; cation channel; Voltage dependence; Acetylcholin receptor......Erythrocyte; Patch-clamp; Non-specific; cation channel; Voltage dependence; Acetylcholin receptor...

  17. Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca²⁺ channels.

    Science.gov (United States)

    Takada, Yoshinori; Hirano, Mitsuru; Kiyonaka, Shigeki; Ueda, Yoshifumi; Yamaguchi, Kazuma; Nakahara, Keiko; Mori, Masayuki X; Mori, Yasuo

    2015-09-01

    Autism is a neurodevelopmental psychiatric disorder characterized by impaired reciprocal social interaction, disrupted communication, and restricted and stereotyped patterns of interests. Autism is known to have a strong genetic component. Although mutations in several genes account for only a small proportion of individuals with autism, they provide insight into potential biological mechanisms that underlie autism, such as dysfunction in Ca(2+) signaling, synaptic dysfunction, and abnormal brain connectivity. In autism patients, two mutations have been reported in the Rab3 interacting molecule 3 (RIM3) gene. We have previously demonstrated that RIM3 physically and functionally interacts with voltage-dependent Ca(2+) channels (VDCCs) expressed in neurons via the β subunits, and increases neurotransmitter release. Here, by introducing corresponding autism-associated mutations that replace glutamic acid residue 176 with alanine (E176A) and methionine residue 259 with valine (M259V) into the C2B domain of mouse RIM3, we demonstrate that both mutations partly cancel the suppressive RIM3 effect on voltage-dependent inactivation of Ba(2+) currents through P/Q-type CaV2.1 recombinantly expressed in HEK293 cells. In recombinant N-type CaV2.2 VDCCs, the attenuation of the suppressive RIM3 effect on voltage-dependent inactivation is conserved for M259V but not E176A. Slowing of activation speed of P/Q-type CaV2.1 currents by RIM3 is abolished in E176A, while the physical interaction between RIM3 and β subunits is significantly attenuated in M259V. Moreover, increases by RIM3 in depolarization-induced Ca(2+) influx and acetylcholine release are significantly attenuated by E176A in rat pheochromocytoma PC12 cells. Thus, our data raise the interesting possibility that autism phenotypes are elicited by synaptic dysfunction via altered regulation of presynaptic VDCC function and neurotransmitter release.

  18. The N-Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities.

    Science.gov (United States)

    Guardiani, Carlo; Scorciapino, Mariano Andrea; Amodeo, Giuseppe Federico; Grdadolnik, Joze; Pappalardo, Giuseppe; De Pinto, Vito; Ceccarelli, Matteo; Casu, Mariano

    2015-09-15

    The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms.

  19. 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

  20. Voltage-dependent BK and Hv1 channels expressed in non-excitable tissues: New therapeutics opportunities as targets in human diseases.

    Science.gov (United States)

    Morera, Francisco J; Saravia, Julia; Pontigo, Juan Pablo; Vargas-Chacoff, Luis; Contreras, Gustavo F; Pupo, Amaury; Lorenzo, Yenisleidy; Castillo, Karen; Tilegenova, Cholpon; Cuello, Luis G; Gonzalez, Carlos

    2015-11-01

    Voltage-gated ion channels are the molecular determinants of cellular excitability. This group of ion channels is one of the most important pharmacological targets in excitable tissues such as nervous system, cardiac and skeletal muscle. Moreover, voltage-gated ion channels are expressed in non-excitable cells, where they mediate key cellular functions through intracellular biochemical mechanisms rather than rapid electrical signaling. This review aims at illustrating the pharmacological impact of these ion channels, highlighting in particular the structural details and physiological functions of two of them - the high conductance voltage- and Ca(2+)-gated K(+) (BK) channels and voltage-gated proton (Hv1) channels- in non-excitable cells. BK channels have been implicated in a variety of physiological processes ranging from regulation of smooth muscle tone to modulation of hormone and neurotransmitter release. Interestingly, BK channels are also involved in modulating K(+) transport in the mammalian kidney and colon epithelium with a potential role in the hyperkalemic phenotype observed in patients with familial hyperkalemic hypertension type 2, and in the pathophysiology of hypertension. In addition, BK channels are responsible for resting and stimulated Ca(2+)-activated K(+) secretion in the distal colon. Hv1 channels have been detected in many cell types, including macrophages, blood cells, lung epithelia, skeletal muscle and microglia. These channels have a central role in the phagocytic system. In macrophages, Hv1 channels participate in the generation of reactive oxygen species in the respiratory burst during the process of phagocytosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Voltage-Dependent Anion Channel 2 of Arabidopsis thaliana (AtVDAC2 Is Involved in ABA-Mediated Early Seedling Development

    Directory of Open Access Journals (Sweden)

    Xufeng Li

    2009-05-01

    Full Text Available The voltage-dependent anion channel (VDAC is the major transport protein in the outer membrane of mitochondria and plays crucial roles in energy metabolism, apoptosis, and metabolites transport. In plants, the expression of VDACs can be affected by different stresses, including drought, salinity and pathogen defense. In this study, we investigated the expression pattern of AtVDAC2 in A. thaliana and found ABA suppressed the accumulation of AtVDAC2 transcripts. Further, phenotype analysis of this VDAC deregulated-expression transgenic Arabidopsis plants indicated that AtVDAC2 anti-sense line showed an ABA-insensitivity phenotype during the early seedling development under ABA treatment. The results suggested that AtVDAC2 might be involved in ABA signaling in A. thaliana.

  2. Quantum entanglement in the voltage dependent sodium channel can reproduce the salient features of neuronal action potential initiation

    CERN Document Server

    Summhammer, Johann

    2007-01-01

    We investigate the effects of a quantum entanglement regime within an ion conducting molecule (ion channel) of the neuronal plasma membrane on the onset dynamics of propagating nerve pulses (action potentials). In particular, we model the onset parameters of the sodium current in the Hodgkin Huxley equation as three similar but independent probabilistic mechanisms which become quantum entangled. The underlying physics is general and can involve entanglement between various degrees of freedom underlaying ion transition states or 'gating states' during conduction, e.g. Na$^+$ ions in different channel locations, or different 'affinity' states of ions with atoms lining the sub-regions of the channel protein ('filter-states'). We find that the 'quantum corrected' Hodgkin Huxley equation incorporating entangled systems states can reproduce action potential pulses with the critical onset dynamics observed recently in neocortical neurons in vivo by Naundorf et al. [Nature {\\bf 440}, 1060 (20 April 2006)]. Interestin...

  3. A CACNA1C variant associated with reduced voltage-dependent inactivation, increased CaV1.2 channel window current, and arrhythmogenesis.

    Directory of Open Access Journals (Sweden)

    Jessica A Hennessey

    Full Text Available Mutations in CACNA1C that increase current through the CaV1.2 L-type Ca2+ channel underlie rare forms of long QT syndrome (LQTS, and Timothy syndrome (TS. We identified a variant in CACNA1C in a male child of Filipino descent with arrhythmias and extracardiac features by candidate gene sequencing and performed functional expression studies to electrophysiologically characterize the effects of the variant on CaV1.2 channels. As a baby, the subject developed seizures and displayed developmental delays at 30 months of age. At age 5 years, he displayed a QTc of 520 ms and experienced recurrent VT. Physical exam at 17 years of age was notable for microcephaly, short stature, lower extremity weakness and atrophy with hyperreflexia, spastic diplegia, multiple dental caries and episodes of rhabdomyolysis. Candidate gene sequencing identified a G>C transversion at position 5731 of CACNA1C (rs374528680 predicting a glycine>arginine substitution at residue 1911 (p.G1911R of CaV1.2. The allele frequency of this variant is 0.01 in Malays, but absent in 984 Caucasian alleles and in the 1000 genomes project. In electrophysiological analyses, the variant decreased voltage-dependent inactivation, thus causing a gain of function of CaV1.2. We also observed a negative shift of V1/2 of activation and positive shift of V1/2 of channel inactivation, resulting in an increase of the window current. Together, these suggest a gain-of-function effect on CaV1.2 and suggest increased susceptibility for arrhythmias in certain clinical settings. The p.G1911R variant was also identified in a case of sudden unexplained infant death (SUID, for which an increasing number of clinical observations have demonstrated can be associated with arrhythmogenic mutations in cardiac ion channels. In summary, the combined effects of the CACNA1C variant to diminish voltage-dependent inactivation of CaV1.2 and increase window current expand our appreciation of mechanisms by which a gain of

  4. Alterations of voltage-dependent calcium channel currents in basilar artery smooth muscle cells at early stage of subarachnoid hemorrhage in a rabbit model.

    Directory of Open Access Journals (Sweden)

    Xianqing Shi

    Full Text Available OBJECTIVE: To investigate the changes in the currents of voltage-dependent calcium channels (VDCCs in smooth muscle cells of basilar artery in a rabbit model of subarachnoid hemorrhage (SAH. METHODS: New Zealand white rabbits were randomly divided into five groups: sham (C, normal (N, 24 hours (S1, 48 hours (S2 and 72 hours (S3 after SAH. Non-heparinized autologous arterial blood (1 ml/kg was injected into the cisterna magna to create SAH after intravenous anesthesia, and 1 ml/kg of saline was injected into cisterna magna in the sham group. Rabbits in group N received no injections. Basilar artery in S1, S2, S3 group were isolated at 24, 48, 72 hours after SAH. Basilar artery in group C was isolated at 72 hours after physiological saline injection. Basilar artery smooth muscle cells were isolated for all groups. Whole-cell patch-clamp technique was utilized to record cell membrane capacitance and VDCCs currents. The VDCCs antagonist nifedipine was added to the bath solution to block the Ca(++ channels currents. RESULTS: There were no significant differences in the number of cells isolated, the cell size and membrane capacitance among all the five groups. VDCC currents in the S1-S3 groups had higher amplitudes than those in control and sham groups. The significant change of current amplitude was observed at 72 hours after SAH, which was higher than those of 24 and 48 hours. The VDCCs were shown to expression in human artery smooth muscle cells. CONCLUSIONS: The changes of activation characteristics and voltage-current relationship at 72 hours after SAH might be an important event which leads to a series of molecular events in the microenvironment of the basilar artery smooth muscle cells. This may be the key time point for potential therapeutic intervention against subarachnoid hemorrhage.

  5. 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....... The physiological significance of proper Kv channel localization is emphasized by the fact that defects in the trafficking of Kv channels are observed in several neurological disorders including epilepsy. In this review, we will summarize the current understanding of the mechanisms of Kv channel trafficking...... is regulated by voltage-gated potassium (Kv) channels, such as Kv4.2, which are specifically localized in the dendritic membrane. The synaptic potentials eventually depolarize the membrane of the axon initial segment, thereby activating voltage-gated sodium channels to generate action potentials. Specific Kv...

  6. Identification of phosphorylation sites for adenosine 3',5'-cyclic phosphate dependent protein kinase on the voltage-sensitive sodium channel from Electrophorus electricus.

    Science.gov (United States)

    Emerick, M C; Agnew, W S

    1989-10-17

    The voltage-sensitive sodium channel from the electroplax of Electrophorus electricus is selectively phosphorylated by the catalytic subunit of cyclic-AMP-dependent protein kinase (protein kinase A) but not by protein kinase C. Under identical limiting conditions, the protein was phosphorylated 20% as rapidly as the synthetic model substrate kemptamide. A maximum of 1.7 +/- 0.6 equiv of phosphate is incorporated per mole. Phosphoamino acid analysis revealed labeled phosphoserine and phosphothreonine at a constant ratio of 3.3:1. Seven distinct phosphopeptides were identified among tryptic fragments prepared from radiolabeled, affinity-purified protein and resolved by HPLC. The three most rapidly labeled fragments were further purified and sequenced. Four phosphorylated amino acids were identified deriving from three consensus phosphorylation sites. These were serine 6, serine 7, and threonine 17 from the amino terminus and a residue within 47 amino acids of the carboxyl terminus, apparently serine 1776. The alpha-subunits of brain sodium channels, like the electroplax protein, are readily phosphorylated by protein kinase A. However, these are also phosphorylated by protein kinase C and exhibit a markedly different pattern of incorporation. Each of three brain alpha-subunits displays an approximately 200 amino acid segment between homologous repeat domains I and II, which is missing from the electroplax and skeletal muscle proteins [Noda et al. (1986) Nature (London) 320, 188; Kayano et al. (1988) FEBS Lett. 228, 1878; Trimmer et al. (1989) Neuron 3, 33]. Most of the phosphorylation of the brain proteins occurs on a cluster of consensus phosphorylation sites located in this segment. This contrasts with the pattern of highly active sites on the amino and carboxyl termini of the electroplax protein. The detection of seven labeled tryptic phosphopeptides compared to the maximal labeling stoichiometry of approximately 2 suggests that many of the acceptor sites on the

  7. 线粒体电压依赖性阴离子通道与心血管疾病%Voltage-dependent Anion Channel and Cardiovascular Diseases

    Institute of Scientific and Technical Information of China (English)

    夏晶

    2013-01-01

    电压依赖性阴离子通道(VDAC)是位于线粒体外膜的通道蛋白,是线粒体与细胞质之间转运ATP以及其他代谢产物的主要通道,在线粒体代谢和细胞生长中发挥重要调控作用.近期研究发现,在心肌缺血再灌、糖尿病、心衰、高血压和动脉粥样硬化时,VDAC表达明显增加,引起细胞内钙离子循环紊乱、氧化应激,进而导致细胞凋亡,已成为心血管疾病研究的新热点.本文就VDAC的分子功能,调控及其在心血管疾病中的作用和相关机制进行综述.%The voltage-dependent anion channel (VDAC),a mitochondrial membrane channel protein located in the outer of mitochondrial membrane,is the main pathway between mitochondria and cytoplasm exchanging ADP,ATP,and other metabolites,and plays an important role in mitochondrial metabolism and cell growth.A growing evidence showed that VDAC was increased in cardiovascular diseases including myocardial ischemia and reperfusion,diabetes,heart failure,hypertension and atherosclerosis.The abnormal state of VDAC will result in cell death by inducing calcium cycling dysfunction and oxidative stress.And VDAC has become a hot topic in the field of cardiovascular diseases research.In this article,we will introduce the molecular function and regulation of VDAC and its role in cardiovascular diseases.

  8. Transcriptional upregulation of α2δ-1 elevates arterial smooth muscle cell voltage-dependent Ca2+ channel surface expression and cerebrovascular constriction in genetic hypertension.

    Science.gov (United States)

    Bannister, John P; Bulley, Simon; Narayanan, Damodaran; Thomas-Gatewood, Candice; Luzny, Patrik; Pachuau, Judith; Jaggar, Jonathan H

    2012-10-01

    A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca2+ (CaV1.2) currents that induces pathological vasoconstriction. CaV1.2 channels are heteromeric complexes composed of a pore-forming CaV1.2α1 with auxiliary α2δ and β subunits. Molecular mechanisms that elevate CaV1.2 currents during hypertension and the potential contribution of CaV1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of α2δ subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats was associated with an unequal elevation in α2δ-1 and CaV1.2α1 mRNA and protein in cerebral artery myocytes, with α2δ-1 increasing more than CaV1.2α1. Other α2δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive spontaneously hypertensive rats displayed higher surface-localized α2δ-1 and CaV1.2α1 proteins, surface α2δ-1:CaV1.2α1 ratio, CaV1.2 current density and noninactivating current, and pressure- and depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α2δ-1 ligand, did not alter α2δ-1 or CaV1.2α1 total protein but normalized α2δ-1 and CaV1.2α1 surface expression, surface α2δ-1:CaV1.2α1, CaV1.2 current density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels. Genetic hypertension is associated with an elevation in α2δ-1 expression that promotes surface trafficking of CaV1.2 channels in cerebral artery myocytes. This leads to an increase in CaV1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that α2δ-1 targeting is a novel strategy that may be used to reverse pathological CaV1.2 channel trafficking to induce cerebrovascular dilation in hypertension.

  9. Forgetting of long-term memory requires activation of NMDA receptors, L-type voltage-dependent Ca2+ channels, and calcineurin

    Science.gov (United States)

    Sachser, Ricardo Marcelo; Santana, Fabiana; Crestani, Ana Paula; Lunardi, Paula; Pedraza, Lizeth Katherine; Quillfeldt, Jorge Alberto; Hardt, Oliver; de Oliveira Alvares, Lucas

    2016-01-01

    In the past decades, the cellular and molecular mechanisms underlying memory consolidation, reconsolidation, and extinction have been well characterized. However, the neurobiological underpinnings of forgetting processes remain to be elucidated. Here we used behavioral, pharmacological and electrophysiological approaches to explore mechanisms controlling forgetting. We found that post-acquisition chronic inhibition of the N-methyl-D-aspartate receptor (NMDAR), L-type voltage-dependent Ca2+ channel (LVDCC), and protein phosphatase calcineurin (CaN), maintains long-term object location memory that otherwise would have been forgotten. We further show that NMDAR activation is necessary to induce forgetting of object recognition memory. Studying the role of NMDAR activation in the decay of the early phase of long-term potentiation (E-LTP) in the hippocampus, we found that ifenprodil infused 30 min after LTP induction in vivo blocks the decay of CA1-evoked postsynaptic plasticity, suggesting that GluN2B-containing NMDARs activation are critical to promote LTP decay. Taken together, these findings indicate that a well-regulated forgetting process, initiated by Ca2+ influx through LVDCCs and GluN2B-NMDARs followed by CaN activation, controls the maintenance of hippocampal LTP and long-term memories over time. PMID:26947131

  10. Forgetting of long-term memory requires activation of NMDA receptors, L-type voltage-dependent Ca2+ channels, and calcineurin.

    Science.gov (United States)

    Sachser, Ricardo Marcelo; Santana, Fabiana; Crestani, Ana Paula; Lunardi, Paula; Pedraza, Lizeth Katherine; Quillfeldt, Jorge Alberto; Hardt, Oliver; Alvares, Lucas de Oliveira

    2016-03-07

    In the past decades, the cellular and molecular mechanisms underlying memory consolidation, reconsolidation, and extinction have been well characterized. However, the neurobiological underpinnings of forgetting processes remain to be elucidated. Here we used behavioral, pharmacological and electrophysiological approaches to explore mechanisms controlling forgetting. We found that post-acquisition chronic inhibition of the N-methyl-D-aspartate receptor (NMDAR), L-type voltage-dependent Ca(2+) channel (LVDCC), and protein phosphatase calcineurin (CaN), maintains long-term object location memory that otherwise would have been forgotten. We further show that NMDAR activation is necessary to induce forgetting of object recognition memory. Studying the role of NMDAR activation in the decay of the early phase of long-term potentiation (E-LTP) in the hippocampus, we found that ifenprodil infused 30 min after LTP induction in vivo blocks the decay of CA1-evoked postsynaptic plasticity, suggesting that GluN2B-containing NMDARs activation are critical to promote LTP decay. Taken together, these findings indicate that a well-regulated forgetting process, initiated by Ca(2+) influx through LVDCCs and GluN2B-NMDARs followed by CaN activation, controls the maintenance of hippocampal LTP and long-term memories over time.

  11. Differential rescue of spatial memory deficits in aged rats by L-type voltage-dependent calcium channel and ryanodine receptor antagonism.

    Science.gov (United States)

    Hopp, S C; D'Angelo, H M; Royer, S E; Kaercher, R M; Adzovic, L; Wenk, G L

    2014-11-01

    Age-associated memory impairments may result as a consequence of neuroinflammatory induction of intracellular calcium (Ca(+2)) dysregulation. Altered L-type voltage-dependent calcium channel (L-VDCC) and ryanodine receptor (RyR) activity may underlie age-associated learning and memory impairments. Various neuroinflammatory markers are associated with increased activity of both L-VDCCs and RyRs, and increased neuroinflammation is associated with normal aging. In vitro, pharmacological blockade of L-VDCCs and RyRs has been shown to be anti-inflammatory. Here, we examined whether pharmacological blockade of L-VDCCs or RyRs with the drugs nimodipine and dantrolene, respectively, could improve spatial memory and reduce age-associated increases in microglia activation. Dantrolene and nimodipine differentially attenuated age-associated spatial memory deficits but were not anti-inflammatory in vivo. Furthermore, RyR gene expression was inversely correlated with spatial memory, highlighting the central role of Ca(+2) dysregulation in age-associated memory deficits.

  12. Cocaine disinhibits dopamine neurons in the ventral tegmental area via use-dependent blockade of GABA neuron voltage-sensitive sodium channels.

    Science.gov (United States)

    Steffensen, Scott C; Taylor, Seth R; Horton, Malia L; Barber, Elise N; Lyle, Laura T; Stobbs, Sarah H; Allison, David W

    2008-11-01

    The aim of this study was to evaluate the effects of cocaine on gamma-aminobutyric acid (GABA) and dopamine (DA) neurons in the ventral tegmental area (VTA). Utilizing single-unit recordings in vivo, microelectrophoretic administration of DA enhanced the firing rate of VTA GABA neurons via D2/D3 DA receptor activation. Lower doses of intravenous cocaine (0.25-0.5 mg/kg), or the DA transporter (DAT) blocker methamphetamine, enhanced VTA GABA neuron firing rate via D2/D3 receptor activation. Higher doses of cocaine (1.0-2.0 mg/kg) inhibited their firing rate, which was not sensitive to the D2/D3 antagonist eticlopride. The voltage-sensitive sodium channel (VSSC) blocker lidocaine inhibited the firing rate of VTA GABA neurons at all doses tested (0.25-2.0 mg/kg). Cocaine or lidocaine reduced VTA GABA neuron spike discharges induced by stimulation of the internal capsule (ICPSDs) at dose levels 0.25-2 mg/kg (IC(50) 1.2 mg/kg). There was no effect of DA or methamphetamine on ICPSDs, or of DA antagonists on cocaine inhibition of ICPSDs. In VTA GABA neurons in vitro, cocaine reduced (IC(50) 13 microm) current-evoked spikes and TTX-sensitive sodium currents in a use-dependent manner. In VTA DA neurons, cocaine reduced IPSCs (IC(50) 13 microm), increased IPSC paired-pulse facilitation and decreased spontaneous IPSC frequency, without affecting miniature IPSC frequency or amplitude. These findings suggest that cocaine acts on GABA neurons to reduce activity-dependent GABA release on DA neurons in the VTA, and that cocaine's use-dependent blockade of VTA GABA neuron VSSCs may synergize with its DAT inhibiting properties to enhance mesolimbic DA transmission implicated in cocaine reinforcement.

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

    Science.gov (United States)

    Rebolledo, Santiago; Perez, Marta E.

    2013-01-01

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

  14. 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.

  15. Inflammatory cytokine signaling in insulin producing beta-cells enhances the colocalization correlation coefficient between L-type voltage-dependent calcium channel and calcium-sensing receptor.

    Science.gov (United States)

    Parkash, Jai

    2008-08-01

    The immunological processes in type 1 diabetes and metabolic/inflammatory disorder in type 2 diabetes converge on common signaling pathway(s) leading to beta-cell death in these two diseases. The cytokine-mediated beta-cell death seems to be dependent on voltage-dependent calcium channel (VDCC)-mediated Ca2+ entry. The Ca2+ handling molecular networks control the homeostasis of [Ca2+]i in the beta-cell. The activity and membrane density of VDCC are regulated by several mechanisms including G protein-coupled receptors (GPCRs). CaR is a 123-kDa seven transmembrane extracellular Ca2+ sensing protein that belongs to GPCR family C. Tumor necrosis factor-alpha (TNF-alpha), is a cytokine widely known to activate nuclear factor-kappaB (NF-kappaB) transcription in beta-cells. To obtain a better understanding of TNF-alpha-induced molecular interactions between CaR and VDCC, confocal fluorescence measurements were performed on insulin-producing beta-cells exposed to varying concentrations of TNF-alpha and the results are discussed in the light of increased colocalization correlation coefficient. The insulin producing beta-cells were exposed to 5, 10, 20, 30, and 50 ng/ml TNF-alpha for 24 h at 37 degrees . The cells were then immunolabelled with antibodies directed against CaR, VDCC, and NF-kappaB. The confocal fluorescence imaging data showed enhancement in the colocalization correlation coefficient between CaR and VDCC in beta-cells exposed to TNF-alpha thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. TNF-alpha-induced colocalization of VDCC with CaR was inhibited by nimodipine, an inhibitor of L-type VDCC thereby suggesting that VDCC activity is required for spatial interactions with CaR. The 3-D confocal fluorescence imaging data also demonstrated that addition of TNF-alpha to RIN cells led to the translocation of NF-kappaB from the cytoplasm to the nucleus. Such molecular interactions between CaR and VDCC in tissues

  16. Uncoupling charge movement from channel opening in voltage-gated potassium channels by ruthenium complexes.

    Science.gov (United States)

    Jara-Oseguera, Andrés; Ishida, Itzel G; Rangel-Yescas, Gisela E; Espinosa-Jalapa, Noel; Pérez-Guzmán, José A; Elías-Viñas, David; Le Lagadec, Ronan; Rosenbaum, Tamara; Islas, León D

    2011-05-06

    The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K(+)-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K(+)-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling.

  17. Characterization of the Ca2+-gated and voltage-dependent K+-channel Slo-1 of nematodes and its interaction with emodepside.

    Science.gov (United States)

    Kulke, Daniel; von Samson-Himmelstjerna, Georg; Miltsch, Sandra M; Wolstenholme, Adrian J; Jex, Aaron R; Gasser, Robin B; Ballesteros, Cristina; Geary, Timothy G; Keiser, Jennifer; Townson, Simon; Harder, Achim; Krücken, Jürgen

    2014-12-01

    The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among

  18. Characterization of the Ca2+-gated and voltage-dependent K+-channel Slo-1 of nematodes and its interaction with emodepside.

    Directory of Open Access Journals (Sweden)

    Daniel Kulke

    2014-12-01

    Full Text Available The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in

  19. Delta receptors are required for full inhibitory coupling of mu-receptors to voltage-dependent Ca(2+) channels in dorsal root ganglion neurons.

    Science.gov (United States)

    Walwyn, Wendy; John, Scott; Maga, Matthew; Evans, Christopher J; Hales, Tim G

    2009-07-01

    Recombinant micro and delta opioid receptors expressed in cell lines can form heterodimers with distinctive properties and trafficking. However, a role for opioid receptor heterodimerization in neurons has yet to be identified. The inhibitory coupling of opioid receptors to voltage-dependent Ca(2+) channels (VDCCs) is a relatively inefficient process and therefore provides a sensitive assay of altered opioid receptor function and expression. We examined micro-receptor coupling to VDCCs in dorsal root ganglion neurons of delta(+/+), delta(+/-), and delta(-/-) mice. Neurons deficient in delta receptors exhibited reduced inhibition of VDCCs by morphine and [D-Ala(2),Phe(4),Gly(5)-ol]-enkephalin (DAMGO). An absence of delta receptors caused reduced efficacy of DAMGO without affecting potency. An absence of delta receptors reduced neither the density of VDCCs nor their inhibition by either the GABA(B) receptor agonist baclofen or intracellular guanosine 5'-O-(3-thio)triphosphate. Flow cytometry revealed a reduction in micro-receptor surface expression in delta(-/-) neurons without altered DAMGO-induced internalization. There was no change in micro-receptor mRNA levels. D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)-sensitive mu-receptor-coupling efficacy was fully restored to delta(+/+) levels in delta(-/-) neurons by expression of recombinant delta receptors. However, the dimerization-deficient delta-15 construct expressed in delta(-/-) neurons failed to fully restore the inhibitory coupling of micro-receptors compared with that seen in delta(+/+) neurons, suggesting that, although not essential for micro-receptor function, micro-delta receptor dimerization contributes to full micro-agonist efficacy. Because DAMGO exhibited a similar potency in delta(+/+) and delta(-/-) neurons and caused similar levels of internalization, the role for heterodimerization is probably at the level of receptor biosynthesis.

  20. IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

    Science.gov (United States)

    Nakatani, Yoshihiko; Nagaoka, Takumi; Hotta, Sayako; Utsunomiya, Iku; Yoshino, Hiide; Miyatake, Tadashi; Hoshi, Keiko; Taguchi, Kyoji

    2007-03-01

    We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

  1. Voltage-dependent anion channels (VDACs) promote mitophagy to protect neuron from death in an early brain injury following a subarachnoid hemorrhage in rats.

    Science.gov (United States)

    Li, Jian; Lu, Jianfei; Mi, Yongjie; Shi, Zhao; Chen, Chunhua; Riley, John; Zhou, Changman

    2014-07-21

    The term mitophagy is coined to describe the selective removal of mitochondria by autophagy but the process itself is still contentious, especially in the early period following subarachnoid hemorrhage (SAH). In the present study, we investigated the role of mitophagy following 48h after SAH injury in rats. Specifically evaluating whether mitophagy, through voltage dependant anion channels (VDACs) interacting with microtubule-associated protein 1 light chain 3, could orchestrate the induction of apoptotic and necrotic cell death in neurons, a VDAC1siRNA and an activitor Rapamycian (RAPA), were engaged. One hundred and twelve male Sprague-Dawley rats were randomly divided into 4 groups: Sham, SAH, SAH+VDAC1siRNA, and SAH+RAPA. Outcomes measured included mortality rate, brain edema, BBB disruption, and neurobehavioral testing. We also used western blotting techniques to analyze the expressions of key mitophagic/autophagic proteins and pro-apoptotic protein such as ROS, VDAC1, LC-3II and Caspase-3. Rapamycin treatment significantly improved the mortality rate, cerebral edema, and neurobehavioral deficits; apoptotic and necrotic cell death in neurons were reduced by Rapamycin following SAH injury. However, VDAC1siRNA worsened the brain injury following SAH. Immunohistochemical staining and western blot analysis demonstrated a decreased expression of VDAC1, LC3II, and an increase of ROS and Caspase-3 followed by VDAC1siRNA administration. In conclusion, mitophagy induced by VDAC1 following SAH injury may in fact play a significant role in neuroprotection, the mechanism which may be through the attenuation of the apoptosic and necrosic molecular pathways. This translates a preservation of functional integrity and an improvement in mortality.

  2. Arrangement and mobility of the voltage sensor domain in prokaryotic voltage-gated sodium channels.

    Science.gov (United States)

    Shimomura, Takushi; Irie, Katsumasa; Nagura, Hitoshi; Imai, Tomoya; Fujiyoshi, Yoshinori

    2011-03-04

    Prokaryotic voltage-gated sodium channels (Na(V)s) form homotetramers with each subunit contributing six transmembrane α-helices (S1-S6). Helices S5 and S6 form the ion-conducting pore, and helices S1-S4 function as the voltage sensor with helix S4 thought to be the essential element for voltage-dependent activation. Although the crystal structures have provided insight into voltage-gated K channels (K(V)s), revealing a characteristic domain arrangement in which the voltage sensor domain of one subunit is close to the pore domain of an adjacent subunit in the tetramer, the structural and functional information on Na(V)s remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic Na(V), is similar to that in K(V)s. Cysteine substitutions of three residues in helix S4, Q107C, T110C, and R113C, effectively induced intersubunit disulfide bond formation with a cysteine introduced in helix S5, M164C, of the adjacent subunit. In addition, substituting two acidic residues with lysine, E43K and D60K, shifted the activation of the channel to more positive membrane potentials and consistently shifted the preferentially formed disulfide bond from T110C/M164C to Q107C/M164C. Because Gln-107 is located closer to the extracellular side of helix S4 than Thr-110, this finding suggests that the functional shift in the voltage dependence of activation is related to a restriction of the position of helix S4 in the lipid bilayer. The domain arrangement and vertical mobility of helix S4 in NaChBac indicate that the structure and the mechanism of voltage-dependent activation in prokaryotic Na(V)s are similar to those in canonical K(V)s.

  3. Polarity-dependent conformational switching of a peptide mimicking the S4-S5 linker of the voltage-sensitive sodium channel.

    Science.gov (United States)

    Helluin, O; Breed, J; Duclohier, H

    1996-02-21

    The S4-S5 linker (or S45) in voltage-sensitive sodium channels was previously shown to be involved in the permeation pathway. The secondary structure, investigated by circular dichroism, of a S4-S45 peptide from domain IV and its fragments (including S45) is reported here and compared with that of the homologous peptide from domain II as a function of the solvent dielectric constant. The reduction in helicity seen for S4-S45 (II) in polar media is cancelled in membrane-like environment. The most striking result-- a sharp alpha-helix --> beta-sheet transition upon exposure of the S45 moiety to aqueous solvents-- is discussed as regards channel activation and selectivity.

  4. Gαi2- and Gαi3-specific regulation of voltage-dependent L-type calcium channels in cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Sara Dizayee

    Full Text Available BACKGROUND: Two pertussis toxin sensitive G(i proteins, G(i2 and G(i3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G(i isoforms are functionally distinct. To test for isoform-specific functions of G(i proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC. METHODS: Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gα(i2 (Gα(i2 (-/- or Gα(i3 (Gα(i3 (-/-. mRNA levels of Gα(i/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gα(i and Ca(vα(1 protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2 phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings. RESULTS: In cardiac tissue from Gα(i2 (-/- mice, Gα(i3 mRNA and protein expression was upregulated to 187 ± 21% and 567 ± 59%, respectively. In Gα(i3 (-/- mouse hearts, Gα(i2 mRNA (127 ± 5% and protein (131 ± 10% levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gα(i2 (-/- mice was lowered (-7.9 ± 0.6 pA/pF, n = 11, p<0.05 compared to wild-type cells (-10.7 ± 0.5 pA/pF, n = 22, whereas it was increased in myocytes from Gα(i3 (-/- mice (-14.3 ± 0.8 pA/pF, n = 14, p<0.05. Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gα(i2 (but not of Gα(i3 and following treatment with pertussis toxin in Gα(i3 (-/-. The pore forming Ca(vα(1 protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca(vα(1 and Ca(vβ(2 subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gα(i2. CONCLUSION: Our data provide novel evidence for an isoform

  5. Actions of Ethanol on Voltage-Sensitive Sodium Channels: Effects on Neurotoxin Binding

    Science.gov (United States)

    1987-01-01

    sodium inhibitory effect of ethanol on channel - mediated sodium influx channels ...Exprnmantal Trherpeutics Ped in I.SA. Actions of Ethanol on Voltage-Sensitive Sodium Channels : Effects on Neurotoxin Binding1 MICHAEL J. MULLIN 2 and... sodium channels . This indirect allosteric mechanism for inhibition of [H]BTX-B binding. effect orethanol was concentration-dependent and was

  6. Allele-dependent changes of olivocerebellar circuit properties in the absence of the voltage-gated potassium channels Kv3.1 and Kv3.3.

    Science.gov (United States)

    McMahon, Anne; Fowler, Stephen C; Perney, Teresa M; Akemann, Walther; Knöpfel, Thomas; Joho, Rolf H

    2004-06-01

    Double-mutant mice (DKO) lacking the two voltage-gated K(+) channels Kv3.1 and Kv3.3 display a series of phenotypic alterations that include ataxia, myoclonus, tremor and alcohol hypersensitivity. The prominent cerebellar expression of mRNAs encoding Kv3.1 and Kv3.3 subunits raised the question as to whether altered electrical activity resulting from the lack of these K(+) channels might be related to the dramatic motor changes. We used the tremorogenic agent harmaline to probe mutant mice lacking different K(+) channel alleles for altered olivocerebellar circuit properties. Harmaline induced the characteristic 13-Hz tremor in wildtype mice (WT); however, no tremor was observed in DKO suggesting that the ensemble properties of the olivocerebellar circuitry are altered in the absence of Kv3.1 and Kv3.3 subunits. Harmaline induced tremor in Kv3.1-single mutants, but it was of smaller amplitude and at a lower frequency indicating the participation of Kv3.1 subunits in normal olivocerebellar system function. In contrast, harmaline tremor was virtually absent in Kv3.3-single mutants indicating an essential role for Kv3.3 subunits in tremor induction by harmaline. Immunohistochemical staining for Kv3.3 showed clear expression in the somata and proximal dendrites of Purkinje cells and in their axonal projections to the deep cerebellar nuclei (DCN). In DCN, both Kv3.1 and Kv3.3 subunits are expressed. Action potential duration is increased by approximately 100% in Purkinje cells from Kv3.3-single mutants compared to WT or Kv3.1-single mutants. We conclude that Kv3.3 channel subunits are essential for the olivocerebellar system to generate and sustain normal harmaline tremor whereas Kv3.1 subunits influence tremor amplitude and frequency.

  7. Modulation of voltage-gated sodium channels hyperpolarizes the voltage threshold for activation in spinal motoneurones.

    Science.gov (United States)

    Power, Kevin E; Carlin, Kevin P; Fedirchuk, Brent

    2012-03-01

    Previous work has shown that motoneurone excitability is enhanced by a hyperpolarization of the membrane potential at which an action potential is initiated (V(th)) at the onset, and throughout brainstem-evoked fictive locomotion in the adult decerebrate cat and neonatal rat. Modeling work has suggested the modulation of Na(+) conductance as a putative mechanism underlying this state-dependent change in excitability. This study sought to determine whether modulation of voltage-gated sodium channels could induce V(th) hyperpolarization. Whole-cell patch-clamp recordings were made from antidromically identified lumbar spinal motoneurones in an isolated neonatal rat spinal cord preparation. Recordings were made with and without the bath application of veratridine, a plant alkaloid neurotoxin that acts as a sodium channel modulator. As seen in HEK 293 cells expressing Nav1.2 channels, veratridine-modified channels demonstrated a hyperpolarizing shift in their voltage-dependence of activation and a slowing of inactivation that resulted in an enhanced inward current in response to voltage ramp stimulations. In the native rat motoneurones, veratridine-modified sodium channels induced a hyperpolarization of V(th) in all 29 neonatal rat motoneurones examined (mean hyperpolarization: -6.6 ± 4.3 mV). V(th) hyperpolarization was not due to the effects on Ca(2+) and/or K(+) channels as blockade of these currents did not alter V(th). Veratridine also significantly increased the amplitude of persistent inward currents (PICs; mean increase: 72.5 ± 98.5 pA) evoked in response to slow depolarizing current ramps. However, the enhancement of the PIC amplitude had a slower time course than the hyperpolarization of V(th), and the PIC onset voltage could be either depolarized or hyperpolarized, suggesting that PIC facilitation did not mediate the V(th) hyperpolarization. We therefore suggest that central neuronal circuitry in mammals could affect V(th) in a mechanism similar to that of

  8. Cnidarian Toxins Acting on Voltage-Gated Ion Channels

    Directory of Open Access Journals (Sweden)

    Robert M. Greenberg

    2006-04-01

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

  9. Voltage-Gated Channels as Causative Agents for Epilepsies

    Directory of Open Access Journals (Sweden)

    Mutasem Abuhamed

    2008-01-01

    Full Text Available Problem statement: Epilepsy is a common neurological disorder that afflicts 1-2% of the general population worldwide. It encompasses a variety of disorders with seizures. Approach: Idiopathic epilepsies were defined as a heterogeneous group of seizure disorders that show no underlying cause .Voltage-gated ion channels defect were recognized etiology of epilepsy in the central nervous system. The aim of this article was to provide an update on voltage-gated channels and their mutation as causative agents for epilepsies. We described the structures of the voltage-gated channels, discuss their current genetic studies, and then review the effects of voltage-gated channels as causative agents for epilepsies. Results: Channels control the flow of ions in and out of the cell causing depolarization and hyper polarization of the cell. Voltage-gated channels were classified into four types: Sodium, potassium calcium ands chloride. Voltage-gated channels were macromolecular protein complexes within the lipid membrane. They were divided into subunits. Each subunit had a specific function and was encoded by more than one gen. Conclusion: Current genetic studies of idiopathic epilepsies show the importance of genetic influence on Voltage-gated channels. Different genes may regulate a function in a channel; the channel defect was directly responsible for neuronal hyper excitability and seizures.

  10. Chronic electroconvulsive stimulation but not chronic restraint stress modulates mRNA expression of voltage-dependent potassium channels Kv7.2 and Kv11.1 in the rat piriform cortex

    DEFF Research Database (Denmark)

    Hjæresen, Marie-Louise; Hageman, Ida; Plenge, Per

    2008-01-01

    The mechanisms by which stress and electroconvulsive therapy exert opposite effects on the course of major depression are not known. Potential candidates might include the voltage-dependent potassium channels. Potassium channels play an important role in maintaining the resting membrane potential...... and controlling neuronal excitability. To explore this hypothesis, we examined the effects of one or several electroconvulsive stimulations and chronic restraint stress (6 h/day for 21 days) on the expression of voltage-dependent potassium channel Kv7.2, Kv11.1, and Kv11.3 mRNA in the rat brain using in situ...... hybridization. Repeated, but not acute, electroconvulsive stimulation increased Kv7.2 and Kv11.1 mRNA levels in the piriform cortex. In contrast, restraint stress had no significant effect on mRNA expression of Kv7.2, Kv11.1, or Kv11.3 in any of the brain regions examined. Thus, it appears that the investigated...

  11. Multimeric nature of voltage-gated proton channels

    OpenAIRE

    Koch, Hans P.; Kurokawa, Tatsuki; Okochi, Yoshifumi; Sasaki, Mari; Okamura, Yasushi; Larsson, H. Peter

    2008-01-01

    Voltage-gated potassium channels are comprised of four subunits, and each subunit has a pore domain and a voltage-sensing domain (VSD). The four pore domains assemble to form one single central pore, and the four individual VSDs control the gate of the pore. Recently, a family of voltage-gated proton channels, such as HV or voltage sensor only protein (VSOP), was discovered that contain a single VSD but no pore domain. It has been assumed that VSOP channels are monomeric and contain a single ...

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

    Directory of Open Access Journals (Sweden)

    Aldo A Rodríguez Menchaca

    2012-09-01

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

  13. Dual Regulation of Voltage-Sensitive Ion Channels by PIP(2).

    Science.gov (United States)

    Rodríguez-Menchaca, Aldo A; Adney, Scott K; Zhou, Lei; Logothetis, Diomedes E

    2012-01-01

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

  14. 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

  15. Voltage-gated Calcium Channels and Autism Spectrum Disorders.

    Science.gov (United States)

    Breitenkamp, Alexandra F; Matthes, Jan; Herzig, Stefan

    2015-01-01

    Autism spectrum disorder is a complex-genetic disease and its etiology is unknown for the majority of cases. So far, more than one hundred different susceptibility genes were detected. Voltage-gated calcium channels are among the candidates linked to autism spectrum disorder by results of genetic studies. Mutations of nearly all pore-forming and some auxiliary subunits of voltage gated calcium channels have been revealed from investigations of autism spectrum disorder patients and populations. Though there are only few electrophysiological characterizations of voltage-gated calcium channel mutations found in autistic patients these studies suggest their functional relevance. In summary, both genetic and functional data suggest a potential role of voltage-gated calcium channels in autism spectrum disorder. Future studies require refinement of the clinical and systems biological concepts of autism spectrum disorder and an appropriate holistic approach at the molecular level, e.g. regarding all facets of calcium channel functions.

  16. Temperature and voltage coupling to channel opening in transient receptor potential melastatin 8 (TRPM8).

    Science.gov (United States)

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

    2014-12-19

    Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca(2+)-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening.

  17. Voltage- and calcium-dependent motility of saccular hair bundles

    Science.gov (United States)

    Quiñones, Patricia M.; Meenderink, Sebastiaan W. F.; Bozovic, Dolores

    2015-12-01

    Active bundle motility, which is hypothesized to supply feedback for mechanical amplification of signals, is thought to enhance sensitivity and sharpen tuning in vestibular and auditory organs. To study active hair bundle motility, we combined high-speed camera recordings of bullfrog sacculi, which were mounted in a two-compartment chamber, and voltage-clamp of the hair cell membrane potential. Using this paradigm, we measured three types of bundle motions: 1) spontaneous oscillations which can be analyzed to measure the physiological operating range of the transduction channel; 2) a sustained quasi-static movement of the bundle that depends on membrane potential; and 3) a fast, transient and asymmetric movement that resets the bundle position and depends on changes in the membrane potential. These data support a role for both calcium and voltage in the transduction-channel function.

  18. Multimeric nature of voltage-gated proton channels.

    Science.gov (United States)

    Koch, Hans P; Kurokawa, Tatsuki; Okochi, Yoshifumi; Sasaki, Mari; Okamura, Yasushi; Larsson, H Peter

    2008-07-01

    Voltage-gated potassium channels are comprised of four subunits, and each subunit has a pore domain and a voltage-sensing domain (VSD). The four pore domains assemble to form one single central pore, and the four individual VSDs control the gate of the pore. Recently, a family of voltage-gated proton channels, such as H(V) or voltage sensor only protein (VSOP), was discovered that contain a single VSD but no pore domain. It has been assumed that VSOP channels are monomeric and contain a single VSD that functions as both the VSD and the pore domain. It remains unclear, however, how a protein that contains only a VSD and no pore domain can conduct ions. Using fluorescence measurements and immunoprecipitation techniques, we show here that VSOP channels are expressed as multimeric channels. Further, FRET experiments on constructs with covalently linked subunits show that VSOP channels are dimers. Truncation of the cytoplasmic regions of VSOP reduced the dimerization, suggesting that the dimerization is caused mainly by cytoplasmic protein-protein interactions. However, these N terminus- and C terminus-deleted channels displayed large proton currents. Therefore, we conclude that even though VSOP channels are expressed mainly as dimers in the cell membrane, single VSOP subunits could function independently as proton channels.

  19. Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

    Energy Technology Data Exchange (ETDEWEB)

    Kozuka, M.; Ito, T.; Hirose, S.; Takahashi, K.; Hagiwara, H.

    1989-02-28

    Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using /sup 125/I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.

  20. Plural voltage minima in an arc-heated channel flow

    Science.gov (United States)

    Sasoh, A.

    2001-04-01

    In flows through a channel with varying cross-sectional area, the impulse and total enthalpy can be increased by superimposing an electrical discharge. The flow field is determined from the inlet flow condition, channel geometry, and discharge specifications. In this study, steady-state, quasi-one-dimensional flows interacting with an arc discharge are computed numerically. Once the arc column configuration is given, the discharge voltage is computed from the solution of flow field variables. For a constant discharge current, there exist plural column configurations which yield a minimum discharge voltage. This result explains the fluid-dynamic mechanisms of the existence of plural voltage modes in an arcjet operation.

  1. Voltage-gated proton channel is expressed on phagosomes.

    Science.gov (United States)

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

    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.

  2. Effects of acidic pH on voltage-gated ion channels in rat trigeminal mesencephalic nucleus neurons.

    Science.gov (United States)

    Han, Jin-Eon; Cho, Jin-Hwa; Choi, In-Sun; Kim, Do-Yeon; Jang, Il-Sung

    2017-03-01

    The effects of acidic pH on several voltage-dependent ion channels, such as voltage-dependent K(+) and Ca(2+) channels, and hyperpolarization-gated and cyclic nucleotide-activated cation (HCN) channels, were examined using a whole-cell patch clamp technique on mechanically isolated rat mesencephalic trigeminal nucleus neurons. The application of a pH 6.5 solution had no effect on the peak amplitude of voltage-dependent K(+) currents. A pH 6.0 solution slightly, but significantly inhibited the peak amplitude of voltage-dependent K(+) currents. The pH 6.0 also shifted both the current-voltage and conductance-voltage relationships to the depolarization range. The application of a pH 6.5 solution scarcely affected the peak amplitude of membrane currents mediated by HCN channels, which were profoundly inhibited by the general HCN channel blocker Cs(+) (1 mM). However, the pH 6.0 solution slightly, but significantly inhibited the peak amplitude of HCN-mediated currents. Although the pH 6.0 solution showed complex modulation of the current-voltage and conductance-voltage relationships, the midpoint voltages for the activation of HCN channels were not changed by acidic pH. On the other hand, voltage-dependent Ca(2+) channels were significantly inhibited by an acidic pH. The application of an acidic pH solution significantly shifted the current-voltage and conductance-voltage relationships to the depolarization range. The modulation of several voltage-dependent ion channels by an acidic pH might affect the excitability of mesencephalic trigeminal nucleus neurons, and thus physiological functions mediated by the mesencephalic trigeminal nucleus could be affected in acidic pH conditions.

  3. Electrophysiology of lead intoxication: effects on voltage-sensitive ion channels.

    Science.gov (United States)

    Audesirk, G

    1993-01-01

    Neuronal function depends on the activity of a variety of voltage-sensitive, ion-specific membrane channels, including channels permeable chiefly to sodium, potassium, and calcium. The plasma membranes of many neurons contain several types of each class of channel. In general, heavy metal ions exert little effect on voltage-sensitive sodium or potassium channels, but inhibit ion flow through voltage-sensitive calcium channels (VSCC). The literature abounds with descriptions of different types of calcium channels in vertebrate neurons. These descriptions suggest that there are many physiologically and pharmacologically distinct calcium channels, some of them possibly cell-type specific. Among the heavy metals, Pb2+ is one of the most potent inhibitors of VSCC in both vertebrate and invertebrate neurons. Some heavy metals, including Ni2+ and Cd2+, are fairly selective against certain types of calcium channels. Limited evidence suggests that Pb2+ inhibits all calcium channel types within a given cell, with only minor differences in potency. However, there appear to be substantial differences among cell types in the concentration dependence of calcium channel inhibition by Pb2+. Therefore, to appreciate the range of effects of Pb2+ on calcium channels throughout the nervous system, it will be important to examine a large number of cell types. Pb2+ is highly permeable through at least some types of VSCC. Entry of Pb2+ into the neuronal cytoplasm through VSCC, followed by disturbance of intracellular functions, may be a major mechanism of Pb2+ neurotoxicity.

  4. 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.

  5. The Position of the Fast-Inactivation Gate during Lidocaine Block of Voltage-gated Na+ Channels

    OpenAIRE

    Vedantham, Vasanth; Cannon, Stephen C.

    1999-01-01

    Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na+ channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na+ channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has been difficult to probe because gating of drug-bound channel...

  6. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide

    Energy Technology Data Exchange (ETDEWEB)

    Gordon, R.D.; Fieles, W.E.; Schotland, D.L.; Hogue-Angeletti, R.; Barchi, R.L.

    1987-01-01

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies. The membrane topology of the peptide recognized by this antiserum was proved in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% right-side-out as determined by (/sup 3/H)saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. The data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by the anitserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.

  7. Channel protein engineering: Synthetic 22-mer peptide from the primary structure of the voltage-sensitive sodium channel forms ionic channels in lipid bilayers

    OpenAIRE

    1988-01-01

    A synthetic 22-mer peptide that mimics the sequence of a putative pore segment of the voltage-dependent sodium channel forms transmembrane ionic channels in lipid bilayers. Several features of the authentic sodium channel are exhibited by the synthetic peptide: (i) The single channel conductance of the most frequent event is 20 pS in 0.5 M NaCl. (ii) The single channel open and closed lifetimes are in the ms time range. (iii) The synthetic channel discriminates cations over anions but is nons...

  8. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide.

    Science.gov (United States)

    Gordon, R D; Fieles, W E; Schotland, D L; Hogue-Angeletti, R; Barchi, R L

    1987-01-01

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies; noninnervated membrane was not labeled, consistent with the known distribution of sodium channels in this tissue. The membrane topology of the peptide recognized by this antiserum was probed in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% "right-side-out" as determined by [3H]saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. Gold particles identifying the antibody were found almost exclusively associated with the cytoplasmic surface of the innervated membrane. Our data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by our antiserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure. Images PMID:2432607

  9. The S4-S5 linker couples voltage sensing and activation of pacemaker channels.

    Science.gov (United States)

    Chen, J; Mitcheson, J S; Tristani-Firouzi, M; Lin, M; Sanguinetti, M C

    2001-09-25

    Voltage-gated channels are normally opened by depolarization and closed by repolarization of the membrane. Despite sharing significant sequence homology with voltage-gated K(+) channels, the gating of hyperpolarization-activated, cyclic-nucleotide-gated (HCN) pacemaker channels has the opposite dependence on membrane potential: hyperpolarization opens, whereas depolarization closes, these channels. The mechanism and structural basis of the process that couples voltage sensor movement to HCN channel opening and closing is not understood. On the basis of our previous studies of a mutant HERG (human ether-a-go-go-related gene) channel, we hypothesized that the intracellular linker that connects the fourth and fifth transmembrane domains (S4-S5 linker) of HCN channels might be important for channel gating. Here, we used alanine-scanning mutagenesis of the HCN2 S4-S5 linker to identify three residues, E324, Y331, and R339, that when mutated disrupted normal channel closing. Mutation of a basic residue in the S4 domain (R318Q) prevented channel opening, presumably by disrupting S4 movement. However, channels with R318Q and Y331S mutations were constitutively open, suggesting that these channels can open without a functioning S4 domain. We conclude that the S4-S5 linker mediates coupling between voltage sensing and HCN channel activation. Our findings also suggest that opening of HCN and related channels corresponds to activation of a gate located near the inner pore, rather than recovery of channels from a C-type inactivated state.

  10. Voltage-gated sodium channels: mutations, channelopathies and targets.

    Science.gov (United States)

    Andavan, G S B; Lemmens-Gruber, R

    2011-01-01

    Voltage-gated sodium channels produce fast depolarization, which is responsible for the rising phase of the action potential in neurons, muscles and heart. These channels are very large membrane proteins and are encoded by ten genes in mammals. Sodium channels are a crucial component of excitable tissues; hence, they are a target for various neurotoxins that are produced by plants and animals for defence and protection, such as tetrodotoxin, scorpion toxins and batrachotoxin. Several mutations in various sodium channel subtypes cause multiple inherited diseases known as channelopathies. When these mutated sodium channel subtypes are expressed in various tissues, channelopathies in brain, skeletal muscle and cardiac muscle develop as well as neuropathic pain. In this review, we discuss aspects of voltage-gated sodium channel genes with an emphasis on cardiac muscle sodium channels. In addition, we report novel mutations that underlie a spectrum of diseases, such as Brugada, long QT syndrome and inherited conduction disorders. Furthermore, this review explains commonalities and differences among the channel subtypes, the channelopathies caused by the sodium channel gene mutation and the specificity of toxins and blockers of the channel subtypes.

  11. A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom

    Science.gov (United States)

    Lee, Seok-Yong; MacKinnon, Roderick

    2004-07-01

    Venomous animals produce small protein toxins that inhibit ion channels with high affinity. In several well-studied cases the inhibitory proteins are water-soluble and bind at a channel's aqueous-exposed extracellular surface. Here we show that a voltage-sensor toxin (VSTX1) from the Chilean Rose Tarantula (Grammostola spatulata) reaches its target by partitioning into the lipid membrane. Lipid membrane partitioning serves two purposes: to localize the toxin in the membrane where the voltage sensor resides and to exploit the free energy of partitioning to achieve apparent high-affinity inhibition. VSTX1, small hydrophobic poisons and anaesthetic molecules reveal a common theme of voltage sensor inhibition through lipid membrane access. The apparent requirement for such access is consistent with the recent proposal that the sensor in voltage-dependent K+ channels is located at the membrane-protein interface.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  13. Flufenamic acid decreases neuronal excitability through modulation of voltage-gated sodium channel gating.

    Science.gov (United States)

    Yau, Hau-Jie; Baranauskas, Gytis; Martina, Marco

    2010-10-15

    The electrophysiological phenotype of individual neurons critically depends on the biophysical properties of the voltage-gated channels they express. Differences in sodium channel gating are instrumental in determining the different firing phenotypes of pyramidal cells and interneurons; moreover, sodium channel modulation represents an important mechanism of action for many widely used CNS drugs. Flufenamic acid (FFA) is a non-steroidal anti-inflammatory drug that has been long used as a blocker of calcium-dependent cationic conductances. Here we show that FFA inhibits voltage-gated sodium currents in hippocampal pyramidal neurons; this effect is dose-dependent with IC(50) = 189 μm. We used whole-cell and nucleated patch recordings to investigate the mechanisms of FFA modulation of TTX-sensitive voltage-gated sodium current. Our data show that flufenamic acid slows down the inactivation process of the sodium current, while shifting the inactivation curve ~10 mV toward more hyperpolarized potentials. The recovery from inactivation is also affected in a voltage-dependent way, resulting in slower recovery at hyperpolarized potentials. Recordings from acute slices demonstrate that FFA reduces repetitive- and abolishes burst-firing in CA1 pyramidal neurons. A computational model based on our data was employed to better understand the mechanisms of FFA action. Simulation data support the idea that FFA acts via a novel mechanism by reducing the voltage dependence of the sodium channel fast inactivation rates. These effects of FFA suggest that it may be an effective anti-epileptic drug.

  14. Voltage-Gated Channels as Causative Agents for Epilepsies

    OpenAIRE

    2008-01-01

    Problem statement: Epilepsy is a common neurological disorder that afflicts 1-2% of the general population worldwide. It encompasses a variety of disorders with seizures. Approach: Idiopathic epilepsies were defined as a heterogeneous group of seizure disorders that show no underlying cause .Voltage-gated ion channels defect were recognized etiology of epilepsy in the central nervous system. The aim of this article was to provide an update on voltage-gated ...

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

    Science.gov (United States)

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

    2013-05-15

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

  16. Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca2+ channels

    DEFF Research Database (Denmark)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin

    2015-01-01

    M), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin–agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1...... rate and led to appearance of long-lasting bursts in the same manner as treatment with the inhibitor of L-VDCC diltiazem. Thus, heparan sulfate proteoglycans may bind to and regulate L-VDCC inactivation and network activity....

  17. Genotypic to expression profiling of bovine calcium channel, voltage-dependent, alpha-2/delta subunit 1 gene, and their association with bovine mastitis among Frieswal (HFX Sahiwal) crossbred cattle of Indian origin.

    Science.gov (United States)

    Deb, Rajib; Singh, Umesh; Kumar, Sushil; Kumar, Arun; Singh, Rani; Sengar, Gyanendra; Mann, Sandeep; Sharma, Arjava

    2014-04-01

    Calcium channel, voltage-dependent, alpha-2/delta subunit 1 (CACNA2D1) gene is considered to be an important noncytokine candidate gene influencing mastitis. Scanty of reports are available until today regarding the role play of CACNA2D1 gene on the susceptibility of bovine mastitis. We interrogated the CACNA2D1 G519663A [A>G] SNP by PCR-RFLP among two hundreds Frieswal (HF X Sahiwal) crossbred cattle of Indian origin. Genotypic frequency of AA (51.5, n=101) was comparatively higher than AG (35, n=70) and GG (14.5, n=29). Association of Somatic cell score (SCS) with genotypes revealed that, GG genotypes showing lesser count (less susceptible to mastitis) compare to AA and AG. Relative expression of CACNA2D1 transcript (in milk samples) was significantly higher among GG than AG and AA. Further we have also isolated blood sample from the all groups and PBMCs were cultured from each blood sample as per the standard protocol. They were treated with Calcium channel blocker and the expression level of the CACNA2D1 gene was evaluated by Real Time PCR. Results show that expression level decline in each genotypic group after treatment and expression level of GG are again significantly higher than AA and AG. Thus, it may be concluded that GG genotypic animals are favorable for selecting disease resistant breeds.

  18. PIP2 in pancreatic β-cells regulates voltage-gated calcium channels by a voltage-independent pathway.

    Science.gov (United States)

    de la Cruz, Lizbeth; Puente, Erika I; Reyes-Vaca, Arturo; Arenas, Isabel; Garduño, Julieta; Bravo-Martínez, Jorge; Garcia, David E

    2016-10-01

    Phosphatidylinositol-4,5-bisphosphate (PIP2) is a membrane phosphoinositide that regulates the activity of many ion channels. Influx of calcium primarily through voltage-gated calcium (CaV) channels promotes insulin secretion in pancreatic β-cells. However, whether CaV channels are regulated by PIP2, as is the case for some non-insulin-secreting cells, is unknown. The purpose of this study was to investigate whether CaV channels are regulated by PIP2 depletion in pancreatic β-cells through activation of a muscarinic pathway induced by oxotremorine methiodide (Oxo-M). CaV channel currents were recorded by the patch-clamp technique. The CaV current amplitude was reduced by activation of the muscarinic receptor 1 (M1R) in the absence of kinetic changes. The Oxo-M-induced inhibition exhibited the hallmarks of voltage-independent regulation and did not involve PKC activation. A small fraction of the Oxo-M-induced CaV inhibition was diminished by a high concentration of Ca(2+) chelator, whereas ≥50% of this inhibition was prevented by diC8-PIP2 dialysis. Localization of PIP2 in the plasma membrane was examined by transfecting INS-1 cells with PH-PLCδ1, which revealed a close temporal association between PIP2 hydrolysis and CaV channel inhibition. Furthermore, the depletion of PIP2 by a voltage-sensitive phosphatase reduced CaV currents in a way similar to that observed following M1R activation. These results indicate that activation of the M1R pathway inhibits the CaV channel via PIP2 depletion by a Ca(2+)-dependent mechanism in pancreatic β- and INS-1 cells and thereby support the hypothesis that membrane phospholipids regulate ion channel activity by interacting with ion channels.

  19. VGIchan: Prediction and Classification of Voltage-Gated Ion Channels

    Institute of Scientific and Technical Information of China (English)

    Sudipto Saha; Jyoti Zack; Balvinder Singh; G.P.S. Raghava

    2006-01-01

    This study describes methods for predicting and classifying voltage-gated ion channels. Firstly, a standard support vector machine (SVM) method was developed for predicting ion channels by using amino acid composition and dipeptide composition, with an accuracy of 82.89% and 85.56%, respectively. The accuracy of this SVM method was improved from 85.56% to 89.11% when combined with PSIBLAST similarity search. Then we developed an SVM method for classifying ion channels (potassium, sodium, calcium, and chloride) by using dipeptide composition and achieved an overall accuracy of 96.89%. We further achieved a classification accuracy of 97.78% by using a hybrid method that combines dipeptidebased SVM and hidden Markov model methods. A web server VGIchan has been developed for predicting and classifying voltage-gated ion channels using the above approaches. VGIchan is freely available at www.imtech.res.in/raghava/vgichan/.

  20. Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.

    Science.gov (United States)

    Kubota, Tomoya; Durek, Thomas; Dang, Bobo; Finol-Urdaneta, Rocio K; Craik, David J; Kent, Stephen B H; French, Robert J; Bezanilla, Francisco; Correa, Ana M

    2017-03-07

    Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

  1. Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

    Science.gov (United States)

    Kubota, Tomoya; Durek, Thomas; Dang, Bobo; Finol-Urdaneta, Rocio K.; Craik, David J.; Kent, Stephen B. H.; French, Robert J.; Bezanilla, Francisco; Correa, Ana M.

    2017-01-01

    Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating. PMID:28202723

  2. Single-channel properties of the reconstituted voltage-regulated Na channel isolated from the electroplax of Electrophorus electricus.

    Science.gov (United States)

    Rosenberg, R L; Tomiko, S A; Agnew, W S

    1984-01-01

    The tetrodotoxin-binding protein purified from electroplax of Electrophorus electricus has been reincorporated into multilamellar vesicles that were used for patch recording. When excised patches of these reconstituted membranes were voltage clamped in the absence of neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties qualitatively and quantitatively similar to those reported for Na channels from nerve and muscle cells, including uniform single-channel conductances of the appropriate magnitude (approximately equal to 11 pS in 95 mM Na+), mean open times of approximately equal to 1.9 msec, and 7-fold selectively for Na+ over K+. Currents averaged from many depolarizations showed initial voltage-dependent activation and subsequent inactivation. In the presence of batrachotoxin, channels were observed with markedly different properties, including conductances of 20-25 pS (95 mM Na+), mean open times of approximately equal to 28 msec, and no indication of inactivation. Collectively, these findings indicate that the tetrodotoxin-binding protein of electroplax is a voltage-regulated sodium channel. PMID:6089214

  3. KCNE1 divides the voltage sensor movement in KCNQ1/KCNE1 channels into two steps

    Science.gov (United States)

    Barro-Soria, Rene; Rebolledo, Santiago; Liin, Sara I.; Perez, Marta E.; Sampson, Kevin J.; Kass, Robert S.; Larsson, H. Peter

    2014-04-01

    The functional properties of KCNQ1 channels are highly dependent on associated KCNE-β subunits. Mutations in KCNQ1 or KCNE subunits can cause congenital channelopathies, such as deafness, cardiac arrhythmias and epilepsy. The mechanism by which KCNE1-β subunits slow the kinetics of KCNQ1 channels is a matter of current controversy. Here we show that KCNQ1/KCNE1 channel activation occurs in two steps: first, mutually independent voltage sensor movements in the four KCNQ1 subunits generate the main gating charge movement and underlie the initial delay in the activation time course of KCNQ1/KCNE1 currents. Second, a slower and concerted conformational change of all four voltage sensors and the gate, which opens the KCNQ1/KCNE1 channel. Our data show that KCNE1 divides the voltage sensor movement into two steps with widely different voltage dependences and kinetics. The two voltage sensor steps in KCNQ1/KCNE1 channels can be pharmacologically isolated and further separated by a disease-causing mutation.

  4. Actions and Mechanisms of Polyunsaturated Fatty Acids on Voltage-Gated Ion Channels.

    Science.gov (United States)

    Elinder, Fredrik; Liin, Sara I

    2017-01-01

    Polyunsaturated fatty acids (PUFAs) act on most ion channels, thereby having significant physiological and pharmacological effects. In this review we summarize data from numerous PUFAs on voltage-gated ion channels containing one or several voltage-sensor domains, such as voltage-gated sodium (NaV), potassium (KV), calcium (CaV), and proton (HV) channels, as well as calcium-activated potassium (KCa), and transient receptor potential (TRP) channels. Some effects of fatty acids appear to be channel specific, whereas others seem to be more general. Common features for the fatty acids to act on the ion channels are at least two double bonds in cis geometry and a charged carboxyl group. In total we identify and label five different sites for the PUFAs. PUFA site 1: The intracellular cavity. Binding of PUFA reduces the current, sometimes as a time-dependent block, inducing an apparent inactivation. PUFA site 2: The extracellular entrance to the pore. Binding leads to a block of the channel. PUFA site 3: The intracellular gate. Binding to this site can bend the gate open and increase the current. PUFA site 4: The interface between the extracellular leaflet of the lipid bilayer and the voltage-sensor domain. Binding to this site leads to an opening of the channel via an electrostatic attraction between the negatively charged PUFA and the positively charged voltage sensor. PUFA site 5: The interface between the extracellular leaflet of the lipid bilayer and the pore domain. Binding to this site affects slow inactivation. This mapping of functional PUFA sites can form the basis for physiological and pharmacological modifications of voltage-gated ion channels.

  5. Actions and Mechanisms of Polyunsaturated Fatty Acids on Voltage-Gated Ion Channels

    Science.gov (United States)

    Elinder, Fredrik; Liin, Sara I.

    2017-01-01

    Polyunsaturated fatty acids (PUFAs) act on most ion channels, thereby having significant physiological and pharmacological effects. In this review we summarize data from numerous PUFAs on voltage-gated ion channels containing one or several voltage-sensor domains, such as voltage-gated sodium (NaV), potassium (KV), calcium (CaV), and proton (HV) channels, as well as calcium-activated potassium (KCa), and transient receptor potential (TRP) channels. Some effects of fatty acids appear to be channel specific, whereas others seem to be more general. Common features for the fatty acids to act on the ion channels are at least two double bonds in cis geometry and a charged carboxyl group. In total we identify and label five different sites for the PUFAs. PUFA site 1: The intracellular cavity. Binding of PUFA reduces the current, sometimes as a time-dependent block, inducing an apparent inactivation. PUFA site 2: The extracellular entrance to the pore. Binding leads to a block of the channel. PUFA site 3: The intracellular gate. Binding to this site can bend the gate open and increase the current. PUFA site 4: The interface between the extracellular leaflet of the lipid bilayer and the voltage-sensor domain. Binding to this site leads to an opening of the channel via an electrostatic attraction between the negatively charged PUFA and the positively charged voltage sensor. PUFA site 5: The interface between the extracellular leaflet of the lipid bilayer and the pore domain. Binding to this site affects slow inactivation. This mapping of functional PUFA sites can form the basis for physiological and pharmacological modifications of voltage-gated ion channels. PMID:28220076

  6. Emerging roles of L-type voltage gated and other calcium channels in T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Abdallah eBadou

    2013-08-01

    Full Text Available In T lymphocytes, calcium ion controls a variety of biological processes including development, survival, proliferation, and effector functions. These distinct and specific roles are regulated by different calcium signals, which are generated by various plasma membrane calcium channels. The repertoire of calcium-conducting proteins in T lymphocytes includes store-operated CRAC channels, transient receptor potential (TRP channels, P2X channels, and L-type voltage-gated calcium (Cav1 channels. In this paper, we will focus mainly on the role of the Cav1 channels found expressed by T lymphocytes, where these channels appear to operate in a TCR stimulation-dependent and voltage-sensor independent manner. We will review their expression profile at various differentiation stages of CD4 and CD8 T lymphocytes. Then, we will present crucial genetic evidence in favor of a role of these Cav1 channels and related regulatory proteins in both CD4 and CD8 T cell functions such as proliferation, survival, cytokine production and cytolysis. Finally, we will provide evidence and speculate on how these voltage-gated channels might function in the T lymphocyte, a non-excitable cell.

  7. Regulation of Voltage-Activated K(+) Channel Gating by Transmembrane β Subunits.

    Science.gov (United States)

    Sun, Xiaohui; Zaydman, Mark A; Cui, Jianmin

    2012-01-01

    Voltage-activated K(+) (K(V)) channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. K(V) channels contain a central pore-gate domain (PGD) surrounded by four voltage-sensing domains (VSDs). The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many K(V) channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the K(V) β subunits that contain transmembrane (TM) segments including the KCNE family and the β subunits of large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels. These TM β subunits affect the voltage-dependent activation of K(V) α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening, and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into K(V) channel modulation by TM β subunits.

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

    Science.gov (United States)

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

    2016-07-05

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

  9. A Ca(2+) chelator ameliorates chromium (VI)-induced hepatocyte L-02 injury via down-regulation of voltage-Dependent anion channel 1 (VDAC1) expression.

    Science.gov (United States)

    Yi, Xing; Xiao, Fang; Zhong, Xiali; Duan, Yujie; Liu, Kaihua; Zhong, Caigao

    2017-01-01

    Hexavalent chromium could result in cell malfunctions. Intracellular Ca(2+) ([Ca(2+)]i) content and VDAC1 expression are both important features related to cell survial. This study aimed to explore the mechanism of cell injury induced by Cr(VI) and tentatively offer clues to repairing this cell damage using [Ca(2+)]i and VDAC1. L-02 hepatocytes were treated with Cr(VI)/BAPTA, and the levels of [Ca(2+)]i and cell injury associated with Cr(VI) were determined in addition to the effect of BAPTA. The expression of VDAC1 in Cr(VI)-induced cells was evaluated. The results showed a dose-dependent elevation of the level of VDAC1 and the mRNA level of the VDAC1 biogenesis-related gene Sam50. BAPTA could ameliorate less severe damage induced by 4μM Cr(VI) via reducing VDAC1 and Sam50. Additionally, cell injury caused by less than 4μM Cr(VI) could be ameliorated by VDAC1 knockdown. Taken together, the findings of this study suggest that inhibition of intracellular Ca(2±) overload could protect cells from damage and that VDAC1 plays a considerable role in Cr(VI)-induced liver injury.

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

    Directory of Open Access Journals (Sweden)

    Richard J. Lewis

    2006-04-01

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

  11. Voltage Dependence of Conformational Dynamics and Subconducting States of VDAC-1.

    Science.gov (United States)

    Briones, Rodolfo; Weichbrodt, Conrad; Paltrinieri, Licia; Mey, Ingo; Villinger, Saskia; Giller, Karin; Lange, Adam; Zweckstetter, Markus; Griesinger, Christian; Becker, Stefan; Steinem, Claudia; de Groot, Bert L

    2016-09-20

    The voltage-dependent anion channel 1 (VDAC-1) is an important protein of the outer mitochondrial membrane that transports energy metabolites and is involved in apoptosis. The available structures of VDAC proteins show a wide β-stranded barrel pore, with its N-terminal α-helix (N-α) bound to its interior. Electrophysiology experiments revealed that voltage, its polarity, and membrane composition modulate VDAC currents. Experiments with VDAC-1 mutants identified amino acids that regulate the gating process. However, the mechanisms for how these factors regulate VDAC-1, and which changes they trigger in the channel, are still unknown. In this study, molecular dynamics simulations and single-channel experiments of VDAC-1 show agreement for the current-voltage relationships of an "open" channel and they also show several subconducting transient states that are more cation selective in the simulations. We observed voltage-dependent asymmetric distortions of the VDAC-1 barrel and the displacement of particular charged amino acids. We constructed conformational models of the protein voltage response and the pore changes that consistently explain the protein conformations observed at opposite voltage polarities, either in phosphatidylethanolamine or phosphatidylcholine membranes. The submicrosecond VDAC-1 voltage response shows intrinsic structural changes that explain the role of key gating amino acids and support some of the current gating hypotheses. These voltage-dependent protein changes include asymmetric barrel distortion, its interaction with the membrane, and significant displacement of N-α amino acids.

  12. The Voltage-Dependent Anion Channel 1 (AtVDAC1 Negatively Regulates Plant Cold Responses during Germination and Seedling Development in Arabidopsis and Interacts with Calcium Sensor CBL1

    Directory of Open Access Journals (Sweden)

    Zhi-Yong Li

    2013-01-01

    Full Text Available The voltage-dependent anion channel (VDAC, a highly conserved major mitochondrial outer membrane protein, plays crucial roles in energy metabolism and metabolite transport. However, knowledge about the roles of the VDAC family in plants is limited. In this study, we investigated the expression pattern of VDAC1 in Arabidopsis and found that cold stress promoted the accumulation of VDAC1 transcripts in imbibed seeds and mature plants. Overexpression of VDAC1 reduced tolerance to cold stress in Arabidopsis. Phenotype analysis of VDAC1 T-DNA insertion mutant plants indicated that a vdac1 mutant line had faster germination kinetics under cold treatment and showed enhanced tolerance to freezing. The yeast two-hybrid system revealed that VDAC1 interacts with CBL1, a calcium sensor in plants. Like the vdac1, a cbl1 mutant also exhibited a higher seed germination rate. We conclude that both VDAC1 and CBL1 regulate cold stress responses during seed germination and plant development.

  13. 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

  14. PROPERTIES OF VOLTAGE-GATED SODIUM CHANNELS IN DEVELOPING AUDITORY NEURONS OF THE MOUSE IN VITRO

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective. To investigate the properties of voltage-gated sodium (Na+) channels in developing auditoryneurons during early postnatal stages in the mammalian central nervous system.Methods. Using the whole-cell voltage-clamp technique, we have studied changes in the electrophysi-ological properties of Na+ channels in the principal neurons of the medial nucleus of the trapezoid body (MNTB).Results. We found that MNTB neurons already express functional Na+ channels at postnatal day 1 (P1),and that channel density begins to increase at P5 when the neurons receive synaptic innervation andreach its maximum (~3 fold) at P11 when functional hearing onsets. These changes were paralleled byan age-dependent acceleration in both inactivation and recovery from inactivation. In contrast, there wasvery little alteration in the voltage-dependence of inactivation.Conclusion. These profound changes in the properties of voltage-gated Na+ channels may increase theexcitability of MNTB neurons and enhance their phase-locking fidelity and capacity during high-frequencysynaptic transmission.

  15. Structure and function of the voltage sensor of sodium channels probed by a beta-scorpion toxin.

    Science.gov (United States)

    Cestèle, Sandrine; Yarov-Yarovoy, Vladimir; Qu, Yusheng; Sampieri, François; Scheuer, Todd; Catterall, William A

    2006-07-28

    Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-scorpion toxin binding but alter voltage dependence of activation and enhance beta-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.

  16. Structure and Function of the Voltage Sensor of Sodium Channels Probed by a β-Scorpion Toxin*S

    Science.gov (United States)

    Cestèle, Sandrine; Yarov-Yarovoy, Vladimir; Qu, Yusheng; Sampieri, François; Scheuer, Todd; Catterall, William A.

    2006-01-01

    Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. β-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that β-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu779 in extracellular loop IIS1–S2 and both Glu837 and Leu840 in extracellular loop IIS3–S4 reduce the binding affinity of β-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect β-scorpion toxin binding but alter voltage dependence of activation and enhance β-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states. PMID:16679310

  17. Heterogeneity of Calcium Channel/cAMP-Dependent Transcriptional Activation.

    Science.gov (United States)

    Kobrinsky, Evgeny

    2015-01-01

    The major function of the voltage-gated calcium channels is to provide the Ca(2+) flux into the cell. L-type voltage-gated calcium channels (Cav1) serve as voltage sensors that couple membrane depolarization to many intracellular processes. Electrical activity in excitable cells affects gene expression through signaling pathways involved in the excitation-transcription (E-T) coupling. E-T coupling starts with activation of the Cav1 channel and results in initiation of the cAMP-response element binding protein (CREB)-dependent transcription. In this review we discuss the new quantitative approaches to measuring E-T signaling events. We describe the use of wavelet transform to detect heterogeneity of transcriptional activation in nuclei. Furthermore, we discuss the properties of discovered microdomains of nuclear signaling associated with the E-T coupling and the basis of the frequency-dependent transcriptional regulation.

  18. Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

    Energy Technology Data Exchange (ETDEWEB)

    Rosenberg, R.L.

    1985-01-01

    The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based on molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.

  19. Towards a Unified Theory of Calmodulin Regulation (Calmodulation) of Voltage-Gated Calcium and Sodium Channels.

    Science.gov (United States)

    Ben-Johny, Manu; Dick, Ivy E; Sang, Lingjie; Limpitikul, Worawan B; Kang, Po Wei; Niu, Jacqueline; Banerjee, Rahul; Yang, Wanjun; Babich, Jennifer S; Issa, John B; Lee, Shin Rong; Namkung, Ho; Li, Jiangyu; Zhang, Manning; Yang, Philemon S; Bazzazi, Hojjat; Adams, Paul J; Joshi-Mukherjee, Rosy; Yue, Daniel N; Yue, David T

    2015-01-01

    Voltage-gated Na and Ca(2+) channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca(2+) and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.

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

    Science.gov (United States)

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

    2017-01-01

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

  1. Voltage-Sensitive Ion Channels Biophysics of Molecular Excitability

    CERN Document Server

    Leuchtag, H. Richard

    2008-01-01

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

  2. 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.

  3. Simulating the Activation of Voltage Sensing Domain for a Voltage-Gated Sodium Channel Using Polarizable Force Field.

    Science.gov (United States)

    Sun, Rui-Ning; Gong, Haipeng

    2017-03-02

    Voltage-gated sodium (NaV) channels play vital roles in the signal transduction of excitable cells. Upon activation of a NaV channel, the change of transmembrane voltage triggers conformational change of the voltage sensing domain, which then elicits opening of the pore domain and thus allows an influx of Na(+) ions. Description of this process with atomistic details is in urgent demand. In this work, we simulated the partial activation process of the voltage sensing domain of a prokaryotic NaV channel using a polarizable force field. We not only observed the conformational change of the voltage sensing domain from resting to preactive state, but also rigorously estimated the free energy profile along the identified reaction pathway. Comparison with the control simulation using an additive force field indicates that voltage-gating thermodynamics of NaV channels may be inaccurately described without considering the electrostatic polarization effect.

  4. 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.

  5. Transcriptional regulation of voltage-gated Ca(2+) channels.

    Science.gov (United States)

    González-Ramírez, Ricardo; Felix, Ricardo

    2017-03-31

    The transcriptional regulation of voltage-gated Ca(2+) (CaV ) channels is an emerging research area that promises to improve our understanding of how many relevant physiological events are shaped in the central nervous system, the skeletal muscle, and other tissues. Interestingly, a picture of how transcription of CaV channel subunit genes is controlled is evolving with the identification of the promoter regions required for tissue-specific expression, and the identification of transcription factors that control their expression. These promoters share several characteristics that include multiple transcriptional start sites, lack of a TATA box, and the presence of elements conferring tissue-selective expression. Likewise, changes in CaV channel expression occur throughout development, following ischemia, seizures, or chronic drug administration. This review focuses on insights achieved regarding the control of CaV channel gene expression. To further understand the complexities of expression and to increase the possibilities of detecting CaV channel alterations causing human disease, a deeper knowledge on the structure of the 5' upstream regions of the genes encoding these remarkable proteins will be necessary. This article is protected by copyright. All rights reserved.

  6. Voltage-independent inhibition of Cav2.2 channels is delimited to a specific region of the membrane potential in rat SCG neurons

    Institute of Scientific and Technical Information of China (English)

    Oscar Vivas; Isabel Arenas; David E.García

    2012-01-01

    Neurotransmitters and hormones regulate Cav2.2 channels through a voltage-independent pathway which is not well understood.It has been suggested that this voltageindependent inhibition is constant at all membrane voltages.However,changes in the percent of voltageindependent inhibition of Cav2.2 have not been tested within a physiological voltage range.Here,we used a double-pulse protocol to isolate the voltage-independent inhibition of Cav2.2 channels induced by noradrenaline in rat superior cervical ganglion neurons.To assess changes in the percent of the voltage-independent inhibition,the activation voltage of the channels was tested between -40 and +40 mV.We found that the percent of voltage-independent inhibition induced by noradrenaline changed with the activation voltage used.In addition,voltage-independent inhibition induced by oxo-M,a muscarinic agonist,exhibited thesame dependence on activation voltage,which supports that this pattern is not exclusive for adrenergic activation.Our results suggested that voltage-independent inhibition of Cav2.2 channels depends on the activation voltage of the channel in a physiological voltage range.This may have relevant implications in the understanding of the mechanism involved in voltage-independent inhibition.

  7. 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.

  8. Voltage dependence of the Na-K pump.

    Science.gov (United States)

    De Weer, P; Gadsby, D C; Rakowski, R F

    1988-01-01

    Present evidence demonstrates that the Na-K pump rate is voltage dependent, whereas early work was largely inconclusive. The I-V relationship has a positive slope over a wide voltage range, and the existence of a negative slope region is now doubtful. Monotonic voltage dependence is consistent with the reaction cycle containing a single voltage-dependent step. Recent measurements suggest that this voltage-dependent step occurs during Na translocation and may be deocclusion of Na+. In addition, two results suggest that K translocation is voltage insensitive: (a) large positive potentials appear to have no influence on Rb-Rb exchange or associated conformational transitions; and (b) transient currents associated with Na translocation appear to involve movement of a single charge, which is sufficient for a 3Na-2K cycle. The simplest interpretation is that the pump's cation binding sites supply two negative charges. Pre-steady-state measurements demonstrate that Na translocation precedes the pump cycle's rate-limiting step, presumably K translocation. But, because K translocation seems voltage insensitive, the voltage dependence of the steady-state pump rate probably reflects that of the concentration of the intermediate entering this slow step. Further pump current and flux data (both transient and steady-state), carefully determined over a range of conditions, should increase our understanding of the voltage-dependent step(s) in the Na-K pump cycle.

  9. Activity-dependent Phosphorylation of Neuronal Kv2.1 Potassium Channels by CDK5*

    OpenAIRE

    Cerda, Oscar; Trimmer, James S.

    2011-01-01

    Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible,...

  10. 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.

  11. 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.

  12. Dynamic memory of a single voltage-gated potassium ion channel: A stochastic nonequilibrium thermodynamic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Kinshuk, E-mail: kbpchem@gmail.com [Department of Chemistry, University of Calcutta, 92 A.P.C. Road, Kolkata 700 009 (India)

    2015-05-14

    In this work, we have studied the stochastic response of a single voltage-gated potassium ion channel to a periodic external voltage that keeps the system out-of-equilibrium. The system exhibits memory, resulting from time-dependent driving, that is reflected in terms of dynamic hysteresis in the current-voltage characteristics. The hysteresis loop area has a maximum at some intermediate voltage frequency and disappears in the limits of low and high frequencies. However, the (average) dissipation at long-time limit increases and finally goes to saturation with rising frequency. This raises the question: how diminishing hysteresis can be associated with growing dissipation? To answer this, we have studied the nonequilibrium thermodynamics of the system and analyzed different thermodynamic functions which also exhibit hysteresis. Interestingly, by applying a temporal symmetry analysis in the high-frequency limit, we have analytically shown that hysteresis in some of the periodic responses of the system does not vanish. On the contrary, the rates of free energy and internal energy change of the system as well as the rate of dissipative work done on the system show growing hysteresis with frequency. Hence, although the current-voltage hysteresis disappears in the high-frequency limit, the memory of the ion channel is manifested through its specific nonequilibrium thermodynamic responses.

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

    Science.gov (United States)

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

    2016-06-01

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

  14. Voltage-gated calcium channel subunits from platyhelminths: Potential role in praziquantel action✩

    Science.gov (United States)

    Jeziorski, Michael C.; Greenberg, Robert M.

    2013-01-01

    Voltage-gated calcium (Ca2+) channels provide the pathway for Ca2+ influxes that underlie Ca2+-dependent responses in muscles, nerves and other excitable cells. They are also targets of a wide variety of drugs and toxins. Ca2+ channels are multisubunit protein complexes consisting of a pore-forming α1 subunit and other modulatory subunits, including the β subunit. Here, we review the structure and function of schistosome Ca2+ channel subunits, with particular emphasis on variant Ca2+ channel β subunits (Cavβvar) found in these parasites. In particular, we examine the role these β subunits may play in the action of praziquantel, the current drug of choice against schistosomiasis. We also present evidence that Cavβvar homologs are found in other praziquantel-sensitive platyhelminths such as the pork tapeworm, Taenia solium, and that these variant β subunits may thus represent a platyhelminth-specific gene family. PMID:16545816

  15. Voltage-gated sodium channels: biophysics, pharmacology, and related channelopathies

    Directory of Open Access Journals (Sweden)

    Eleonora eSavio Galimberti

    2012-07-01

    Full Text Available Voltage-gated sodium channels (VGSC are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit that can be associated with one or more regulatory β-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (INa and can also function as cell-adhesion molecules (CAMs. In-vitro some of the cell-adhesive functions of the β-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named channel partners or channel interacting proteins (ChiPs like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin, and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics. Mutations in genes that encode α- and/or β-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium channelopathies. This review will outline the structure, function and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field

  16. Voltage-Gated Sodium Channels: Biophysics, Pharmacology, and Related Channelopathies

    Science.gov (United States)

    Savio-Galimberti, Eleonora; Gollob, Michael H.; Darbar, Dawood

    2012-01-01

    Voltage-gated sodium channels (VGSC) are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit) that can be associated with one or more regulatory β-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (INa) and can also function as cell adhesion molecules. In vitro some of the cell-adhesive functions of the β-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named “channel partners” or “channel interacting proteins” (ChiPs) like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII) can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin), and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics). Mutations in genes that encode α- and/or β-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium “channelopathies”.  This review will outline the structure, function, and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field. PMID:22798951

  17. Nonequilibrium response of a voltage gated sodium ion channel and biophysical characterization of dynamic hysteresis.

    Science.gov (United States)

    Pal, Krishnendu; Das, Biswajit; Gangopadhyay, Gautam

    2017-02-21

    Here we have studied the dynamic as well as the non-equilibrium thermodynamic response properties of voltage-gated Na-ion channel. Using sinusoidally oscillating external voltage protocol we have both kinetically and energetically studied the non-equilibrium steady state properties of dynamic hysteresis in details. We have introduced a method of estimating the work done associated with the dynamic memory due to a cycle of oscillating voltage. We have quantitatively characterised the loop area of ionic current which gives information about the work done to sustain the dynamic memory only for ion conduction, while the loop area of total entropy production rate gives the estimate of work done for overall gating dynamics. The maximum dynamic memory of Na-channel not only depends on the frequency and amplitude but it also depends sensitively on the mean of the oscillating voltage and here we have shown how the system optimize the dynamic memory itself in the biophysical range of field parameters. The relation between the average ionic current with increasing frequency corresponds to the nature of the average dissipative work done at steady state. It is also important to understand that the utilization of the energy from the external field can not be directly obtained only from the measurement of ionic current but also requires nonequilibrium thermodynamic study.

  18. Deciphering voltage-gated Na(+) and Ca(2+) channels by studying prokaryotic ancestors.

    Science.gov (United States)

    Catterall, William A; Zheng, Ning

    2015-09-01

    Voltage-gated sodium channels (NaVs) and calcium channels (CaVs) are involved in electrical signaling, contraction, secretion, synaptic transmission, and other physiological processes activated in response to depolarization. Despite their physiological importance, the structures of these closely related proteins have remained elusive because of their size and complexity. Bacterial NaVs have structures analogous to a single domain of eukaryotic NaVs and CaVs and are their likely evolutionary ancestor. Here we review recent work that has led to new understanding of NaVs and CaVs through high-resolution structural studies of their prokaryotic ancestors. New insights into their voltage-dependent activation and inactivation, ion conductance, and ion selectivity provide realistic structural models for the function of these complex membrane proteins at the atomic level. Published by Elsevier Ltd.

  19. Voltage-gated K+ channels contain multiple intersubunit association sites.

    Science.gov (United States)

    Tu, L; Santarelli, V; Sheng, Z; Skach, W; Pain, D; Deutsch, C

    1996-08-02

    A domain in the cytoplasmic NH2 terminus of voltage-gated K+ channels supervises the proper assembly of specific tetrameric channels (Li, M., Jan, J. M., and Jan, L. Y.(1992) Science 257, 1225-1230; Shen, N. V., Chen X., Boyer, M. M., and Pfaffinger, P. (1993) Neuron 11, 67-76). It is referred to as a first tetramerization domain, or T1 (Shen, N. V., Chen X., Boyer, M. M., and Pfaffinger, P.(1993) Neuron 11, 67-76). However, a deletion mutant of Kv1.3 that lacks the first 141 amino acids, Kv1.3 (T1(-)) forms functional channels, suggesting that additional association sites in the central core of Kv1.3 mediate oligomerization. To characterize these sites, we have tested the abilities of cRNA Kv1.3 (T1(-)) fragments co-injected with Kv1.3 (T1(-)) to suppress current in Xenopus oocytes. The fragments include portions of the six putative transmembrane segments, S1 through S6, specifically: S1, S1-S2, S1-S2-S3, S2-S3, S2-S3-S4, S3-S4, S3-S4-S5, S2 through COOH, S3 through COOH, S4 through COOH, and S5-S6-COOH. Electrophysiologic experiments show that the fragments S1-S2-S3, S3-S4-S5, S2 through COOH, and S3 through COOH strongly suppress Kv1.3 (T1(-)) current, while others do not. Suppression of expressed current is due to specific effects of the translated peptide Kv1.3 fragments, as validated by in vivo immunoprecipitation studies of a strong suppressor and a nonsuppressor. Pulse-chase experiments indicate that translation of truncated peptide fragments neither prevents translation of Kv1.3 (T1(-)) nor increases its rate of degradation. Co-immunoprecipitation experiments suggest that suppression involves direct association of a peptide fragment with Kv1.3 (T1(-)). Fragments that strongly suppress Kv1.3 (T1(-)) also suppress an analogous NH2-terminal deletion mutant of Kv2.1 (Kv2.1 (DeltaN139)), an isoform belonging to a different subfamily. Our results indicate that sites in the central core of Kv1.3 facilitate intersubunit association and that there are suppression

  20. Hypotonicity activates a voltage-dependent membrane conductance in N2a neuroblastoma cells.

    Science.gov (United States)

    Taruno, Akiyuki; Marunaka, Yoshinori

    2017-03-04

    To maintain cellular and bodily homeostasis, cells respond to extracellular stimuli including osmotic stress by activating various ion channels, which have been implicated in many physiological and pathophysiological conditions. However, cellular osmosensory mechanisms remain elusive. Here, we report a novel voltage-dependent current in N2a cells activated by exposure to hypotonic stress. After a hypotonic challenge, N2a cells sequentially develop two distinct currents. The volume-regulated anion channel (VRAC) current emerges first and, after a delay, activation of a previously uncharacterized strongly outwardly rectifying current follows. The latter, delayed current (Id) is insensitive to NPPB, a nonspecific blocker of Cl(-) channels, and intracellular Mg(2+), which inhibits VRAC and swelling-activated TRPM3 and TRPM7 channels. Replacement of extracellular Na(+) with NMDG(+) reduces inward tail currents, suggesting that Id is mediated by cations. Finally, Id shows voltage-dependent activation with slow activation kinetics and half-maximal activation at +76 mV. These pharmacological and biophysical characteristics of Id are distinct from those of known osmotic cell swelling-activated ion channels. In conclusion, our data identify and characterize a novel osmotically-activated, voltage-dependent ion channel in N2a cells.

  1. Sleep disturbances in voltage-gated potassium channel antibody syndrome.

    Science.gov (United States)

    Barone, Daniel A; Krieger, Ana C

    2016-05-01

    Voltage-gated potassium channels (VGKCs) are a family of membrane proteins responsible for controlling cell membrane potential. The presence of antibodies (Ab) against neuronal VGKC complexes aids in the diagnosis of idiopathic and paraneoplastic autoimmune neurologic disorders. The diagnosis of VGKC Ab-associated encephalopathy (VCKC Ab syndrome) should be suspected in patients with subacute onset of disorientation, confusion, and memory loss in the presence of seizures or a movement disorder. VGKC Ab syndrome may present with sleep-related symptoms, and the purpose of this communication is to alert sleep and neurology clinicians of this still-under-recognized condition. In this case, we are presenting the VGKC Ab syndrome which improved after treatment with solumedrol. The prompt recognition and treatment of this condition may prevent the morbidity associated with cerebral atrophy and the mortality associated with intractable seizures and electrolyte disturbances.

  2. 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.

  3. Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation.

    Science.gov (United States)

    Zhou, Meng-Hua; Bavencoffe, Alexis; Pan, Hui-Lin

    2015-12-18

    Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and synaptic transmission. We have shown previously that NO inhibits high voltage-activated Ca(2+) channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca(2+) channels. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavβ1 or Cavβ3 subunits in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavβ subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys-805, Cys-930, and Cys-1045 in the II-III intracellular loop, Cys-1835 and Cys-2145 in the C terminus of Cav2.2, and Cys-346 in the Cavβ3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca(2+) channels by NO. S-Nitrosylation mediates the feedback regulation of N-type channels by NO.

  4. Differential regulation of voltage- and calcium-activated potassium channels in human B lymphocytes.

    Science.gov (United States)

    Partiseti, M; Choquet, D; Diu, A; Korn, H

    1992-06-01

    The expression and characteristics of K+ channels of human B lymphocytes were studied by using single and whole-cell patch-clamp recordings. They were gated by depolarization (voltage-gated potassium current, IKv, 11-20 pS) and by an increase in intracellular Ca2+ concentration (calcium-activated potassium current, IKCa, 26 pS), respectively. The level of expression of these channels was correlated with the activational status of the cell. Both conductances are blocked by tetraethylammonium, verapamil, and charybdotoxin, and are insensitive to apamin; 4-aminopyridine blocks IK, preferentially. We used a protein kinase C activator (PMA) or antibodies to membrane Ig (anti-mu) to activate resting splenocytes in culture. Although IKv was recorded in the majority of the resting lymphocytic population, less than 20% of the activated cells expressed this conductance. However, in this subset the magnitude of IKv was 20-fold larger than in resting cells. On the other hand, IKCa was detected in nearly one half of the resting cells, whereas all activated cells expressed this current. The magnitude of IKCa was, on average, 30 times larger in activated than in nonactivated cells. These results probably reflect that during the course of activation 1) the number of voltage-dependent K+ channels per cell decreases and increases in a small subset and 2) the number of Ca(2+)-dependent K+ channels per cell increases in all cells. We suggest that the expression of functional Ca(2+)- and voltage-activated K+ channels are under the control of different regulatory signals.

  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. Regulation of cough and action potentials by voltage-gated Na channels.

    Science.gov (United States)

    Carr, Michael J

    2013-10-01

    The classical role ascribed to voltage-gated Na channels is the conduction of action potentials. Some excitable tissues such as cardiac muscle and skeletal muscle predominantly express a single voltage-gated Na channels isoform. Of the nine voltage-gated Na channels, seven are expressed in neurons, of these Nav 1.7, 1.8 and 1.9 are expressed in sensory neurons including vagal sensory neurons that innervate the airways and initiate cough. Nav 1.7 and Nav 1.9 are of particular interest as they represent two extremes in the functional diversity of voltage-gated Na channels. Voltage-gated Na channel isoforms expressed in airway sensory neurons produce multiple distinct Na currents that underlie distinct aspects of sensory neuron function. The interaction between voltage-gated Na currents underlies the characteristic ability of airway sensory nerves to encode encounters with irritant stimuli into action potential discharge and evoke the cough reflex.

  7. [Potential-dependent Cation Selective Ion Channels Formed by Peroxiredoxin 6 in the Lipid Bilayer].

    Science.gov (United States)

    Grigoriev, P A; Sharapov, M G; Novoselov, V I

    2015-01-01

    The antioxidant enzyme peroxiredoxin 6 forms cation selective ion cluster-type channels in the lipid bilayer. Channel clustering as oligomeric structure consists of three or more subunits--channels with conductance of about 350 pS in the 200 mM KCl. Mean dwell time of the channel's open states decreases with increasing membrane voltage. A possible molecular mechanism of the observed potential-dependent inactivation of the channel cluster is discussed.

  8. The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels.

    Science.gov (United States)

    Bende, Niraj S; Kang, Eunji; Herzig, Volker; Bosmans, Frank; Nicholson, Graham M; Mobli, Mehdi; King, Glenn F

    2013-05-15

    One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Endogenous polyamines regulate cortical neuronal excitability by blocking voltage-gated Na+ channels.

    Science.gov (United States)

    Fleidervish, Ilya A; Libman, Lior; Katz, Efrat; Gutnick, Michael J

    2008-12-02

    Because the excitable properties of neurons in the neocortex depend on the characteristics of voltage-gated Na(+) channels, factors which regulate those characteristics can fundamentally modify the dynamics of cortical circuits. Here, we report on a novel neuromodulatory mechanism that links the availability of Na(+) channels to metabolism of polyamines (PAs) in the cerebral cortex. Using single channel and whole-cell recordings, we found that products of PA metabolism, the ubiquitous aliphatic polycations spermine and spermidine, are endogenous blockers of Na(+) channels in layer 5 pyramidal cells. Because the blockade is activity-dependent, it is particularly effective against Na(+) channels which fail to inactivate rapidly and thus underlie the persistent Na(+) current. At the level of the local cortical circuit, pharmacological depletion of PAs led to increased spontaneous spiking and periods of hypersynchronous discharge. Our data suggest that changes in PA levels, whether associated with normal brain states or pathological conditions, profoundly modify Na(+) channel availability and thereby shape the integrative behavior of single neurons and neocortical circuits.

  10. 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.

  11. Calcium binding protein-mediated regulation of voltage-gated calcium channels linked to human diseases

    Institute of Scientific and Technical Information of China (English)

    Nasrin NFJATBAKHSH; Zhong-ping FENG

    2011-01-01

    Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kariev, Alisher M.; Green, Michael E.

    2012-02-26

    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

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

    Directory of Open Access Journals (Sweden)

    Alisher M. Kariev

    2012-02-01

    Full Text Available 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

  14. Two-pore channels (TPCs): Novel voltage-gated ion channels with pleiotropic functions.

    Science.gov (United States)

    Feijóo-Bandín, Sandra; García-Vence, María; García-Rúa, Vanessa; Roselló-Lletí, Esther; Portolés, Manuel; Rivera, Miguel; González-Juanatey, José Ramón; Lago, Francisca

    2017-01-02

    Two-pore channels (TPC1-3) comprise a subfamily of the eukaryotic voltage-gated ion channels (VGICs) superfamily that are mainly expressed in acidic stores in plants and animals. TPCS are widespread across the animal kingdom, with primates, mice and rats lacking TPC3, and mainly act as Ca(+) and Na(+) channels, although it was also suggested that they could be permeable to other ions. Nowadays, TPCs have been related to the development of different diseases, including Parkinson´s disease, obesity or myocardial ischemia. Due to this, their study has raised the interest of the scientific community to try to understand their mechanism of action in order to be able to develop an efficient drug that could regulate TPCs activity. In this review, we will provide an updated view regarding TPCs structure, function and activation, as well as their role in different pathophysiological processes.

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

    DEFF Research Database (Denmark)

    Hansen, Pernille B L

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Alison R Taylor

    2011-06-01

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

  17. Tetrahydroacridine inhibits voltage-dependent Na+ current in guinea-pig ventricular myocytes

    Institute of Scientific and Technical Information of China (English)

    Wei WANG; Yi-ping WANG; Guo-yuan HU

    2004-01-01

    AIM: To study the effects of tetrahydroacridine (tacrine) on voltage-gated Na+ channels in cardiac tissues.METHODS: Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltagedependent Na+ current was recorded using whole cell voltage-clamp technique. RESULTS: (1) Tacrine reversibly inhibited Na+ current with an IC50 value of 120 μmol/L (95 % confidence range: 108-133 μmol/L). (2) The inhibitory effects of tacrine on Na+ current exhibited both a tonic nature and use-dependence. (3) Tacrine at 100 μmol/L caused a negative shift (about 10 mV) in the voltage-dependence of steady-state inactivation of Na+ current, and retarded its recovery from inactivation, but did not affect its activation curve. (4) Intracellular application of tacrine significantly inhibited Na+ current. CONCLUSION: In addition to blocking other voltage-gated ion channels,tacrine blocked Na+ channels in guinea-pig ventricular myocytes. Tactine acted as inactivation stabilizer of Na+channels in cardiac tissues.

  18. Molecular Model of Anticonvulsant Drug Binding to the Voltage-Gated Sodium Channel Inner Pore

    Science.gov (United States)

    Lipkind, Gregory M.

    2010-01-01

    The tricyclic anticonvulsant drugs phenytoin, carbamazepine, and lamotrigine block neuronal voltage-gated Na+ channels, and their binding sites to domain IV-S6 in the channel's inner pore overlap with those of local anesthetic drugs. These anticonvulsants are neutral, in contrast to the mostly positively charged local anesthetics, but their open/inactivated-state blocking affinities are similar. Using a model of the open pore of the Na+ channel that we developed by homology with the crystal structures of potassium channels, we have docked these three anticonvulsants with residues identified by mutagenesis as important for their binding energy. The three drugs show a common pharmacophore, including an aromatic ring that has an aromatic-aromatic interaction with Tyr-1771 of NaV1.2 and a polar amide or imide that interacts with the aromatic ring of Phe-1764 by a low-energy amino-aromatic hydrogen bond. The second aromatic ring is nearly at a right angle to the pharmacophore and fills the pore lumen, probably interacting with the other S6 segments and physically occluding the inner pore to block Na+ permeation. Hydrophobic interactions with this second aromatic ring may contribute an important component to binding for anticonvulsants, which compensates energetically for the absence of positive charge in their structures. Voltage dependence of block, their important therapeutic property, results from their interaction with Phe-1764, which connects them to the voltage sensors. Their use dependence is modest and this results from being neutral, with a fast drug off-rate after repolarization, allowing a normal action potential rate in the presence of the drugs. PMID:20643904

  19. CaV1.1: The atypical prototypical voltage-gated Ca2+ channel

    Science.gov (United States)

    Bannister, Roger A.; Beam, Kurt G.

    2012-01-01

    CaV1.1 is the prototype for the other nine known CaV channel isoforms, yet it has functional properties that make it truly atypical of this group. Specifically, CaV1.1 is expressed solely in skeletal muscle where it serves multiple purposes; it is the voltage sensor for excitation-contraction (EC) coupling and it is an L-type Ca2+ channel which contributes to a form of activity-dependent Ca2+ entry that has been termed Excitation-Coupled Ca2+ Entry (ECCE). The ability of CaV1.1 to serve as voltage-sensor for EC coupling appears to be unique amongst CaV channels, whereas the physiological role of its more conventional function as a Ca2+ channel has been a matter of uncertainty for nearly 50 years. In this chapter, we discuss how CaV1.1 supports EC coupling, the possible relevance of Ca2+ entry through CaV1.1 and how alterations of CaV1.1 function can have pathophysiological consequences. PMID:22982493

  20. Distance measurements reveal a common topology of prokaryotic voltage-gated ion channels in the lipid bilayer.

    Science.gov (United States)

    Richardson, Jessica; Blunck, Rikard; Ge, Pinghua; Selvin, Paul R; Bezanilla, Francisco; Papazian, Diane M; Correa, Ana M

    2006-10-24

    Voltage-dependent ion channels are fundamental to the physiology of excitable cells because they underlie the generation and propagation of the action potential and excitation-contraction coupling. To understand how ion channels work, it is important to determine their structures in different conformations in a membrane environment. The validity of the crystal structure for the prokaryotic K(+) channel, K(V)AP, has been questioned based on discrepancies with biophysical data from functional eukaryotic channels, underlining the need for independent structural data under native conditions. We investigated the structural organization of two prokaryotic voltage-gated channels, NaChBac and K(V)AP, in liposomes by using luminescence resonance energy transfer. We describe here a transmembrane packing representation of the voltage sensor and pore domains of the prokaryotic Na channel, NaChBac. We find that NaChBac and K(V)AP share a common arrangement in which the structures of the Na and K selective pores and voltage-sensor domains are conserved. The packing arrangement of the voltage-sensing region as determined by luminescence resonance energy transfer differs significantly from that of the K(V)AP crystal structure, but resembles that of the eukaryotic K(V)1.2 crystal structure. However, the voltage-sensor domain in prokaryotic channels is closer to the pore domain than in the K(V)1.2 structure. Our results indicate that prokaryotic and eukaryotic channels that share similar functional properties have similar helix arrangements, with differences arising likely from the later introduction of additional structural elements.

  1. The voltage dependence of Ih in human myelinated axons

    Science.gov (United States)

    Howells, James; Trevillion, Louise; Bostock, Hugh; Burke, David

    2012-01-01

    HCN channels are responsible for Ih, a voltage-gated inwardly rectifying current activated by hyperpolarization. This current appears to be more active in human sensory axons than motor and may play a role in the determination of threshold. Differences in Ih are likely to be responsible for the high variability in accommodation to hyperpolarization seen in different subjects. The aim of this study was to characterise this current in human axons, both motor and sensory. Recordings of multiple axonal excitability properties were performed in 10 subjects, with a focus on the changes in threshold evoked by longer and stronger hyperpolarizing currents than normally studied. The findings confirm that accommodation to hyperpolarization is greater in sensory than motor axons in all subjects, but the variability between subjects was greater than the modality difference. An existing model of motor axons was modified to take into account the behaviour seen with longer and stronger hyperpolarization, and a mathematical model of human sensory axons was developed based on the data collected. The differences in behaviour of sensory and motor axons and the differences between different subjects are best explained by modulation of the voltage dependence, along with a modest increase of expression of the underlying conductance of Ih. Accommodation to hyperpolarization for the mean sensory data is fitted well with a value of −94.2 mV for the mid-point of activation (V0.5) of Ih as compared to −107.3 mV for the mean motor data. The variation in response to hyperpolarization between subjects is accounted for by varying this parameter for each modality (sensory: −89.2 to −104.2 mV; motor −87.3 to −127.3 mV). These voltage differences are within the range that has been described for physiological modulation of Ih function. The presence of slowly activated Ih isoforms on both motor and sensory axons was suggested by modelling a large internodal leak current and a masking of

  2. Zn2+ regulates Kv2.1 voltage-dependent gating and localization following ischemia

    OpenAIRE

    Aras, Mandar A.; Saadi, Robert A.; Aizenman, Elias

    2009-01-01

    The delayed-rectifier K+ channel Kv2.1 exists in highly phosphorylated somatodendritic clusters. Ischemia induces rapid Kv2.1 dephosphorylation and a dispersal of these clusters, accompanied by a hyperpolarizing shift in their voltage-dependent activation kinetics. Transient modulation of Kv2.1 activity and localization following ischemia is dependent on a rise in intracellular Ca2+and the protein phosphatase calcineurin. Here, we show that neuronal free Zn2+also plays a critical role in the ...

  3. Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels

    Science.gov (United States)

    Lau, Carus H. Y.; King, Glenn F.; Mobli, Mehdi

    2016-01-01

    Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels. PMID:27677715

  4. Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels

    Science.gov (United States)

    Lau, Carus H. Y.; King, Glenn F.; Mobli, Mehdi

    2016-09-01

    Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels.

  5. Kinetics properties of voltage induced colicin Ia channels into a lipid bilayer

    CERN Document Server

    Cassia-Moura, R

    1998-01-01

    The activation kinetics of the ion channels formed by colicin Ia incorporated into a planar bilayer lipid membrane (BLM) was investigated by the voltage clamp technique using different step voltage stimuli. The temporal behaviour of ion channels put in evidence a gain or a loss of memory, revealed by a specific sequence of electrical pulses used for stimulation.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    Voltage-gated potassium (Kv) channels enable potassium efflux and membrane repolarization in excitable tissues. Many Kv channels undergo a progressive loss of ion conductance in the presence of a prolonged voltage stimulus, termed slow inactivation, but the atomic determinants that regulate the k...

  7. Functionality of the voltage-gated proton channel truncated in S4.

    Science.gov (United States)

    Sakata, Souhei; Kurokawa, Tatsuki; Nørholm, Morten H H; Takagi, Masahiro; Okochi, Yoshifumi; von Heijne, Gunnar; Okamura, Yasushi

    2010-02-02

    The voltage sensor domain (VSD) is the key module for voltage sensing in voltage-gated ion channels and voltage-sensing phosphatases. Structurally, both the VSD and the recently discovered voltage-gated proton channels (Hv channels) voltage sensor only protein (VSOP) and Hv1 contain four transmembrane segments. The fourth transmembrane segment (S4) of Hv channels contains three periodically aligned arginines (R1, R2, R3). It remains unknown where protons permeate or how voltage sensing is coupled to ion permeation in Hv channels. Here we report that Hv channels truncated just downstream of R2 in the S4 segment retain most channel properties. Two assays, site-directed cysteine-scanning using accessibility of maleimide-reagent as detected by Western blotting and insertion into dog pancreas microsomes, both showed that S4 inserts into the membrane, even if it is truncated between the R2 and R3 positions. These findings provide important clues to the molecular mechanism underlying voltage sensing and proton permeation in Hv channels.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Voltage-sensor domains couple membrane potential to conformational changes in voltage-gated ion channels and phosphatases. Highly coevolved acidic and aromatic side chains assist the transfer of cationic side chains across the transmembrane electric field during voltage sensing. We investigated...

  9. Mapping of scorpion toxin receptor sites at voltage-gated sodium channels.

    Science.gov (United States)

    Gurevitz, Michael

    2012-09-15

    Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Na(v)s) at two pharmacologically distinct sites. Alpha toxins bind at receptor site-3 and inhibit channel inactivation, whereas beta toxins bind at receptor site-4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and ability to compete for the receptor sites at Na(v) subtypes. To elucidate the toxin-channel surface of interaction at both receptor sites and clarify the molecular basis of varying toxin preferences, an efficient bacterial system for their expression in recombinant form was established. Mutagenesis accompanied by toxicity, binding and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography uncovered a bipartite bioactive surface in toxin representatives of all pharmacological groups. Exchange of external loops between the mammalian brain channel rNa(v)1.2a and the insect channel DmNa(v)1 highlighted channel regions involved in the varying sensitivity to assorted toxins. In parallel, thorough mutagenesis of channel external loops illuminated points of putative interaction with the toxins. Amino acid substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain II of rNa(v)1.2a had prominent impact on the activity of the beta-toxin Css4 (from Centruroides suffusus suffusus), and substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain IV affected the activity of the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). Rosetta modeling of toxin-Na(v) interaction using the voltage sensor module of the potassium channel as template raises commonalities in the way alpha and beta toxins interact with the channel. Css4 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain

  10. Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

    Science.gov (United States)

    Taft, William C.; Delorenzo, Robert J.

    1984-05-01

    Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

  11. Regulation of voltage gated calcium channels by GPCRs and post-translational modification.

    Science.gov (United States)

    Huang, Junting; Zamponi, Gerald W

    2016-10-18

    Calcium entry via voltage gated calcium channels mediates a wide range of physiological functions, whereas calcium channel dysregulation has been associated with numerous pathophysiological conditions. There are myriad cell signaling pathways that act on voltage gated calcium channels to fine tune their activities and to regulate their cell surface expression. These regulatory mechanisms include the activation of G protein-coupled receptors and downstream phosphorylation events, and their control over calcium channel trafficking through direct physical interactions. Calcium channels also undergo post-translational modifications that alter both function and density of the channels in the plasma membrane. Here we focus on select aspects of these regulatory mechanisms and highlight recent developments.

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

    Science.gov (United States)

    Riedelsberger, Janin; Dreyer, Ingo; Gonzalez, Wendy

    2015-01-01

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

  13. Current view on regulation of voltage-gated sodium channels by calcium and auxiliary proteins.

    Science.gov (United States)

    Pitt, Geoffrey S; Lee, Seok-Yong

    2016-09-01

    In cardiac and skeletal myocytes, and in most neurons, the opening of voltage-gated Na(+) channels (NaV channels) triggers action potentials, a process that is regulated via the interactions of the channels' intercellular C-termini with auxiliary proteins and/or Ca(2+) . The molecular and structural details for how Ca(2+) and/or auxiliary proteins modulate NaV channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca(2+) acts directly upon the NaV channel or through interacting proteins, such as the Ca(2+) binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of NaV intracellular C-termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca(2+) affects NaV function, and how altered Ca(2+) -dependent or FHF-mediated regulation of NaV channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.

  14. Differential effects of K(+) channel blockers on frequency-dependent action potential broadening in supraoptic neurons.

    Science.gov (United States)

    Hlubek, M D; Cobbett, P

    2000-09-15

    Recordings were made from magnocellular neuroendocrine cells dissociated from the supraoptic nucleus of the adult guinea pig to determine the role of voltage gated K(+) channels in controlling the duration of action potentials and in mediating frequency-dependent action potential broadening exhibited by these neurons. The K(+) channel blockers charybdotoxin (ChTx), tetraethylammonium (TEA), and 4-aminopyridine (4-AP) increased the duration of individual action potentials indicating that multiple types of K(+) channel are important in controlling action potential duration. The effect of these K(+) channel blockers was almost completely reversed by simultaneous blockade of voltage gated Ca(2+) channels with Cd(2+). Frequency-dependent action potential broadening was exhibited by these neurons during trains of action potentials elicited by membrane depolarizing current pulses presented at 10 Hz but not at 1 Hz. 4-AP but not ChTx or TEA inhibited frequency-dependent action potential broadening indicating that frequency-dependent action potential broadening is dependent on increasing steady-state inactivation of A-type K(+) channels (which are blocked by 4-AP). A model of differential contributions of voltage gated K(+) channels and voltage gated Ca(2+) channels to frequency-dependent action potential broadening, in which an increase of Ca(2+) current during each successive action potential is permitted as a result of the increasing steady-state inactivation of A-type K(+) channels, is presented.

  15. 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.

  16. The position of the fast-inactivation gate during lidocaine block of voltage-gated Na+ channels.

    Science.gov (United States)

    Vedantham, V; Cannon, S C

    1999-01-01

    Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na+ channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na+ channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has been difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For that reason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substituted at phenylalanine 1304 in the rat adult skeletal muscle sodium channel alpha subunit (rSkM1) with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of the fast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence of site 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. More significantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from a slowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activation pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action.

  17. Role of low voltage activated calcium channels in neuritogenesis and active migration of embryonic neural progenitor cells.

    Science.gov (United States)

    Louhivuori, Lauri M; Louhivuori, Verna; Wigren, Henna-Kaisa; Hakala, Elina; Jansson, Linda C; Nordström, Tommy; Castrén, Maija L; Akerman, Karl E

    2013-04-15

    The central role of calcium influx and electrical activity in embryonic development raises important questions about the role and regulation of voltage-dependent calcium influx. Using cultured neural progenitor cell (NPC) preparations, we recorded barium currents through voltage-activated channels using the whole-cell configuration of the patch-clamp technique and monitored intracellular free calcium concentrations with Fura-2 digital imaging. We found that NPCs as well as expressing high-voltage-activated (HVA) calcium channels express functional low-threshold voltage-dependent calcium channels in the very early stages of differentiation (5 h to 1 day). The size of the currents recorded at -50 versus -20 mV after 1 day in differentiation was dependent on the nature of the charge carrier. Peak currents measured at -20 mV in the presence 10 mM Ca2+ instead of 10 mM Ba2+ had a tendency to be smaller, whereas the nature of the divalent species did not influence the amplitude measured at -50 mV. The T-type channel blockers mibefradil and NNC 55-0396 significantly reduced the calcium responses elicited by depolarizing with extracellular potassium, while the overall effect of the HVA calcium channel blockers was small at differentiation day 1. At differentiation day 20, the calcium responses were effectively blocked by nifedipine. Time-lapse imaging of differentiating neurospheres cultured in the presence of low-voltage-activated (LVA) blockers showed a significant decrease in the number of active migrating neuron-like cells and neurite extensions. Together, these data provide evidence that LVA calcium channels are involved in the physiology of differentiating and migrating NPCs.

  18. 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...

  19. Interface Engineering for Precise Threshold Voltage Control in Multilayer-Channel Thin Film Transistors

    KAUST Repository

    Park, Jihoon

    2016-11-29

    Multilayer channel structure is used to effectively manipulate the threshold voltage of zinc oxide transistors without degrading its field-effect mobility. Transistors operating in enhancement mode with good mobility are fabricated by optimizing the structure of the multilayer channel. The optimization is attributed to the formation of additional channel and suppression of the diffusion of absorbed water molecules and oxygen vacancies.

  20. The role of voltage-gated potassium channels in the regulation of mouse uterine contractility.

    Science.gov (United States)

    Smith, Ryan C; McClure, Marisa C; Smith, Margaret A; Abel, Peter W; Bradley, Michael E

    2007-11-02

    Uterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv) channels and large-conductance, calcium-activated potassium (BK) channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium. Functional assays which evaluated the effects of channel blockers and various contractile agonists were accomplished by quantifying contractility of isolated uterine smooth muscle obtained from nonpregnant mice as well as mice at various stages of pregnancy. Expression of Kv channel proteins in isolated uterine smooth muscle was evaluated by Western blots. The Kv channel blocker 4-aminopyridine (4-AP) caused contractions in nonpregnant mouse myometrium (EC50 = 54 micromolar, maximal effect at 300 micromolar) but this effect disappeared in pregnant mice; similarly, the Kv4.2/Kv4.3 blocker phrixotoxin-2 caused contractions in nonpregnant, but not pregnant, myometrium. Contractile responses to 4-AP were not dependent upon nerves, as neither tetrodotoxin nor storage of tissues at room temperature significantly altered these responses, nor were responses dependent upon the presence of the endometrium. Spontaneous contractions and contractions in response to 4-AP did not appear to be mediated by BK, as the BK channel-selective blockers iberiotoxin, verruculogen, or tetraethylammonium failed to affect either spontaneous contractions or 4-AP-elicited responses. A number of different Kv channel alpha subunit proteins were found in isolated myometrium from both nonpregnant and

  1. The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

    Directory of Open Access Journals (Sweden)

    Abel Peter W

    2007-11-01

    Full Text Available Abstract Background Uterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv channels and large-conductance, calcium-activated potassium (BK channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium. Methods Functional assays which evaluated the effects of channel blockers and various contractile agonists were accomplished by quantifying contractility of isolated uterine smooth muscle obtained from nonpregnant mice as well as mice at various stages of pregnancy. Expression of Kv channel proteins in isolated uterine smooth muscle was evaluated by Western blots. Results The Kv channel blocker 4-aminopyridine (4-AP caused contractions in nonpregnant mouse myometrium (EC50 = 54 micromolar, maximal effect at 300 micromolar but this effect disappeared in pregnant mice; similarly, the Kv4.2/Kv4.3 blocker phrixotoxin-2 caused contractions in nonpregnant, but not pregnant, myometrium. Contractile responses to 4-AP were not dependent upon nerves, as neither tetrodotoxin nor storage of tissues at room temperature significantly altered these responses, nor were responses dependent upon the presence of the endometrium. Spontaneous contractions and contractions in response to 4-AP did not appear to be mediated by BK, as the BK channel-selective blockers iberiotoxin, verruculogen, or tetraethylammonium failed to affect either spontaneous contractions or 4-AP-elicited responses. A number of different Kv channel alpha subunit proteins were

  2. Membrane-localized β-subunits alter the PIP2 regulation of high-voltage activated Ca2+ channels.

    Science.gov (United States)

    Suh, Byung-Chang; Kim, Dong-Il; Falkenburger, Björn H; Hille, Bertil

    2012-02-21

    The β-subunits of voltage-gated Ca(2+) (Ca(V)) channels regulate the functional expression and several biophysical properties of high-voltage-activated Ca(V) channels. We find that Ca(V) β-subunits also determine channel regulation by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)). When Ca(V)1.3, -2.1, or -2.2 channels are cotransfected with the β3-subunit, a cytosolic protein, they can be inhibited by activating a voltage-sensitive lipid phosphatase to deplete PIP(2). When these channels are coexpressed with a β2a-subunit, a palmitoylated peripheral membrane protein, the inhibition is much smaller. PIP(2) sensitivity could be increased by disabling the two palmitoylation sites in the β2a-subunit. To further test effects of membrane targeting of Ca(V) β-subunits on PIP(2) regulation, the N terminus of Lyn was ligated onto the cytosolic β3-subunit to confer lipidation. This chimera, like the Ca(V) β2a-subunit, displayed plasma membrane localization, slowed the inactivation of Ca(V)2.2 channels, and increased the current density. In addition, the Lyn-β3 subunit significantly decreased Ca(V) channel inhibition by PIP(2) depletion. Evidently lipidation and membrane anchoring of Ca(V) β-subunits compete with the PIP(2) regulation of high-voltage-activated Ca(V) channels. Compared with expression with Ca(V) β3-subunits alone, inhibition of Ca(V)2.2 channels by PIP(2) depletion could be significantly attenuated when β2a was coexpressed with β3. Our data suggest that the Ca(V) currents in neurons would be regulated by membrane PIP(2) to a degree that depends on their endogenous β-subunit combinations.

  3. Effects of inorganic lead on voltage-sensitive calcium channels in N1E-115 neuroblastoma cells.

    Science.gov (United States)

    Audesirk, G; Audesirk, T

    1991-01-01

    N1E-115 mouse neuroblastoma cells have been reported to possess two types of voltage-sensitive calcium channels: Low voltage activated, rapidly inactivating T-type (type I) and high voltage activated, slowly inactivating L-type (type II). We studied the effects of acute in vitro exposure to inorganic lead on these calcium channels, using the whole-cell variant of patch clamping. Using salines with a high lead-buffering capacity, we found that both T-type and L-type channels are reversibly inhibited in a dose-dependent manner at free Pb2+ concentrations ranging from 20 nM to 14 microM. L-type channels are somewhat more sensitive to Pb2+ than T-type channels are (L-type: IC50 approx. 0.7 microM; T-type: IC50 approx. 1.3 microM). Both channels show small but significant inhibition (approx. 10%) at 20 nM free Pb2+. Pb2+ affects neither activation nor inactivation of T-type channels, but enhances inactivation of L-type channels at holding potentials around -60 to -40 mV. A peculiar phenomenon was observed in cells exposed to 2.3 microM free Pb2+. T-type channels were inhibited in all 20 cells studied. In 15 cells, L-type channels were also inhibited, but in the remaining 5 cells, current flow through L-type channels was enhanced by Pb2+ exposure.

  4. 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.

  5. The effects of inorganic lead on voltage-sensitive calcium channels differ among cell types and among channel subtypes.

    Science.gov (United States)

    Audesirk, G; Audesirk, T

    1993-01-01

    The whole-cell version of patch clamping was used to compare the effects of acute in vitro exposure to inorganic lead (Pb2+) on voltage-sensitive calcium channels in cultured N1E-115 mouse neuroblastoma cells and E18 rat hippocampal neurons. Free Pb2+ concentrations in salines with a high lead-buffering capacity were measured with a calibrated Pb(2+)-selective electrode. Previously, we found that N1E-115 neurons contain low voltage activated, rapidly inactivating (T) channels and high voltage activated, slowly inactivating (L) channels. Pb2+ inhibits both channel subtypes in N1E-115 cells, with some selectivity against L-type channels (IC50 approximately 700 nM free Pb2+ for L-type channels, 1300 nM free Pb2+ for T-type channels; Audesirk and Audesirk, 1991). In addition to T-type and L-type channels, cultured E18 rat hippocampal neurons have been reported to contain high voltage-activated, rapidly inactivating (N) channels. In our experiments with 5 to 20 day old cultures, almost all neurons showed substantial L-type current, approximately half showed significant N-type current, and fewer than 5% showed significant T-type current. We found that Pb2+ is somewhat selective against L-type channels (IC50 approximately 30 nM free Pb2+ in 10 mM Ba2+ as the charge carrier, 55 nM in 50 mM Ba2+) compared to N-channels (IC50 approximately 80 nM free Pb2+ in 10 mM Ba2+, 200 nM in 50 mM Ba2+). These results suggest that the effects of Pb2+ on calcium channels of vertebrate neurons vary both among cell types and among channel subtypes.

  6. Dampening of hyperexcitability in CA1 pyramidal neurons by polyunsaturated fatty acids acting on voltage-gated ion channels.

    Directory of Open Access Journals (Sweden)

    Jenny Tigerholm

    Full Text Available A ketogenic diet is an alternative treatment of epilepsy in infants. The diet, rich in fat and low in carbohydrates, elevates the level of polyunsaturated fatty acids (PUFAs in plasma. These substances have therefore been suggested to contribute to the anticonvulsive effect of the diet. PUFAs modulate the properties of a range of ion channels, including K and Na channels, and it has been hypothesized that these changes may be part of a mechanistic explanation of the ketogenic diet. Using computational modelling, we here study how experimentally observed PUFA-induced changes of ion channel activity affect neuronal excitability in CA1, in particular responses to synaptic input of high synchronicity. The PUFA effects were studied in two pathological models of cellular hyperexcitability associated with epileptogenesis. We found that experimentally derived PUFA modulation of the A-type K (K(A channel, but not the delayed-rectifier K channel, restored healthy excitability by selectively reducing the response to inputs of high synchronicity. We also found that PUFA modulation of the transient Na channel was effective in this respect if the channel's steady-state inactivation was selectively affected. Furthermore, PUFA-induced hyperpolarization of the resting membrane potential was an effective approach to prevent hyperexcitability. When the combined effect of PUFA on the K(A channel, the Na channel, and the resting membrane potential, was simulated, a lower concentration of PUFA was needed to restore healthy excitability. We therefore propose that one explanation of the beneficial effect of PUFAs lies in its simultaneous action on a range of ion-channel targets. Furthermore, this work suggests that a pharmacological cocktail acting on the voltage dependence of the Na-channel inactivation, the voltage dependences of K(A channels, and the resting potential can be an effective treatment of epilepsy.

  7. Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker.

    Science.gov (United States)

    Thouta, Samrat; Hull, Christina M; Shi, Yu Patrick; Sergeev, Valentine; Young, James; Cheng, Yen M; Claydon, Thomas W

    2017-01-24

    Slow deactivation of hERG channels is critical for preventing cardiac arrhythmia yet the mechanistic basis for the slow gating transition is unclear. Here, we characterized the temporal sequence of events leading to voltage sensor stabilization upon membrane depolarization. Progressive increase in step depolarization duration slowed voltage-sensor return in a biphasic manner (τfast = 34 ms, τslow = 2.5 s). The faster phase of voltage-sensor return slowing correlated with the kinetics of pore opening. The slower component occurred over durations that exceeded channel activation and was consistent with voltage sensor relaxation. The S4-S5 linker mutation, G546L, impeded the faster phase of voltage sensor stabilization without attenuating the slower phase, suggesting that the S4-S5 linker is important for communications between the pore gate and the voltage sensor during deactivation. These data also demonstrate that the mechanisms of pore gate-opening-induced and relaxation-induced voltage-sensor stabilization are separable. Deletion of the distal N-terminus (Δ2-135) accelerated off-gating current, but did not influence the relative contribution of either mechanism of stabilization of the voltage sensor. Lastly, we characterized mode-shift behavior in hERG channels, which results from stabilization of activated channel states. The apparent mode-shift depended greatly on recording conditions. By measuring slow activation and deactivation at steady state we found the "true" mode-shift to be ∼15 mV. Interestingly, the "true" mode-shift of gating currents was ∼40 mV, much greater than that of the pore gate. This demonstrates that voltage sensor return is less energetically favorable upon repolarization than pore gate closure. We interpret this to indicate that stabilization of the activated voltage sensor limits the return of hERG channels to rest. The data suggest that this stabilization occurs as a result of reconfiguration of the pore gate upon opening by

  8. Pyrethroids differentially alter voltage-gated sodium channels from the honeybee central olfactory neurons.

    Directory of Open Access Journals (Sweden)

    Aklesso Kadala

    Full Text Available The sensitivity of neurons from the honey bee olfactory system to pyrethroid insecticides was studied using the patch-clamp technique on central 'antennal lobe neurons' (ALNs in cell culture. In these neurons, the voltage-dependent sodium currents are characterized by negative potential for activation, fast kinetics of activation and inactivation, and the presence of cumulative inactivation during train of depolarizations. Perfusion of pyrethroids on these ALN neurons submitted to repetitive stimulations induced (1 an acceleration of cumulative inactivation, and (2 a marked slowing of the tail current recorded upon repolarization. Cypermethrin and permethrin accelerated cumulative inactivation of the sodium current peak in a similar manner and tetramethrin was even more effective. The slow-down of channel deactivation was markedly dependent on the type of pyrethroid. With cypermethrin, a progressive increase of the tail current amplitude along with successive stimulations reveals a traditionally described use-dependent recruitment of modified sodium channels. However, an unexpected decrease in this tail current was revealed with tetramethrin. If one considers the calculated percentage of modified channels as an index of pyrethroids effects, ALNs are significantly more susceptible to tetramethrin than to permethrin or cypermethrin for a single depolarization, but this difference attenuates with repetitive activity. Further comparison with peripheral neurons from antennae suggest that these modifications are neuron type specific. Modeling the sodium channel as a multi-state channel with fast and slow inactivation allows to underline the effects of pyrethroids on a set of rate constants connecting open and inactivated conformations, and give some insights to their specificity. Altogether, our results revealed a differential sensitivity of central olfactory neurons to pyrethroids that emphasize the ability for these compounds to impair detection and

  9. Kinetic changes and modulation by carbamazepine on voltage-gated sodium channels in rat CA1 neurons after epilepsy

    Institute of Scientific and Technical Information of China (English)

    Guang-chun SUN; Taco WERKMAN; Wytse J WADMAN

    2006-01-01

    Aim: To study whether the functional properties of sodium channels, and subsequently the channel modulation by carbamazepine (CBZ) in hippocampal CA1 neurons can be changed after epileptic seizures. Methods: We used the acutely dissociated hippocampal CA1 pyramidal cells from epilepsy model rats 3 weeks and 3 months respectively after kainate injection, and whole-cell voltage-clamp techniques. Results: After long-term epileptic seizures, both sodium channel voltage-dependence of activation and steady-state inactivation shifted to more hyperpolarizing potentials, which resulted in the enlarged window current; the membrane density of sodium current decreased and the time constant of recovery from inactivation increased. CBZ displayed unchanged efficacy on sodium channels, with a similar binding rate to them, except that at higher concentrations, the voltage shift of inactivation was reduced. For the short-term kainate model rats, no differences were detected between the control and epilepsy groups. Conclusion: These results indicate that the properties of sodium channels in acutely dissociated hippocampal neurons could be changed following long-term epilepsy, but the alternation might not be enough to induce the channel resistance to CBZ.

  10. Involvement of the S4-S5 linker and the C-linker domain regions to voltage-gating in plant Shaker channels: comparison with animal HCN and Kv channels.

    Science.gov (United States)

    Nieves-Cordones, Manuel; Gaillard, Isabelle

    2014-01-01

    Among the different transport systems present in plant cells, Shaker channels constitute the major pathway for K(+) in the plasma membrane. Plant Shaker channels are members of the 6 transmembrane-1 pore (6TM-1P) cation channel superfamily as the animal Shaker (Kv) and HCN channels. All these channels are voltage-gated K(+) channels: Kv channels are outward-rectifiers, opened at depolarized voltages and HCN channels are inward-rectifiers, opened by membrane hyperpolarization. Among plant Shaker channels, we can find outward-rectifiers, inward-rectifiers and also weak-rectifiers, with weak voltage dependence. Despite the absence of crystal structures of plant Shaker channels, functional analyses coupled to homology modeling, mostly based on Kv and HCN crystals, have permitted the identification of several regions contributing to plant Shaker channel gating. In the present mini-review, we make an update on the voltage-gating mechanism of plant Shaker channels which seem to be comparable to that proposed for HCN channels.

  11. hERG S4-S5 linker acts as a voltage-dependent ligand that binds to the activation gate and locks it in a closed state.

    Science.gov (United States)

    Malak, Olfat A; Es-Salah-Lamoureux, Zeineb; Loussouarn, Gildas

    2017-12-01

    Delayed-rectifier potassium channels (hERG and KCNQ1) play a major role in cardiac repolarization. These channels are formed by a tetrameric pore (S5-S6) surrounded by four voltage sensor domains (S1-S4). Coupling between voltage sensor domains and the pore activation gate is critical for channel voltage-dependence. However, molecular mechanisms remain elusive. Herein, we demonstrate that covalently binding, through a disulfide bridge, a peptide mimicking the S4-S5 linker (S4-S5L) to the channel S6 C-terminus (S6T) completely inhibits hERG. This shows that channel S4-S5L is sufficient to stabilize the pore activation gate in its closed state. Conversely, covalently binding a peptide mimicking S6T to the channel S4-S5L prevents its inhibiting effect and renders the channel almost completely voltage-independent. This shows that the channel S4-S5L is necessary to stabilize the activation gate in its closed state. Altogether, our results provide chemical evidence that S4-S5L acts as a voltage-controlled ligand that binds S6T to lock the channel in a closed state, elucidating the coupling between voltage sensors and the gate in delayed rectifier potassium channels and potentially other voltage-gated channels.

  12. The sorting receptor Rer1 controls Purkinje cell function via voltage gated sodium channels

    Science.gov (United States)

    Valkova, Christina; Liebmann, Lutz; Krämer, Andreas; Hübner, Christian A.; Kaether, Christoph

    2017-01-01

    Rer1 is a sorting receptor in the early secretory pathway that controls the assembly and the cell surface transport of selected multimeric membrane protein complexes. Mice with a Purkinje cell (PC) specific deletion of Rer1 showed normal polarization and differentiation of PCs and normal development of the cerebellum. However, PC-specific loss of Rer1 led to age-dependent motor deficits in beam walk, ladder climbing and gait. Analysis of brain sections revealed a specific degeneration of PCs in the anterior cerebellar lobe in old animals. Electrophysiological recordings demonstrated severe deficits in spontaneous action potential generation. Measurements of resurgent currents indicated decreased surface densities of voltage-gated sodium channels (Nav), but not changes in individual channels. Analysis of mice with a whole brain Rer1-deletion demonstrated a strong down-regulation of Nav1.6 and 1.1 in the absence of Rer1, whereas protein levels of the related Cav2.1 and of Kv3.3 and 7.2 channels were not affected. The data suggest that Rer1 controls the assembly and transport of Nav1.1 and 1.6, the principal sodium channels responsible for recurrent firing, in PCs. PMID:28117367

  13. Transient Receptor Potential Vanilloid 4-Induced Modulation of Voltage-Gated Sodium Channels in Hippocampal Neurons.

    Science.gov (United States)

    Hong, Zhiwen; Jie, Pinghui; Tian, Yujing; Chen, Tingting; Chen, Lei; Chen, Ling

    2016-01-01

    Transient receptor potential vanilloid 4 (TRPV4) is reported to control the resting membrane potential and increase excitability in many types of cells. Voltage-gated sodium channels (VGSCs) play an important role in initiating action potentials in neurons. However, whether VGSCs can be modulated by the activation of TRPV4 in hippocampal pyramidal neurons remains unknown. In this study, we tested the effect of TRPV4 agonists (GSK1016790A and 4α-PDD) on voltage-gated sodium current (I Na) in hippocampal CA1 pyramidal neurons and the protein levels of α/β-subunit of VGSCs in the hippocampus of mice subjected to intracerebroventricular (icv.) injection of GSK1016790A (GSK-injected mice). Herein, we report that I Na was inhibited by acute application of GSK1016790A or 4α-PDD. In the presence of TRPV4 agonists, the voltage-dependent inactivation curve shifted to the hyperpolarization, whereas the voltage-dependent activation curve remained unchanged. The TRPV4 agonist-induced inhibition of I Na was blocked by the TRPV4 antagonist or tetrodotoxin. Moreover, blocking protein kinase A (PKA) markedly attenuated the GSK1016790A-induced inhibition of I Na, whereas antagonism of protein kinase C or p38 mitogen-activated protein kinase did not change GSK1016790A action. Finally, the protein levels of Nav1.1, Nav1.2, and Nav1.6 in the hippocampus increased in GSK-injected mice, whereas those of Nav1.3 and Navβ1 remained nearly unchanged. We conclude that I Na is inhibited by the acute activation of TRPV4 through PKA signaling pathway in hippocampal pyramidal neurons, but protein expression of α-subunit of VGSCs is increased by sustained TRPV4 activation, which may compensate for the acute inhibition of I Na and provide a possibility for hyper-excitability upon sustained TRPV4 activation.

  14. 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.

  15. Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

    DEFF Research Database (Denmark)

    Rougier, Jean-Sébastien; van Bemmelen, Miguel X; Bruce, M Christine

    2004-01-01

    -ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na(v)1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na(v) proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na(v)1.2 and Na......The voltage-gated Na(+) channels (Na(v)) form a family composed of 10 genes. The COOH termini of Na(v) contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein...... that Nedd4-dependent ubiquitination of Na(v) channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane....

  16. Hlf is a genetic modifier of epilepsy caused by voltage-gated sodium channel mutations.

    Science.gov (United States)

    Hawkins, Nicole A; Kearney, Jennifer A

    2016-01-01

    Mutations in voltage-gated sodium channel genes cause several types of human epilepsies. Often, individuals with the same sodium channel mutation exhibit diverse phenotypes. This suggests that factors beyond the primary mutation influence disease severity, including genetic modifiers. Mouse epilepsy models with voltage-gated sodium channel mutations exhibit strain-dependent phenotype variability, supporting a contribution of genetic modifiers in epilepsy. The Scn2a(Q54) (Q54) mouse model has a strain-dependent epilepsy phenotype. Q54 mice on the C57BL/6J (B6) strain exhibit delayed seizure onset and improved survival compared to [B6xSJL/J]F1.Q54 mice. We previously mapped two dominant modifier loci that influence Q54 seizure susceptibility and identified Hlf (hepatic leukemia factor) as a candidate modifier gene at one locus. Hlf and other PAR bZIP transcription factors had previously been associated with spontaneous seizures in mice thought to be caused by down-regulation of the pyridoxine pathway. An Hlf targeted knockout mouse model was used to evaluate the effect of Hlf deletion on Q54 phenotype severity. Hlf(KO/KO);Q54 double mutant mice exhibited elevated frequency and reduced survival compared to Q54 controls. To determine if direct modulation of the pyridoxine pathway could alter the Q54 phenotype, mice were maintained on a pyridoxine-deficient diet for 6 weeks. Dietary pyridoxine deficiency resulted in elevated seizure frequency and decreased survival in Q54 mice compared to control diet. To determine if Hlf could modify other epilepsies, Hlf(KO/+) mice were crossed with the Scn1a(KO/+) Dravet syndrome mouse model to examine the effect on premature lethality. Hlf(KO/+);Scn1a(KO/+) offspring exhibited decreased survival compared to Scn1a(KO/+) controls. Together these results demonstrate that Hlf is a genetic modifier of epilepsy caused by voltage-gated sodium channel mutations and that modulation of the pyridoxine pathway can also influence phenotype

  17. Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins.

    Science.gov (United States)

    Pinto, Bernardo I; García, Isaac E; Pupo, Amaury; Retamal, Mauricio A; Martínez, Agustín D; Latorre, Ramón; González, Carlos

    2016-07-22

    Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity.

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

    Directory of Open Access Journals (Sweden)

    Francesco eMiceli

    2011-02-01

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

  19. Manipulating the voltage dependence of tunneling spin torques

    KAUST Repository

    Manchon, Aurelien

    2012-10-01

    Voltage-driven spin transfer torques in magnetic tunnel junctions provide an outstanding tool to design advanced spin-based devices for memory and reprogrammable logic applications. The non-linear voltage dependence of the torque has a direct impact on current-driven magnetization dynamics and on devices performances. After a brief overview of the progress made to date in the theoretical description of the spin torque in tunnel junctions, I present different ways to alter and control the bias dependence of both components of the spin torque. Engineering the junction (barrier and electrodes) structural asymmetries or controlling the spin accumulation profile in the free layer offer promising tools to design effcient spin devices.

  20. The hitchhiker's guide to the voltage-gated sodium channel galaxy.

    Science.gov (United States)

    Ahern, Christopher A; Payandeh, Jian; Bosmans, Frank; Chanda, Baron

    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.

  1. Upregulation of voltage-activated potassium channels in hippocampus of Aβ25.35-treated rats

    Institute of Scientific and Technical Information of China (English)

    Xiao-liangWANG; Ya-pingPAN

    2004-01-01

    AIM: Potassium channels dysfunction has been indicated in Alzheimer disease. In the present study, the mRNA and protein expression alterations and the functional changes ot VOltage- activated potassium channels were studied in rat hippocampus after a single intracerebro- ventricular injection of β-amyloid peptide 25-35 (Aβ25.35). METHODS: The expressions of mRNA

  2. Functional Expression Profile of Voltage-Gated K(+) Channel Subunits in Rat Small Mesenteric Arteries.

    Science.gov (United States)

    Cox, Robert H; Fromme, Samantha

    2016-06-01

    Multiple K v channel complexes contribute to total K v current in numerous cell types and usually subserve different physiological functions. Identifying the complete compliment of functional K v channel subunits in cells is a prerequisite to understanding regulatory function. It was the goal of this work to determine the complete K v subunit compliment that contribute to functional K v currents in rat small mesenteric artery (SMA) myocytes as a prelude to studying channel regulation. Using RNA prepared from freshly dispersed myocytes, high levels of K v 1.2, 1.5, and 2.1 and lower levels of K v 7.4 α-subunit expressions were demonstrated by quantitative PCR and confirmed by Western blotting. Selective inhibitors correolide (K v 1; COR), stromatoxin (K v 2.1; ScTx), and linopirdine (K v 7.4; LINO) decreased K v current at +40 mV in SMA by 46 ± 4, 48 ± 4, and 6.5 ± 2 %, respectively, and K v current in SMA was insensitive to α-dendrotoxin. Contractions of SMA segments pretreated with 100 nmol/L phenylephrine were enhanced by 27 ± 3, 30 ± 8, and 7 ± 3 % of the response to 120 mmol/L KCl by COR, ScTX, and LINO, respectively. The presence of K v 6.1, 9.3, β1.1, and β1.2 was demonstrated by RT-PCR using myocyte RNA with expressions of K vβ1.2 and K v 9.3 about tenfold higher than K vβ1.1 and K v 6.1, respectively. Selective inhibitors of K v 1.3, 3.4, 4.1, and 4.3 channels also found at the RNA and/or protein level had no significant effect on K v current or contraction. These results suggest that K v current in rat SMA myocytes are dominated equally by two major components consisting of K v 1.2-1.5-β1.2 and K v 2.1-9.3 channels along with a smaller contribution from K v 7.4 channels but differences in voltage dependence of activation allows all three to provide significant contributions to SMA function at physiological voltages.

  3. Dynamical characterization of inactivation path in voltage-gated Na(+) ion channel by non-equilibrium response spectroscopy.

    Science.gov (United States)

    Pal, Krishnendu; Gangopadhyay, Gautam

    2016-11-01

    Inactivation path of voltage gated sodium channel has been studied here under various voltage protocols as it is the main governing factor for the periodic occurrence and shape of the action potential. These voltage protocols actually serve as non-equilibrium response spectroscopic tools to study the ion channel in non-equilibrium environment. In contrast to a lot of effort in finding the crystal structure based molecular mechanism of closed-state(CSI) and open-state inactivation(OSI); here our approach is to understand the dynamical characterization of inactivation. The kinetic flux as well as energetic contribution of the closed and open- state inactivation path is compared here for voltage protocols, namely constant, pulsed and oscillating. The non-equilibrium thermodynamic quantities used in response to these voltage protocols serve as improved characterization tools for theoretical understanding which not only agrees with the previously known kinetic measurements but also predict the energetically optimum processes to sustain the auto-regulatory mechanism of action potential and the consequent inactivation steps needed. The time dependent voltage pattern governs the population of the conformational states which when couple with characteristic rate parameters, the CSI and OSI selectivity arise dynamically to control the inactivation path. Using constant, pulsed and continuous oscillating voltage protocols we have shown that during depolarization the OSI path is more favored path of inactivation however, in the hyper-polarized situation the CSI is favored. It is also shown that the re-factorisation of inactivated sodium channel to resting state occurs via CSI path. Here we have shown how the subtle energetic and entropic cost due to the change in the depolarization magnitude determines the optimum path of inactivation. It is shown that an efficient CSI and OSI dynamical profile in principle can characterize the open-state drug blocking phenomena.

  4. On Channels with Action-Dependent States

    CERN Document Server

    Ahmadi, Behzad

    2012-01-01

    Action-dependent channels model scenarios in which transmission takes place in two successive phases. In the first phase, the encoder selects an "action" sequence, with the twofold aim of conveying information to the receiver and of affecting in a desired way the state of the channel to be used in the second phase. In the second phase, communication takes place in the presence the mentioned action-dependent state. In this work, two extensions of the original action-dependent channel are studied. In the first, the decoder is interested in estimating not only the message, but also the state sequence within an average per-letter distortion. Under the constraint of common knowledge (i.e., the decoder's estimate of the state must be recoverable also at the encoder) and assuming non-causal state knowledge at the encoder in the second phase, we obtain a single-letter characterization of the achievable rate-distortion-cost trade-off. In the second extension, we study an action-dependent degraded broadcast channel. Un...

  5. Hydrophobic plug functions as a gate in voltage-gated proton channels.

    Science.gov (United States)

    Chamberlin, Adam; Qiu, Feng; Rebolledo, Santiago; Wang, Yibo; Noskov, Sergei Y; Larsson, H Peter

    2014-01-14

    Voltage-gated proton (Hv1) channels play important roles in the respiratory burst, in pH regulation, in spermatozoa, in apoptosis, and in cancer metastasis. Unlike other voltage-gated cation channels, the Hv1 channel lacks a centrally located pore formed by the assembly of subunits. Instead, the proton permeation pathway in the Hv1 channel is within the voltage-sensing domain of each subunit. The gating mechanism of this pathway is still unclear. Mutagenic and fluorescence studies suggest that the fourth transmembrane (TM) segment (S4) functions as a voltage sensor and that there is an outward movement of S4 during channel activation. Using thermodynamic mutant cycle analysis, we find that the conserved positively charged residues in S4 are stabilized by countercharges in the other TM segments both in the closed and open states. We constructed models of both the closed and open states of Hv1 channels that are consistent with the mutant cycle analysis. These structural models suggest that electrostatic interactions between TM segments in the closed state pull hydrophobic residues together to form a hydrophobic plug in the center of the voltage-sensing domain. Outward S4 movement during channel activation induces conformational changes that remove this hydrophobic plug and instead insert protonatable residues in the center of the channel that, together with water molecules, can form a hydrogen bond chain across the channel for proton permeation. This suggests that salt bridge networks and the hydrophobic plug function as the gate in Hv1 channels and that outward movement of S4 leads to the opening of this gate.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation-contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar...... tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium....... Low concentrations of nickel, an agent that blocks Ca(v)3.2, had a similar effect. Thus, T-type voltage-gated calcium channels are functionally important for depolarization-induced vasoconstriction and subsequent dilatation in mouse cortical efferent arterioles.Kidney International advance online...

  7. Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Elstone, Fisal D; Niciforovic, Ana P

    2014-01-01

    functional phenotypes that are different from those observed previously in Kv VSDs. In contrast, and similar to results obtained with Kv channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect......Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain...... largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (NaV1...

  8. The Structural Basis and Functional Consequences of Interactions Between Tetrodotoxin and Voltage-Gated Sodium Channels

    Directory of Open Access Journals (Sweden)

    C. Ruben

    2006-04-01

    Full Text Available Abstract: Tetrodotoxin (TTX is a highly specific blocker of voltage-gated sodium channels. The dissociation constant of block varies with different channel isoforms. Until recently, channel resistance was thought to be primarily imparted by amino acid substitutions at a single position in domain I. Recent work reveals a novel site for tetrodotoxin resistance in the P-region of domain IV.

  9. Differential CaMKII regulation by voltage-gated calcium channels in the striatum.

    Science.gov (United States)

    Pasek, Johanna G; Wang, Xiaohan; Colbran, Roger J

    2015-09-01

    Calcium signaling regulates synaptic plasticity and many other functions in striatal medium spiny neurons to modulate basal ganglia function. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a major calcium-dependent signaling protein that couples calcium entry to diverse cellular changes. CaMKII activation results in autophosphorylation at Thr286 and sustained calcium-independent CaMKII activity after calcium signals dissipate. However, little is known about the mechanisms regulating striatal CaMKII. To address this, mouse brain slices were treated with pharmacological modulators of calcium channels and punches of dorsal striatum were immunoblotted for CaMKII Thr286 autophosphorylation as an index of CaMKII activation. KCl depolarization increased levels of CaMKII autophosphorylation ~2-fold; this increase was blocked by an LTCC antagonist and was mimicked by treatment with pharmacological LTCC activators. The chelation of extracellular calcium robustly decreased basal CaMKII autophosphorylation within 5min and increased levels of total CaMKII in cytosolic fractions, in addition to decreasing the phosphorylation of CaMKII sites in the GluN2B subunit of NMDA receptors and the GluA1 subunit of AMPA receptors. We also found that the maintenance of basal levels of CaMKII autophosphorylation requires low-voltage gated T-type calcium channels, but not LTCCs or R-type calcium channels. Our findings indicate that CaMKII activity is dynamically regulated by multiple calcium channels in the striatum thus coupling calcium entry to key downstream substrates.

  10. Non-silent story on synonymous sites in voltage-gated ion channel genes.

    Science.gov (United States)

    Zhou, Tong; Ko, Eun A; Gu, Wanjun; Lim, Inja; Bang, Hyoweon; Ko, Jae-Hong

    2012-01-01

    Synonymous mutations are usually referred to as "silent", but increasing evidence shows that they are not neutral in a wide range of organisms. We looked into the relationship between synonymous codon usage bias and residue importance of voltage-gated ion channel proteins in mice, rats, and humans. We tested whether translationally optimal codons are associated with transmembrane or channel-forming regions, i.e., the sites that are particularly likely to be involved in the closing and opening of an ion channel. Our hypothesis is that translationally optimal codons are preferred at the sites within transmembrane domains or channel-forming regions in voltage-gated ion channel genes to avoid mistranslation-induced protein misfolding or loss-of-function. Using the Mantel-Haenszel procedure, which applies to categorical data, we found that translationally optimal codons are more likely to be used at transmembrane residues and the residues involved in channel-forming. We also found that the conservation level at synonymous sites in the transmembrane region is significantly higher than that in the non-transmembrane region. This study provides evidence that synonymous sites in voltage-gated ion channel genes are not neutral. Silent mutations at channel-related sites may lead to dysfunction of the ion channel.

  11. Non-silent story on synonymous sites in voltage-gated ion channel genes.

    Directory of Open Access Journals (Sweden)

    Tong Zhou

    Full Text Available Synonymous mutations are usually referred to as "silent", but increasing evidence shows that they are not neutral in a wide range of organisms. We looked into the relationship between synonymous codon usage bias and residue importance of voltage-gated ion channel proteins in mice, rats, and humans. We tested whether translationally optimal codons are associated with transmembrane or channel-forming regions, i.e., the sites that are particularly likely to be involved in the closing and opening of an ion channel. Our hypothesis is that translationally optimal codons are preferred at the sites within transmembrane domains or channel-forming regions in voltage-gated ion channel genes to avoid mistranslation-induced protein misfolding or loss-of-function. Using the Mantel-Haenszel procedure, which applies to categorical data, we found that translationally optimal codons are more likely to be used at transmembrane residues and the residues involved in channel-forming. We also found that the conservation level at synonymous sites in the transmembrane region is significantly higher than that in the non-transmembrane region. This study provides evidence that synonymous sites in voltage-gated ion channel genes are not neutral. Silent mutations at channel-related sites may lead to dysfunction of the ion channel.

  12. Localized Calcineurin Confers Ca2+-Dependent Inactivation Upon Neuronal L-Type Ca2+ Channels

    OpenAIRE

    2012-01-01

    Excitation-driven entry of Ca2+ through L-type voltage-gated Ca2+ channels controls gene expression in neurons and a variety of fundamental activities in other kinds of excitable cells. The probability of opening of CaV1.2 L-type channels is subject to pronounced enhancement by cAMP-dependent protein kinase (PKA), which is scaffolded to CaV1.2 channels by A-kinase anchoring proteins (AKAPs). CaV1.2 channels also undergo negative autoregulation via Ca2+-dependent inactivation (CDI), which stro...

  13. 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.

  14. Characterization of single voltage-gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons.

    Science.gov (United States)

    Magee, J C; Johnston, D

    1995-08-15

    1. We have used dendrite-attached patch-clamp techniques to record single Na+ and Ca2+ channel activity from the apical dendrites (up to 350 microns away from soma) of CA1 pyramidal neurons in rat hippocampal slices (ages: 2-8 weeks). 2. Na+ channels were found in every patch examined (range: 2 to > 20 channels per patch). Channel openings, which had a slope conductance of 15 +/- 0.3 pS (mean +/- S.E.M.), began with test commands to around -50 mV and consisted of both early transient channel activity and also later occurring prolonged openings of 5-15 ms. All Na+ channel activity was suppressed by inclusion of TTX (1 microM) in the recording pipette. 3. Ca2+ channel activity was recorded in about 80% of the patches examined (range: 1 to > 10 channels per patch). Several types of channel behaviour were observed in these patches. Single channel recordings in 110 mM BaCl2, revealed an approximately 10 pS channel of small unitary current amplitude (-0.5 pA at -20 mV). These channels began activating at relatively hyperpolarized potentials (-50 mV) and ensemble averages of this low voltage-activated (LVA) channel activity showed rapid inactivation. 4. A somewhat heterogeneous population of high voltage-activated, moderate conductance (HVAm; approximately 17 pS), Ca2+ channel activity was also encountered. These channels exhibited a relatively large unitary amplitude (-0.8 pA at 0 mV) and ensemble averages demonstrated moderate inactivation. The HVAm population of channels could be tentatively subdivided into two separate groups based upon mean channel open times. 5. Less frequently, HVA, large conductance (27 pS) Ca2+ channel activity (HVA1) was also observed. This large unitary amplitude (-1.5 pA at 0 mV) channel activity began with steps to approximately 0 mV and ensemble averages did not show any time-dependent inactivation. The dihydropyridine Ca2+ channel agonist Bay K 8644 (0.5 or 1 microM) was found to characteristically prolong these channel openings. 6. omega

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

    Science.gov (United States)

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

    2016-09-01

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

  16. Voltage-gated calcium channel and antisense oligonucleotides thereto

    Science.gov (United States)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  17. Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: a periodic paralysis mutation in Na(V)1.4 (L689I).

    Science.gov (United States)

    Silva, Jonathan R; Goldstein, Steve A N

    2013-03-01

    In skeletal muscle, slow inactivation (SI) of Na(V)1.4 voltage-gated sodium channels prevents spontaneous depolarization and fatigue. Inherited mutations in Na(V)1.4 that impair SI disrupt activity-induced regulation of channel availability and predispose patients to hyperkalemic periodic paralysis. In our companion paper in this issue (Silva and Goldstein. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210909), the four voltage sensors in Na(V)1.4 responsible for activation of channels over microseconds are shown to slowly immobilize over 1-160 s as SI develops and to regain mobility on recovery from SI. Individual sensor movements assessed via attached fluorescent probes are nonidentical in their voltage dependence, time course, and magnitude: DI and DII track SI onset, and DIII appears to reflect SI recovery. A causal link was inferred by tetrodotoxin (TTX) suppression of both SI onset and immobilization of DI and DII sensors. Here, the association of slow sensor immobilization and SI is verified by study of Na(V)1.4 channels with a hyperkalemic periodic paralysis mutation; L689I produces complex changes in SI, and these are found to manifest directly in altered sensor movements. L689I removes a component of SI with an intermediate time constant (~10 s); the mutation also impedes immobilization of the DI and DII sensors over the same time domain in support of direct mechanistic linkage. A model that recapitulates SI attributes responsibility for intermediate SI to DI and DII (10 s) and a slow component to DIII (100 s), which accounts for residual SI, not impeded by L689I or TTX.

  18. Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: A periodic paralysis mutation in NaV1.4 (L689I)

    Science.gov (United States)

    Silva, Jonathan R.

    2013-01-01

    In skeletal muscle, slow inactivation (SI) of NaV1.4 voltage-gated sodium channels prevents spontaneous depolarization and fatigue. Inherited mutations in NaV1.4 that impair SI disrupt activity-induced regulation of channel availability and predispose patients to hyperkalemic periodic paralysis. In our companion paper in this issue (Silva and Goldstein. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210909), the four voltage sensors in NaV1.4 responsible for activation of channels over microseconds are shown to slowly immobilize over 1–160 s as SI develops and to regain mobility on recovery from SI. Individual sensor movements assessed via attached fluorescent probes are nonidentical in their voltage dependence, time course, and magnitude: DI and DII track SI onset, and DIII appears to reflect SI recovery. A causal link was inferred by tetrodotoxin (TTX) suppression of both SI onset and immobilization of DI and DII sensors. Here, the association of slow sensor immobilization and SI is verified by study of NaV1.4 channels with a hyperkalemic periodic paralysis mutation; L689I produces complex changes in SI, and these are found to manifest directly in altered sensor movements. L689I removes a component of SI with an intermediate time constant (∼10 s); the mutation also impedes immobilization of the DI and DII sensors over the same time domain in support of direct mechanistic linkage. A model that recapitulates SI attributes responsibility for intermediate SI to DI and DII (10 s) and a slow component to DIII (100 s), which accounts for residual SI, not impeded by L689I or TTX. PMID:23401572

  19. Upregulation of Voltage-Gated Calcium Channel Cav1.3 in Bovine Somatotropes Treated with Ghrelin

    Directory of Open Access Journals (Sweden)

    V. M. Salinas Zarate

    2013-01-01

    Full Text Available Activation of the growth hormone (GH secretagogue receptor (GHS-R by synthetic GH releasing peptides (GHRP or its endogenous ligand (Ghrelin stimulates GH release. Though much is known about the signal transduction underlying short-term regulation, there is far less information on the mechanisms that produce long-term effects. In the current report, using an enzyme-linked immunosorbent assay for GH detection and whole-cell patch-clamp recordings, we assessed the long-term actions of such regulatory factors on voltage-activated Ca2+ currents in bovine somatotropes (BS separated on a Percoll gradient and detected by immunohistochemistry. After 24 h of treatment with Ghrelin (10 nM or GHRP-6 (100 nM enhanced BS secretory activity; GH secretion stimulated by GHS through the activation of GHS-R because treatment with the antagonist of GHS-R (D-Lys3-GHRP-6, 10 μM blocked the GH secretion, and the effect was dose and time dependent (24, 48, and 72 h. GH secretion stimulated by GHRP-6 was abolished by nifedipine (0.5 μM, a blocker of L-type HVA Ca2+ channels, and KN-62 (10 μM, an inhibitor of Ca2+/CaM-KII. After 72 h in culture, all recorded BS exhibited two main Ca2+ currents: a low voltage-activated (LVA; T-type and a high voltage-activated (HVA; mostly dihydropyridine-sensitive L-type current. Interestingly, HVA and LVA channels were differentially upregulated by Ghrelin. Chronic treatment with the GHS induced a significant selective increase on the Ba2+ current through HVA Ca2+ channels, and caused only a small increase of currents through LVA channels. The stimulatory effect on HVA current density was accompanied by an augment in maximal conductance with no apparent changes in the kinetics and the voltage dependence of the Ca2+ currents, suggesting an increase in the number of functional channels in the cell membrane. Lastly, in consistency with the functional data, quantitative real-time RT-PCR revealed transcripts encoding for

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

    Science.gov (United States)

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

    2016-09-01

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

  1. An update on transcriptional and post-translational regulation of brain voltage-gated sodium channels.

    Science.gov (United States)

    Onwuli, Donatus O; Beltran-Alvarez, Pedro

    2016-03-01

    Voltage-gated sodium channels are essential proteins in brain physiology, as they generate the sodium currents that initiate neuronal action potentials. Voltage-gated sodium channels expression, localisation and function are regulated by a range of transcriptional and post-translational mechanisms. Here, we review our understanding of regulation of brain voltage-gated sodium channels, in particular SCN1A (NaV1.1), SCN2A (NaV1.2), SCN3A (NaV1.3) and SCN8A (NaV1.6), by transcription factors, by alternative splicing, and by post-translational modifications. Our focus is strongly centred on recent research lines, and newly generated knowledge.

  2. A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina.

    Science.gov (United States)

    Ozaita, Ander; Petit-Jacques, Jerome; Völgyi, Béla; Ho, Chi Shun; Joho, Rolf H; Bloomfield, Stewart A; Rudy, Bernardo

    2004-08-18

    Direction-selective retinal ganglion cells show an increased activity evoked by light stimuli moving in the preferred direction. This selectivity is governed by direction-selective inhibition from starburst amacrine cells occurring during stimulus movement in the opposite or null direction. To understand the intrinsic membrane properties of starburst cells responsible for direction-selective GABA release, we performed whole-cell recordings from starburst cells in mouse retina. Voltage-clamp recordings revealed prominent voltage-dependent K(+) currents. The currents were mostly blocked by 1 mm TEA, activated rapidly at voltages more positive than -20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K+ channels. Immunoblots confirmed the presence of Kv3.1 and Kv3.2 proteins in retina and immunohistochemistry revealed their expression in starburst cell somata and dendrites. The Kv3-like current in starburst cells was absent in Kv3.1-Kv3.2 knock-out mice. Current-clamp recordings showed that the fast activation of the Kv3 channels provides a voltage-dependent shunt that limits depolarization of the soma to potentials more positive than -20 mV. This provides a mechanism likely to contribute to the electrical isolation of individual starburst cell dendrites, a property thought essential for direction selectivity. This function of Kv3 channels differs from that in other neurons where they facilitate high-frequency repetitive firing. Moreover, we found a gradient in the intensity of Kv3.1b immunolabeling favoring proximal regions of starburst cells. We hypothesize that this Kv3 channel gradient contributes to the preference for centrifugal signal flow in dendrites underlying direction-selective GABA release from starburst amacrine cells

  3. Being flexible: the voltage-controllable activation gate of Kv channels

    Directory of Open Access Journals (Sweden)

    Alain J. Labro

    2012-09-01

    Full Text Available Kv channels form voltage-dependent potassium selective pores in the outer cell membrane and are composed out of four -subunits, each having six membrane-spanning -helices (S1-S6. The -subunits tetramerize such that the S5-S6 pore domains co-assemble into a centrally located K+ pore which is surrounded by four operational voltage sensing domains (VSD that are each formed by the S1-S4 segments. Consequently, each subunit is capable of responding to changes in membrane potential and dictates whether the pore should be conductive or not. K+ permeation through the pore can be sealed off by two separate gates in series: (a at the inner S6 bundle crossing (BC gate and (b at the level of the selectivity-filter (SF gate located at the extracellular entrance of the pore. Within the last years a general consensus emerged that a direct communication between the S4S5-linker and the bottom part of S6 (S6c constitutes the coupling with the VSD thus making the BC gate the main voltage-controllable activation gate. While the BC gate listens to the VSD, the SF changes its conformation depending on the status of the BC gate. Through the eyes of an entering K+ ion, the operation of the BC gate apparatus can be compared with the iris-like motion of the diaphragm from a camera whereby its diameter widens. Two main gating motions have been proposed to create this BC gate widening: (1 tilting of the helix whereby the S6 converts from a straight -helix to a tilted one or (2 swiveling of the S6c whereby the S6 remains bent. Such motions require a flexible hinge that decouples the pre- and post-hinge segment. Roughly at the middle of the S6 there exists a highly conserved glycine residue and a tandem proline motif that seem to fulfill the role of a gating hinge which allows for tilting/swiveling/rotations of the post-hinge S6 segment. In this review we delineate our current view on the operation of the BC gate for controlling K+ permeation in Kv channels.

  4. Voltage-gated calcium channels and their auxiliary subunits: physiology and pathophysiology and pharmacology.

    Science.gov (United States)

    Dolphin, Annette C

    2016-10-01

    Voltage-gated calcium channels are essential players in many physiological processes in excitable cells. There are three main subdivisions of calcium channel, defined by the pore-forming α1 subunit, the CaV 1, CaV 2 and CaV 3 channels. For all the subtypes of voltage-gated calcium channel, their gating properties are key for the precise control of neurotransmitter release, muscle contraction and cell excitability, among many other processes. For the CaV 1 and CaV 2 channels, their ability to reach their required destinations in the cell membrane, their activation and the fine tuning of their biophysical properties are all dramatically influenced by the auxiliary subunits that associate with them. Furthermore, there are many diseases, both genetic and acquired, involving voltage-gated calcium channels. This review will provide a general introduction and then concentrate particularly on the role of auxiliary α2 δ subunits in both physiological and pathological processes involving calcium channels, and as a therapeutic target.

  5. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    Science.gov (United States)

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.

  6. Taurine-induced modulation of voltage-sensitive Na+ channels in rat dorsal root ganglion neurons.

    Science.gov (United States)

    Yu, Shan-Shan; Yu, Kuai; Gu, Yan; Ruan, Di-Yun

    2005-08-15

    The physiological role of taurine, an abundant free amino acid in the neural system, is still poorly understood. The aim of this study was to investigate its effect on TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ currents in enzymatically dissociated neurons from rat dorsal root ganglion (DRG) with conventional whole-cell recording manner under voltage-clamp conditions. A TTX-S Na+ current was recorded preferentially from large DRG neurons and a TTX-R Na+ current preferentially from small ones. For TTX-S Na+ channel, taurine of the concentration > or = 10 mM shifted the activation curve in the depolarizing direction and the inactivation curve in the hyperpolarizing direction. There was no change in the activation curve for TTX-R Na+ channel and the inactivation curve was shifted in the hyperpolarizing direction slightly in the presence of taurine > or = 20 mM. When the recovery kinetics was examined, the presence of taurine resulted in a slower recovery from inactivation of TTX-S currents and no change of TTX-R ones. All the effects of taurine were weakly concentration-dependent and partly recovered quite slowly after washout. Our data indicate that taurine alters the properties of Na+ currents in intact DRG neurons. These may contribute to the understanding of taurine as a natural neuroprotectant and the potential of taurine as a useful medicine for the treatment of sensory neuropathies.

  7. BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis.

    Science.gov (United States)

    Béguin, Pascal; Nagashima, Kazuaki; Mahalakshmi, Ramasubbu N; Vigot, Réjan; Matsunaga, Atsuko; Miki, Takafumi; Ng, Mei Yong; Ng, Yu Jin Alvin; Lim, Chiaw Hwee; Tay, Hock Soon; Hwang, Le-Ann; Firsov, Dmitri; Tang, Bor Luen; Inagaki, Nobuya; Mori, Yasuo; Seino, Susumu; Launey, Thomas; Hunziker, Walter

    2014-04-28

    Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca(2+)-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca(2+) overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain-binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca(2+) channel activity at the plasma membrane, resulting in the inhibition of Ca(2+)-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.

  8. 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.

  9. Effects of in vitro lead exposure on voltage-sensitive calcium channels differ among cell types in central neurons of Lymnaea stagnalis.

    Science.gov (United States)

    Audesirk, G; Audesirk, T

    1989-01-01

    The effects of acute in vitro lead exposure on slowly inactivating voltage-sensitive calcium channels in central neurons of the freshwater pond snail Lymnaea stagnalis were studied under voltage clamp. Three physiologically distinct cell types were used: two subsets of the B cell cluster (Bpos and Bneg) and the pedal giant neuron (RPeD1). In Bpos neurons, 5 nM free Pb2+ irreversibly inhibited current flow through calcium channels by 38 +/- 10%. In Bneg neurons, 5 nM free Pb2+ slightly inhibited inward currents (12 +/- 6%) and may have shifted their voltage dependence to more depolarized voltages. The inhibition and voltage shift were irreversible. In RPeD1 neurons, Pb2+ caused a small, statistically insignificant inhibition of inward current (5 nM free Pb2+; 18 +/- 19%; 30 nM free Pb2+: 31 +/- 23%). The effects of Pb2+ were fully reversible. These data indicate that (1) voltage-sensitive calcium channels in Lymnaea neurons are inhibited by nanomolar concentrations of free Pb2+; (2) there are multiple types of calcium channels in Lymnaea neurons; and (3) the effects of in vitro lead exposure differ qualitatively among channel types.

  10. Comparative study of the gating motif and C-type inactivation in prokaryotic voltage-gated sodium channels.

    Science.gov (United States)

    Irie, Katsumasa; Kitagawa, Kazuya; Nagura, Hitoshi; Imai, Tomoya; Shimomura, Takushi; Fujiyoshi, Yoshinori

    2010-02-05

    Prokaryotic voltage-gated sodium channels (Na(V)s) are homotetramers and are thought to inactivate through a single mechanism, named C-type inactivation. Here we report the voltage dependence and inactivation rate of the NaChBac channel from Bacillus halodurans, the first identified prokaryotic Na(V), as well as of three new homologues cloned from Bacillus licheniformis (Na(V)BacL), Shewanella putrefaciens (Na(V)SheP), and Roseobacter denitrificans (Na(V)RosD). We found that, although activated by a lower membrane potential, Na(V)BacL inactivates as slowly as NaChBac. Na(V)SheP and Na(V)RosD inactivate faster than NaChBac. Mutational analysis of helix S6 showed that residues corresponding to the "glycine hinge" and "PXP motif" in voltage-gated potassium channels are not obligatory for channel gating in these prokaryotic Na(V)s, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in Na(V)BacL (A214G), Na(V)SheP (A216G), and NaChBac (G219A) accelerated inactivation in these channels, whereas mutation of glycine to alanine in the lower part of helix S6 in NaChBac (G229A), Na(V)BacL (G224A), and Na(V)RosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic Na(V)s does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels.

  11. Scorpion beta-toxins and voltage-gated sodium channels: interactions and effects.

    Science.gov (United States)

    Pedraza Escalona, Martha; Possani, Lourival D

    2013-01-01

    Scorpion beta-toxins (beta-ScTxs) modify the activity of voltage-gated sodium (Nav) channels, thereby producing neurotoxic effects in diverse organisms. For this reason, beta-ScTxs are essential tools not only for discriminating among different channel sub-types but also for studying the mechanisms of channel gating and the structure-function relationship involved in this process. This review considers both the structural and the functional implications of the beta-ScTxs after they bind to their receptor sites, in accord with their classification into a) anti-mammalian beta-ScTxs, b) anti-insect selective excitatory beta-ScTxs, c) anti-insect selective depressant beta-ScTxs and d) beta-ScTxs active on both insect and mammals Nav channels. Additionally, the molecular mechanism of toxin action by the "voltage sensor trapping" model is discussed, and the systemic effects produced by these toxins are reviewed.

  12. 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.

  13. Adaptive evolution of voltage-gated sodium channels: the first 800 million years.

    Science.gov (United States)

    Zakon, Harold H

    2012-06-26

    Voltage-gated Na(+)-permeable (Nav) channels form the basis for electrical excitability in animals. Nav channels evolved from Ca(2+) channels and were present in the common ancestor of choanoflagellates and animals, although this channel was likely permeable to both Na(+) and Ca(2+). Thus, like many other neuronal channels and receptors, Nav channels predated neurons. Invertebrates possess two Nav channels (Nav1 and Nav2), whereas vertebrate Nav channels are of the Nav1 family. Approximately 500 Mya in early chordates Nav channels evolved a motif that allowed them to cluster at axon initial segments, 50 million years later with the evolution of myelin, Nav channels "capitalized" on this property and clustered at nodes of Ranvier. The enhancement of conduction velocity along with the evolution of jaws likely made early gnathostomes fierce predators and the dominant vertebrates in the ocean. Later in vertebrate evolution, the Nav channel gene family expanded in parallel in tetrapods and teleosts (∼9 to 10 genes in amniotes, 8 in teleosts). This expansion occurred during or after the late Devonian extinction, when teleosts and tetrapods each diversified in their respective habitats, and coincided with an increase in the number of telencephalic nuclei in both groups. The expansion of Nav channels may have allowed for more sophisticated neural computation and tailoring of Nav channel kinetics with potassium channel kinetics to enhance energy savings. Nav channels show adaptive sequence evolution for increasing diversity in communication signals (electric fish), in protection against lethal Nav channel toxins (snakes, newts, pufferfish, insects), and in specialized habitats (naked mole rats).

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

    Directory of Open Access Journals (Sweden)

    Fernando Lazcano-Pérez

    2016-05-01

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

  15. Voltage-dependent K+ currents contribute to heterogeneity of olfactory ensheathing cells

    Science.gov (United States)

    Rela, Lorena; Piantanida, Ana Paula; Bordey, Angelique; Greer, Charles A.

    2015-01-01

    The olfactory nerve is permissive for axon growth throughout life. This has been attributed in part to the olfactory ensheathing glial cells that encompass the olfactory sensory neuron fascicles. Olfactory ensheathing cells also promote axon growth in vitro and when transplanted in vivo to sites of injury. The mechanisms involved remain largely unidentified owing in part to the limited knowledge of the physiological properties of ensheathing cells. Glial cells rely for many functions on the properties of the potassium channels expressed; however, those expressed in ensheathing cells are unknown. Here we show that olfactory ensheathing cells express voltage-dependent potassium currents compatible with inward rectifier (Kir) and delayed rectifier (KDR) channels. Together with gap junction coupling, these contribute to the heterogeneity of membrane properties observed in olfactory ensheathing cells. The relevance of K+ currents expressed by ensheathing cells is discussed in relation to plasticity of the olfactory nerve. PMID:25856239

  16. Molecular Dynamics Simulations of Voltage Gated Cation Channels: Insights on Voltage-Sensor Domain Function and Modulation

    Directory of Open Access Journals (Sweden)

    Lucie eDelemotte

    2012-05-01

    Full Text Available Since their discovery in the 1950s, the structure and function of voltage gated cation channels (VGCC has been largely understood thanks to results stemming from electrophysiology, pharmacology, spectroscopy and structural biology. Over the past decade, computational methods such as molecular dynamics (MD simulations have also contributed, providing molecular level information that can be tested against experimental results, thereby allowing the validation of the models and protocols. Importantly, MD can shed light on elements of VGCC function that cannot be easily accessed through classical experiments. Here, we review the results of recent MD simulations addressing key questions that pertain to the function and modulation of the VGCC’s voltage sensor domain (VSD highlighting: 1 the movement of the S4-helix basic residues during channel activation, articulating how the electrical driving force acts upon them; 2 the nature of the VSD intermediate states on transitioning between open and closed states of the VGCC; and 3 the molecular level effects on the VSD arising from mutations of specific S4 positively charged residues involved in certain genetic diseases.

  17. L-type Voltage-Gated Calcium Channels in Conditioned Fear: A Genetic and Pharmacological Analysis

    Science.gov (United States)

    McKinney, Brandon C.; Sze, Wilson; White, Jessica A.; Murphy, Geoffrey G.

    2008-01-01

    Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear…

  18. [Progress in sodium channelopathies and biological functions of voltage-gated sodium channel blockers].

    Science.gov (United States)

    Wang, Hongyan; Gou, Meng; Xiao, Rong; Li, Qingwei

    2014-06-01

    Voltage-gated sodium channels (VGSCs), which are widely distributed in the excitable cells, are the primary mediators of electrical signal amplification and propagation. They play important roles in the excitative conduction of the neurons and cardiac muscle cells. The abnormalities of the structures and functions of VGSCs can change the excitability of the cells, resulting in a variety of diseases such as neuropathic pain, epilepsy and arrhythmia. At present, some voltage-gated sodium channel blockers are used for treating those diseases. In the recent years, several neurotoxins have been purified from the venom of the animals, which could inhibit the current of the voltage-gated sodium channels. Usually, these neurotoxins are compounds or small peptides that have been further designed and modified for targeted drugs of sodium channelopathies in the clinical treatment. In addition, a novel cysteine-rich secretory protein (CRBGP) has been isolated and purified from the buccal gland of the lampreys (Lampetra japonica), and it could inhibit the Na+ current of the hippocampus and dorsal root neurons for the first time. In the present study, the progress of the sodium channelopathies and the biological functions of voltage-gated sodium channel blockers are analyzed and summarized.

  19. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels.

    Science.gov (United States)

    Castillo, Karen; Contreras, Gustavo F; Pupo, Amaury; Torres, Yolima P; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-04-14

    Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

  20. Chronic ciguatoxin treatment induces synaptic scaling through voltage gated sodium channels in cortical neurons.

    Science.gov (United States)

    Martín, Víctor; Vale, Carmen; Rubiolo, Juan A; Roel, Maria; Hirama, Masahiro; Yamashita, Shuji; Vieytes, Mercedes R; Botana, Luís M

    2015-06-15

    Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) cortical neurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in cortical neurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of cortical neurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin

  1. Neutralization of Gating Charges in Domain II of the Sodium Channel α Subunit Enhances Voltage-Sensor Trapping by a β-Scorpion Toxin

    Science.gov (United States)

    Cestèle, Sandrine; Scheuer, Todd; Mantegazza, Massimo; Rochat, Hervé; Catterall, William A.

    2001-01-01

    β-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3–S4 loop at the extracellular end of the S4 voltage sensor in domain II of the α subunit. Here, we probe the role of gating charges in the IIS4 segment in β-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances β-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the β-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from −80 to −140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor–trapping model in which the β-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently

  2. Molecular insights into the local anesthetic receptor within voltage-gated sodium channels using hydroxylated analogues of mexiletine

    Directory of Open Access Journals (Sweden)

    Jean-François eDesaphy

    2012-02-01

    Full Text Available We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003, suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogues by adding one or two hydroxyl groups to the aryl moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration-response relationships were constructed using an holding potential of -120 mV at 0.1 Hz (tonic block and 10 Hz (use-dependent block stimulation frequencies. The half-maximum inhibitory concentrations (IC50 were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. Alteration of tonic block suggests that the aryl moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryl moiety may modify high-affinity binding of the drug ammine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion, these results confirm our former hypothesis showing that the presence of hydroxyl groups to the aryl moiety of mexiletine greatly reduced sodium channel block, and provide molecular insights into the intimate interaction of local anesthetics with

  3. The voltage dependence of GABAA receptor gating depends on extracellular pH.

    Science.gov (United States)

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W

    2005-11-28

    Recent studies have indicated that changes in extracellular pH and in membrane voltage affect the gamma-amino-n-butyric acid type A receptor gating mainly by altering desensitization and binding. To test whether the effects of membrane potential and pH are additive, their combined actions were investigated. By analyzing the current responses to rapid gamma-amino-n-butyric acid applications, we found that the current to voltage relationship was close to linear at acid pH but the increasing pH induced an inward rectification. Desensitization was enhanced at depolarizing potentials, but this strongly depended on pH, being weak at acidic and strong at basic pH values. A similar trend was observed for the onset rate of responses to saturating gamma-amino-n-butyric acid concentration. These data provide evidence that the voltage sensitivity of GABAA receptors depends on extracellular pH.

  4. The voltage dependence of GABAA receptor gating depends on extracellular pH

    Science.gov (United States)

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W.

    2007-01-01

    Recent studies have indicated that changes in extracellular pH and in membrane voltage affect the γ-amino-n-butyric acid type A receptor gating mainly by altering desensitization and binding. To test whether the effects of membrane potential and pH are additive, their combined actions were investigated. By analyzing the current responses to rapid γ-amino-n-butyric acid applications, we found that the current to voltage relationship was close to linear at acid pH but the increasing pH induced an inward rectification. Desensitization was enhanced at depolarizing potentials, but this strongly depended on pH, being weak at acidic and strong at basic pH values. A similar trend was observed for the onset rate of responses to saturating γ-amino-n-butyric acid concentration. These data provide evidence that the voltage sensitivity of GABAA receptors depends on extracellular pH. PMID:16272885

  5. The voltage-gated proton channel Hv1 enhances brain damage from ischemic stroke.

    Science.gov (United States)

    Wu, Long-Jun; Wu, Gongxiong; Akhavan Sharif, M Reza; Baker, Amanda; Jia, Yonghui; Fahey, Frederic H; Luo, Hongbo R; Feener, Edward P; Clapham, David E

    2012-03-04

    Phagocytic cell NADPH oxidase (NOX) generates reactive oxygen species (ROS) as part of innate immunity. Unfortunately, ischemia can also induce this pathway and inflict damage on native cells. The voltage-gated proton channel Hv1 enables NOX function by compensating cellular loss of electrons with protons. Accordingly, we investigated whether NOX-mediated brain damage in stroke can be inhibited by suppression of Hv1. We found that mouse and human brain microglia, but not neurons or astrocytes, expressed large Hv1-mediated currents. Hv1 was required for NOX-dependent ROS generation in brain microglia in situ and in vivo. Mice lacking Hv1 were protected from NOX-mediated neuronal death and brain damage 24 h after stroke. These results indicate that Hv1-dependent ROS production is responsible for a substantial fraction of brain damage at early time points after ischemic stroke and provide a rationale for Hv1 as a therapeutic target for the treatment of ischemic stroke.

  6. 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.

  7. S1-S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation.

    Science.gov (United States)

    Groome, James R; Winston, Vern

    2013-05-01

    The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1-S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1-S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I-III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

  8. 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

  9. A novel mechanism for fine-tuning open-state stability in a voltage-gated potassium channel

    DEFF Research Database (Denmark)

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

    2013-01-01

    Voltage-gated potassium channels elicit membrane hyperpolarization through voltage-sensor domains that regulate the conductive status of the pore domain. To better understand the inherent basis for the open-closed equilibrium in these channels, we undertook an atomistic scan using synthetic fluor...... that the intrinsic open-state destabilization via aromatic repulsion represents a new mechanism by which ion channels, and likely other proteins, fine-tune conformational equilibria.......Voltage-gated potassium channels elicit membrane hyperpolarization through voltage-sensor domains that regulate the conductive status of the pore domain. To better understand the inherent basis for the open-closed equilibrium in these channels, we undertook an atomistic scan using synthetic...... fluorinated derivatives of aromatic residues previously implicated in the gating of Shaker potassium channels. Here we show that stepwise dispersion of the negative electrostatic surface potential of only one site, Phe481, stabilizes the channel open state. Furthermore, these data suggest that this apparent...

  10. Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus.

    Science.gov (United States)

    DeCaen, Paul G; Takahashi, Yuka; Krulwich, Terry A; Ito, Masahiro; Clapham, David E

    2014-11-11

    Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na(+) to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na(+) channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na(+), Ca(2+) and K(+) ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na(+) or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

  11. Omega currents in voltage-gated ion channels: what can we learn from uncovering the voltage-sensing mechanism using MD simulations?

    Science.gov (United States)

    Tarek, Mounir; Delemotte, Lucie

    2013-12-17

    Ion channels conduct charged species through otherwise impermeable biological membranes. Their activity supports a number of physiological processes, and genetic mutations can disrupt their function dramatically. Among these channels, voltage gated cation channels (VGCCs) are ubiquitous transmembrane proteins involved in electrical signaling. In addition to their selectivity for ions, their function requires membrane-polarization-dependent gating. Triggered by changes in the transmembrane voltage, the activation and deactivation of VGCCs proceed through a sensing mechanism that prompts motion of conserved positively charged (basic) residues within the S4 helix of a four-helix bundle, the voltage sensor domain (VSD). Decades of experimental investigations, using electrophysiology, molecular biology, pharmacology, and spectroscopy, have revealed details about the function of VGCCs. However, in 2005, the resolution of the crystal structure of the activated state of one member of the mammalian voltage gated potassium (Kv) channels family (the Kv1.2) enabled researchers to make significant progress in understanding the structure-function relationship in these proteins on a molecular level. In this Account, we review the use of a complementary technique, molecular dynamics (MD) simulations, that has offered new insights on this timely issue. Starting from the "open-activated state" crystal structure, we have carried out large-scale all atom MD simulations of the Kv1.2 channel embedded in its lipidic environment and submitted to a hyperpolarizing (negative) transmembrane potential. We then used steered MD simulations to complete the full transition to the resting-closed state. Using these procedures, we have followed the operation of the VSDs and uncovered three intermediate states between their activated and deactivated conformations. Each conformational state is characterized by its network of salt bridges and by the occupation of the gating charge transfer center by a

  12. Modeling of the Binding of Peptide Blockers to Voltage-Gated Potassium Channels: Approaches and Evidence.

    Science.gov (United States)

    Novoseletsky, V N; Volyntseva, A D; Shaitan, K V; Kirpichnikov, M P; Feofanov, A V

    2016-01-01

    Modeling of the structure of voltage-gated potassium (KV) channels bound to peptide blockers aims to identify the key amino acid residues dictating affinity and provide insights into the toxin-channel interface. Computational approaches open up possibilities for in silico rational design of selective blockers, new molecular tools to study the cellular distribution and functional roles of potassium channels. It is anticipated that optimized blockers will advance the development of drugs that reduce over activation of potassium channels and attenuate the associated malfunction. Starting with an overview of the recent advances in computational simulation strategies to predict the bound state orientations of peptide pore blockers relative to KV-channels, we go on to review algorithms for the analysis of intermolecular interactions, and then take a look at the results of their application.

  13. Intron retention in mRNA encoding ancillary subunit of insect voltage-gated sodium channel modulates channel expression, gating regulation and drug sensitivity.

    Science.gov (United States)

    Bourdin, Céline M; Moignot, Bénédicte; Wang, Lingxin; Murillo, Laurence; Juchaux, Marjorie; Quinchard, Sophie; Lapied, Bruno; Guérineau, Nathalie C; Dong, Ke; Legros, Christian

    2013-01-01

    Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation "temperature-induced-paralysis locus E." The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na(+) currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3'UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an

  14. Effect of dexamethasone on voltage-gated Na+ channel in cultured human bronchial smooth muscle cells.

    Science.gov (United States)

    Nakajima, Toshiaki; Jo, Taisuke; Meguro, Kentaro; Oonuma, Hitoshi; Ma, Ji; Kubota, Nami; Imuta, Hiroyuki; Takano, Haruhito; Iida, Haruko; Nagase, Takahide; Nagata, Taiji

    2008-06-06

    Voltage-gated Na(+) channel (I(Na)) encoded by SCN9A mRNA is expressed in cultured human bronchial smooth muscle cells. We investigated the effects of dexamethasone on I(Na), by using whole-cell voltage clamp techniques, reverse transcriptase/polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR. Acute application of dexamethasone (10(-6) M) did not affect I(Na). However, the percentage of the cells with I(Na) was significantly less in cells pretreated with dexamethasone for 48 h, and the current-density of I(Na) adjusted by cell capacitance in cells with I(Na) was also decreased in cells treated with dexamethasone. RT-PCR analysis showed that alpha and beta subunits mRNA of I(Na) mainly consisted of SCN9A and SCN1beta, respectively. Treatment with dexamethasone for 24-48 h inhibited the expression of SCN9A mRNA. The inhibitory effect of dexamethasone was concentration-dependent, and was observed at a concentration higher than 0.1 nM. The effect of dexamethasone on SCN9A mRNA was not blocked by spironolactone, but inhibited by mifepristone. The inhibitory effects of dexamethasone on SCN9A mRNA could not be explained by the changes of the stabilization of mRNA measured by using actinomycin D. These results suggest that dexamethasone inhibited I(Na) encoded by SCN9A mRNA in cultured human bronchial smooth muscle cells by inhibiting the transcription via the glucocorticoid receptor.

  15. Designing a C84 fullerene as a specific voltage-gated sodium channel blocker

    Science.gov (United States)

    Hilder, Tamsyn A.; Chung, Shin-Ho

    2013-07-01

    Fullerene derivatives demonstrate considerable potential for numerous biological applications, such as the effective inhibition of HIV protease. Recently, they were identified for their ability to indiscriminately block biological ion channels. A fullerene derivative which specifically blocks a particular ion channel could lead to a new set of drug leads for the treatment of various ion channel-related diseases. Here, we demonstrate their extraordinary potential by designing a fullerene which mimics some of the functions of μ-conotoxin, a peptide derived from cone snail venom which potently binds to the bacterial voltage-gated sodium channel (NavAb). We show, using molecular dynamics simulations, that the C84 fullerene with six lysine derivatives uniformly attached to its surface is selective to NavAb over a voltage-gated potassium channel (Kv1.3). The side chain of one of the lysine residues protrudes into the selectivity filter of the channel, while the methionine residues located just outside of the channel form hydrophobic contacts with the carbon atoms of the fullerene. The modified C84 fullerene strongly binds to the NavAb channel with an affinity of 46 nM but binds weakly to Kv1.3 with an affinity of 3 mM. This potent blocker of NavAb may serve as a structural template from which potent compounds can be designed for the targeting of mammalian Nav channels. There is a genuine need to target mammalian Nav channels as a form of treatment of various diseases which have been linked to their malfunction, such as epilepsy and chronic pain.

  16. The L-Type Voltage-Gated Calcium Channel Ca[subscript v]1.3 Mediates Consolidation, but Not Extinction, of Contextually Conditioned Fear in Mice

    Science.gov (United States)

    McKinney, Brandon C.; Murphy, Geoffrey G.

    2006-01-01

    Using pharmacological techniques, it has been demonstrated that both consolidation and extinction of Pavlovian fear conditioning are dependent to some extent upon L-type voltage-gated calcium channels (LVGCCs). Although these studies have successfully implicated LVGCCs in Pavlovian fear conditioning, they do not provide information about the…

  17. Like Extinction, Latent Inhibition of Conditioned Fear in Mice Is Blocked by Systemic Inhibition of L-Type Voltage-Gated Calcium Channels

    Science.gov (United States)

    Blouin, Ashley M.; Cain, Chris K.; Barad, Mike

    2004-01-01

    Having recently shown that extinction of conditioned fear depends on L-type voltage-gated calcium channels (LVGCCs), we have been seeking other protocols that require this unusual induction mechanism. We tested latent inhibition (LI) of fear, because LI resembles extinction except that cue exposures precede, rather than follow, cue-shock pairing.…

  18. Deltamethrin Inhibits the Human T-type Voltage-Sensitive Calcium Channel (Cav3.2

    Directory of Open Access Journals (Sweden)

    Steven B. Symington

    2009-01-01

    Full Text Available The goal of this study was to determine the effect of deltamethrin, a pyrethroid insecticide, on CaV3.2, a human T-type voltage-sensitive calcium channel expressed in Xenopus laevis (X.laevis oocytes. Cav3.2 cDNA was transcribed into cRNA; the cRNA was then injected into X.laevis oocytes and electrophysiologically characterized using the two-electrode voltage clamp technique with Ba2+ as a charge carrier. Deltamethrin (10-7 M reduced peak current in a nonreversible manner compared to the untreated control, but had no effect on the voltagedependent activation and inactivation kinetics. These findings confirm that human CaV3.2 is a target for deltamethrin and quite possibly other pyrethroid insecticides. These studies provide insight into the molecular mechanisms of the effect that pyrethroids have on voltage-sensitive calcium channels in general. This information will allow a more complete understanding of the molecular and cellular nature of pyrethroid-induced toxicity and expand our knowledge of the structure-activity relationships of pyrethroids with regard to their action on voltage-sensitive calcium channels.

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

    Science.gov (United States)

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

    2017-03-06

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

  20. Voltage-gated ion channel dysfunction precedes cardiomyopathy development in the dystrophic heart.

    Directory of Open Access Journals (Sweden)

    Xaver Koenig

    Full Text Available Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene, is associated with severe cardiac complications including cardiomyopathy and cardiac arrhythmias. Recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. It is, however, unknown if the ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiac pathology.To address this question, we first investigated sodium channel impairments in cardiomyocytes derived from dystrophic neonatal mice prior to cardiomyopahty development, by using the whole cell patch clamp technique. Besides the most common model for DMD, the dystrophin-deficient mdx mouse, we also used mice additionally carrying an utrophin mutation. In neonatal cardiomyocytes, dystrophin-deficiency generated a 25% reduction in sodium current density. In addition, extra utrophin-deficiency significantly altered sodium channel gating parameters. Moreover, also calcium channel inactivation was considerably reduced in dystrophic neonatal cardiomyocytes, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. To assess developmental changes, we also studied sodium channel impairments in cardiomyocytes derived from dystrophic adult mice, and compared them with the respective abnormalities in dystrophic neonatal cells. Here, we found a much stronger sodium current reduction in adult cardiomyocytes. The described sodium channel impairments slowed the upstroke of the action potential in adult cardiomyocytes, and only in dystrophic adult mice, the QRS interval of the electrocardiogram was prolonged.Ion channel impairments precede pathology development in the dystrophic heart, and may thus be considered potential cardiomyopathy triggers.

  1. A novel ZVS high voltage power supply for micro-channel plate photomultiplier tubes

    Science.gov (United States)

    Pei, Chengquan; Tian, Jinshou; Liu, Zhen; Qin, Hong; Wu, Shengli

    2017-04-01

    A novel resonant high voltage power supply (HVPS) with zero voltage switching (ZVS), to reduce the voltage stress on switching devices and improve conversion efficiency, is proposed. The proposed HVPS includes a drive circuit, a transformer, several voltage multiplying circuits, and a regulator circuit. The HVPS contains several secondary windings that can be precisely regulated. The proposed HVPS performed better than the traditional resistor voltage divider, which requires replacing matching resistors resulting in resistor dispersibility in the Micro-Channel Plate (MCP). The equivalent circuit of the proposed HVPS was established and the operational principle analyzed. The entire switching element can achieve ZVS, which was validated by a simulation and experiments. The properties of this HVPS were tested including minimum power loss (240 mW), maximum power loss (1 W) and conversion efficiency (85%). The results of this research are that the proposed HVPS was suitable for driving the micro-channel plate photomultiplier tube (MCP-PMT). It was therefore adopted to test the MCP-PMT, which will be used in Daya Bay reactor neutrino experiment II in China.

  2. Functional expression of voltage-gated calcium channels in human melanoma.

    Science.gov (United States)

    Das, A; Pushparaj, C; Bahí, N; Sorolla, A; Herreros, J; Pamplona, R; Vilella, R; Matias-Guiu, X; Martí, R M; Cantí, C

    2012-03-01

    The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression. © 2012 John Wiley & Sons A/S.

  3. The action of a phorbol ester on voltage-dependent parameters of the sodium current in isolated hippocampal neurons.

    Science.gov (United States)

    Chizhmakov, I V; Klee, M R

    1994-03-01

    The action of a phorbol ester (phorbol-12,13-diacetate) on the voltage-activated sodium current has been investigated by the voltage-clamp method in acutely isolated pyramidal neurons from rat hippocampus. The intracellular perfusion of isolated pyramidal neurons for 30-40 min induced a gradual 10-15 mV shift in both the current-voltage relationship and voltage-dependent steady-state inactivation to more negative potentials. The application of phorbol ester (1-10 microM) to isolated neurons for the same time increased the amplitude of sodium current by 15-20%, shifted the above-mentioned voltage-dependent parameters for an additional 10-15 mV in the same direction and changed the slope of the steady-state inactivation curve. In contrast, after prolonged incubation of slices in the phorbol ester-containing solution (1-10 microM) for 0.5-3 h, subsequent application of phorbol ester at the same concentration caused neither the addition shift of the voltage-dependent characteristics of sodium channels nor the change of the slope of the steady-state inactivation curve. However, in this case an increase in the amplitude of sodium current by 15-20% during 30-40 min intracellular perfusion was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    Directory of Open Access Journals (Sweden)

    Francesco eMiceli

    2015-07-01

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

  5. Voltage-dependent modulation of cardiac ryanodine receptors (RyR2 by protamine.

    Directory of Open Access Journals (Sweden)

    Paula L Diaz-Sylvester

    Full Text Available It has been reported that protamine (>10 microg/ml blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca2+ release from sarcoplasmic reticulum microsomes. We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02-20 microg/ml added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage (V(m; SR lumen-cytosol = 0 mV, protamine induced conductance transitions to several intermediate states (substates as well as full block of RyR2. At V(m>10 mV, the substate with the highest level of conductance was predominant. Increasing V(m from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate had the same magnitude at 0 and +80 mV despite the approximately 3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers (ryanoids, imperatoxin. A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations is the activation of RyR2 in the presence of nanomolar cytosolic Ca2+ and millimolar Mg2+ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca2+ leak observed in cardiac diseases.

  6. State-dependent blocker interactions with the CFTR chloride channel: implications for gating the pore.

    Science.gov (United States)

    Linsdell, Paul

    2014-12-01

    Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is subject to voltage-dependent open-channel block by a diverse range of cytoplasmic anions. However, in most cases the ability of these blocking substances to influence the pore opening and closing process has not been reported. In the present work, patch clamp recording was used to investigate the state-dependent block of CFTR by cytoplasmic Pt(NO2)4(2-) ions. Two major effects of Pt(NO2)4(2-) were identified. First, this anion caused fast, voltage-dependent block of open channels, leading to an apparent decrease in single-channel current amplitude. Secondly, Pt(NO2)4(2-) also decreased channel open probability due to an increase in interburst closed times. Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO2)4(2-) altered blocker effects both on Cl(-) permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO2)4(2-) interaction with a single site within the pore. Experiments at reduced extracellular Cl(-) concentration hinted that Pt(NO2)4(2-) may have a third effect, possibly increasing channel activity by interfering with channel closure. These results suggest that Pt(NO2)4(2-) can enter from the cytoplasm into the pore inner vestibule of both open and closed CFTR channels, and that Pt(NO2)4(2-) bound in the inner vestibule blocks Cl(-) permeation as well as interfering with channel opening and, perhaps, channel closure. Implications for the location of the channel gate in the pore, and the operation of this gate, are discussed.

  7. A specialized molecular motion opens the Hv1 voltage-gated proton channel.

    Science.gov (United States)

    Mony, Laetitia; Berger, Thomas K; Isacoff, Ehud Y

    2015-04-01

    The Hv1 proton channel is unique among voltage-gated channels for containing the pore and gate within its voltage-sensing domain. Pore opening has been proposed to include assembly of the selectivity filter between an arginine (R3) of segment S4 and an aspartate (D1) of segment S1. We determined whether gating involves motion of S1, using Ciona intestinalis Hv1. We found that channel opening is concomitant with solution access to the pore-lining face of S1, from the cytoplasm to deep inside the pore. Voltage- and patch-clamp fluorometry showed that this involves a motion of S1 relative to its surroundings. S1 motion and the S4 motion that precedes it are each influenced by residues on the other helix, thus suggesting a dynamic interaction between S1 and S4. Our findings suggest that the S1 of Hv1 has specialized to function as part of the channel's gate.

  8. 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.

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

    Science.gov (United States)

    Cheng, Lan; Sanguinetti, Michael C

    2009-05-01

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

  10. Tolperisone-type drugs inhibit spinal reflexes via blockade of voltage-gated sodium and calcium channels.

    Science.gov (United States)

    Kocsis, Pál; Farkas, Sándor; Fodor, László; Bielik, Norbert; Thán, Márta; Kolok, Sándor; Gere, Anikó; Csejtei, Mónika; Tarnawa, István

    2005-12-01

    The spinal reflex depressant mechanism of tolperisone and some of its structural analogs with central muscle relaxant action was investigated. Tolperisone (50-400 microM), eperisone, lanperisone, inaperisone, and silperisone (25-200 microM) dose dependently depressed the ventral root potential of isolated hemisected spinal cord of 6-day-old rats. The local anesthetic lidocaine (100-800 microM) produced qualitatively similar depression of spinal functions in the hemicord preparation, whereas its blocking effect on afferent nerve conduction was clearly stronger. In vivo, tolperisone and silperisone as well as lidocaine (10 mg/kg intravenously) depressed ventral root reflexes and excitability of motoneurons. However, in contrast with lidocaine, the muscle relaxant drugs seemed to have a more pronounced action on the synaptic responses than on the excitability of motoneurons. Whole-cell measurements in dorsal root ganglion cells revealed that tolperisone and silperisone depressed voltage-gated sodium channel conductance at concentrations that inhibited spinal reflexes. Results obtained with tolperisone and its analogs in the [3H]batrachotoxinin A 20-alpha-benzoate binding in cortical neurons and in a fluorimetric membrane potential assay in cerebellar neurons further supported the view that blockade of sodium channels may be a major component of the action of tolperisone-type centrally acting muscle relaxant drugs. Furthermore, tolperisone, eperisone, and especially silperisone had a marked effect on voltage-gated calcium channels, whereas calcium currents were hardly influenced by lidocaine. These data suggest that tolperisone-type muscle relaxants exert their spinal reflex inhibitory action predominantly via a presynaptic inhibition of the transmitter release from the primary afferent endings via a combined action on voltage-gated sodium and calcium channels.

  11. Voltage-dependent potassium currents during fast spikes of rat cerebellar Purkinje neurons: inhibition by BDS-I toxin.

    Science.gov (United States)

    Martina, Marco; Metz, Alexia E; Bean, Bruce P

    2007-01-01

    We characterized the kinetics and pharmacological properties of voltage-activated potassium currents in rat cerebellar Purkinje neurons using recordings from nucleated patches, which allowed high resolution of activation and deactivation kinetics. Activation was exceptionally rapid, with 10-90% activation in about 400 mus at +30 mV, near the peak of the spike. Deactivation was also extremely rapid, with a decay time constant of about 300 mus near -80 mV. These rapid activation and deactivation kinetics are consistent with mediation by Kv3-family channels but are even faster than reported for Kv3-family channels in other neurons. The peptide toxin BDS-I had very little blocking effect on potassium currents elicited by 100-ms depolarizing steps, but the potassium current evoked by action potential waveforms was inhibited nearly completely. The mechanism of inhibition by BDS-I involves slowing of activation rather than total channel block, consistent with the effects described in cloned Kv3-family channels and this explains the dramatically different effects on currents evoked by short spikes versus voltage steps. As predicted from this mechanism, the effects of toxin on spike width were relatively modest (broadening by roughly 25%). These results show that BDS-I-sensitive channels with ultrafast activation and deactivation kinetics carry virtually all of the voltage-dependent potassium current underlying repolarization during normal Purkinje cell spikes.

  12. Actions of Ethanol on Voltage-Sensitive Sodium Channels. Effects on Neurotoxin-Stimulated Sodium Uptake in Synaptosomes

    Science.gov (United States)

    1985-01-01

    concentration in the nonaqueuus (membrane) phase (Lyon et aL, 1981). Concentration- effect summarized in table 1 . When sodium channels were activated curves were...Voltage-Sensitive Sodium Channels : Effects on Neurotoxin-Stimulated Sodium Uptake in DT (7 Synaptosomes E L C MICHAEL J. MULLIN’ and WALTER A. HUNT...1984). At the present time, the 8 1 structural and functional properties of the voltage-sensitive sodium channels are understood most completely

  13. VOLTAGE STABILITY ASSESSMENT FOR WIND FARMS INTEGRATION INTO ELECTRICITY GRIDS WITH AND WITHOUT CONSIDERATION OF VOLTAGE DEPENDENT LOADS

    Directory of Open Access Journals (Sweden)

    TOMA R.

    2016-09-01

    Full Text Available The paper presents a comparative study between the effects on voltage stability of the integration of a wind farm into the electricity grid with or without voltage dependent loads in the context of different locations of a synchronous compensator from the grid. The P-V curves are built by using the PowerFactory DigSilent 15.2.2 and a DPL script that implements a simplified form of the Continuation Power Flow method.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sherman, S.J.

    1985-01-01

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

  15. Current-dependent block of rabbit sino-atrial node I(f) channels by ivabradine.

    Science.gov (United States)

    Bucchi, Annalisa; Baruscotti, Mirko; DiFrancesco, Dario

    2002-07-01

    "Funny" (f-) channels have a key role in generation of spontaneous activity of pacemaker cells and mediate autonomic control of cardiac rate; f-channels and the related neuronal h-channels are composed of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel subunits. We have investigated the block of f-channels of rabbit cardiac sino-atrial node cells by ivabradine, a novel heart rate-reducing agent. Ivabradine is an open-channel blocker; however, block is exerted preferentially when channels deactivate on depolarization, and is relieved by long hyperpolarizing steps. These features give rise to use-dependent behavior. In this, the action of ivabradine on f-channels is similar to that reported of other rate-reducing agents such as UL-FS49 and ZD7288. However, other features of ivabradine-induced block are peculiar and do not comply with the hypothesis that the voltage-dependence of block is entirely attributable to either the sensitivity of ivabradine-charged molecules to the electrical field in the channel pore, or to differential affinity to different channel states, as has been proposed for UL-FS49 (DiFrancesco, D. 1994. Pflugers Arch. 427:64-70) and ZD7288 (Shin, S.K., B.S. Rotheberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91-101), respectively. Experiments where current flows through channels is modified without changing membrane voltage reveal that the ivabradine block depends on the current driving force, rather than voltage alone, a feature typical of block induced in inwardly rectifying K(+) channels by intracellular cations. Bound drug molecules do not detach from the binding site in the absence of inward current through channels, even if channels are open and the drug is therefore not "trapped" by closed gates. Our data suggest that permeation through f-channel pores occurs according to a multiion, single-file mechanism, and that block/unblock by ivabradine is coupled to ionic flow. The use-dependence resulting from specific features of

  16. X-ray crystal structure of voltage-gated proton channel.

    Science.gov (United States)

    Takeshita, Kohei; Sakata, Souhei; Yamashita, Eiki; Fujiwara, Yuichiro; Kawanabe, Akira; Kurokawa, Tatsuki; Okochi, Yoshifumi; Matsuda, Makoto; Narita, Hirotaka; Okamura, Yasushi; Nakagawa, Atsushi

    2014-04-01

    The voltage-gated proton channel Hv1 (or VSOP) has a voltage-sensor domain (VSD) with dual roles of voltage sensing and proton permeation. Its gating is sensitive to pH and Zn(2+). Here we present a crystal structure of mouse Hv1 in the resting state at 3.45-Å resolution. The structure showed a 'closed umbrella' shape with a long helix consisting of the cytoplasmic coiled coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. Two out of three arginines in S4 were located below the phenylalanine constituting the gating charge-transfer center. The extracellular region of each protomer coordinated a Zn(2+), thus suggesting that Zn(2+) stabilizes the resting state of Hv1 by competing for acidic residues that otherwise form salt bridges with voltage-sensing positive charges on S4. These findings provide a platform for understanding the general principles of voltage sensing and proton permeation.

  17. Lacosamide Inhibition of Nav1.7 Voltage-Gated Sodium Channels: Slow Binding to Fast-Inactivated States.

    Science.gov (United States)

    Jo, Sooyeon; Bean, Bruce P

    2017-04-01

    Lacosamide is an antiseizure agent that targets voltage-dependent sodium channels. Previous experiments have suggested that lacosamide is unusual in binding selectively to the slow-inactivated state of sodium channels, in contrast to drugs like carbamazepine and phenytoin, which bind tightly to fast-inactivated states. Using heterologously expressed human Nav1.7 sodium channels, we examined the state-dependent effects of lacosamide. Lacosamide induced a reversible shift in the voltage dependence of fast inactivation studied with 100-millisecond prepulses, suggesting binding to fast-inactivated states. Using steady holding potentials, lacosamide block was very weak at -120 mV (3% inhibition by 100 µM lacosamide) but greatly enhanced at -80 mV (43% inhibition by 100 µM lacosamide), where there is partial fast inactivation but little or no slow inactivation. During long depolarizations, lacosamide slowly (over seconds) put channels into states that recovered availability slowly (hundreds of milliseconds) at -120 mV. This resembles enhancement of slow inactivation, but the effect was much more pronounced at -40 mV, where fast inactivation is complete, but slow inactivation is not, than at 0 mV, where slow inactivation is maximal, more consistent with slow binding to fast-inactivated states than selective binding to slow-inactivated states. Furthermore, inhibition by lacosamide was greatly reduced by pretreatment with 300 µM lidocaine or 300 µM carbamazepine, suggesting that lacosamide, lidocaine, and carbamazepine all bind to the same site. The results suggest that lacosamide binds to fast-inactivated states in a manner similar to other antiseizure agents but with slower kinetics of binding and unbinding.

  18. Voltage-dependent Calcium Channel Plays a Role in the Formation of Large-amplitude Miniature Excitatory Postsynaptic Current%电压依赖性钙通道参与大振幅微小兴奋性突触后电流形成的实验研究

    Institute of Scientific and Technical Information of China (English)

    黄福森; 杨小娟; 王儒蓉; 吴超然

    2012-01-01

    目的 观察电压依赖性钙通道是否作用于大鼠脊髓背角胶状质层(SG)神经元大振幅微小兴奋性突触后电流的形成.方法 选用成年雄性Sprague-Dawley (SD)大鼠,2%~3%异氟烷麻醉后,分离其腰骶部的脊髓,然后切片.采用全细胞电压钳技术,玻璃微电极的电阻为4~6 MΩ,钳制电压为-70 mV,记录胶状质层神经元微小兴奋性突触后电流( mEPSC)电流.将电流信号用Axopatch 200来放大并储存于电脑.对照组和用药结束后,持续采样mEPSC电流30 s.mEPSC电流的频率和振幅用Clampfit 8.1进行分析.结果 钳制电压为-70 mV时,所有SG神经元均有自发性的EPSC.辣椒素增加mEPSC发生的频率和波幅.钴离子抑制辣椒素诱导的大振幅mEPSC.钴离子抑制辣椒素诱导的mEPSC的平均振幅,而不抑制其发生频率.结论 电压依赖性钙离子通道参与了辣椒素引起的痛觉形成.%Objective To observe whether the voltage-dependent calcium channel contributes to the formation of capsaicin-induced miniature excitatory postsynaptic current (mEPSC) in rats. Methods Experiments were performed in adult male Sprague-Dawley rats. The lumbosacral portion of the spinal cord were separated after anesthesia by 2%-3% isoflurane, and the spinal cord slices were prepared. Whole-cell voltage-clamp technique was applied to substantia gelatinosa (SG) neurons with a glass patch-pipette having a resistance of 4-6 MΩ, holding potential -70 mV. Signals were amplified with an Axopatch 200 amplifier and then stored in a personal computer. The mEPSC in controls and immediately after the end of drug applications were sampled for 30 seconds and the frequency and amplitude were analyzed using Clampfit 8.1. Results All SG neurons in this database had spontaneous mEPSC with the holding potential of-70 mV. Capsaicin increased the frequency and mean amplitude of mEPSC. Cobalt inhibited the capsaicin-induced large-amplitude mEPSC, as well as the mean amplitude but

  19. 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.

  20. 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.

  1. Leaky sodium channels from voltage sensor mutations in periodic paralysis, but not paramyotonia

    Science.gov (United States)

    Francis, David G.; Rybalchenko, Volodymyr; Struyk, Arie

    2011-01-01

    Background: Hypokalemic periodic paralysis (HypoPP) is associated with mutations in either the CaV1.1 calcium channel or the NaV1.4 sodium channel. Some NaV1.4 HypoPP mutations have been shown to cause an anomalous inward current that may contribute to the attacks of paralysis. Herein, we test whether disease-associated NaV1.4 mutations in previously untested homologous regions of the channel also give rise to the anomalous current. Methods: The functional properties of mutant NaV1.4 channels were studied with voltage-clamp techniques in an oocyte expression system. Results: The HypoPP mutation NaV1.4-R1132Q conducts an anomalous gating pore current, but the homologous R1448C mutation in paramyotonia congenita does not. Conclusions: Gating pore currents arising from missense mutations at arginine residues in the voltage sensor domains of NaV1.4 are a common feature of HypoPP mutant channels and contribute to the attacks of paralysis. PMID:21490317

  2. 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.

  3. Effect of angiotensin II-induced arterial hypertension on the voltage-dependent contractions of mouse arteries.

    Science.gov (United States)

    Fransen, Paul; Van Hove, Cor E; Leloup, Arthur J A; Schrijvers, Dorien M; De Meyer, Guido R Y; De Keulenaer, Gilles W

    2016-02-01

    Arterial hypertension (AHT) affects the voltage dependency of L-type Ca(2+) channels in cardiomyocytes. We analyzed the effect of angiotensin II (AngII)-induced AHT on L-type Ca(2+) channel-mediated isometric contractions in conduit arteries. AHT was induced in C57Bl6 mice with AngII-filled osmotic mini-pumps (4 weeks). Normotensive mice treated with saline-filled osmotic mini-pumps were used for comparison. Voltage-dependent contractions mediated by L-type Ca(2+) channels were studied in vaso-reactive studies in vitro in isolated aortic and femoral arteries by using extracellular K(+) concentration-response (KDR) experiments. In aortic segments, AngII-induced AHT significantly sensitized isometric contractions induced by elevated extracellular K(+) and depolarization. This sensitization was partly prevented by normalizing blood pressure with hydralazine, suggesting that it was caused by AHT rather than by direct AngII effects on aortic smooth muscle cells. The EC50 for extracellular K(+) obtained in vitro correlated significantly with the rise in arterial blood pressure induced by AngII in vivo. The AHT-induced sensitization persisted when aortic segments were exposed to levcromakalim or to inhibitors of basal nitric oxide release. Consistent with these observations, AngII-treatment also sensitized the vaso-relaxing effects of the L-type Ca(2+) channel blocker diltiazem during K(+)-induced contractions. Unlike aorta, AngII-treatment desensitized the isometric contractions to depolarization in femoral arteries pointing to vascular bed specific responses of arteries to hypertension. AHT affects the voltage-dependent L-type Ca(2+) channel-mediated contraction of conduit arteries. This effect may contribute to the decreased vascular compliance in AHT and explain the efficacy of Ca(2+) channel blockers to reduce vascular stiffness and central blood pressure in AHT.

  4. Amitriptyline and carbamazepine utilize voltage-gated ion channel suppression to impair excitability of sensory dorsal horn neurons in thin tissue slice: An in vitro study.

    Science.gov (United States)

    Wolff, Matthias; Czorlich, Patrick; Nagaraj, Chandran; Schnöbel-Ehehalt, Rose; Li, Yingji; Kwapiszewska, Grazyna; Olschewski, Horst; Heschl, Stefan; Olschewski, Andrea

    2016-08-01

    Amitriptyline, carbamazepine and gabapentin are often used for the treatment of neuropathic pain. However, their analgesic action on central sensory neurons is still not fully understood. Moreover, the expression pattern of their target ion channels is poorly elucidated in the dorsal horn of the spinal cord. Thus, we performed patch-clamp investigations in visualized neurons of lamina I-III of the spinal cord. The expression of the different voltage-gated ion channels, as the targets of these drugs, was detected by RT-PCR and immunohistochemistry. Neurons of the lamina I-III express the TTX-sensitive voltage-gated Na(+) as well as voltage-gated K(+) subunits assembling the fast inactivating (A-type) currents and the delayed rectifier K(+) currents. Our pharmacological studies show that tonically-firing, adapting-firing and single spike neurons responded dose-dependently to amitriptyline and carbamazepine. The ion channel inhibition consecutively reduced the firing rate of tonically-firing and adapting-firing neurons. This study provides evidence for the distribution of voltage-gated Na(+) and K(+) subunits in lamina I-III of the spinal cord and for the action of drugs used for the treatment of neuropathic pain. Our work confirms that modulation of voltage-gated ion channels in the central nervous system contributes to the antinociceptive effects of these drugs.

  5. Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence.

    Science.gov (United States)

    Keum, Dongil; Kruse, Martin; Kim, Dong-Il; Hille, Bertil; Suh, Byung-Chang

    2016-06-28

    Voltage-sensing phosphatases (VSPs) are homologs of phosphatase and tensin homolog (PTEN), a phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] 3-phosphatase. However, VSPs have a wider range of substrates, cleaving 3-phosphate from PI(3,4)P2 and probably PI(3,4,5)P3 as well as 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 in response to membrane depolarization. Recent proposals say these reactions have differing voltage dependence. Using Förster resonance energy transfer probes specific for different PIs in living cells with zebrafish VSP, we quantitate both voltage-dependent 5- and 3-phosphatase subreactions against endogenous substrates. These activities become apparent with different voltage thresholds, voltage sensitivities, and catalytic rates. As an analytical tool, we refine a kinetic model that includes the endogenous pools of phosphoinositides, endogenous phosphatase and kinase reactions connecting them, and four exogenous voltage-dependent 5- and 3-phosphatase subreactions of VSP. We show that apparent voltage threshold differences for seeing effects of the 5- and 3-phosphatase activities in cells are not due to different intrinsic voltage dependence of these reactions. Rather, the reactions have a common voltage dependence, and apparent differences arise only because each VSP subreaction has a different absolute catalytic rate that begins to surpass the respective endogenous enzyme activities at different voltages. For zebrafish VSP, our modeling revealed that 3-phosphatase activity against PI(3,4,5)P3 is 55-fold slower than 5-phosphatase activity against PI(4,5)P2; thus, PI(4,5)P2 generated more slowly from dephosphorylating PI(3,4,5)P3 might never accumulate. When 5-phosphatase activity was counteracted by coexpression of a phosphatidylinositol 4-phosphate 5-kinase, there was accumulation of PI(4,5)P2 in parallel to PI(3,4,5)P3 dephosphorylation

  6. 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.

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

    Science.gov (United States)

    Langille, Megan M; Desai, Jay

    2015-01-01

    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.

  8. A Korean Family of Hypokalemic Periodic Paralysis with Mutation in a Voltage-gated Calcium Channel (R1239G)

    Science.gov (United States)

    Kim, June-Bum; Lee, Kyung-Yil

    2005-01-01

    Hypokalemic periodic paralysis (HOPP) is a rare disease characterized by reversible attacks of muscle weakness accompanied by episodic hypokalemia. Recent molecular work has revealed that the majority of familial HOPP is due to mutations in a skeletal muscle voltage-dependent calcium-channel: the dihydropyridine receptor. We report a 13-yr old boy with HOPP from a family in which 6 members are affected in three generations. Genetic examination identified a nucleotide 3705 C to G mutation in exon 30 of the calcium channel gene, CACNA1S. This mutation predicts a codon change from arginine to glycine at the amino acid position #1239 (R1239G). Among the three known mutations of the CACNA1S gene, the R1239G mutation was rarely reported. This boy and the other family members who did not respond to acetazolamide, showed a marked improvement of the paralytic symptoms after spironolactone treatment. PMID:15716625

  9. A robust CELP coder with source-dependent channel coding

    Science.gov (United States)

    Sukkar, Rafid A.; Kleijn, W. Bastiaan

    1990-01-01

    A CELP coder using Source Dependent Channel Encoding (SDCE) for optimal channel error protection is introduced. With SDCE, each of the CELP parameters are encoded by minimizing a perceptually meaningful error criterion under prevalent channel conditions. Unlike conventional channel coding schemes, SDCE allows for optimal balance between error detection and correction. The experimental results show that the CELP system is robust under various channel bit error rates and displays a graceful degradation in SSNR as the channel error rate increases. This is a desirable property to have in a coder since the exact channel conditions cannot usually be specified a priori.

  10. KCNE1 and KCNE3: The yin and yang of voltage-gated K(+) channel regulation.

    Science.gov (United States)

    Abbott, Geoffrey W

    2016-01-15

    The human KCNE gene family comprises five genes encoding single transmembrane-spanning ion channel regulatory subunits. The primary function of KCNE subunits appears to be regulation of voltage-gated potassium (Kv) channels, and the best-understood KCNE complexes are with the KCNQ1 Kv α subunit. Here, we review the often opposite effects of KCNE1 and KCNE3 on Kv channel biology, with an emphasis on regulation of KCNQ1. Slow-activating IKs channel complexes formed by KCNQ1 and KCNE1 are essential for human ventricular myocyte repolarization, while constitutively active KCNQ1-KCNE3 channels are important in the intestine. Inherited sequence variants in human KCNE1 and KCNE3 cause cardiac arrhythmias but by different mechanisms, and each is important for hearing in unique ways. Because of their contrasting effects on KCNQ1 function, KCNE1 and KCNE3 have proved invaluable tools in the mechanistic understanding of how channel gating can be manipulated, and each may also provide a window into novel insights and new therapeutic opportunities in K(+) channel pharmacology. Finally, findings from studies of Kcne1(-/-) and Kcne3(-/-) mouse lines serve to illustrate the complexity of KCNE biology and KCNE-linked disease states.

  11. Crystal structure of a voltage-gated sodium channel in two potentially inactivated states.

    Science.gov (United States)

    Payandeh, Jian; Gamal El-Din, Tamer M; Scheuer, Todd; Zheng, Ning; Catterall, William A

    2012-05-20

    In excitable cells, voltage-gated sodium (Na(V)) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of Na(V) channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na(V)Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of Na(V)Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na(V)Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na(V)Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na(V) channel gating and inactivation. These potential inactivated-state structures provide new insights into Na(V) channel gating and novel avenues to drug development and therapy for a range of debilitating Na(V) channelopathies.

  12. 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...... releases CGRP, and the release is regulated by Ca2+ ions and voltage-gated calcium channels.......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...

  13. Caution Is Required in Interpretation of Mutations in the Voltage Sensing Domain of Voltage Gated Channels as Evidence for Gating Mechanisms

    Directory of Open Access Journals (Sweden)

    Alisher M. Kariev

    2015-01-01

    Full Text Available The gating mechanism of voltage sensitive ion channels is generally considered to be the motion of the S4 transmembrane segment of the voltage sensing domains (VSD. The primary supporting evidence came from R→C mutations on the S4 transmembrane segment of the VSD, followed by reaction with a methanethiosulfonate (MTS reagent. The cys side chain is –SH (reactive form –S−; the arginine side chain is much larger, leaving space big enough to accommodate the MTS sulfonate head group. The cavity created by the mutation has space for up to seven more water molecules than were present in wild type, which could be displaced irreversibly by the MTS reagent. Our quantum calculations show there is major reorientation of three aromatic residues that face into the cavity in response to proton displacement within the VSD. Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain –OH. These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone. The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations.

  14. Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

    Institute of Scientific and Technical Information of China (English)

    Xu Zhang; Mengdie Xia; Yang Li; Huihui Liu; Xin Jiang; Wenlin Ren; Jianping Wu

    2013-01-01

    NaChBac is a bacterial voltage-gated sodium (Nav) channel that shows sequence similarity to voltage-gated calcium channels.To understand the ion-permeation mechanism of Nav channels,we combined molecular dynamics simulation,structural biology and electrophysiological approaches to investigate the recently determined structure of NavRh,a marine bacterial NaChBac ortholog.Two Na+ binding sites are identified in the selectivity filter (SF) in our simulations:The extracellular Na+ ion first approaches site 1 constituted by the side groups of Ser181 and Glu183,and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyi oxygens of Leu179 and Thr178.In contrast,Ca2+ ions are prone to being trapped by Glu183 at site 1,which then blocks the entrance of both Na+ and Ca2+ to the vestibule of the SF.In addition,Na+ permeates through the selective filter in an asymmetrical manner,a feature that resembles that of the mammalian Nav orthologs.The study reported here provides insights into the mechanism of ion selectivity on Na+ over Ca2+ in mammalian Nav channels.

  15. Regulation of KCNQ/Kv7 family voltage-gated K(+) channels by lipids.

    Science.gov (United States)

    Taylor, Keenan C; Sanders, Charles R

    2017-04-01

    Many years of studies have established that lipids can impact membrane protein structure and function through bulk membrane effects, by direct but transient annular interactions with the bilayer-exposed surface of protein transmembrane domains, and by specific binding to protein sites. Here, we focus on how phosphatidylinositol 4,5-bisphosphate (PIP2) and polyunsaturated fatty acids (PUFAs) impact ion channel function and how the structural details of the interactions of these lipids with ion channels are beginning to emerge. We focus on the Kv7 (KCNQ) subfamily of voltage-gated K(+) channels, which are regulated by both PIP2 and PUFAs and play a variety of important roles in human health and disease. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

  16. Ion conduction and conformational flexibility of a bacterial voltage-gated sodium channel.

    Science.gov (United States)

    Boiteux, Céline; Vorobyov, Igor; Allen, Toby W

    2014-03-04

    Voltage-gated Na(+) channels play an essential role in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. The recent solution of X-ray structures for the bacterial channel NavAb has provided an opportunity to study functional mechanisms at the atomic level. This channel's selectivity filter exhibits an EEEE ring sequence, characteristic of mammalian Ca(2+), not Na(+), channels. This raises the fundamentally important question: just what makes a Na(+) channel conduct Na(+) ions? Here we explore ion permeation on multimicrosecond timescales using the purpose-built Anton supercomputer. We isolate the likely protonation states of the EEEE ring and observe a striking flexibility of the filter that demonstrates the necessity for extended simulations to study conduction in this channel. We construct free energy maps to reveal complex multi-ion conduction via knock-on and "pass-by" mechanisms, involving concerted ion and glutamate side chain movements. Simulations in mixed ionic solutions reveal relative energetics for Na(+), K(+), and Ca(2+) within the pore that are consistent with the modest selectivity seen experimentally. We have observed conformational changes in the pore domain leading to asymmetrical collapses of the activation gate, similar to proposed inactivated structures of NavAb, with helix bending involving conserved residues that are critical for slow inactivation. These structural changes are shown to regulate access to fenestrations suggested to be pathways for lipophilic drugs and provide deeper insight into the molecular mechanisms connecting drug activity and slow inactivation.

  17. Functional and molecular characterization of voltage-gated sodium channels in uteri from nonpregnant rats.

    Science.gov (United States)

    Seda, Marian; Pinto, Francisco M; Wray, Susan; Cintado, Cristina G; Noheda, Pedro; Buschmann, Helmut; Candenas, Luz

    2007-11-01

    We investigated the function and expression of voltage-gated Na(+) channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca(2+)] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50-60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca(2+)](i), which were abolished by treatment with 0.1 microM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.

  18. Potential L-Type Voltage-Operated Calcium Channel Blocking Effect of Drotaverine on Functional Models.

    Science.gov (United States)

    Patai, Zoltán; Guttman, András; Mikus, Endre G

    2016-12-01

    Drotaverine is considered an inhibitor of cyclic-3',5'-nucleotide-phophodiesterase (PDE) enzymes; however, published receptor binding data also support the potential L-type voltage- operated calcium channel (L-VOCC) blocking effect of drotaverine. Hence, in this work, we focus on the potential L-VOCC blocking effect of drotaverine by using L-VOCC-associated functional in vitro models. Accordingly, drotaverine and reference agents were tested on KCl-induced guinea pig tracheal contraction. Drotaverine, like the L-VOCC blockers nifedipine or diltiazem, inhibited the KCl-induced inward Ca(2+)- induced contraction in a concentration- dependent fashion. The PDE inhibitor theophylline had no effect on the KCl-evoked contractions, indicating its lack of inhibition on inward Ca(2+) flow. Drotaverine was also tested on the L-VOCC-mediated resting Ca(2+) refill model. In this model, the extracellular Ca(2+) enters the cells to replenish the emptied intracellular Ca(2+) stores. Drotaverine and L-VOCC blocker reference molecules inhibited Ca(2+) replenishment of Ca(2+)-depleted preparations detected by agonist-induced contractions in post-Ca(2+) replenishment Ca(2+)-free medium. Theophylline did not modify the Ca(2+) store replenishment after contraction. It seems that drotaverine, but not theophylline, inhibits inward Ca(2+) flux. The addition of CaCl2 to Ca(2+)-free medium containing the agonist induced inward Ca(2+) flow and subsequent contraction of Ca(2+)-depleted tracheal preparations. Drotaverine, similar to the L-VOCC blockers, inhibited inward Ca(2+) flow and blunted the slope of CaCl2-induced contraction in agonist containing Ca(2+)-free medium with Ca(2+)-depleted tracheal preparations. These results show that drotaverine behaves like L-VOCC blockers but, unlike PDE inhibitors using L-VOCC associated in vitro experimental models.

  19. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    Science.gov (United States)

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-06-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  20. Development of a voltage-dependent current noise algorithm for conductance-based stochastic modelling of auditory nerve fibres.

    Science.gov (United States)

    Badenhorst, Werner; Hanekom, Tania; Hanekom, Johan J

    2016-12-01

    This study presents the development of an alternative noise current term and novel voltage-dependent current noise algorithm for conductance-based stochastic auditory nerve fibre (ANF) models. ANFs are known to have significant variance in threshold stimulus which affects temporal characteristics such as latency. This variance is primarily caused by the stochastic behaviour or microscopic fluctuations of the node of Ranvier's voltage-dependent sodium channels of which the intensity is a function of membrane voltage. Though easy to implement and low in computational cost, existing current noise models have two deficiencies: it is independent of membrane voltage, and it is unable to inherently determine the noise intensity required to produce in vivo measured discharge probability functions. The proposed algorithm overcomes these deficiencies while maintaining its low computational cost and ease of implementation compared to other conductance and Markovian-based stochastic models. The algorithm is applied to a Hodgkin-Huxley-based compartmental cat ANF model and validated via comparison of the threshold probability and latency distributions to measured cat ANF data. Simulation results show the algorithm's adherence to in vivo stochastic fibre characteristics such as an exponential relationship between the membrane noise and transmembrane voltage, a negative linear relationship between the log of the relative spread of the discharge probability and the log of the fibre diameter and a decrease in latency with an increase in stimulus intensity.

  1. Opposite Effects of the S4-S5 Linker and PIP(2) on Voltage-Gated Channel Function: KCNQ1/KCNE1 and Other Channels.

    Science.gov (United States)

    Choveau, Frank S; Abderemane-Ali, Fayal; Coyan, Fabien C; Es-Salah-Lamoureux, Zeineb; Baró, Isabelle; Loussouarn, Gildas

    2012-01-01

    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 (S4S5(L)) and of the S6 C-terminal part (S6(T)) 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 S4S5(L) is acting like a ligand binding to S6(T) 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 S4S5(L), the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP(2)), stabilizes KCNQ1 channel in an open state. Many other ion channels (not only voltage-gated) require PIP(2) to function properly, confirming its crucial importance as an ion channel cofactor. This is highlighted in cases in which an altered regulation of ion channels by PIP(2) 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 (PIP(2) and S4S5(L)), and assesses their potential physiological and pathophysiological roles.

  2. 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.

  3. A cell model study of calcium influx mechanism regulated by calcium-dependent potassium channels in Purkinje cell dendrites.

    Science.gov (United States)

    Chono, Koji; Takagi, Hiroshi; Koyama, Shozo; Suzuki, Hideo; Ito, Etsuro

    2003-10-30

    The present study was designed to elucidate the roles of dendritic voltage-gated K+ channels in Ca2+ influx mechanism of a rat Purkinje cell using a computer simulation program. First, we improved the channel descriptions and the maximum conductance in the Purkinje cell model to mimic both the kinetics of ion channels and the Ca2+ spikes, which had failed in previous studies. Our cell model is, therefore, much more authentic than those in previous studies. Second, synaptic inputs that mimic stimulation of parallel fibers and induce sub-threshold excitability were simultaneously applied to the spiny dendrites. As a result, transient Ca2+ responses were observed in the stimulation points and they decreased with the faster decay rate in the cell model including high-threshold Ca2+-dependent K+ channels than in those excluding these channels. Third, when a single synaptic input was applied into a spiny dendrite, Ca2+-dependent K+ channels suppressed Ca2+ increases at stimulation and recording points. Finally, Ca2+-dependent K+ channels were also found to suppress the time to peak Ca2+ values in the recording points. These results suggest that the opening of Ca2+-dependent K+ channels by Ca2+ influx through voltage-gated Ca2+ channels hyperpolarizes the membrane potentials and deactivates these Ca2+ channels in a negative feedback manner, resulting in local, weak Ca2+ responses in spiny dendrites of Purkinje cells.

  4. Scorpion toxin prolongs an inactivation phase of the voltage-dependent sodium current in rat isolated single hippocampal neurons.

    Science.gov (United States)

    Kaneda, M; Oyama, Y; Ikemoto, Y; Akaike, N

    1989-05-15

    The effects of scorpion toxin on the voltage-dependent sodium current (INa) of CA1 pyramidal neurons isolated from rat hippocampus were studied under the single-electrode voltage-clamp condition using a 'concentration-clamp' technique. The toxin increased the peak amplitude of INa and prolonged its inactivation phase in a time- and dose-dependent manner. Inactivation phase of INa proceeded with two exponential components in the absence (control) and presence of the toxin. In the toxin-treated neurons, both the time constant of slow component and its fractional contribution to the total current increased dose-dependently while the fractional contribution of the fast one decreased in a dose-dependent fashion without changing its time constant. Actions of scorpion toxin on the sodium channels of hippocampal pyramidal neurons were essentially similar to those of peripheral preparations. Therefore, it can be concluded that the sodium channels of mammalian brain neurons have structures and functions similar to peripheral channels.

  5. Regulation of voltage-gated sodium channel expression in cancer: hormones, growth factors and auto-regulation.

    Science.gov (United States)

    Fraser, Scott P; Ozerlat-Gunduz, Iley; Brackenbury, William J; Fitzgerald, Elizabeth M; Campbell, Thomas M; Coombes, R Charles; Djamgoz, Mustafa B A

    2014-03-19

    Although ion channels are increasingly being discovered in cancer cells in vitro and in vivo, and shown to contribute to different aspects and stages of the cancer process, much less is known about the mechanisms controlling their expression. Here, we focus on voltage-gated Na(+) channels (VGSCs) which are upregulated in many types of carcinomas where their activity potentiates cell behaviours integral to the metastatic cascade. Regulation of VGSCs occurs at a hierarchy of levels from transcription to post-translation. Importantly, mainstream cancer mechanisms, especially hormones and growth factors, play a significant role in the regulation. On the whole, in major hormone-sensitive cancers, such as breast and prostate cancer, there is a negative association between genomic steroid hormone sensitivity and functional VGSC expression. Activity-dependent regulation by positive feedback has been demonstrated in strongly metastatic cells whereby the VGSC is self-sustaining, with its activity promoting further functional channel expression. Such auto-regulation is unlike normal cells in which activity-dependent regulation occurs mostly via negative feedback. Throughout, we highlight the possible clinical implications of functional VGSC expression and regulation in cancer.

  6. Fine-tuning of voltage sensitivity of the Kv1.2 potassium channel by interhelix loop dynamics.

    Science.gov (United States)

    Sand, Rheanna; Sharmin, Nazlee; Morgan, Carla; Gallin, Warren J

    2013-04-01

    Many proteins function by changing conformation in response to ligand binding or changes in other factors in their environment. Any change in the sequence of a protein, for example during evolution, which alters the relative free energies of the different functional conformations changes the conditions under which the protein will function. Voltage-gated ion channels are membrane proteins that open and close an ion-selective pore in response to changes in transmembrane voltage. The charged S4 transmembrane helix transduces changes in transmembrane voltage into a change in protein internal energy by interacting with the rest of the channel protein through a combination of non-covalent interactions between adjacent helices and covalent interactions along the peptide backbone. However, the structural basis for the wide variation in the V50 value between different voltage-gated potassium channels is not well defined. To test the role of the loop linking the S3 helix and the S4 helix in voltage sensitivity, we have constructed a set of mutants of the rat Kv1.2 channel that vary solely in the length and composition of the extracellular loop that connects S4 to S3. We evaluated the effect of these different loop substitutions on the voltage sensitivity of the channel and compared these experimental results with molecular dynamics simulations of the loop structures. Here, we show that this loop has a significant role in setting the precise V50 of activation in Kv1 family channels.

  7. Bidirectional regulation of dendritic voltage-gated potassium channels by the fragile X mental retardation protein.

    Science.gov (United States)

    Lee, Hye Young; Ge, Woo-Ping; Huang, Wendy; He, Ye; Wang, Gordon X; Rowson-Baldwin, Ashley; Smith, Stephen J; Jan, Yuh Nung; Jan, Lily Yeh

    2011-11-17

    How transmitter receptors modulate neuronal signaling by regulating voltage-gated ion channel expression remains an open question. Here we report dendritic localization of mRNA of Kv4.2 voltage-gated potassium channel, which regulates synaptic plasticity, and its local translational regulation by fragile X mental retardation protein (FMRP) linked to fragile X syndrome (FXS), the most common heritable mental retardation. FMRP suppression of Kv4.2 is revealed by elevation of Kv4.2 in neurons from fmr1 knockout (KO) mice and in neurons expressing Kv4.2-3'UTR that binds FMRP. Moreover, treating hippocampal slices from fmr1 KO mice with Kv4 channel blocker restores long-term potentiation induced by moderate stimuli. Surprisingly, recovery of Kv4.2 after N-methyl-D-aspartate receptor (NMDAR)-induced degradation also requires FMRP, likely due to NMDAR-induced FMRP dephosphorylation, which turns off FMRP suppression of Kv4.2. Our study of FMRP regulation of Kv4.2 deepens our knowledge of NMDAR signaling and reveals a FMRP target of potential relevance to FXS.

  8. Thalamic microinfusion of antibody to a voltage-gated potassium channel restores consciousness during anesthesia.

    Science.gov (United States)

    Alkire, Michael T; Asher, Christopher D; Franciscus, Amanda M; Hahn, Emily L

    2009-04-01

    The Drosophila Shaker mutant fruit-fly, with its malfunctioning voltage-gated potassium channel, exhibits anesthetic requirements that are more than twice normal. Shaker mutants with an abnormal Kv1.2 channel also demonstrate significantly reduced sleep. Given the important role the thalamus plays in both sleep and arousal, the authors investigated whether localized central medial thalamic (CMT) microinfusion of an antibody designed to block the pore of the Kv1.2 channel might awaken anesthetized rats. Male Sprague-Dawley rats were implanted with a cannula aimed at the CMT or lateral thalamus. One week later, unconsciousness was induced with either desflurane (3.6 +/- 0.2%; n = 55) or sevoflurane (1.2 +/- 0.1%; n = 51). Arousal effects of a single 0.5-microl infusion of Kv1.2 potassium channel blocking antibody (0.1- 0.2 mg/ml) or a control infusion of Arc-protein antibody (0.2 mg/ml) were then determined. The Kv1.2 antibody, but not the control antibody, temporarily restored consciousness in 17% of all animals and in 75% of those animals where infusions occurred within the CMT (P Consciousness returned on average (+/- SD) 170 +/- 99 s after infusion and lasted a median time of 398 s (interquartile range: 279-510 s). Temporary seizures, without apparent consciousness, predominated in 33% of all animals. These findings support the idea that the CMT plays a role in modulating levels of arousal during anesthesia and further suggest that voltage-gated potassium channels in the CMT may contribute to regulating arousal or may even be relevant targets of anesthetic action.

  9. A numerical approach to ion channel modelling using whole-cell voltage-clamp recordings and a genetic algorithm.

    Directory of Open Access Journals (Sweden)

    Meron Gurkiewicz

    2007-08-01

    Full Text Available The activity of trans-membrane proteins such as ion channels is the essence of neuronal transmission. The currently most accurate method for determining ion channel kinetic mechanisms is single-channel recording and analysis. Yet, the limitations and complexities in interpreting single-channel recordings discourage many physiologists from using them. Here we show that a genetic search algorithm in combination with a gradient descent algorithm can be used to fit whole-cell voltage-clamp data to kinetic models with a high degree of accuracy. Previously, ion channel stimulation traces were analyzed one at a time, the results of these analyses being combined to produce a picture of channel kinetics. Here the entire set of traces from all stimulation protocols are analysed simultaneously. The algorithm was initially tested on simulated current traces produced by several Hodgkin-Huxley-like and Markov chain models of voltage-gated potassium and sodium channels. Currents were also produced by simulating levels of noise expected from actual patch recordings. Finally, the algorithm was used for finding the kinetic parameters of several voltage-gated sodium and potassium channels models by matching its results to data recorded from layer 5 pyramidal neurons of the rat cortex in the nucleated outside-out patch configuration. The minimization scheme gives electrophysiologists a tool for reproducing and simulating voltage-gated ion channel kinetics at the cellular level.

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

    Directory of Open Access Journals (Sweden)

    Lei Wen

    2006-04-01

    Full Text Available Selective blockers of the N-type voltage-sensitive calcium (CaV channels are useful in the management of severe chronic pain. Here, the structure and function characteristics of a novel N-type CaV channel blocker, SO-3, are reviewed. SO-3 is a 25-amino acid conopeptide originally derived from the venom of Conus striatus, and contains the same 4-loop, 6-cysteine framework (C-C-CC-C-C as O-superfamily conotoxins. The synthetic SO-3 has high analgesic activity similar to ω-conotoxin MVIIA (MVIIA, a selective N-type CaV channel blocker approved in the USA and Europe for the alleviation of persistent pain states. In electrophysiological studies, SO-3 shows more selectivity towards the N-type CaV channels than MVIIA. The dissimilarity between SO-3 and MVIIA in the primary and tertiary structures is further discussed in an attempt to illustrate the difference in selectivity of SO-3 and MVIIA towards N-type CaV channels.

  11. Action of insecticidal N-alkylamides at site 2 of the voltage-sensitive sodium channel

    Energy Technology Data Exchange (ETDEWEB)

    Ottea, J.A.; Payne, G.T.; Soderlund, D.M. (Cornell Univ., Geneva, NY (USA))

    1990-08-01

    Nine synthetic N-alkylamides were examined as inhibitors of the specific binding of ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate (({sup 3}H)BTX-B) to sodium channels and as activators of sodium uptake in mouse brain synaptoneurosomes. In the presence of scorpion (Leiurus quinquestriatus) venom, the six insecticidal analogues were active as both inhibitors of ({sup 3}H)BTX-B binding and stimulators of sodium uptake. These findings are consistent with an action of these compounds at the alkaloid activator recognition site (site 2) of the voltage-sensitive sodium channel. The three noninsecticidal N-alkylamides also inhibited ({sup 3}H)BTX-B binding but were ineffective as activators of sodium uptake. Concentration-response studies revealed that some of the insecticidal amides also enhanced sodium uptake through a second, high-affinity interaction that does not involve site 2, but this secondary effect does not appear to be correlated with insecticidal activity. The activities of N-alkylamides as sodium channel activators were influenced by the length of the alkenyl chain and the location of unsaturation within the molecule. These results further define the actions of N-alkylamides on sodium channels and illustrate the significance of the multiple binding domains of the sodium channel as target sites for insect control agents.

  12. Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle.

    Science.gov (United States)

    Hristov, Kiril L; Smith, Amy C; Parajuli, Shankar P; Malysz, John; Petkov, Georgi V

    2014-03-01

    Large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca(2+) imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca(2+) sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca(2+) levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca(2+)-dependent mechanism, thus increasing DSM contractility.

  13. Kv3 voltage-gated potassium channels regulate neurotransmitter release from mouse motor nerve terminals.

    Science.gov (United States)

    Brooke, Ruth E; Moores, Thomas S; Morris, Neil P; Parson, Simon H; Deuchars, Jim

    2004-12-01

    Voltage-gated potassium (Kv) channels are critical to regulation of neurotransmitter release throughout the nervous system but the roles and identity of the subtypes involved remain unclear. Here we show that Kv3 channels regulate transmitter release at the mouse neuromuscular junction (NMJ). Light- and electron-microscopic immunohistochemistry revealed Kv3.3 and Kv3.4 subunits within all motor nerve terminals of muscles examined [transversus abdominus, lumbrical and flexor digitorum brevis (FDB)]. To determine the roles of these Kv3 subunits, intracellular recordings were made of end-plate potentials (EPPs) in FDB muscle fibres evoked by electrical stimulation of tibial nerve. Tetraethylammonium (TEA) applied at low concentrations (0.05-0.5 mM), which blocks only a few known potassium channels including Kv3 channels, did not affect muscle fibre resting potential but significantly increased the amplitude of all EPPs tested. Significantly, this effect of TEA was still observed in the presence of the large-conductance calcium-activated potassium channel blockers iberiotoxin (25-150 nM) and Penitrem A (100 nM), suggesting a selective action on Kv3 subunits. Consistent with this, 15-microM 4-aminopyridine, which blocks Kv3 but not large-conductance calcium-activated potassium channels, enhanced evoked EPP amplitude. Unexpectedly, blood-depressing substance-I, a toxin selective for Kv3.4 subunits, had no effect at 0.05-1 microM. The combined presynaptic localization of Kv3 subunits and pharmacological enhancement of EPP amplitude indicate that Kv3 channels regulate neurotransmitter release from presynaptic terminals at the NMJ.

  14. Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels.

    Science.gov (United States)

    Corzo, Gerardo; Sabo, Jennifer K; Bosmans, Frank; Billen, Bert; Villegas, Elba; Tytgat, Jan; Norton, Raymond S

    2007-02-16

    Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion beta-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded beta-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion beta-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.

  15. Role of voltage-gated calcium channels in the regulation of aldosterone production from zona glomerulosa cells of the adrenal cortex.

    Science.gov (United States)

    Barrett, Paula Q; Guagliardo, Nick A; Klein, Peter M; Hu, Changlong; Breault, David T; Beenhakker, Mark P

    2016-10-15

    Zona glomerulosa cells (ZG) of the adrenal gland constantly integrate fluctuating ionic, hormonal and paracrine signals to control the synthesis and secretion of aldosterone. These signals modulate Ca(2+) levels, which provide the critical second messenger to drive steroid hormone production. Angiotensin II is a hormone known to modulate the activity of voltage-dependent L- and T-type Ca(2+) channels that are expressed on the plasma membrane of ZG cells in many species. Because the ZG cell maintains a resting membrane voltage of approximately -85 mV and has been considered electrically silent, low voltage-activated T-type Ca(2+) channels are assumed to provide the primary Ca(2+) signal that drives aldosterone production. However, this view has recently been challenged by human genetic studies identifying somatic gain-of-function mutations in L-type CaV 1.3 channels in aldosterone-producing adenomas of patients with primary hyperaldosteronism. We provide a review of these assumptions and challenges, and update our understanding of the state of the ZG cell in a layer in which native cellular associations are preserved. This updated view of Ca(2+) signalling in ZG cells provides a unifying mechanism that explains how transiently activating CaV 3.2 channels can generate a significant and recurring Ca(2+) signal, and how CaV 1.3 channels may contribute to the Ca(2+) signal that drives aldosterone production.

  16. Voltage-gated sodium channels: pharmaceutical targets via anticonvulsants to treat epileptic syndromes.

    Science.gov (United States)

    Abdelsayed, Mena; Sokolov, Stanislav

    2013-01-01

    Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel's fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment.

  17. SCN9A mutations define primary erythermalgia as a neuropathic disorder of voltage gated sodium channels.

    Science.gov (United States)

    Drenth, Joost P H; te Morsche, Rene H M; Guillet, Gerard; Taieb, Alain; Kirby, R Lee; Jansen, Jan B M J

    2005-06-01

    Primary erythermalgia is a rare disorder characterized by recurrent attacks of red, warm and painful hands, and/or feet. We previously localized the gene for primary erythermalgia to a 7.94 cM region on chromosome 2q. Recently, Yang et al identified two missense mutations of the sodium channel alpha subunit SCN9A in patients with erythermalgia. The presence of voltage-gated sodium channels in sensory neurons is thought to play a crucial role in several chronic painful neuropathies. We examined four different families and two sporadic cases and detected missense sequence variants in SCN9A to be present in primary erythermalgia patients. A total of five of six mutations were located in highly conserved regions. One family with autosomal dominantly inherited erythermalgia was double heterozygous for two separate SCN9A mutations. These data establish primary erythermalgia as a neuropathic disorder and offers hope for treatment of this incapacitating painful disorder.

  18. Characterization of a novel radiolabeled peptide selective for a subpopulation of voltage-gated potassium channels in mammalian brain.

    Science.gov (United States)

    Racapé, Judith; Lecoq, Alain; Romi-Lebrun, Régine; Liu, Jessica; Kohler, Martin; Garcia, Maria L; Ménez, André; Gasparini, Sylvaine

    2002-02-08

    BgK, a 37-amino acid voltage-gated potassium (Kv) 1 channel blocker isolated from the sea anemone Bunodosoma granulifera, can be modified at certain positions to alter its pharmacological profile (Alessandri-Haber, N., Lecoq, A., Gasparini, S., Grangier-Macmath, G., Jacquet, G., Harvey, A. L., de Medeiros, C., Rowan, E. G., Gola, M., Ménez, A., and Crest, M. (1999) J. Biol. Chem. 274, 35653-35661). In the present study, we report the design of two BgK analogs that have been radiolabeled with (125)INa. Whereas BgK(W5Y/Y26F) and its radiolabeled derivative, (125)I-BgK(W5Y/Y26F), bind to Kv1.1, Kv1.2, and Kv1.6 channels with potencies similar to those for the parent peptide, BgK, BgK(W5Y/F6A/Y26F) and its monoiodo-tyrosine derivative, (125)I-BgK(W5Y/F6A/Y26F), display a distinctive and unique pharmacological profile; they bind with high affinity to homomultimeric Kv1.1 and Kv1.6 channels, but not to Kv1.2 channels. Interaction of BgK(W5Y/F6A/Y26F) with potassium channels depends on the nature of a residue in the mouth of the channel, at a position that determines channel sensitivity to external tetraethylammonium. In native brain tissue, (125)I-BgK(W5Y/F6A/Y26F) binds to a population of Kv1 channels that appear to consist of at least two sensitive (Kv1.1 and/or Kv1.6) subunits, in adjacent position. Given its unique pharmacological properties, (125)I-BgK(W5Y/F6A/Y26F) represents a new tool for studying subpopulations of Kv1 channels in native tissues.

  19. Elimination of the channel current effect on the characterization of MOSFET threshold voltage using junction capacitance measurements

    Science.gov (United States)

    Tomaszewski, Daniel; Głuszko, Grzegorz; Łukasiak, Lidia; Kucharski, Krzysztof; Malesińska, Jolanta

    2017-02-01

    An alternative method for an extraction of the MOSFET threshold voltage has been proposed. It is based on an analysis of the MOSFET source-bulk junction capacitance behavior as a function of the gate-source voltage. The effect of the channel current on the threshold voltage extraction is fully eliminated. For the threshold voltage and junction capacitance model parameters non-iterative methods have been used. The proposed method has been demonstrated using a series of MOS transistors manufactured using a standard CMOS technology.

  20. 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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    We investigate the I(V) characteristics (current versus bias voltage) of side-gated quantum-point contacts, defined in GaAs/AlxGa1-xAs heterostructures. These point contacts are operated in the closed-channel regime, that is, at fixed gate voltages below zero-bias pinch-off for conductance. Our...... analysis is based on a single scaling factor, extracted from the experimental I(V) characteristics. For both polarities, this scaling factor transforms the change of bias voltage into a change of electron energy. The latter is determined with respect to the top of the potential barrier of the contact....... Such a built-in energy-voltage calibration allows us to distinguish between the different contributions to the electron transport across the pinched-off contact due to thermal activation or quantum tunneling. The first involves the height of the barrier, and the latter also its length. In the model that we...

  2. Inhibition of voltage-gated calcium channels by sequestration of beta subunits.

    Science.gov (United States)

    Cuchillo-Ibañez, Inmaculada; Aldea, Marcos; Brocard, Jacques; Albillos, Almudena; Weiss, Norbert; Garcia, Antonio G; De Waard, Michel

    2003-11-28

    The auxiliary Ca(v)beta subunit is essential for functional expression of high-voltage activated Ca(2+) channels. Here, we describe a lure sequence designed to sequester the Ca(v)beta subunits in transfected bovine chromaffin cells. This sequence is composed of the extracellular and transmembrane domains of the alpha chain of the human CD8, the I-II loop of Ca(v)2.1 subunit, and EGFP. We showed that expressing the CD8-I-II-EGFP sequence in chromaffin cells led to a >50% decrease in overall Ca(2+) current density. Although this decrease involved all the Ca(2+) channel types (L, N, P/Q, R), the proportion of each type supporting the remaining current was altered. A similar effect was observed after transfection when measuring the functional role of Ca(2+) channels in catecholamine release by chromaffin cells: global decrease of release and change of balance between the different channel types supporting it. Possible explanations for this apparent discrepancy are further discussed.

  3. 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.

  4. ¬cAMP promotes the differentiation of neural progenitor cells in vitro via modulation of voltage-gated calcium channels

    Directory of Open Access Journals (Sweden)

    Guilherme eLepski

    2013-09-01

    Full Text Available The molecular mechanisms underlying the differentiation of neural progenitor cells (NPCs remain poorly understood. In this study we investigated the role of Ca2+ and cAMP (cyclic adenosine monophosphate in the differentiation of NPCs extracted from the subventricular zone of E14.5 rat embryos. Patch clamp recordings revealed that increasing cAMP-signaling with Forskolin or IBMX (3-isobutyl-1-methylxantine significantly facilitated neuronal functional maturation. A continuous application of IBMX to the differentiation medium substantially increased the functional expression of voltage-gated Na+ and K+ channels, as well as neuronal firing frequency. Furthermore, we observed an increase in the frequency of spontaneous synaptic currents and in the amplitude of evoked glutamatergic and GABAergic synaptic currents. The most prominent acute effect of applying IBMX was an increase in L-type Ca2+currents. Conversely, blocking L-type channels strongly inhibited dendritic outgrowth and synapse formation even in the presence of IBMX, indicating that voltage-gated Ca2+ influx plays a major role in neuronal differentiation. Finally, we found that nifedipine completely blocks IBMX-induced CREB phosphorylation (cAMP-response-element-binding protein, indicating that the activity of this important transcription factor equally depends on both enhanced cAMP and voltage-gated Ca2+-signaling. Taken together, these data indicate that the up-regulation of voltage-gated L-type Ca2+-channels and early electrical excitability are critical steps in the cAMP-dependent differentiation of SVZ-derived NPCs into functional neurons. To our knowledge, this is the first demonstration of the acute effects of cAMP on voltage-gated Ca+2channels in NPC-derived developing neurons.

  5. The cytoplasmic coiled-coil mediates cooperative gating temperature sensitivity in the voltage-gated H(+) channel Hv1.

    Science.gov (United States)

    Fujiwara, Yuichiro; Kurokawa, Tatsuki; Takeshita, Kohei; Kobayashi, Megumi; Okochi, Yoshifumi; Nakagawa, Atsushi; Okamura, Yasushi

    2012-05-08

    Hv1/VSOP is a dimeric voltage-gated H(+) channel in which the gating of one subunit is reportedly coupled to that of the other subunit within the dimer. The molecular basis for dimer formation and intersubunit coupling, however, remains unknown. Here we show that the carboxy terminus ends downstream of the S4 voltage-sensor helix twist in a dimer coiled-coil architecture, which mediates cooperative gating. We also show that the temperature-dependent activation of H(+) current through Hv1/VSOP is regulated by thermostability of the coiled-coil domain, and that this regulation is altered by mutation of the linker between S4 and the coiled-coil. Cooperative gating within the dimer is also dependent on the linker structure, which circular dichroism spectrum analysis suggests is α-helical. Our results indicate that the cytoplasmic coiled-coil strands form continuous α-helices with S4 and mediate cooperative gating to adjust the range of temperatures over which Hv1/VSOP operates.

  6. Electrical coupling between the human serotonin transporter and voltage-gated Ca(2+) channels.

    Science.gov (United States)

    Ruchala, Iwona; Cabra, Vanessa; Solis, Ernesto; Glennon, Richard A; De Felice, Louis J; Eltit, Jose M

    2014-07-01

    Monoamine transporters have been implicated in dopamine or serotonin release in response to abused drugs such as methamphetamine or ecstasy (MDMA). In addition, monoamine transporters show substrate-induced inward currents that may modulate excitability and Ca(2+) mobilization, which could also contribute to neurotransmitter release. How monoamine transporters modulate Ca(2+) permeability is currently unknown. We investigate the functional interaction between the human serotonin transporter (hSERT) and voltage-gated Ca(2+) channels (CaV). We introduce an excitable expression system consisting of cultured muscle cells genetically engineered to express hSERT. Both 5HT and S(+)MDMA depolarize these cells and activate the excitation-contraction (EC)-coupling mechanism. However, hSERT substrates fail to activate EC-coupling in CaV1.1-null muscle cells, thus implicating Ca(2+) channels. CaV1.3 and CaV2.2 channels are natively expressed in neurons. When these channels are co-expressed with hSERT in HEK293T cells, only cells expressing the lower-threshold L-type CaV1.3 channel show Ca(2+) transients evoked by 5HT or S(+)MDMA. In addition, the electrical coupling between hSERT and CaV1.3 takes place at physiological 5HT concentrations. The electrical coupling between monoamine neurotransmitter transporters and Ca(2+) channels such as CaV1.3 is a novel mechanism by which endogenous substrates (neurotransmitters) or exogenous substrates (like ecstasy) could modulate Ca(2+)-driven signals in excitable cells.

  7. Mutation in the neuronal voltage-gated sodium channel SCN1A in familial hemiplegic migraine.

    Science.gov (United States)

    Dichgans, Martin; Freilinger, Tobias; Eckstein, Gertrud; Babini, Elena; Lorenz-Depiereux, Bettina; Biskup, Saskia; Ferrari, Michel D; Herzog, Jürgen; van den Maagdenberg, Arn M J M; Pusch, Michael; Strom, Tim M

    Familial hemiplegic migraine is an autosomal dominant severe subtype of migraine with aura characterised by some degree of hemiparesis during the attacks. So far, mutations in two genes regulating ion translocation-CACNA1A and ATP1A2-have been identified in pedigrees with this disease. To identify additional genes for familial hemiplegic migraine, we did a genome-wide linkage analysis of two disease pedigrees without mutations in CACNA1A and ATP1A2. Ion channel genes in the candidate interval were analysed for mutations, and the functional consequences of the recorded sequence alteration were determined. We identified a novel locus for familial hemiplegic migraine on chromosome 2q24. Sequencing of candidate genes in this region revealed a heterozygous missense mutation (Gln1489Lys) in the neuronal voltage-gated sodium channel gene SCN1A, mutations of which have been associated with epilepsy. This same mutation was present in three families with familial hemiplegic migraine. It results in a charge-altering aminoacid exchange in the so-called hinged-lid domain of the protein, which is critical for fast inactivation of the channel. Whole-cell recordings in transiently transfected tsA201 cells expressing the highly homologous SCN5A sodium channel showed that the mutation induces a two-fold to four-fold accelerated recovery from fast inactivation without altering any of the other channel parameters investigated. Dysfunction of the neuronal sodium channel SCN1A can cause familial hemiplegic migraine. Our findings have implications for the understanding of migraine aura. Moreover, our study reinforces the molecular links between migraine and epilepsy, two common paroxysmal disorders.

  8. Regulation of persistent Na current by interactions between beta subunits of voltage-gated Na channels.

    Science.gov (United States)

    Aman, Teresa K; Grieco-Calub, Tina M; Chen, Chunling; Rusconi, Raffaella; Slat, Emily A; Isom, Lori L; Raman, Indira M

    2009-02-18

    The beta subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming alpha subunits, as well as their trafficking and localization. In heterologous expression systems, beta1, beta2, and beta3 subunits influence inactivation and persistent current in different ways. To test how the beta4 protein regulates Na channel gating, we transfected beta4 into HEK (human embryonic kidney) cells stably expressing Na(V)1.1. Unlike a free peptide with a sequence from the beta4 cytoplasmic domain, the full-length beta4 protein did not block open channels. Instead, beta4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of beta1 or chimeric subunits including the beta1 extracellular domain, however, favored inactivation. Coexpressing Na(V)1.1 and beta4 with beta1 produced tiny persistent currents, indicating that beta1 overcomes the effects of beta4 in heterotrimeric channels. In contrast, beta1(C121W), which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by beta4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with beta4, persistent current was slightly but significantly increased. Moreover, in beta4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that beta1 and beta4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted beta1 subunits.

  9. Regulation of voltage-gated potassium channels by PI(4,5)P2.

    Science.gov (United States)

    Kruse, Martin; Hammond, Gerald R V; Hille, Bertil

    2012-08-01

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) regulates activities of numerous ion channels including inwardly rectifying potassium (K(ir)) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K(V)) channels might be regulated by PI(4,5)P(2). Wide expression of K(V) channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K(V) channels by PI(4,5)P(2), we have coexpressed several of them in tsA-201 cells with a G protein-coupled receptor (M(1)R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P(2) with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P(2) at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K(V)7.1, K(V)7.2/7.3, and K(ir)2.1 channel current by 90-95%. Activation of M(1)R inhibited K(V)7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P(2) regulation of activity of K(V)1.1/K(V)β1.1, K(V)1.3, K(V)1.4, and K(V)1.5/K(V)β1.3, K(V)2.1, K(V)3.4, K(V)4.2, K(V)4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K(V)1.1/K(V)β1.1 and K(V)3.4, resulting in up-regulation of current density upon activation of M(1)R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P(2) at the plasma membrane by enzymes does not seem to influence activity of most tested K(V) channels, whereas it does strongly inhibit members of the K(V)7 and K(ir) families.

  10. Vector spin modeling for magnetic tunnel junctions with voltage dependent effects

    Energy Technology Data Exchange (ETDEWEB)

    Manipatruni, Sasikanth, E-mail: sasikanth.manipatruni@intel.com; Nikonov, Dmitri E.; Young, Ian A. [Exploratory Integrated Circuits, Components Research, Intel Corp., Hillsboro, Oregon 97124 (United States)

    2014-05-07

    Integration and co-design of CMOS and spin transfer devices requires accurate vector spin conduction modeling of magnetic tunnel junction (MTJ) devices. A physically realistic model of the MTJ should comprehend the spin torque dynamics of nanomagnet interacting with an injected vector spin current and the voltage dependent spin torque. Vector spin modeling allows for calculation of 3 component spin currents and potentials along with the charge currents/potentials in non-collinear magnetic systems. Here, we show 4-component vector spin conduction modeling of magnetic tunnel junction devices coupled with spin transfer torque in the nanomagnet. Nanomagnet dynamics, voltage dependent spin transport, and thermal noise are comprehended in a self-consistent fashion. We show comparison of the model with experimental magnetoresistance (MR) of MTJs and voltage degradation of MR with voltage. Proposed model enables MTJ circuit design that comprehends voltage dependent spin torque effects, switching error rates, spin degradation, and back hopping effects.

  11. Voltage-gated potassium channel Kvl.3 in rabbit ciliary epithelium regulates the membrane potential via coupling intracellular calcium

    Institute of Scientific and Technical Information of China (English)

    LI Yan-feng; ZHUO Ye-hong; BI Wei-na; BAI Yu-jing; LI Yan-na; WANG Zhi-jian

    2008-01-01

    Background The cell layer of the ciliary epithelium is responsible for aqueous humor secretion and maintenance.Ion channels play an important role in these processes.The main aim of this study was to determine whether the well-characterized members of the Kvl family (Kv1.3) contribute to the Kv currents in ciliary epithelium.Methods New Zealand White rabbits were maintained in a 12 hours light/dark cycle.Ciliary epithelium samples were isolated from the rabbits.We used Western blotting and immunocytochemistry to identify the expression and location of a voltage-gated potassium channel Kvl.3 in ciliary body epithelium.Membrane potential change after adding of Kv1.3 inhibitor margatoxin (MgTX) was observed with a fluorescence method.Results Western blotting and immunocytochemical studies showed that the Kv1.3 protein expressed in pigment ciliary epithelium and nonpigment ciliary epithelium,however it seemed to express more in the apical membrane of the nonpigmented epithelial cells.One nmol/L margatoxin,a specific inhibitor of Kv1.3 channels caused depolarization of the cultured nonpigmented epithelium (NPE) membrane potential.The cytosotic calcium increased after NPE cell depolarization,this increase of cytosolic calcium was partially blocked by 12.5 μmol/L dantrolene and 10 μmol/L nifedipine.These observations suggest that Kv1.3 channels modulate ciliary epithelium potential and effect calcium dependent mechanisms.Conclusion Kv1.3 channels contribute to K+ efflux at the membrane of rabbit ciliary epithelium.

  12. Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

    Energy Technology Data Exchange (ETDEWEB)

    James, W.M.; Emerick, M.C.; Agnew, W.S. (Yale Univ. School of medicine, New Haven, CT (USA))

    1989-07-11

    The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

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

    Science.gov (United States)

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

    2016-01-01

    The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30–60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control. PMID:27411529

  14. Coexpression of high-voltage-activated ion channels Kv3.4 and Cav1.2 in pioneer axons during pathfinding in the developing rat forebrain.

    Science.gov (United States)

    Huang, Chia-Yi; Chu, Dachen; Hwang, Wei-Chao; Tsaur, Meei-Ling

    2012-11-01

    Precise axon pathfinding is crucial for establishment of the initial neuronal network during development. Pioneer axons navigate without the help of preexisting axons and pave the way for follower axons that project later. Voltage-gated ion channels make up the intrinsic electrical activity of pioneer axons and regulate axon pathfinding. To elucidate which channel molecules are present in pioneer axons, immunohistochemical analysis was performed to examine 14 voltage-gated ion channels (Kv1.1-Kv1.3, Kv3.1-Kv3.4, Kv4.3, Cav1.2, Cav1.3, Cav2.2, Nav1.2, Nav1.6, and Nav1.9) in nine axonal tracts in the developing rat forebrain, including the optic nerve, corpus callosum, corticofugal fibers, thalamocortical axons, lateral olfactory tract, hippocamposeptal projection, anterior commissure, hippocampal commissure, and medial longitudinal fasciculus. We found A-type K⁺ channel Kv3.4 in both pioneer axons and early follower axons and L-type Ca²⁺ channel Cav1.2 in pioneer axons and early and late follower axons. Spatially, Kv3.4 and Cav1.2 were colocalized with markers of pioneer neurons and pioneer axons, such as deleted in colorectal cancer (DCC), in most fiber tracts examined. Temporally, Kv3.4 and Cav1.2 were expressed abundantly in most fiber tracts during axon pathfinding but were downregulated beginning in synaptogenesis. By contrast, delayed rectifier Kv channels (e.g., Kv1.1) and Nav channels (e.g., Nav1.2) were absent from these fiber tracts (except for the corpus callosum) during pathfinding of pioneer axons. These data suggest that Kv3.4 and Cav1.2, two high-voltage-activated ion channels, may act together to control Ca²⁺ -dependent electrical activity of pioneer axons and play important roles during axon pathfinding.

  15. Elevated neuronal excitability due to modulation of the voltage-gated sodium channel Nav1.6 by Aβ1-42

    Directory of Open Access Journals (Sweden)

    Xi eWang

    2016-03-01

    Full Text Available Aberrant increases in neuronal network excitability may contribute to the cognitive deficits in Alzheimer’s disease (AD. However, the mechanisms underlying hyperexcitability are not fully understood. Such overexcitation of neuronal networks has been detected in the brains of APP/PS1 mice. In the present study, using current-clamp recording techniques, we observed that 12 days in vitro (DIV primary cultured pyramidal neurons from P0 APP/PS1 mice exhibited a more prominent action potential burst and a lower threshold than WT littermates. Moreover, after treatment with Aβ1-42 peptide, 12 DIV primary cultured neurons showed similar changes, to a greater degree than in controls. Voltage-clamp recordings revealed that the voltage-dependent sodium current density of neurons incubated with Aβ1-42 was significantly increased, without change in the voltage-dependent sodium channel kinetic characteristics. Immunohistochemistry and western blot results showed that, after treatment with Aβ1-42, expressions of Nav and Nav1.6 subtype increased in cultured neurons or APP/PS1 brains compared to control groups. The intrinsic neuronal hyperexcitability of APP/PS1 mice might thus be due to an increased expression of voltage-dependent sodium channels induced by Aβ1-42. These results may illuminate the mechanism of aberrant neuronal networks in AD.

  16. 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.

  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. Voltage-Gated R-Type Calcium Channel Inhibition via Human μ-, δ-, and κ-opioid Receptors Is Voltage-Independently Mediated by Gβγ Protein Subunits.

    Science.gov (United States)

    Berecki, Géza; Motin, Leonid; Adams, David J

    2016-01-01

    Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Cav2.2) are inhibited by activation of G protein-coupled opioid receptors (ORs). However, inhibition of R-type (Cav2.3) channels by μ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human μ-, δ-, or κ-ORs with human Cav2.3 or Cav2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba(2+) currents (IBa). Selective agonists of μ-, δ-, and κ-ORs inhibited IBa through Cav2.3 channels by 35%. Cav2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via μ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated IBa amplitude mediated by Cav2.3 and Cav2.2 channels. Consistent with G protein βγ (Gβγ) interaction, modulation of Cav2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Cav2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Cav2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5'-[β-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gβγ-specific scavengers-namely, the carboxyl terminus of the G protein-coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin-attenuated or abolished Cav2.3 modulation. Our study reveals the diversity of OR-mediated signaling at Cav2 channels and identifies neuronal Cav2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gβγ subunits in a voltage-independent manner.

  19. Alcohol modulation of BK channel gating depends on β subunit composition.

    Science.gov (United States)

    Kuntamallappanavar, Guruprasad; Dopico, Alex M

    2016-11-01

    In most mammalian tissues, Ca(2+)i/voltage-gated, large conductance K(+) (BK) channels consist of channel-forming slo1 and auxiliary (β1-β4) subunits. When Ca(2+)i (3-20 µM) reaches the vicinity of BK channels and increases their activity at physiological voltages, β1- and β4-containing BK channels are, respectively, inhibited and potentiated by intoxicating levels of ethanol (50 mM). Previous studies using different slo1s, lipid environments, and Ca(2+)i concentrations-all determinants of the BK response to ethanol-made it impossible to determine the specific contribution of β subunits to ethanol action on BK activity. Furthermore, these studies measured ethanol action on ionic current under a limited range of stimuli, rendering no information on the gating processes targeted by alcohol and their regulation by βs. Here, we used identical experimental conditions to obtain single-channel and macroscopic currents of the same slo1 channel ("cbv1" from rat cerebral artery myocytes) in the presence and absence of 50 mM ethanol. First, we assessed the role five different β subunits (1,2,2-IR, 3-variant d, and 4) in ethanol action on channel function. Thus, two phenotypes were identified: (1) ethanol potentiated cbv1-, cbv1+β3-, and cbv1+β4-mediated currents at low Ca(2+)i while inhibiting current at high Ca(2+)i, the potentiation-inhibition crossover occurring at 20 µM Ca(2+)i; (2) for cbv1+β1, cbv1+wt β2, and cbv1+β2-IR, this crossover was shifted to ∼3 µM Ca(2+)i Second, applying Horrigan-Aldrich gating analysis on both phenotypes, we show that ethanol fails to modify intrinsic gating and the voltage-dependent parameters under examination. For cbv1, however, ethanol (a) drastically increases the channel's apparent Ca(2+) affinity (nine-times decrease in Kd) and (b) very mildly decreases allosteric coupling between Ca(2+) binding and channel opening (C). The decreased Kd leads to increased channel activity. For cbv1+β1, ethanol (a) also decreases Kd

  20. Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

    Directory of Open Access Journals (Sweden)

    Cédric James Laedermann

    2015-11-01

    Full Text Available In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs is a very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential. Navs are composed of 9 different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8 and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the action potential and consequently modify pain transmission, a process that is observed in persistent pain conditions.Post-translational modification (PTM of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e. peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with the subunit of Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological

  1. 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

  2. Frequency-dependent modulation of KCNQ1 and HERG1 potassium channels

    DEFF Research Database (Denmark)

    Diness, Thomas Goldin; Hansen, Rie Schultz; Olesen, Søren-Peter

    2006-01-01

    of the beta-subunits KCNE1 and KCNE2. In addition, the functional role of HERG1 in native guinea pig cardiac myocytes was demonstrated at different pacing frequencies by application of 10microM of the new HERG1 activator, NS1643. In conclusion, we have demonstrated that HERG1 and hKCNQ1 channels are inversely......To obtain information about a possible frequency-dependent modulation of HERG1 and hKCNQ1 channels, we performed heterologous expression in Xenopus laevis oocytes. Channel activation was obtained by voltage protocols roughly imitating cardiac action potentials at frequencies of 1, 3, 5.8, and 8.3Hz....... The activity of HERG1 channels was inhibited down to 65% at high frequencies. In contrast, hKCNQ1 channel activity was increased up to 525% at high frequencies. The general frequency-dependent modulation of the channels was unaffected by both co-expression of hKCNQ1 and HERG1 channels, and by the presence...

  3. Effect of genistein on voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Shi-Ying Li; Bin-Bin Huang; Shou Ouyang

    2006-01-01

    AIM: To investigate the action of genistein (GST), a broad spectrum tyrosine kinase inhibitor, on voltagegated potassium channels in guinea pig proximal colon smooth muscle cells.METHODS: Smooth muscle cells in guinea pig proximal colon were enzymatically isolate