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Sample records for a-type kv channels

  1. Localization of A-type K+ channel subunit Kv4.2 in rat brain.

    PubMed

    Tsaur, M L; Wu, Y L; Huang, F L; Shih, Y H

    2001-09-30

    Kv4.2, a voltage-gated K+ (Kv) channel subunit, has been suggested to be the key component of the subthreshold A-type K+ currents (I(SA)s) recorded from the specific subcellular compartments of certain CNS neurons. To correlate Kv4.2 localization with the I(SA)s detected, immunohistochemistry will be useful. Although the Kv4.2 immunostaining pattern in the hippocampus and cerebellum has been reported, the Kv4.2 antibody used was not specific. Furthermore, Kv4.2 localization in other brain regions remains unclear. In this report, we first demonstrated the specificity of a new Kv4.2 antibody, and then used it to examine Kv4.2 localization throughout adult rat brain by immunohistochemistry. At the cellular level, Kv4.2 was found in neurons but not glias. At the subcellular level, Kv4.2 was localized in the somatodendritic compartment of most neurons examined. Nevertheless, our preliminary data indicated that Kv4.2 might be also present in the axon/terminal compartment. At the functional level, our data indicates that Kv4.2 localization and I(SA) correlate quite well in some CNS neurons, supporting that Kv4.2 is the key component of some I(SA)s recorded in vivo.

  2. Postnatal development of A-type and Kv1- and Kv2-mediated potassium channel currents in neocortical pyramidal neurons

    PubMed Central

    Guan, Dongxu; Horton, Leslie R.; Armstrong, William E.

    2011-01-01

    Potassium channels regulate numerous aspects of neuronal excitability, and several voltage-gated K+ channel subunits have been identified in pyramidal neurons of rat neocortex. Previous studies have either considered the development of outward current as a whole or divided currents into transient, A-type and persistent, delayed rectifier components but did not differentiate between current components defined by α-subunit type. To facilitate comparisons of studies reporting K+ currents from animals of different ages and to understand the functional roles of specific current components, we characterized the postnatal development of identified Kv channel-mediated currents in pyramidal neurons from layers II/III from rat somatosensory cortex. Both the persistent/slowly inactivating and transient components of the total K+ current increased in density with postnatal age. We used specific pharmacological agents to test the relative contributions of putative Kv1- and Kv2-mediated currents (100 nM α-dendrotoxin and 600 nM stromatoxin, respectively). A combination of voltage protocol, pharmacology, and curve fitting was used to isolate the rapidly inactivating A-type current. We found that the density of all identified current components increased with postnatal age, approaching a plateau at 3–5 wk. We found no significant changes in the relative proportions or kinetics of any component between postnatal weeks 1 and 5, except that the activation time constant for A-type current was longer at 1 wk. The putative Kv2-mediated component was the largest at all ages. Immunocytochemistry indicated that protein expression for Kv4.2, Kv4.3, Kv1.4, and Kv2.1 increased between 1 wk and 4–5 wk of age. PMID:21451062

  3. Postnatal development of A-type and Kv1- and Kv2-mediated potassium channel currents in neocortical pyramidal neurons.

    PubMed

    Guan, Dongxu; Horton, Leslie R; Armstrong, William E; Foehring, Robert C

    2011-06-01

    Potassium channels regulate numerous aspects of neuronal excitability, and several voltage-gated K(+) channel subunits have been identified in pyramidal neurons of rat neocortex. Previous studies have either considered the development of outward current as a whole or divided currents into transient, A-type and persistent, delayed rectifier components but did not differentiate between current components defined by α-subunit type. To facilitate comparisons of studies reporting K(+) currents from animals of different ages and to understand the functional roles of specific current components, we characterized the postnatal development of identified Kv channel-mediated currents in pyramidal neurons from layers II/III from rat somatosensory cortex. Both the persistent/slowly inactivating and transient components of the total K(+) current increased in density with postnatal age. We used specific pharmacological agents to test the relative contributions of putative Kv1- and Kv2-mediated currents (100 nM α-dendrotoxin and 600 nM stromatoxin, respectively). A combination of voltage protocol, pharmacology, and curve fitting was used to isolate the rapidly inactivating A-type current. We found that the density of all identified current components increased with postnatal age, approaching a plateau at 3-5 wk. We found no significant changes in the relative proportions or kinetics of any component between postnatal weeks 1 and 5, except that the activation time constant for A-type current was longer at 1 wk. The putative Kv2-mediated component was the largest at all ages. Immunocytochemistry indicated that protein expression for Kv4.2, Kv4.3, Kv1.4, and Kv2.1 increased between 1 wk and 4-5 wk of age.

  4. Modeling-independent elucidation of inactivation pathways in recombinant and native A-type Kv channels.

    PubMed

    Fineberg, Jeffrey D; Ritter, David M; Covarrubias, Manuel

    2012-11-01

    A-type voltage-gated K(+) (Kv) channels self-regulate their activity by inactivating directly from the open state (open-state inactivation [OSI]) or by inactivating before they open (closed-state inactivation [CSI]). To determine the inactivation pathways, it is often necessary to apply several pulse protocols, pore blockers, single-channel recording, and kinetic modeling. However, intrinsic hurdles may preclude the standardized application of these methods. Here, we implemented a simple method inspired by earlier studies of Na(+) channels to analyze macroscopic inactivation and conclusively deduce the pathways of inactivation of recombinant and native A-type Kv channels. We investigated two distinct A-type Kv channels expressed heterologously (Kv3.4 and Kv4.2 with accessory subunits) and their native counterparts in dorsal root ganglion and cerebellar granule neurons. This approach applies two conventional pulse protocols to examine inactivation induced by (a) a simple step (single-pulse inactivation) and (b) a conditioning step (double-pulse inactivation). Consistent with OSI, the rate of Kv3.4 inactivation (i.e., the negative first derivative of double-pulse inactivation) precisely superimposes on the profile of the Kv3.4 current evoked by a single pulse because the channels must open to inactivate. In contrast, the rate of Kv4.2 inactivation is asynchronous, already changing at earlier times relative to the profile of the Kv4.2 current evoked by a single pulse. Thus, Kv4.2 inactivation occurs uncoupled from channel opening, indicating CSI. Furthermore, the inactivation time constant versus voltage relation of Kv3.4 decreases monotonically with depolarization and levels off, whereas that of Kv4.2 exhibits a J-shape profile. We also manipulated the inactivation phenotype by changing the subunit composition and show how CSI and CSI combined with OSI might affect spiking properties in a full computational model of the hippocampal CA1 neuron. This work unambiguously

  5. Modeling-independent elucidation of inactivation pathways in recombinant and native A-type Kv channels

    PubMed Central

    Fineberg, Jeffrey D.; Ritter, David M.

    2012-01-01

    A-type voltage-gated K+ (Kv) channels self-regulate their activity by inactivating directly from the open state (open-state inactivation [OSI]) or by inactivating before they open (closed-state inactivation [CSI]). To determine the inactivation pathways, it is often necessary to apply several pulse protocols, pore blockers, single-channel recording, and kinetic modeling. However, intrinsic hurdles may preclude the standardized application of these methods. Here, we implemented a simple method inspired by earlier studies of Na+ channels to analyze macroscopic inactivation and conclusively deduce the pathways of inactivation of recombinant and native A-type Kv channels. We investigated two distinct A-type Kv channels expressed heterologously (Kv3.4 and Kv4.2 with accessory subunits) and their native counterparts in dorsal root ganglion and cerebellar granule neurons. This approach applies two conventional pulse protocols to examine inactivation induced by (a) a simple step (single-pulse inactivation) and (b) a conditioning step (double-pulse inactivation). Consistent with OSI, the rate of Kv3.4 inactivation (i.e., the negative first derivative of double-pulse inactivation) precisely superimposes on the profile of the Kv3.4 current evoked by a single pulse because the channels must open to inactivate. In contrast, the rate of Kv4.2 inactivation is asynchronous, already changing at earlier times relative to the profile of the Kv4.2 current evoked by a single pulse. Thus, Kv4.2 inactivation occurs uncoupled from channel opening, indicating CSI. Furthermore, the inactivation time constant versus voltage relation of Kv3.4 decreases monotonically with depolarization and levels off, whereas that of Kv4.2 exhibits a J-shape profile. We also manipulated the inactivation phenotype by changing the subunit composition and show how CSI and CSI combined with OSI might affect spiking properties in a full computational model of the hippocampal CA1 neuron. This work unambiguously

  6. Open channel block of A-type, kv4.3, and delayed rectifier K+ channels, Kv1.3 and Kv3.1, by sibutramine.

    PubMed

    Kim, Sung Eun; Ahn, Hye Sook; Choi, Bok Hee; Jang, Hyun-Jong; Kim, Myung-Jun; Rhie, Duck-Joo; Yoon, Shin-Hee; Jo, Yang-Hyeok; Kim, Myung-Suk; Sung, Ki-Wug; Hahn, Sang June

    2007-05-01

    The effects of sibutramine on voltage-gated K+ channel (Kv)4.3, Kv1.3, and Kv3.1, stably expressed in Chinese hamster ovary cells, were investigated using the whole-cell patch-clamp technique. Sibutramine did not significantly decrease the peak Kv4.3 currents, but it accelerated the rate of decay of current inactivation in a concentration-dependent manner. This phenomenon was effectively characterized by integrating the total current over the duration of a depolarizing pulse to +40 mV. The IC50 value for the sibutramine block of Kv4.3 was 17.3 microM. Under control conditions, the inactivation of Kv4.3 currents could be fit to a biexponential function, and the time constants for the fast and slow components were significantly decreased after the application of sibutramine. The association (k+1) and dissociation (k-1) rate constants for the sibutramine block of Kv 4.3 were 1.51 microM-1s-1 and 27.35 s-1, respectively. The theoretical KD value, derived from k-1/k+1, yielded a value of 18.11 microM. The block of Kv4.3 by sibutramine displayed a weak voltage dependence, increasing at more positive potentials, and it was use-dependent at 2 Hz. Sibutramine did not affect the time course for the deactivating tail currents. Neither steady-state activation and inactivation nor the recovery from inactivation was affected by sibutramine. Sibutramine caused the concentration-dependent block of the Kv1.3 and Kv3.1 currents with an IC50 value of 3.7 and 32.7 microM, respectively. In addition, sibutramine reduced the tail current amplitude and slowed the deactivation of the tail currents of Kv1.3 and Kv3.1, resulting in a crossover phenomenon. These results indicate that sibutramine acts on Kv4.3, Kv1.3, and Kv3.1 as an open channel blocker.

  7. Elimination of fast inactivation in Kv4 A-type potassium channels by an auxiliary subunit domain.

    PubMed

    Holmqvist, Mats H; Cao, Jie; Hernandez-Pineda, Ricardo; Jacobson, Michael D; Carroll, Karen I; Sung, M Amy; Betty, Maria; Ge, Pei; Gilbride, Kevin J; Brown, Melissa E; Jurman, Mark E; Lawson, Deborah; Silos-Santiago, Inmaculada; Xie, Yu; Covarrubias, Manuel; Rhodes, Kenneth J; Distefano, Peter S; An, W Frank

    2002-01-22

    The Kv4 A-type potassium currents contribute to controlling the frequency of slow repetitive firing and back-propagation of action potentials in neurons and shape the action potential in heart. Kv4 currents exhibit rapid activation and inactivation and are specifically modulated by K-channel interacting proteins (KChIPs). Here we report the discovery and functional characterization of a modular K-channel inactivation suppressor (KIS) domain located in the first 34 aa of an additional KChIP (KChIP4a). Coexpression of KChIP4a with Kv4 alpha-subunits abolishes fast inactivation of the Kv4 currents in various cell types, including cerebellar granule neurons. Kinetic analysis shows that the KIS domain delays Kv4.3 opening, but once the channel is open, it disrupts rapid inactivation and slows Kv4.3 closing. Accordingly, KChIP4a increases the open probability of single Kv4.3 channels. The net effects of KChIP4a and KChIP1-3 on Kv4 gating are quite different. When both KChIP4a and KChIP1 are present, the Kv4.3 current shows mixed inactivation profiles dependent on KChIP4a/KChIP1 ratios. The KIS domain effectively converts the A-type Kv4 current to a slowly inactivating delayed rectifier-type potassium current. This conversion is opposite to that mediated by the Kv1-specific "ball" domain of the Kv beta 1 subunit. Together, these results demonstrate that specific auxiliary subunits with distinct functions actively modulate gating of potassium channels that govern membrane excitability.

  8. KChIPs and Kv4 alpha subunits as integral components of A-type potassium channels in mammalian brain.

    PubMed

    Rhodes, Kenneth J; Carroll, Karen I; Sung, M Amy; Doliveira, Lisa C; Monaghan, Michael M; Burke, Sharon L; Strassle, Brian W; Buchwalder, Lynn; Menegola, Milena; Cao, Jie; An, W Frank; Trimmer, James S

    2004-09-08

    Voltage-gated potassium (Kv) channels from the Kv4, or Shal-related, gene family underlie a major component of the A-type potassium current in mammalian central neurons. We recently identified a family of calcium-binding proteins, termed KChIPs (Kv channel interacting proteins), that bind to the cytoplasmic N termini of Kv4 family alpha subunits and modulate their surface density, inactivation kinetics, and rate of recovery from inactivation (An et al., 2000). Here, we used single and double-label immunohistochemistry, together with circumscribed lesions and coimmunoprecipitation analyses, to examine the regional and subcellular distribution of KChIPs1-4 and Kv4 family alpha subunits in adult rat brain. Immunohistochemical staining using KChIP-specific monoclonal antibodies revealed that the KChIP polypeptides are concentrated in neuronal somata and dendrites where their cellular and subcellular distribution overlaps, in an isoform-specific manner, with that of Kv4.2 and Kv4.3. For example, immunoreactivity for KChIP1 and Kv4.3 is concentrated in the somata and dendrites of hippocampal, striatal, and neocortical interneurons. Immunoreactivity for KChIP2, KChIP4, and Kv4.2 is concentrated in the apical and basal dendrites of hippocampal and neocortical pyramidal cells. Double-label immunofluorescence labeling revealed that throughout the forebrain, KChIP2 and KChIP4 are frequently colocalized with Kv4.2, whereas in cortical, hippocampal, and striatal interneurons, KChIP1 is frequently colocalized with Kv4.3. Coimmunoprecipitation analyses confirmed that all KChIPs coassociate with Kv4 alpha subunits in brain membranes, indicating that KChIPs 1-4 are integral components of native A-type Kv channel complexes and are likely to play a major role as modulators of somatodendritic excitability.

  9. PKA modulation of Kv4.2-encoded A-type potassium channels requires formation of a supramolecular complex.

    PubMed

    Schrader, Laura A; Anderson, Anne E; Mayne, Amber; Pfaffinger, Paul J; Sweatt, John David

    2002-12-01

    A-type channels, encoded by the pore-forming alpha-subunits of the Kv4.x family, are particularly important in regulating membrane excitability in the CNS and the heart. Given the key role of modulation of A currents by kinases, we sought to investigate the protein structure-function relationships underlying the regulation of these currents by PKA. We have previously shown the existence of two PKA phosphorylation sites in the Kv4.2 sequence; therefore, we focused this study on the Kv4.2 primary subunit. In the present studies we made the surprising finding that PKA phosphorylation of the Kv4.2 alpha-subunit is necessary but not sufficient for channel modulation; channel modulation by PKA required the presence of an ancillary subunit, the K+ channel interacting protein (KChIP3). Therefore, these findings indicate a surprising complexity to kinase regulation of A currents, in that an interaction of two separate molecular events, alpha-subunit phosphorylation and the association of an ancillary subunit (KChIP3), are necessary for phosphorylation-dependent regulation of Kv4.2-encoded A channels by PKA. Overall, our studies indicate that PKA must of necessity act on a supramolecular complex of pore-forming alpha-subunits plus ancillary subunits to alter channel properties.

  10. Ternary Kv4.2 channels recapitulate voltage-dependent inactivation kinetics of A-type K+ channels in cerebellar granule neurons.

    PubMed

    Amarillo, Yimy; De Santiago-Castillo, Jose A; Dougherty, Kevin; Maffie, Jonathon; Kwon, Elaine; Covarrubias, Manuel; Rudy, Bernardo

    2008-04-15

    Kv4 channels mediate most of the somatodendritic subthreshold operating A-type current (I(SA)) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore-forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX-S, or Kv4.2, KChIP1 and DPPX-S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the I(SA) in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX-S proteins in heterologous cells. The fact that this complex and unique behaviour (among A-type K(+) currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed-state inactivation and a weakly voltage-dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed-state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native I(SA)-mediating channels.

  11. Hippocampal A-type current and Kv4.2 channel modulation by the sulfonylurea compound NS5806.

    PubMed

    Witzel, Katrin; Fischer, Paul; Bähring, Robert

    2012-12-01

    We examined the effects of the sulfonylurea compound NS5806 on neuronal A-type channel function. Using whole-cell patch-clamp we studied the effects of NS5806 on the somatodendritic A-type current (I(SA)) in cultured hippocampal neurons and the currents mediated by Kv4.2 channels coexpressed with different auxiliary β-subunits, including both Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-related proteins (DPPs), in HEK 293 cells. The amplitude of the I(SA) component in hippocampal neurons was reduced in the presence of 20 μM NS5806. I(SA) decay kinetics were slowed and the recovery kinetics accelerated, but the voltage dependence of steady-state inactivation was shifted to more negative potentials by NS5806. The peak amplitudes of currents mediated by ternary Kv4.2 channel complexes, associated with DPP6-S (short splice-variant) and either KChIP2, KChIP3 or KChIP4, were potentiated and their macroscopic inactivation slowed by NS5806, whereas the currents mediated by binary Kv4.2 channels, associated only with DPP6-S, were suppressed, and the NS5806-mediated slowing of macroscopic inactivation was less pronounced. Neither potentiation nor suppression and no effect on current decay kinetics in the presence of NS5806 were observed for Kv4.2 channels associated with KChIP3 and the N-type inactivation-conferring DPP6a splice-variant. For all recombinant channel complexes, NS5806 slowed the recovery from inactivation and shifted the voltage dependence of steady-state inactivation to more negative potentials. Our results demonstrate the activity of NS5806 on native I(SA) and possible molecular correlates in the form of recombinant Kv4.2 channels complexed with different KChIPs and DPPs, and they shed some light on the mechanism of NS5806 action. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Delayed Rectifier and A-Type Potassium Channels Associated with Kv 2.1 and Kv 4.3 Expression in Embryonic Rat Neural Progenitor Cells

    PubMed Central

    Smith, Dean O.; Rosenheimer, Julie L.; Kalil, Ronald E.

    2008-01-01

    Background Because of the importance of voltage-activated K+ channels during embryonic development and in cell proliferation, we present here the first description of these channels in E15 rat embryonic neural progenitor cells derived from the subventricular zone (SVZ). Activation, inactivation, and single-channel conductance properties of recorded progenitor cells were compared with those obtained by others when these Kv gene products were expressed in oocytes. Methodology/Principal Findings Neural progenitor cells derived from the subventricular zone of E15 embryonic rats were cultured under conditions that did not promote differentiation. Immunocytochemical and Western blot assays for nestin expression indicated that almost all of the cells available for recording expressed this intermediate filament protein, which is generally accepted as a marker for uncommitted embryonic neural progenitor cells. However, a very small numbers of the cells expressed GFAP, a marker for astrocytes, O4, a marker for immature oligodendrocytes, and βIII-tubulin, a marker for neurons. Using immunocytochemistry and Western blots, we detected consistently the expression of Kv2.1, and 4.3. In whole-cell mode, we recorded two outward currents, a delayed rectifier and an A-type current. Conclusions/Significance We conclude that Kv2.1, and 4.3 are expressed in E15 SVZ neural progenitor cells, and we propose that they may be associated with the delayed-rectifier and the A-type currents, respectively, that we recorded. These results demonstrate the early expression of delayed rectifier and A-type K+ currents and channels in embryonic neural progenitor cells prior to the differentiation of these cells. PMID:18270591

  13. Delayed rectifier and A-type potassium channels associated with Kv 2.1 and Kv 4.3 expression in embryonic rat neural progenitor cells.

    PubMed

    Smith, Dean O; Rosenheimer, Julie L; Kalil, Ronald E

    2008-02-13

    Because of the importance of voltage-activated K(+) channels during embryonic development and in cell proliferation, we present here the first description of these channels in E15 rat embryonic neural progenitor cells derived from the subventricular zone (SVZ). Activation, inactivation, and single-channel conductance properties of recorded progenitor cells were compared with those obtained by others when these Kv gene products were expressed in oocytes. Neural progenitor cells derived from the subventricular zone of E15 embryonic rats were cultured under conditions that did not promote differentiation. Immunocytochemical and Western blot assays for nestin expression indicated that almost all of the cells available for recording expressed this intermediate filament protein, which is generally accepted as a marker for uncommitted embryonic neural progenitor cells. However, a very small numbers of the cells expressed GFAP, a marker for astrocytes, O4, a marker for immature oligodendrocytes, and betaIII-tubulin, a marker for neurons. Using immunocytochemistry and Western blots, we detected consistently the expression of Kv2.1, and 4.3. In whole-cell mode, we recorded two outward currents, a delayed rectifier and an A-type current. We conclude that Kv2.1, and 4.3 are expressed in E15 SVZ neural progenitor cells, and we propose that they may be associated with the delayed-rectifier and the A-type currents, respectively, that we recorded. These results demonstrate the early expression of delayed rectifier and A-type K(+) currents and channels in embryonic neural progenitor cells prior to the differentiation of these cells.

  14. Interaction of the scorpion toxin discrepin with Kv4.3 channels and A-type K(+) channels in cerebellum granular cells.

    PubMed

    Picco, Cristiana; Corzo, Gerardo; Possani, Lourival D; Prestipino, Gianfranco

    2014-09-01

    The peptide discrepin from the α-KTx15 subfamily of scorpion toxins preferentially affects transient A-type potassium currents, which regulate many aspects of neuronal function in the central nervous system. However, the specific Kv channel targeted by discrepin and the molecular mechanism of interaction are still unknown. Different variant peptides of discrepin were chemically synthesized and their effects were studied using patch clamp technique on rat cerebellum granular cells (CGC) and HEK cells transiently expressing Kv4.3 channels. Functional analysis indicated that nanomolar concentrations of native discrepin blocked Kv4.3 expressed channels, as previously observed in CGC. Similarly, the apparent affinities of all mutated peptides for Kv4.3 expressed channels were analogous to those found in CGC. In particular, in the double variant [V6K, D20K] the apparent affinity increased about 10-fold, whereas in variants carrying a deletion (ΔK13) or substitution (K13A) at position K13, the blockage was removed and the apparent affinity decreased more than 20-fold. These results indicate that Kv4.3 is likely the target of discrepin and highlight the importance of the basic residue K13, located in the α-helix of the toxin, for current blockage. We report the first example of a Kv4 subfamily potassium channel blocked by discrepin and identify the amino acid residues responsible for the blockage. The availability of discrepin variant peptides stimulates further research on the functions and pharmacology of neuronal Kv4 channels and on their possible roles in neurodegenerative disorders. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Nicotine is a potent blocker of the cardiac A-type K(+) channels. Effects on cloned Kv4.3 channels and native transient outward current.

    PubMed

    Wang, H; Shi, H; Zhang, L; Pourrier, M; Yang, B; Nattel, S; Wang, Z

    2000-09-05

    Nicotine is a main constituent of cigarette smoke and smokeless tobacco, known to increase the risk of sudden cardiac death. This study aimed at establishing ionic mechanisms underlying potential electrophysiological effects of nicotine. Effects of nicotine on Kv4.3 and Kv4.2 channels expressed in Xenopus oocytes were studied at the whole-cell and single-channel levels. The effects of nicotine on the transient outward K(+) current (I:(to)) were studied by use of whole-cell patch-clamp techniques in canine ventricular myocytes. Nicotine potently inhibited Kv4 current. The concentration for half-maximal inhibition (IC(50)) was 40+/-4 nmol/L, and the current was abolished by 100 micromol/L nicotine. The IC(50) for block of native I:(to) was 270+/-43 nmol/L. The steady-state activation properties of Kv4.3 and I:(to) were unaltered by nicotine, whereas positive shifts of the inactivation curves were observed. Of the total inhibition of Kv4.3 and I:(to) by nicotine, 40% was due to tonic block and 60% was attributable to use-dependent block. Activation, inactivation, and reactivation kinetics were not significantly changed by nicotine. Nicotine reduced single-channel conductance, open probability, and open time but increased the closed time of Kv4.3. The effects of nicotine were not altered by antagonists to various neurotransmitter receptors, indicating direct effects on I:(to) channels. Nicotine is a potent inhibitor of cardiac A-type K(+) channels, with blockade probably due to block of closed and open channels. This action may contribute to the ability of nicotine to affect cardiac electrophysiology and induce arrhythmias.

  16. Expression of A-type K channel alpha subunits Kv 4.2 and Kv 4.3 in rat spinal lamina II excitatory interneurons and colocalization with pain-modulating molecules.

    PubMed

    Huang, Hsin-Yi; Cheng, Jen-Kun; Shih, Yang-Hsin; Chen, Pei-Hsuan; Wang, Chin-Lin; Tsaur, Meei-Ling

    2005-09-01

    Voltage-gated K(+) channel alpha subunits Kv 4.2 and Kv 4.3 are the major contributors of somatodendritic A-type K(+) currents in many CNS neurons. A recent hypothesis suggests that Kv 4 subunits may be involved in pain modulation in dorsal horn neurons. However, whether Kv 4 subunits are expressed in dorsal horn neurons remains unknown. Using immunohistochemistry, we found that Kv 4.2 and Kv 4.3 immunoreactivity was concentrated in the superficial dorsal horn, mainly in lamina II. Both Kv 4.2 and Kv 4.3 appeared on many rostrocaudally orientated dendrites, whereas Kv 4.3 could be also detected from certain neuronal somata. Kv 4.3(+) neurons were a subset of excitatory inerneurons with calretinin(+)/calbindin(-)/PKCgamma(-) markers, and a fraction of them expressed micro-opioid receptors. Kv 4.3(+) neurons also expressed ERK 2 and mGluR 5, which are molecules related to the induction of central sensitization, a mechanism mediating nociceptive plasticity. Together with the expression of Kv 4.3 in VR 1(+) DRG neurons, our data suggest that Kv C4 subunits could be involved in pain modulation.

  17. Differential contribution of Kv4-containing channels to A-type, voltage-gated potassium currents in somatic and visceral dorsal root ganglion neurons.

    PubMed

    Yunoki, Takakazu; Takimoto, Koichi; Kita, Kaori; Funahashi, Yasuhito; Takahashi, Ryosuke; Matsuyoshi, Hiroko; Naito, Seiji; Yoshimura, Naoki

    2014-11-15

    Little is known about electrophysiological differences of A-type transient K(+) (KA) currents in nociceptive afferent neurons that innervate somatic and visceral tissues. Staining with isolectin B4 (IB4)-FITC classifies L6-S1 dorsal root ganglion (DRG) neurons into three populations with distinct staining intensities: negative to weak, moderate, and intense fluorescence signals. All IB4 intensely stained cells are negative for a fluorescent dye, Fast Blue (FB), injected into the bladder wall, whereas a fraction of somatic neurons labeled by FB, injected to the external urethral dermis, is intensely stained with IB4. In whole-cell, patch-clamp recordings, phrixotoxin 2 (PaTx2), a voltage-gated K(+) (Kv)4 channel blocker, exhibits voltage-independent inhibition of the KA current in IB4 intensely stained cells but not the one in bladder-innervating cells. The toxin also shows voltage-independent inhibition of heterologously expressed Kv4.1 current, whereas its inhibition of Kv4.2 and Kv4.3 currents is voltage dependent. The swapping of four amino acids at the carboxyl portion of the S3 region between Kv4.1 and Kv4.2 transfers this characteristic. RT-PCRs detected Kv4.1 and the long isoform of Kv4.3 mRNAs without significant Kv4.2 mRNA in L6-S1 DRGs. Kv4.1 and Kv4.3 mRNA levels were higher in laser-captured, IB4-stained neurons than in bladder afferent neurons. These results indicate that PaTx2 acts differently on channels in the Kv4 family and that Kv4.1 and possibly Kv4.3 subunits functionally participate in the formation of KA channels in a subpopulation of somatic C-fiber neurons but not in visceral C-fiber neurons innervating the bladder. Copyright © 2014 the American Physiological Society.

  18. Differential contribution of Kv4-containing channels to A-type, voltage-gated potassium currents in somatic and visceral dorsal root ganglion neurons

    PubMed Central

    Yunoki, Takakazu; Takimoto, Koichi; Kita, Kaori; Funahashi, Yasuhito; Takahashi, Ryosuke; Matsuyoshi, Hiroko; Naito, Seiji

    2014-01-01

    Little is known about electrophysiological differences of A-type transient K+ (KA) currents in nociceptive afferent neurons that innervate somatic and visceral tissues. Staining with isolectin B4 (IB4)-FITC classifies L6-S1 dorsal root ganglion (DRG) neurons into three populations with distinct staining intensities: negative to weak, moderate, and intense fluorescence signals. All IB4 intensely stained cells are negative for a fluorescent dye, Fast Blue (FB), injected into the bladder wall, whereas a fraction of somatic neurons labeled by FB, injected to the external urethral dermis, is intensely stained with IB4. In whole-cell, patch-clamp recordings, phrixotoxin 2 (PaTx2), a voltage-gated K+ (Kv)4 channel blocker, exhibits voltage-independent inhibition of the KA current in IB4 intensely stained cells but not the one in bladder-innervating cells. The toxin also shows voltage-independent inhibition of heterologously expressed Kv4.1 current, whereas its inhibition of Kv4.2 and Kv4.3 currents is voltage dependent. The swapping of four amino acids at the carboxyl portion of the S3 region between Kv4.1 and Kv4.2 transfers this characteristic. RT-PCRs detected Kv4.1 and the long isoform of Kv4.3 mRNAs without significant Kv4.2 mRNA in L6-S1 DRGs. Kv4.1 and Kv4.3 mRNA levels were higher in laser-captured, IB4-stained neurons than in bladder afferent neurons. These results indicate that PaTx2 acts differently on channels in the Kv4 family and that Kv4.1 and possibly Kv4.3 subunits functionally participate in the formation of KA channels in a subpopulation of somatic C-fiber neurons but not in visceral C-fiber neurons innervating the bladder. PMID:25143545

  19. Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels*

    PubMed Central

    Tang, Yi-Quan; Liang, Ping; Zhou, Jingheng; Lu, Yanxin; Lei, Lei; Bian, Xiling; Wang, KeWei

    2013-01-01

    In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K+ channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1–4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12–17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19–21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation. PMID:23576435

  20. Auxiliary KChIP4a suppresses A-type K+ current through endoplasmic reticulum (ER) retention and promoting closed-state inactivation of Kv4 channels.

    PubMed

    Tang, Yi-Quan; Liang, Ping; Zhou, Jingheng; Lu, Yanxin; Lei, Lei; Bian, Xiling; Wang, KeWei

    2013-05-24

    In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K(+) channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12-17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.

  1. Calcium-calmodulin-dependent kinase II modulates Kv4.2 channel expression and upregulates neuronal A-type potassium currents.

    PubMed

    Varga, Andrew W; Yuan, Li-Lian; Anderson, Anne E; Schrader, Laura A; Wu, Gang-Yi; Gatchel, Jennifer R; Johnston, Daniel; Sweatt, J David

    2004-04-07

    Calcium-calmodulin-dependent kinase II (CaMKII) has a long history of involvement in synaptic plasticity, yet little focus has been given to potassium channels as CaMKII targets despite their importance in repolarizing EPSPs and action potentials and regulating neuronal membrane excitability. We now show that Kv4.2 acts as a substrate for CaMKII in vitro and have identified CaMKII phosphorylation sites as Ser438 and Ser459. To test whether CaMKII phosphorylation of Kv4.2 affects channel biophysics, we expressed wild-type or mutant Kv4.2 and the K(+) channel interacting protein, KChIP3, with or without a constitutively active form of CaMKII in Xenopus oocytes and measured the voltage dependence of activation and inactivation in each of these conditions. CaMKII phosphorylation had no effect on channel biophysical properties. However, we found that levels of Kv4.2 protein are increased with CaMKII phosphorylation in transfected COS cells, an effect attributable to direct channel phosphorylation based on site-directed mutagenesis studies. We also obtained corroborating physiological data showing increased surface A-type channel expression as revealed by increases in peak K(+) current amplitudes with CaMKII phosphorylation. Furthermore, endogenous A-currents in hippocampal pyramidal neurons were increased in amplitude after introduction of constitutively active CaMKII, which results in a decrease in neuronal excitability in response to current injections. Thus CaMKII can directly modulate neuronal excitability by increasing cell-surface expression of A-type K(+) channels.

  2. Coexpression of the KCNA3B gene product with Kv1.5 leads to a novel A-type potassium channel.

    PubMed

    Leicher, T; Bähring, R; Isbrandt, D; Pongs, O

    1998-12-25

    Shaker-related voltage-gated potassium (Kv) channels may be heterooligomers consisting of membrane-integral alpha-subunits associated with auxiliary cytoplasmic beta-subunits. In this study we have cloned the human Kvbeta3.1 subunit and the corresponding KCNA3B gene. Identification of sequence-tagged sites in the gene mapped KCNA3B to band p13.1 of human chromosome 17. Comparison of the KCNA1B, KCNA2B, and KCNA3B gene structures showed that the three Kvbeta genes have very disparate lengths varying from >/=350 kb (KCNA1B) to approximately 7 kb (KCNA3B). Yet, the exon patterns of the three genes, which code for the seven known mammalian Kvbeta subunits, are very similar. The Kvbeta1 and Kvbeta2 splice variants are generated by alternative use of 5'-exons. Mouse Kvbeta4, a potential splice variant of Kvbeta3, is a read-through product where the open reading frame starts within the sequence intervening between Kvbeta3 exons 7 and 8. The human KCNA3B sequence does not contain a mouse Kvbeta4-like open reading frame. Human Kvbeta3 mRNA is specifically expressed in the brain, where it is predominantly detected in the cerebellum. The heterologous coexpression of human Kv1.5 and Kvbeta3.1 subunits in Chinese hamster ovary cells yielded a novel Kv channel mediating very fast inactivating (A-type) outward currents upon depolarization. Thus, the expression of Kvbeta3.1 subunits potentially extends the possibilities to express diverse A-type Kv channels in the human brain.

  3. Solution structure and function of the "tandem inactivation domain" of the neuronal A-type potassium channel Kv1.4.

    PubMed

    Wissmann, Ralph; Bildl, Wolfgang; Oliver, Dominik; Beyermann, Michael; Kalbitzer, Hans-Robert; Bentrop, Detlef; Fakler, Bernd

    2003-05-02

    Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

  4. Effect of tyrphostin AG879 on Kv4.2 and Kv4.3 potassium channels

    PubMed Central

    Yu, Haibo; Zou, Beiyan; Wang, Xiaoliang; Li, Min

    2015-01-01

    Background and Purpose A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv4.2/Kv channel-interacting protein 2 (KChIP2) channels. Experimental Approach To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv4.2/KChIP2 channels using a whole-cell patch-clamp technique. Key Results Tyrphostin AG879 selectively and dose-dependently inhibited Kv4.2 and Kv4.3 channels. In Kv4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons. Conclusion and Implications AG879 was identified as a selective and potent inhibitor the Kv4.2 channel. AG879 inhibited Kv4.2 channels by preferentially interacting with the open state and further accelerating their inactivation. PMID:25752739

  5. Effect of tyrphostin AG879 on Kv 4.2 and Kv 4.3 potassium channels.

    PubMed

    Yu, Haibo; Zou, Beiyan; Wang, Xiaoliang; Li, Min

    2015-07-01

    A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv 4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv 4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv 4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv 4.2/Kv channel-interacting protein 2 (KChIP2) channels. To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv 4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv 4.2/KChIP2 channels using a whole-cell patch-clamp technique. Tyrphostin AG879 selectively and dose-dependently inhibited Kv 4.2 and Kv 4.3 channels. In Kv 4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv 4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons. AG879 was identified as a selective and potent inhibitor the Kv 4.2 channel. AG879 inhibited Kv 4.2 channels by preferentially interacting with the open state and further accelerating their inactivation. © 2015 The British Pharmacological Society.

  6. [Modulation of Kv4 channels by KChIPs clamping].

    PubMed

    Cui, Yuan-Yuan; Wang, Ke-Wei

    2009-01-01

    The rapidly inactivating (A-type) potassium channels regulate membrane excitability that defines the fundamental mechanism of neuronal functions such as pain signaling. Cytosolic Kv channel-interacting proteins KChIPs co-assemble with Kv4 (Shal) alpha subunits to form a native complex. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Based on recent structural efforts, here we attempt to emphasize the interaction between KChIPs and Kv4 channel complex in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner. Greater insights into molecular mechanism between KChIPs and Kv4 interaction may provide therapeutic potentials by structure-based design of chemical compounds aimed at disrupting the protein-protein interaction for treatment of membrane excitability-related disorders.

  7. Tamoxifen Inhibition of Kv7.2/Kv7.3 Channels

    PubMed Central

    Ferrer, Tania; Aréchiga-Figueroa, Ivan Arael; Shapiro, Mark S.; Tristani-Firouzi, Martin; Sanchez-Chapula, José A.

    2013-01-01

    KCNQ genes encode five Kv7 K+ channel subunits (Kv7.1–Kv7.5). Four of these (Kv7.2–Kv7.5) are expressed in the nervous system. Kv7.2 and Kv7.3 are the principal molecular components of the slow voltage-gated M-channel, which regulates neuronal excitability. In this study, we demonstrate that tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits Kv7.2/Kv7.3 currents heterologously expressed in human embryonic kidney HEK-293 cells. Current inhibition by tamoxifen was voltage independent but concentration-dependent. The IC50 for current inhibition was 1.68 ± 0.44 µM. The voltage-dependent activation of the channel was not modified. Tamoxifen inhibited Kv7.2 homomeric channels with a higher potency (IC50 = 0.74 ± 0.16 µM). The mutation Kv7.2 R463E increases phosphatidylinositol- 4,5-bisphosphate (PIP2) - channel interaction and diminished dramatically the inhibitory effect of tamoxifen compared with that for wild type Kv7.2. Conversely, the mutation Kv7.2 R463Q, which decreases PIP2 -channel interaction, increased tamoxifen potency. Similar results were obtained on the heteromeric Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 channels, compared to Kv7.2/Kv7.3 WT. Overexpression of type 2A PI(4)P5-kinase (PIP5K 2A) significantly reduced tamoxifen inhibition of Kv7.2/Kv7.3 and Kv7.2 R463Q channels. Our results suggest that tamoxifen inhibited Kv7.2/Kv7.3 channels by interfering with PIP2-channel interaction because of its documented interaction with PIP2 and the similar effect of tamoxifen on various PIP2 sensitive channels. PMID:24086693

  8. Tamoxifen inhibition of kv7.2/kv7.3 channels.

    PubMed

    Ferrer, Tania; Aréchiga-Figueroa, Ivan Arael; Shapiro, Mark S; Tristani-Firouzi, Martin; Sanchez-Chapula, José A

    2013-01-01

    KCNQ genes encode five Kv7 K(+) channel subunits (Kv7.1-Kv7.5). Four of these (Kv7.2-Kv7.5) are expressed in the nervous system. Kv7.2 and Kv7.3 are the principal molecular components of the slow voltage-gated M-channel, which regulates neuronal excitability. In this study, we demonstrate that tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits Kv7.2/Kv7.3 currents heterologously expressed in human embryonic kidney HEK-293 cells. Current inhibition by tamoxifen was voltage independent but concentration-dependent. The IC50 for current inhibition was 1.68 ± 0.44 µM. The voltage-dependent activation of the channel was not modified. Tamoxifen inhibited Kv7.2 homomeric channels with a higher potency (IC50 = 0.74 ± 0.16 µM). The mutation Kv7.2 R463E increases phosphatidylinositol- 4,5-bisphosphate (PIP2) - channel interaction and diminished dramatically the inhibitory effect of tamoxifen compared with that for wild type Kv7.2. Conversely, the mutation Kv7.2 R463Q, which decreases PIP2 -channel interaction, increased tamoxifen potency. Similar results were obtained on the heteromeric Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 channels, compared to Kv7.2/Kv7.3 WT. Overexpression of type 2A PI(4)P5-kinase (PIP5K 2A) significantly reduced tamoxifen inhibition of Kv7.2/Kv7.3 and Kv7.2 R463Q channels. Our results suggest that tamoxifen inhibited Kv7.2/Kv7.3 channels by interfering with PIP2-channel interaction because of its documented interaction with PIP2 and the similar effect of tamoxifen on various PIP2 sensitive channels.

  9. Decreased expression of Kv7 channels in Hirchsprung's disease.

    PubMed

    O'Donnell, Anne-Marie; Coyle, David; Puri, Prem

    2017-07-01

    Voltage-dependent K + channels (Kv channels) participate in electrical rhythmicity and smooth muscle responses and are regulated by excitatory and inhibitory neurotransmitters. Kv channels also participate in the interstitial cell of Cajal (ICC) and smooth muscle cell (SMC) responses to neural inputs. The Kv family consists of 12 subfamilies, Kv1-Kv12, with five members of the Kv7 family identified to date: Kv7.1-Kv7.5. A recent study identified the potassium channel Kv7.5 as having a role in the excitability of ICC-IM in the mouse colon. We therefore designed this study to test the hypothesis that Kv7 channels are present in the normal human colon and are reduced in Hirschprung's disease (HSCR). HSCR tissue specimens were collected at the time of pull-through surgery (n=10), while normal control tissue specimens were obtained at the time of colostomy closure in patients with imperforate anus (n=10). Kv7.3-Kv7.5 immunohistochemistry was performed and visualized using confocal microscopy to assess their distribution. Western blot analysis was undertaken to determine Kv7.3-Kv7.5 protein quantification. Kv7.3 and Kv7.4-immunoreactivity was co-localized with neuron and ICC markers, while Kv7.5 was found to be expressed on both ICCs and SMCs. Western blot analysis revealed similar levels of Kv7.3 and Kv7.5 expression in the normal colon and HSCR colon, while Kv7.4 proteins were found to be markedly decreased in ganglionic specimens and decreased further in aganglionic specimens. A deficiency of Kv7.4 channels in the ganglionic and aganglionic bowel may place a role in colonic dysmotility in HSCR. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. I(A) channels encoded by Kv1.4 and Kv4.2 regulate neuronal firing in the suprachiasmatic nucleus and circadian rhythms in locomotor activity.

    PubMed

    Granados-Fuentes, Daniel; Norris, Aaron J; Carrasquillo, Yarimar; Nerbonne, Jeanne M; Herzog, Erik D

    2012-07-18

    Neurons in the suprachiasmatic nucleus (SCN) display coordinated circadian changes in electrical activity that are critical for daily rhythms in physiology, metabolism, and behavior. SCN neurons depolarize spontaneously and fire repetitively during the day and hyperpolarize, drastically reducing firing rates, at night. To explore the hypothesis that rapidly activating and inactivating A-type (I(A)) voltage-gated K(+) (Kv) channels, which are also active at subthreshold membrane potentials, are critical regulators of the excitability of SCN neurons, we examined locomotor activity and SCN firing in mice lacking Kv1.4 (Kv1.4(-/-)), Kv4.2 (Kv4.2(-/-)), or Kv4.3 (Kv4.3(-/-)), the pore-forming (α) subunits of I(A) channels. Mice lacking either Kv1.4 or Kv4.2 α subunits have markedly shorter (0.5 h) periods of locomotor activity than wild-type (WT) mice. In vitro extracellular multi-electrode recordings revealed that Kv1.4(-/-) and Kv4.2(-/-) SCN neurons display circadian rhythms in repetitive firing, but with shorter periods (0.5 h) than WT cells. In contrast, the periods of wheel-running activity in Kv4.3(-/-) mice and firing in Kv4.3(-/-) SCN neurons were indistinguishable from WT animals and neurons. Quantitative real-time PCR revealed that the transcripts encoding all three Kv channel α subunits, Kv1.4, Kv4.2, and Kv4.3, are expressed constitutively throughout the day and night in the SCN. Together, these results demonstrate that Kv1.4- and Kv4.2-encoded I(A) channels regulate the intrinsic excitability of SCN neurons during the day and night and determine the period and amplitude of circadian rhythms in SCN neuron firing and locomotor behavior.

  11. Kv7 (KCNQ) channel openers induce hypothermia in the mouse.

    PubMed

    Kristensen, Line V; Sandager-Nielsen, Karin; Hansen, Henrik H

    2011-01-20

    Kv7 channels, encoded by corresponding kcnq genes, are expressed both centrally and peripherally where they serve to dampen neuronal activity. While Kv7 channel openers have shown efficacy in neurological and neuropsychiatric disease models, the impact of Kv7 channel activation on physiological endpoint markers have not been addressed in detail. In this study we assessed the effect of a range of Kv7 channel openers with different affinity for neuronal Kv7.2-5 channel subunits on body temperature regulation in mice. Female NMRI mice were acutely exposed to vehicle (10% Tween-80, i.p.), retigabine (3-30 mg/kg, i.p., pan-Kv7 channel opener), (S)BMS-204352 (60-240 mg/kg, i.p., Kv7.4/5 channel-preferring opener), ICA-27243 (1-10mg/kg, i.p., Kv7.2/3 channel-preferring opener), or S-(1) (10-60 mg/kg, i.p., Kv7.2/3 channel-preferring opener), and rectal body temperature was measured 15-120 min post-injection. Retigabine (>10mg/kg), ICA-27243 (≥ 10 mg/kg), and S-(1) (≥ 30 mg/kg) dose-dependently lowered rectal body temperature with maximal doses of each Kv7 channel opener inducing a marked drop (>4°C) in rectal temperature. The Kv7 channel openers showed differential temporal pharmacodynamics, which likely reflects their different pharmacokinetic profiles. Pretreatment with the pan-Kv7 channel blocker XE-991 (1.0mg/kg, i.p.) completely reversed the hypothermic effect of the pan-Kv7 opener, retigabine (15 mg/kg), whereas ICA-27243-induced hypothermia (10mg/kg) could only be partially prevented by XE-991. Because ICA-27743 and S-(1) are Kv7.2/3 channel subunit-preferring compounds, this suggests that the Kv7.2/3 channel isoform is the predominant substrate for Kv7 channel opener-evoked hypothermia. These data indicate the physiological relevance of Kv7 channel function on body temperature regulation which may potentially reside from central inhibitory Kv7 channel activity. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  12. Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating.

    PubMed

    Bähring, R; Dannenberg, J; Peters, H C; Leicher, T; Pongs, O; Isbrandt, D

    2001-06-29

    Association of Kv channel-interacting proteins (KChIPs) with Kv4 channels leads to modulation of these A-type potassium channels (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). We cloned a KChIP2 splice variant (KChIP2.2) from human ventricle. In comparison with KChIP2.1, coexpression of KChIP2.2 with human Kv4 channels in mammalian cells slowed the onset of Kv4 current inactivation (2-3-fold), accelerated the recovery from inactivation (5-7-fold), and shifted Kv4 steady-state inactivation curves by 8-29 mV to more positive potentials. The features of Kv4.2/KChIP2.2 currents closely resemble those of cardiac rapidly inactivating transient outward currents. KChIP2.2 stimulated the Kv4 current density in Chinese hamster ovary cells by approximately 55-fold. This correlated with a redistribution of immunoreactivity from perinuclear areas to the plasma membrane. Increased Kv4 cell-surface expression and current density were also obtained in the absence of KChIP2.2 when the highly conserved proximal Kv4 N terminus was deleted. The same domain is required for association of KChIP2.2 with Kv4 alpha-subunits. We propose that an efficient transport of Kv4 channels to the cell surface depends on KChIP binding to the Kv4 N-terminal domain. Our data suggest that the binding is necessary, but not sufficient, for the functional activity of KChIPs.

  13. Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells.

    PubMed

    Jerng, Henry H; Pfaffinger, Paul J

    2012-01-01

    Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (I(SA)). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of I(SA) found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native I(SA) channels. Differential expression of DPP6 variants was detected in rat CG cells, with DPP6K (41 ± 3%)>DPP6a (33 ± 3%)>DPP6S (18 ± 2%)>DPP6L (8 ± 3%). To better understand how DPP6 variants shape native neuronal I(SA), we focused on studying interactions between the two dominant variants, DPP6K and DPP6a. Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve. By contrast, DPP6a uses its distinct N-terminus to directly confer rapid N-type inactivation independently of KChIP3a. When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function. The more rapid inactivation from DPP6a dominates during strong depolarization; however, DPP6K produces a negative shift in the steady-state inactivation curve and introduces a slow phase of recovery from inactivation. A direct comparison to the native CG cell I(SA) shows that these mixed effects are present in the native channels. Our results support the hypothesis that the precise expression and co-assembly of different auxiliary subunit variants are important factors in shaping the I

  14. The Kv7 Channel and Cardiovascular Risk Factors.

    PubMed

    Fosmo, Andreas L; Skraastad, Øyvind B

    2017-01-01

    Potassium channels play a pivotal role in the regulation of excitability in cells such as neurons, cardiac myocytes, and vascular smooth muscle cells. The KCNQ (Kv7) family of voltage-activated K + channels hyperpolarizes the cell and stabilizes the membrane potential. Here, we outline how Kv7 channel activity may contribute to the development of the cardiovascular risk factors such as hypertension, diabetes, and obesity. Questions and hypotheses regarding previous and future research have been raised. Alterations in the Kv7 channel may contribute to the development of cardiovascular disease (CVD). Pharmacological modification of Kv7 channels may represent a possible treatment for CVD in the future.

  15. The Kv7 Channel and Cardiovascular Risk Factors

    PubMed Central

    Fosmo, Andreas L.; Skraastad, Øyvind B.

    2017-01-01

    Potassium channels play a pivotal role in the regulation of excitability in cells such as neurons, cardiac myocytes, and vascular smooth muscle cells. The KCNQ (Kv7) family of voltage-activated K+ channels hyperpolarizes the cell and stabilizes the membrane potential. Here, we outline how Kv7 channel activity may contribute to the development of the cardiovascular risk factors such as hypertension, diabetes, and obesity. Questions and hypotheses regarding previous and future research have been raised. Alterations in the Kv7 channel may contribute to the development of cardiovascular disease (CVD). Pharmacological modification of Kv7 channels may represent a possible treatment for CVD in the future. PMID:29259974

  16. Modulation of Kv7 channels and excitability in the brain.

    PubMed

    Greene, Derek L; Hoshi, Naoto

    2017-02-01

    Neuronal Kv7 channels underlie a voltage-gated non-inactivating potassium current known as the M-current. Due to its particular characteristics, Kv7 channels show pronounced control over the excitability of neurons. We will discuss various factors that have been shown to drastically alter the activity of this channel such as protein and phospholipid interactions, phosphorylation, calcium, and numerous neurotransmitters. Kv7 channels locate to key areas for the control of action potential initiation and propagation. Moreover, we will explore the dynamic surface expression of the channel modulated by neurotransmitters and neural activity. We will also focus on known principle functions of neural Kv7 channels: control of resting membrane potential and spiking threshold, setting the firing frequency, afterhyperpolarization after burst firing, theta resonance, and transient hyperexcitability from neurotransmitter-induced suppression of the M-current. Finally, we will discuss the contribution of altered Kv7 activity to pathologies such as epilepsy and cognitive deficits.

  17. Modulation of Kv7 channels and excitability in the brain

    PubMed Central

    Greene, Derek L; Hoshi, Naoto

    2016-01-01

    Neuronal Kv7 channels underlie a voltage-gated non-inactivating potassium current known as the M-current. Due to its particular characteristics, Kv7 channels show pronounced control over the excitability of neurons. We will discuss various factors that have been shown to drastically alter the activity of this channel such as protein and phospholipid interactions, phosphorylation, calcium, and numerous neurotransmitters. Kv7 channels locate to key areas for the control of action potential initiation and propagation. Moreover, we will explore the dynamic surface expression of the channel modulated by neurotransmitters and neural activity. We will also focus on known principle functions of neural Kv7 channels: control of resting membrane potential and spiking threshold, setting the firing frequency, afterhyperpolarization after burst firing, theta resonance, and transient hyperexcitability from neurotransmitter-induced suppression of the M-current. Finally, we will discuss the contribution of altered Kv7 activity to pathologies such as epilepsy and cognitive deficits. PMID:27645822

  18. The tetramerization domain potentiates Kv4 channel function by suppressing closed-state inactivation.

    PubMed

    Tang, Yi-Quan; Zhou, Jing-Heng; Yang, Fan; Zheng, Jie; Wang, KeWei

    2014-09-02

    A-type Kv4 potassium channels undergo a conformational change toward a nonconductive state at negative membrane potentials, a dynamic process known as pre-open closed states or closed-state inactivation (CSI). CSI causes inhibition of channel activity without the prerequisite of channel opening, thus providing a dynamic regulation of neuronal excitability, dendritic signal integration, and synaptic plasticity at resting. However, the structural determinants underlying Kv4 CSI remain largely unknown. We recently showed that the auxiliary KChIP4a subunit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts with Kv4.3 channels to enhance CSI. In this study, we utilized the KChIP4a KID to probe key structural elements underlying Kv4 CSI. Using fluorescence resonance energy transfer two-hybrid mapping and bimolecular fluorescence complementation-based screening combined with electrophysiology, we identified the intracellular tetramerization (T1) domain that functions to suppress CSI and serves as a receptor for the binding of KID. Disrupting the Kv4.3 T1-T1 interaction interface by mutating C110A within the C3H1 motif of T1 domain facilitated CSI and ablated the KID-mediated enhancement of CSI. Furthermore, replacing the Kv4.3 T1 domain with the T1 domain from Kv1.4 (without the C3H1 motif) or Kv2.1 (with the C3H1 motif) resulted in channels functioning with enhanced or suppressed CSI, respectively. Taken together, our findings reveal a novel (to our knowledge) role of the T1 domain in suppressing Kv4 CSI, and that KChIP4a KID directly interacts with the T1 domain to facilitate Kv4.3 CSI, thus leading to inhibition of channel function. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. Functional assembly of Kv7.1/Kv7.5 channels with emerging properties on vascular muscle physiology.

    PubMed

    Oliveras, Anna; Roura-Ferrer, Meritxell; Solé, Laura; de la Cruz, Alicia; Prieto, Angela; Etxebarria, Ainhoa; Manils, Joan; Morales-Cano, Daniel; Condom, Enric; Soler, Concepció; Cogolludo, Angel; Valenzuela, Carmen; Villarroel, Alvaro; Comes, Núria; Felipe, Antonio

    2014-07-01

    Voltage-dependent K(+) (Kv) channels from the Kv7 family are expressed in blood vessels and contribute to cardiovascular physiology. Although Kv7 channel blockers trigger muscle contractions, Kv7 activators act as vasorelaxants. Kv7.1 and Kv7.5 are expressed in many vessels. Kv7.1 is under intense investigation because Kv7.1 blockers fail to modulate smooth muscle reactivity. In this study, we analyzed whether Kv7.1 and Kv7.5 may form functional heterotetrameric channels increasing the channel diversity in vascular smooth muscles. Kv7.1 and Kv7.5 currents elicited in arterial myocytes, oocyte, and mammalian expression systems suggest the formation of heterotetrameric complexes. Kv7.1/Kv7.5 heteromers, exhibiting different pharmacological characteristics, participate in the arterial tone. Kv7.1/Kv7.5 associations were confirmed by coimmunoprecipitation, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching experiments. Kv7.1/Kv7.5 heterotetramers were highly retained at the endoplasmic reticulum. Studies in HEK-293 cells, heart, brain, and smooth and skeletal muscles demonstrated that the predominant presence of Kv7.5 stimulates release of Kv7.1/Kv7.5 oligomers out of lipid raft microdomains. Electrophysiological studies supported that KCNE1 and KCNE3 regulatory subunits further increased the channel diversity. Finally, the analysis of rat isolated myocytes and human blood vessels demonstrated that Kv7.1 and Kv7.5 exhibited a differential expression, which may lead to channel diversity. Kv7.1 and Kv7.5 form heterotetrameric channels increasing the diversity of structures which fine-tune blood vessel reactivity. Because the lipid raft localization of ion channels is crucial for cardiovascular physiology, Kv7.1/Kv7.5 heteromers provide efficient spatial and temporal regulation of smooth muscle function. Our results shed light on the debate about the contribution of Kv7 channels to vasoconstriction and hypertension. © 2014 American

  20. KV1 and KV3 Potassium Channels Identified at Presynaptic Terminals of the Corticostriatal Synapses in Rat

    PubMed Central

    Meneses, David; Vega, Ana V.; Torres-Cruz, Francisco Miguel; Barral, Jaime

    2016-01-01

    In the last years it has been increasingly clear that KV-channel activity modulates neurotransmitter release. The subcellular localization and composition of potassium channels are crucial to understanding its influence on neurotransmitter release. To investigate the role of KV in corticostriatal synapses modulation, we combined extracellular recording of population-spike and pharmacological blockage with specific and nonspecific blockers to identify several families of KV channels. We induced paired-pulse facilitation (PPF) and studied the changes in paired-pulse ratio (PPR) before and after the addition of specific KV blockers to determine whether particular KV subtypes were located pre- or postsynaptically. Initially, the presence of KV channels was tested by exposing brain slices to tetraethylammonium or 4-aminopyridine; in both cases we observed a decrease in PPR that was dose dependent. Further experiments with tityustoxin, margatoxin, hongotoxin, agitoxin, dendrotoxin, and BDS-I toxins all rendered a reduction in PPR. In contrast heteropodatoxin and phrixotoxin had no effect. Our results reveal that corticostriatal presynaptic KV channels have a complex stoichiometry, including heterologous combinations KV1.1, KV1.2, KV1.3, and KV1.6 isoforms, as well as KV3.4, but not KV4 channels. The variety of KV channels offers a wide spectrum of possibilities to regulate neurotransmitter release, providing fine-tuning mechanisms to modulate synaptic strength. PMID:27379187

  1. Differential Protein Kinase C-dependent Modulation of Kv7.4 and Kv7.5 Subunits of Vascular Kv7 Channels*

    PubMed Central

    Brueggemann, Lioubov I.; Mackie, Alexander R.; Cribbs, Leanne L.; Freda, Jessica; Tripathi, Abhishek; Majetschak, Matthias; Byron, Kenneth L.

    2014-01-01

    The Kv7 family (Kv7.1–7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels. PMID:24297175

  2. The phosphoinositide sensitivity of the KV channel family

    PubMed Central

    Kruse, Martin; Hille, Bertil

    2013-01-01

    Recently, we screened several KV channels for possible dependence on plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The channels were expressed in tsA-201 cells and the PI(4,5)P2 was depleted by several manipulations in whole-cell experiments with parallel measurements of channel activity. In contrast to reports on excised-patches using Xenopus laevis oocytes, we found only KV7, but none of the other tested KV channels, to be strongly dependent on PI(4,5)P2. We now have extended our study to KV1.2 channels, a KV channel we had not previously tested, because a new published study on excised patches showed regulation of the voltage-dependence of activation by PI(4,5)P2. In full agreement with those published results, we found a reduction of current amplitude by ~20% after depletion of PI(4,5)P2 and a small left shift in the activation curve of KV1.2 channels. We also found a small reduction of KV11.1 (hERG) currents that was not accompanied by a gating shift. In conclusion, our whole-cell methods yield a PI(4,5)P2-dependence of KV1.2 currents in tsA-201 cells that is comparable to findings from excised patches of Xenopus laevis oocytes. We discuss possible physiological rationales for PI(4,5)P2 sensitivity of some ion channels and insensitivity of others. PMID:23907203

  3. Effects of haloperidol on Kv4.3 potassium channels.

    PubMed

    Lee, Hong Joon; Sung, Ki-Wug; Hahn, Sang June

    2014-10-05

    Haloperidol is commonly used in clinical practice to treat acute and chronic psychosis, but it also has been associated with adverse cardiovascular events. We investigated the effects of haloperidol on Kv4.3 currents stably expressed in CHO cells using a whole-cell patch-clamp technique. Haloperidol did not significantly inhibit the peak amplitude of Kv4.3, but accelerated the decay rate of inactivation of Kv4.3 in a concentration-dependent manner. Thus, the effects of haloperidol on Kv4.3 were estimated from the integral of the Kv4.3 currents during the depolarization pulse. The Kv4.3 was decreased by haloperidol in a concentration-dependent manner with an IC50 value of 3.6 μM. Haloperidol accelerated the decay rate of Kv4.3 inactivation and activation kinetics in a concentration-dependent manner, thereby decreasing the time-to-peak. Haloperidol shifted the voltage dependence of the steady-state activation and inactivation of Kv4.3 in a hyperpolarizing direction. Haloperidol also caused an acceleration of the closed-state inactivation of Kv4.3. Haloperidol produced a use-dependent block of Kv4.3, which was accompanied by a slowing of recovery from the inactivation of Kv4.3. These results suggest that haloperidol blocks Kv4.3 by both interacting with the open state of Kv4.3 channels during depolarization and accelerating the closed-state inactivation at subthreshold membrane potentials. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Differential Activation of Vascular Smooth Muscle Kv7.4, Kv7.5, and Kv7.4/7.5 Channels by ML213 and ICA-069673

    PubMed Central

    Brueggemann, Lyubov I.; Haick, Jennifer M.; Cribbs, Leanne L.

    2014-01-01

    Recent research suggests that smooth muscle cells express Kv7.4 and Kv7.5 voltage-activated potassium channels, which contribute to maintenance of their resting membrane voltage. New pharmacologic activators of Kv7 channels, ML213 (N-mesitybicyclo[2.2.1]heptane-2-carboxamide) and ICA-069673 N-(6-chloropyridin-3-yl)-3,4-difluorobenzamide), have been reported to discriminate among channels formed from different Kv7 subtypes. We compared the effects of ML213 and ICA-069673 on homomeric human Kv7.4, Kv7.5, and heteromeric Kv7.4/7.5 channels exogenously expressed in A7r5 vascular smooth muscle cells. We found that, despite its previous description as a selective activator of Kv7.2 and Kv7.4, ML213 significantly increased the maximum conductance of homomeric Kv7.4 and Kv7.5, as well as heteromeric Kv7.4/7.5 channels, and induced a negative shift of their activation curves. Current deactivation rates decreased in the presence of the ML213 (10 μM) for all three channel combinations. Mutants of Kv7.4 (W242L) and Kv7.5 (W235L), previously found to be insensitive to another Kv7 channel activator, retigabine, were also insensitive to ML213 (10 μM). In contrast to ML213, ICA-069673 robustly activated Kv7.4 channels but was significantly less effective on homomeric Kv7.5 channels. Heteromeric Kv7.4/7.5 channels displayed intermediate responses to ICA-069673. In each case, ICA-069673 induced a negative shift of the activation curves without significantly increasing maximal conductance. Current deactivation rates decreased in the presence of ICA-069673 in a subunit-specific manner. Kv7.4 W242L responded to ICA-069673-like wild-type Kv7.4, but a Kv7.4 F143A mutant was much less sensitive to ICA-069673. Based on these results, ML213 and ICA-069673 likely bind to different sites and are differentially selective among Kv7.4, Kv7.5, and Kv7.4/7.5 channel subtypes. PMID:24944189

  5. Complex expression and localization of inactivating Kv channels in cultured hippocampal astrocytes.

    PubMed

    Bekar, Lane K; Loewen, Matthew E; Cao, Kun; Sun, Xianfeng; Leis, Jerome; Wang, Rui; Forsyth, George W; Walz, Wolfgang

    2005-03-01

    Voltage-gated potassium channels are well established as critical for setting action potential frequency, membrane potential, and neurotransmitter release in neurons. However, their role in the "nonexcitable" glial cell type is yet to be fully understood. We used whole cell current kinetics, pharmacology, immunocytochemistry, and RT-PCR to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that approximately 70, 10, and <5% of total A current is associated with Kv4, Kv3, and Kv1 channels, respectively. In addition, pharmacology and kinetics provide evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Given this complex A-type channel expression pattern, we assessed the role of A currents in membrane voltage oscillations in response to current injections. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. As in neurons, this effect may enable astrocytes to respond rapidly to high-frequency synaptic events. Our results indicate that hippocampal astrocytes in vitro express multiple A-type Kv channel alpha-subunits with accessory, possibly Ca(2+)-sensitive, cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Function of these channels remains to be determined in a physiological setting. However, this study suggests that they enable astrocytes to respond rapidly with membrane voltage oscillations to high-frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity.

  6. Modulation of Kv4.2 channels by a peptide isolated from the venom of the giant bird-eating tarantula Theraphosa leblondi.

    PubMed

    Ebbinghaus, Jan; Legros, Christian; Nolting, Andreas; Guette, Catherine; Celerier, Marie-Louise; Pongs, Olaf; Bähring, Robert

    2004-06-15

    In order to find new peptide inhibitors for voltage-dependent potassium (Kv) channels, we examined the effects of venom from Theraphosa leblondi on Kv channel-mediated currents with the whole-cell patch-clamp technique. Both A-type currents in cultured hippocampal neurons and A-type currents recorded from HEK 293 cells transiently expressing recombinant Kv4.2 channels were selectively inhibited by T. leblondi venom. No venom activity was observed on recombinant Kv1.3, Kv1.4, Kv2.1 or Kv3.4 channels. We purified and sequenced three novel homologous peptides from this venom, which are related to previously identified Kv4 channel-specific peptide inhibitors and were named T. leblondi toxin (TLTx) 1, 2 and 3. The mode of action of TLTx1 on recombinant Kv4.2 channels was studied in more detail. TLTx1 inhibited Kv4.2-mediated currents with an IC50 of approximately 200 nM, and macroscopic current inactivation was slowed in the presence of TLTx1. Notably, TLTx1 also caused a shallower voltage dependence of Kv4.2 peak conductance and a shift of the activation midpoint to more positive potentials (DeltaV1/2 = +35 mV). TLTx1 caused a noticable slowing of Kv4.2 activation kinetics, and Kv4.2 deactivation kinetics were accelerated by TLTx1 as infered from Rb+ tail current measurements. Chimeric Kv2.1(4.2L3-4) channels, in which the linker region between S3 and S4 of the TLTx1-insensitive Kv2.1 channel was replaced by the corresponding Kv4.2 domain, were sensitive to TLTx1. Apparently, TLTx1 can act as a gating modifier of Kv4.2 channels. Copyright 2004 Elsevier Ltd.

  7. Kv1 channels and neural processing in vestibular calyx afferents.

    PubMed

    Meredith, Frances L; Kirk, Matthew E; Rennie, Katherine J

    2015-01-01

    Potassium-selective ion channels are important for accurate transmission of signals from auditory and vestibular sensory end organs to their targets in the central nervous system. During different gravity conditions, astronauts experience altered input signals from the peripheral vestibular system resulting in sensorimotor dysfunction. Adaptation to altered sensory input occurs, but it is not explicitly known whether this involves synaptic modifications within the vestibular epithelia. Future investigations of such potential plasticity require a better understanding of the electrophysiological mechanisms underlying the known heterogeneity of afferent discharge under normal conditions. This study advances this understanding by examining the role of the Kv1 potassium channel family in mediating action potentials in specialized vestibular afferent calyx endings in the gerbil crista and utricle. Pharmacological agents selective for different sub-types of Kv1 channels were tested on membrane responses in whole cell recordings in the crista. Kv1 channels sensitive to α-dendrotoxin and dendrotoxin-K were found to prevail in the central regions, whereas K(+) channels sensitive to margatoxin, which blocks Kv1.3 and 1.6 channels, were more prominent in peripheral regions. Margatoxin-sensitive currents showed voltage-dependent inactivation. Dendrotoxin-sensitive currents showed no inactivation and dampened excitability in calyces in central neuroepithelial regions. The differential distribution of Kv1 potassium channels in vestibular afferents supports their importance in accurately relaying gravitational and head movement signals through specialized lines to the central nervous system. Pharmacological modulation of specific groups of K(+) channels could help alleviate vestibular dysfunction on earth and in space.

  8. A Role for DPPX Modulating External TEA Sensitivity of Kv4 Channels

    PubMed Central

    Colinas, Olaia; Pérez-Carretero, Francisco D.; López-López, José R.; Pérez-García, M. Teresa

    2008-01-01

    Shal-type (Kv4) channels are expressed in a large variety of tissues, where they contribute to transient voltage-dependent K+ currents. Kv4 are the molecular correlate of the A-type current of neurons (ISA), the fast component of ITO current in the heart, and also of the oxygen-sensitive K+ current (KO2) in rabbit carotid body (CB) chemoreceptor cells. The enormous degree of variability in the physiological properties of Kv4-mediated currents can be attributable to the complexity of their regulation together with the large number of ancillary subunits and scaffolding proteins that associate with Kv4 proteins to modify their trafficking and their kinetic properties. Among those, KChIPs and DPPX proteins have been demonstrated to be integral components of ISA and ITO currents, as their coexpression with Kv4 subunits recapitulates the kinetics of native currents. Here, we explore the presence and functional contribution of DPPX to KO2 currents in rabbit CB chemoreceptor cells by using DPPX functional knockdown with siRNA. Additionally, we investigate if the presence of DPPX endows Kv4 channels with new pharmacological properties, as we have observed anomalous tetraethylammonium (TEA) sensitivity in the native KO2 currents. DPPX association with Kv4 channels induced an increased TEA sensitivity both in heterologous expression systems and in CB chemoreceptor cells. Moreover, TEA application to Kv4-DPPX heteromultimers leads to marked kinetic effects that could be explained by an augmented closed-state inactivation. Our data suggest that DPPX proteins are integral components of KO2 currents, and that their association with Kv4 subunits modulate the pharmacological profile of the heteromultimers. PMID:18411327

  9. Proteomic analysis highlights the molecular complexities of native Kv4 channel macromolecular complexes.

    PubMed

    Marionneau, Céline; Townsend, R Reid; Nerbonne, Jeanne M

    2011-04-01

    Voltage-gated K(+) (Kv) channels are key determinants of membrane excitability in the nervous and cardiovascular systems, functioning to control resting membrane potentials, shape action potential waveforms and influence the responses to neurotransmitters and neurohormones. Consistent with this functional diversity, multiple types of Kv currents, with distinct biophysical properties and cellular/subcellular distributions, have been identified. Rapidly activating and inactivating Kv currents, typically referred to as I(A) (A-type) in neurons, for example, regulate repetitive firing rates, action potential back-propagation (into dendrites) and modulate synaptic responses. Currents with similar properties, referred to as I(to,f) (fast transient outward), expressed in cardiomyocytes, control the early phase of myocardial action potential repolarization. A number of studies have demonstrated critical roles for pore-forming (α) subunits of the Kv4 subfamily in the generation of native neuronal I(A) and cardiac I(to,f) channels. Studies in heterologous cells have also suggested important roles for a number of Kv channel accessory and regulatory proteins in the generation of functional I(A) and I(to,f) channels. Quantitative mass spectrometry-based proteomic analysis is increasingly recognized as a rapid and, importantly, unbiased, approach to identify the components of native macromolecular protein complexes. The recent application of proteomic approaches to identify the components of native neuronal (and cardiac) Kv4 channel complexes has revealed even greater complexity than anticipated. The continued emphasis on development of improved biochemical and analytical proteomic methods seems certain to accelerate progress and to provide important new insights into the molecular determinants of native ion channel protein complexes. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. The epilepsy-linked Lgi1 protein assembles into presynaptic Kv1 channels and inhibits inactivation by Kvbeta1.

    PubMed

    Schulte, Uwe; Thumfart, Jörg-Oliver; Klöcker, Nikolaj; Sailer, Claudia A; Bildl, Wolfgang; Biniossek, Martin; Dehn, Doris; Deller, Thomas; Eble, Silke; Abbass, Karen; Wangler, Tanja; Knaus, Hans-Günther; Fakler, Bernd

    2006-03-02

    The voltage-gated potassium (Kv) channel subunit Kv1.1 is a major constituent of presynaptic A-type channels that modulate synaptic transmission in CNS neurons. Here, we show that Kv1.1-containing channels are complexed with Lgi1, the functionally unassigned product of the leucine-rich glioma inactivated gene 1 (LGI1), which is causative for an autosomal dominant form of lateral temporal lobe epilepsy (ADLTE). In the hippocampal formation, both Kv1.1 and Lgi1 are coassembled with Kv1.4 and Kvbeta1 in axonal terminals. In A-type channels composed of these subunits, Lgi1 selectively prevents N-type inactivation mediated by the Kvbeta1 subunit. In contrast, defective Lgi1 molecules identified in ADLTE patients fail to exert this effect resulting in channels with rapid inactivation kinetics. The results establish Lgi1 as a novel subunit of Kv1.1-associated protein complexes and suggest that changes in inactivation gating of presynaptic A-type channels may promote epileptic activity.

  11. Modulation of A-type potassium channels by a family of calcium sensors.

    PubMed

    An, W F; Bowlby, M R; Betty, M; Cao, J; Ling, H P; Mendoza, G; Hinson, J W; Mattsson, K I; Strassle, B W; Trimmer, J S; Rhodes, K J

    2000-02-03

    In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

  12. The Natural Plant Product Rottlerin Activates Kv7.1/KCNE1 Channels.

    PubMed

    Matschke, Veronika; Piccini, Ilaria; Schubert, Janina; Wrobel, Eva; Lang, Florian; Matschke, Johann; Amedonu, Elsie; Meuth, Sven G; Strünker, Timo; Strutz-Seebohm, Nathalie; Greber, Boris; Scherkenbeck, Jürgen; Seebohm, Guiscard

    2016-01-01

    Acquired as well as inherited channelopathies are disorders that are caused by altered ion channel function. A family of channels whose malfunction is associated with different channelopathies is the Kv7 K+ channel family; and restoration of normal Kv7 channel function by small molecule modulators is a promising approach for treatment of these often fatal diseases. Here, we show the modulation of Kv7 channels by the natural compound Rottlerin heterologously expressed in Xenopus laevis oocytes and on iPSC cardiomyocytes overexpressing Kv7.1 channels. We show that currents carried by Kv7.1 (EC50 = 1.48 μM), Kv7.1/KCNE1 (EC50 = 4.9 μM), and Kv7.4 (EC50 = 0.148 μM) are strongly enhanced by the compound, whereas Kv7.2, Kv7.2/Kv7.3, and Kv7.5 are not sensitive to Rottlerin. Studies on Kv7.1/KCNE1 mutants and in silico modelling indicate that Rottlerin binds to the R-L3-activator site. Rottlerin mediated activation of Kv7.1/KCNE1 channels might be a promising approach in long QT syndrome. As a proof of concept, we show that Rottlerin shortens cardiac repolarisation in iPSC-derived cardiomyocytes expressing Kv7.1. Rottlerin or an optimized derivative holds a potential as QT interval correcting drug. © 2016 The Author(s) Published by S. Karger AG, Basel.

  13. S-glutathionylation of an auxiliary subunit confers redox sensitivity to Kv4 channel inactivation.

    PubMed

    Jerng, Henry H; Pfaffinger, Paul J

    2014-01-01

    Reactive oxygen species (ROS) regulate ion channels, modulate neuronal excitability, and contribute to the etiology of neurodegenerative disorders. ROS differentially suppress fast "ball-and-chain" N-type inactivation of cloned Kv1 and Kv3 potassium channels but not of Kv4 channels, likely due to a lack of reactive cysteines in Kv4 N-termini. Recently, we discovered that N-type inactivation of Kv4 channel complexes can be independently conferred by certain N-terminal variants of Kv4 auxiliary subunits (DPP6a, DPP10a). Here, we report that both DPP6a and DPP10a, like Kv subunits with redox-sensitive N-type inactivation, contain a highly conserved cysteine in their N-termini (Cys-13). To test if N-type inactivation mediated by DPP6a or DPP10a is redox sensitive, Xenopus oocyte recordings were performed to examine the effects of two common oxidants, tert-butyl hydroperoxide (tBHP) and diamide. Both oxidants markedly modulate DPP6a- or DPP10a-conferred N-type inactivation of Kv4 channels, slowing the overall inactivation and increasing the peak current. These functional effects are fully reversed by the reducing agent dithiothreitol (DTT) and appear to be due to a selective modulation of the N-type inactivation mediated by these auxiliary subunits. Mutation of DPP6a Cys-13 to serine eliminated the tBHP or diamide effects, confirming the importance of Cys-13 to the oxidative regulation. Biochemical studies designed to elucidate the underlying molecular mechanism show no evidence of protein-protein disulfide linkage formation following cysteine oxidation. Instead, using a biotinylated glutathione (BioGEE) reagent, we discovered that oxidation by tBHP or diamide leads to S-glutathionylation of Cys-13, suggesting that S-glutathionylation underlies the regulation of fast N-type inactivation by redox. In conclusion, our studies suggest that Kv4-based A-type current in neurons may show differential redox sensitivity depending on whether DPP6a or DPP10a is highly expressed

  14. Dysfunction of the Heteromeric KV7.3/KV7.5 Potassium Channel is Associated with Autism Spectrum Disorders.

    PubMed

    Gilling, Mette; Rasmussen, Hanne B; Calloe, Kirstine; Sequeira, Ana F; Baretto, Marta; Oliveira, Guiomar; Almeida, Joana; Lauritsen, Marlene B; Ullmann, Reinhard; Boonen, Susanne E; Brondum-Nielsen, Karen; Kalscheuer, Vera M; Tümer, Zeynep; Vicente, Astrid M; Schmitt, Nicole; Tommerup, Niels

    2013-01-01

    Heterozygous mutations in the KCNQ3 gene on chromosome 8q24 encoding the voltage-gated potassium channel KV7.3 subunit have previously been associated with rolandic epilepsy and idiopathic generalized epilepsy (IGE) including benign neonatal convulsions. We identified a de novo t(3;8) (q21;q24) translocation truncating KCNQ3 in a boy with childhood autism. In addition, we identified a c.1720C > T [p.P574S] nucleotide change in three unrelated individuals with childhood autism and no history of convulsions. This nucleotide change was previously reported in patients with rolandic epilepsy or IGE and has now been annotated as a very rare SNP (rs74582884) in dbSNP. The p.P574S KV7.3 variant significantly reduced potassium current amplitude in Xenopus laevis oocytes when co-expressed with KV7.5 but not with KV7.2 or KV7.4. The nucleotide change did not affect trafficking of heteromeric mutant KV7.3/2, KV7.3/4, or KV7.3/5 channels in HEK 293 cells or primary rat hippocampal neurons. Our results suggest that dysfunction of the heteromeric KV7.3/5 channel is implicated in the pathogenesis of some forms of autism spectrum disorders, epilepsy, and possibly other psychiatric disorders and therefore, KCNQ3 and KCNQ5 are suggested as candidate genes for these disorders.

  15. Dysfunction of the Heteromeric KV7.3/KV7.5 Potassium Channel is Associated with Autism Spectrum Disorders

    PubMed Central

    Gilling, Mette; Rasmussen, Hanne B.; Calloe, Kirstine; Sequeira, Ana F.; Baretto, Marta; Oliveira, Guiomar; Almeida, Joana; Lauritsen, Marlene B.; Ullmann, Reinhard; Boonen, Susanne E.; Brondum-Nielsen, Karen; Kalscheuer, Vera M.; Tümer, Zeynep; Vicente, Astrid M.; Schmitt, Nicole; Tommerup, Niels

    2012-01-01

    Heterozygous mutations in the KCNQ3 gene on chromosome 8q24 encoding the voltage-gated potassium channel KV7.3 subunit have previously been associated with rolandic epilepsy and idiopathic generalized epilepsy (IGE) including benign neonatal convulsions. We identified a de novo t(3;8) (q21;q24) translocation truncating KCNQ3 in a boy with childhood autism. In addition, we identified a c.1720C > T [p.P574S] nucleotide change in three unrelated individuals with childhood autism and no history of convulsions. This nucleotide change was previously reported in patients with rolandic epilepsy or IGE and has now been annotated as a very rare SNP (rs74582884) in dbSNP. The p.P574S KV7.3 variant significantly reduced potassium current amplitude in Xenopus laevis oocytes when co-expressed with KV7.5 but not with KV7.2 or KV7.4. The nucleotide change did not affect trafficking of heteromeric mutant KV7.3/2, KV7.3/4, or KV7.3/5 channels in HEK 293 cells or primary rat hippocampal neurons. Our results suggest that dysfunction of the heteromeric KV7.3/5 channel is implicated in the pathogenesis of some forms of autism spectrum disorders, epilepsy, and possibly other psychiatric disorders and therefore, KCNQ3 and KCNQ5 are suggested as candidate genes for these disorders. PMID:23596459

  16. Mechanism of the modulation of Kv4:KChIP-1 channels by external K+.

    PubMed

    Kaulin, Yu A; De Santiago-Castillo, J A; Rocha, C A; Covarrubias, M

    2008-02-15

    In response to a prolonged membrane depolarization, inactivation autoregulates the activity of voltage-gated ion channels. Slow inactivation involving a localized constriction of the selectivity filter (P/C-type mechanism) is prevalent in many voltage-gated K(+) channels of the Kv1 subfamily. However, the generalization of this mechanism to other Kv channel subfamilies has remained uncertain and controversial. In agreement with a "foot-in-the-door" mechanism and the presence of ion-ion interactions in the pore, elevated external K(+) slows the development of P/C-type inactivation and accelerates its recovery. In sharp contrast and resembling the regulation of the hippocampal A-type K(+) current, we found that Kv4.x channels associated with KChIP-1 (an auxiliary subunit) exhibit accelerated inactivation and unaffected recovery from inactivation when exposed to elevated external K(+). This regulation depends on the ability of a permeant ion to enter the selectivity filter (K(+) = Rb(+) = NH4(+) > Cs(+) > Na(+)); and the apparent equilibrium dissociation constant of a single regulatory site is 8 mM for K(+). By applying a robust quantitative global kinetic modeling approach to all macroscopic properties over a 210-mV range of membrane potentials, we determined that elevated external K(+) inhibits unstable closed states outside the main activation pathway and thereby promotes preferential closed-state inactivation. These results suggest the presence of a vestigial and unstable P/C-type mechanism of inactivation in Kv4 channels and strengthen the concept of novel mechanisms of closed-state inactivation. Regulation of Kv4 channel inactivation by hyperkalemia may help to explain the pathophysiology of electrolyte imbalances in excitable tissues.

  17. Regulation of Kv7.2/Kv7.3 channels by cholesterol: Relevance of an optimum plasma membrane cholesterol content.

    PubMed

    Delgado-Ramírez, Mayra; Sánchez-Armass, Sergio; Meza, Ulises; Rodríguez-Menchaca, Aldo A

    2018-05-01

    Kv7.2/Kv7.3 channels are the molecular correlate of the M-current, which stabilizes the membrane potential and controls neuronal excitability. Previous studies have shown the relevance of plasma membrane lipids on both M-currents and Kv7.2/Kv7.3 channels. Here, we report the sensitive modulation of Kv7.2/Kv7.3 channels by membrane cholesterol level. Kv7.2/Kv7.3 channels transiently expressed in HEK-293 cells were significantly inhibited by decreasing the cholesterol level in the plasma membrane by three different pharmacological strategies: methyl-β-cyclodextrin (MβCD), Filipin III, and cholesterol oxidase treatment. Surprisingly, Kv7.2/Kv7.3 channels were also inhibited by membrane cholesterol loading with the MβCD/cholesterol complex. Depletion or enrichment of plasma membrane cholesterol differentially affected the biophysical parameters of the macroscopic Kv7.2/Kv7.3 currents. These results indicate a complex mechanism of Kv7.2/Kv7.3 channels modulation by membrane cholesterol. We propose that inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol depletion involves a loss of a direct cholesterol-channel interaction. However, the inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol enrichment could include an additional direct cholesterol-channel interaction, or changes in the physical properties of the plasma membrane. In summary, our results indicate that an optimum cholesterol level in the plasma membrane is required for the proper functioning of Kv7.2/Kv7.3 channels. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2018-01-01

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

  19. Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

    SciTech Connect

    Roura-Ferrer, Meritxell; Sole, Laura; Martinez-Marmol, Ramon

    Voltage-dependent K{sup +} channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G{sub 1}-phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletalmore » muscle cell proliferation.« less

  20. Kv channel subunits that contribute to voltage-gated K+ current in renal vascular smooth muscle.

    PubMed

    Fergus, Daniel J; Martens, Jeffrey R; England, Sarah K

    2003-03-01

    The rat renal arterial vasculature displays differences in K(+) channel current phenotypes along its length. Small arcuate to cortical radial arteries express a delayed rectifier phenotype, while the predominant Kv current in larger arcuate and interlobar arteries is composed of both transient and sustained components. We sought to determine whether Kvalpha subunits in the rat renal interlobar and arcuate arteries form heterotetramers, which may account for the unique currents, and whether modulatory Kvbeta subunits are present in renal vascular smooth muscle cells. RT-PCR indicated the presence of several different Kvalpha subunit isoform transcripts. Co-immunoprecipitation with immunoblotting and immunohistochemical evidence suggests that a portion of the K(+) current phenotype is a heteromultimer containing delayed-rectifier Kv1.2 and A-type Kv1.4 channel subunits. RT-PCR and immunoblot analyses also demonstrated the presence of both Kvbeta1.2 and Kvbeta1.3 in renal arteries. These results suggest that heteromultimeric formation of Kvalpha subunits and the presence of modulatory Kvbeta subunits are important factors in mediating Kv currents in the renal microvasculature and suggest a potentially critical role for these channel subunits in blood pressure regulation.

  1. Dendritic A-type potassium channel subunit expression in CA1 hippocampal interneurons.

    PubMed

    Menegola, M; Misonou, H; Vacher, H; Trimmer, J S

    2008-06-26

    Voltage-gated potassium (Kv) channels are important and diverse determinants of neuronal excitability and exhibit specific expression patterns throughout the brain. Among Kv channels, Kv4 channels are major determinants of somatodendritic A-type current and are essential in controlling the amplitude of backpropagating action potentials (BAPs) into neuronal dendrites. BAPs have been well studied in a variety of neurons, and have been recently described in hippocampal and cortical interneurons, a heterogeneous population of GABAergic inhibitory cells that regulate activity of principal cells and neuronal networks. We used well-characterized mouse monoclonal antibodies against the Kv4.3 and potassium channel interacting protein (KChIP) 1 subunits of A-type Kv channels, and antibodies against different interneuron markers in single- and double-label immunohistochemistry experiments to analyze the expression patterns of Kv4.3 and KChIP1 in hippocampal Ammon's horn (CA1) neurons. Immunohistochemistry was performed on 40 mum rat brain sections using nickel-enhanced diaminobenzidine staining or multiple-label immunofluorescence. Our results show that Kv4.3 and KChIP1 component subunits of A-type channels are co-localized in the soma and dendrites of a large number of GABAergic hippocampal interneurons. These subunits co-localize extensively but not completely with markers defining the four major interneuron subpopulations tested (parvalbumin, calbindin, calretinin, and somatostatin). These results suggest that CA1 hippocampal interneurons can be divided in two groups according to the expression of Kv4.3/KChIP1 channel subunits. Antibodies against Kv4.3 and KChIP1 represent an important new tool for identifying a subpopulation of hippocampal interneurons with a unique dendritic A-type channel complement and ability to control BAPs.

  2. Lower KV7.5 Potassium Channel Subunit Expression in an Animal Model of Paroxysmal Dystonia.

    PubMed

    Sander, Svenja E; Diwan, Mustansir; Raymond, Roger; Nobrega, José N; Richter, Angelika

    2016-01-01

    Dystonia is a hyperkinetic disabling movement disorder. In the dt(sz) hamster, a model of paroxysmal dystonia, pronounced antidystonic effects of the KV7.2-5 potassium channel opener retigabine and aggravation of dystonia by a selective KV7.2-5 blocker indicated a pathophysiological role of an abnormal expression of KV7 channels. We therefore investigated the expression of KV7 subunits in brains of dystonic hamsters. While KV7.2 and KV7.3 subunits were unaltered, lower KV7.5 mRNA levels became evident in motor areas and in limbic structures of dystonic hamsters. The KV7.2/3 subunit-preferring channel opener N-(6-chloropyridin-3-yl)-3,4- difluorobenzamide (ICA 27243; 10-30 mg/kg i.p.) failed to reduce the severity of dystonia in mutant hamsters, suggesting that the previously observed antidystonic action of retigabine is mediated by the activation of KV7.5 channels. The experiments indicate a functional relevance for KV7.5 channels in paroxysmal dystonia. We suggest that compounds highly selective for subtypes of KV7 channels, i.e. for KV7.5, may provide new therapeutic approaches.

  3. Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids.

    PubMed

    Oliver, Dominik; Lien, Cheng-Chang; Soom, Malle; Baukrowitz, Thomas; Jonas, Peter; Fakler, Bernd

    2004-04-09

    Voltage-gated potassium (Kv) channels control action potential repolarization, interspike membrane potential, and action potential frequency in excitable cells. It is thought that the combinatorial association between distinct alpha and beta subunits determines whether Kv channels function as non-inactivating delayed rectifiers or as rapidly inactivating A-type channels. We show that membrane lipids can convert A-type channels into delayed rectifiers and vice versa. Phosphoinositides remove N-type inactivation from A-type channels by immobilizing the inactivation domains. Conversely, arachidonic acid and its amide anandamide endow delayed rectifiers with rapid voltage-dependent inactivation. The bidirectional control of Kv channel gating by lipids may provide a mechanism for the dynamic regulation of electrical signaling in the nervous system.

  4. Regulation of Kv2.1 K+ conductance by cell surface channel density

    PubMed Central

    Fox, Philip D.; Loftus, Rob J.; Tamkun, Michael M.

    2013-01-01

    The Kv2.1 voltage-gated K+ channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are non-conducting. Using TIRF microscopy the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared to K+ channel conductance measured by whole-cell voltage-clamp of the same cell. This approach indicated that as channel density increases non-clustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the non-conducting state with 17% conducting K+ at higher surface densities. The non-conducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immuno-fluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared to the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 days, respectively. Together these data indicate that the non-conducting state depends primarily on surface density as opposed to cluster location and that this non-conducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K+ conductance further supports a non-conducting role for Kv2.1 in excitable tissues. PMID:23325261

  5. Phosphatidic acid modulation of Kv channel voltage sensor function.

    PubMed

    Hite, Richard K; Butterwick, Joel A; MacKinnon, Roderick

    2014-10-06

    Membrane phospholipids can function as potent regulators of ion channel function. This study uncovers and investigates the effect of phosphatidic acid on Kv channel gating. Using the method of reconstitution into planar lipid bilayers, in which protein and lipid components are defined and controlled, we characterize two effects of phosphatidic acid. The first is a non-specific electrostatic influence on activation mediated by electric charge density on the extracellular and intracellular membrane surfaces. The second is specific to the presence of a primary phosphate group, acts only through the intracellular membrane leaflet and depends on the presence of a particular arginine residue in the voltage sensor. Intracellular phosphatidic acid accounts for a nearly 50 mV shift in the midpoint of the activation curve in a direction consistent with stabilization of the voltage sensor's closed conformation. These findings support a novel mechanism of voltage sensor regulation by the signaling lipid phosphatidic acid.

  6. Inactivation of the cloned potassium channel mouse Kv1.1 by the human Kv3.4 'ball' peptide and its chemical modification.

    PubMed Central

    Stephens, G J; Robertson, B

    1995-01-01

    1. This study used the whole-cell patch clamp technique to investigate the action of a 28-mer 'inactivation peptide' based on part of the N-terminal sequence of the human Kv3.4 K+ channel (hKv3.4 peptide) on the cloned mouse brain K+ channel mKv1.1 expressed in Chinese hamster ovary (CHO) cells, and compared this with the inactivation produced by Shaker B inactivation peptide (ShB peptide). 2. Inclusion of the hKv3.4 peptide in the patch electrode (320 microM) transformed non-inactivating mKv1.1 into a rapidly inactivating current. The voltage dependence of time constants of decay and steady-state inactivation induced by hKv3.4 peptide were characteristic of an 'A-type' K+ current. 3. The hKv3.4 peptide had no effect on the voltage dependence of activation of mKv1.1, with a mid-point of activation of -8 mV, and a slope factor of 15 mV. Steady-state inactivation curves had a mid-point of inactivation of -36 mV and a slope factor of -7 mV; the time constant of recovery from inactivation at -90 mV was 1.3 s. 4. The chemical modification reagents N-bromoacetamide (NBA, 100 microM) and chloramine-T (CL-T, 500 microM) had no effect on the fast inactivation of mKv1.1 induced by ShB peptide. In contrast, the inactivation caused by hKv3.4 peptide was removed by brief exposure to NBA and CL-T. 5. Chemical modification resulted in a hyperpolarizing shift of -8 mV (CL-T) and -11 mV (NBA) in the voltage dependence of activation of mKv1.1 in the presence of hKv3.4 peptide. 6. Chemical modification was critically dependent on the presence of a cysteine residue at position 6, and not position 24, of hKv3.4 peptide. 7. NBA and CL-T caused only a slight inhibition of unmodified mKv1.1 current with no significant effect on the voltage dependence of mKv1.1 activation, and also had no effect on channel deactivation at -90 mV. 8. Chemical modification experiments were consistent with a selective action on the hKv3.4 peptide itself, specifically at the cysteine residue at position 6

  7. Characteristics and molecular basis of celecoxib modulation on Kv7 potassium channels

    PubMed Central

    Du, XN; Zhang, X; Qi, JL; An, HL; Li, JW; Wan, YM; Fu, Y; Gao, HX; Gao, ZB; Zhan, Y; Zhang, HL

    2011-01-01

    BACKGROUND AND PURPOSE Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used for the treatment of pain and inflammation. Emerging and accumulating evidence suggests that celecoxib can affect cellular targets other than COX, such as ion channels. In this study, we characterized the effects of celecoxib on Kv7 K+ channels and compared its effects with the well-established Kv7 channel opener retigabine. EXPERIMENTAL APPROACH A perforated whole-cell patch technique was used to record Kv7currents expressed in HEK 293 cells and M-type currents from rat superior cervical ganglion neurons. KEY RESULTS Celecoxib enhanced Kv7.2–7.4, Kv7.2/7.3 and Kv7.3/7.5 currents but inhibited Kv7.1 and Kv7.1/KCNE1 currents and these effects were concentration dependent. The IC50 value for inhibition of Kv7.1 channels was approximately 4 µM and the EC50 values for activation of Kv7.2–7.4, Kv7.2/Kv7.3 and Kv7.3/Kv7.5 channels were approximately 2–5 µM. The effects of celecoxib were manifested by increasing current amplitudes, shifting the voltage-dependent activation curve in a more negative direction and slowing the deactivation of Kv7 currents. 2,5-Dimethyl-celecoxib, a celecoxib analogue devoid of COX inhibition activity, has similar but greater effects on Kv7currents. Kv7.2(A235T) and Kv7.2(W236L) mutant channels, which have greatly attenuated responses to retigabine, showed a reversed response to celecoxib, from activation to inhibition. CONCLUSIONS AND IMPLICATIONS These results suggest that Kv7 channels are targets of celecoxib action and provide new mechanistic evidence for understanding the effects of celecoxib. They also provide a new approach to developing Kv7 modulators and for studying the structure–function relationship of Kv7 channels. PMID:21564087

  8. Selective activation of vascular Kv7.4/Kv7.5 K+ channels by fasudil contributes to its vasorelaxant effect

    PubMed Central

    Zhang, Xuan; An, Hailong; Li, Junwei; Zhang, Yuanyuan; Liu, Yang; Jia, Zhanfeng; Zhang, Wei

    2016-01-01

    Background and Purpose Kv7 (Kv7.1–7.5) channels play an important role in the regulation of neuronal excitability and the cardiac action potential. Growing evidence suggests Kv7.4/Kv7.5 channels play a crucial role in regulating vascular smooth muscle contractility. Most of the reported Kv7 openers have shown poor selectivity across these five subtypes. In this study, fasudil – a drug used for cerebral vasospasm – has been found to be a selective opener of Kv7.4/Kv7.5 channels. Experimental Approach A perforated whole‐cell patch technique was used to record the currents and membrane potential. Homology modelling and a docking technique were used to investigate the interaction between fasudil and the Kv7.4 channel. An isometric tension recording technique was used to assess the vascular tension. Key Results Fasudil selectively and potently enhanced Kv7.4 and Kv7.4/Kv7.5 currents expressed in HEK293 cells, and shifted the voltage‐dependent activation curve in a more negative direction. Fasudil did not affect either Kv7.2 and Kv7.2/Kv7.3 currents expressed in HEK293 cells, the native neuronal M‐type K+ currents, or the resting membrane potential in small rat dorsal root ganglia neurons. The Val248 in S5 and Ile308 in S6 segment of Kv7.4 were critical for this activating effect of fasudil. Fasudil relaxed precontracted rat small arteries in a concentration‐dependent fashion; this effect was antagonized by the Kv7 channel blocker XE991. Conclusions and Implications These results suggest that fasudil is a selective Kv7.4/Kv7.5 channel opener and provide a new dimension for developing selective Kv7 modulators and a new prospective for the use, action and mechanism of fasudil. PMID:27677924

  9. Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents.

    PubMed

    Mani, Bharath K; Robakowski, Christina; Brueggemann, Lyubov I; Cribbs, Leanne L; Tripathi, Abhishek; Majetschak, Matthias; Byron, Kenneth L

    2016-03-01

    Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 > Kv7.4/Kv7.5 > Kv7.4. Copyright © 2016 by The American Society for

  10. Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents

    PubMed Central

    Mani, Bharath K.; Robakowski, Christina; Brueggemann, Lyubov I.; Cribbs, Leanne L.; Tripathi, Abhishek; Majetschak, Matthias

    2016-01-01

    Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K+ currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4. PMID:26700561

  11. Inhibition of Kv7/M Channel Currents by the Local Anesthetic Chloroprocaine.

    PubMed

    Zhang, Fan; Cheng, Yanxin; Li, Hong; Jia, Qingzhong; Zhang, Hailin; Zhao, Senming

    2015-01-01

    Chloroprocaine is a local ester anesthetic, producing excellent sensory block in clinical use. The Kv7/M potassium channel plays an important role in the control of neuronal excitability. In this study, we investigated the effects of the local anesthetic chloroprocaine on Kv7/M channels as well as the effect of retigabine on chloroprocaine-induced seizures. A perforated whole-cell patch technique was used to record Kv7 currents from HEK293 cells and M-type currents from rat dorsal root ganglion (DRG) neurons. Chloroprocaine produced a number of effects on Kv7.2/Kv7.3 currents, including a lowering of current amplitudes, a rightward shift in the voltage-dependent activation curves, and a slowing of channel activation. Chloroprocaine had a more selective inhibitory effect on the homomeric Kv7.3 and heteromeric Kv7.2/Kv7.3 channels than on the homomeric Kv7.2 channel. Chloroprocaine also inhibited native M channel currents and induced a depolarization of the DRG neuron membrane potential. Taken together, the findings indicate that chloroprocaine concentration dependently inhibited Kv7/M channel currents. © 2015 S. Karger AG, Basel.

  12. Activation of KV7 channels stimulates vasodilatation of human placental chorionic plate arteries.

    PubMed

    Mills, T A; Greenwood, S L; Devlin, G; Shweikh, Y; Robinson, M; Cowley, E; Hayward, C E; Cottrell, E C; Tropea, T; Brereton, M F; Dalby-Brown, W; Wareing, M

    2015-06-01

    Potassium (K(+)) channels are key regulators of vascular smooth muscle cell (VSMC) excitability. In systemic small arteries, Kv7 channel expression/activity has been noted and a role in vascular tone regulation demonstrated. We aimed to demonstrate functional Kv7 channels in human fetoplacental small arteries. Human placental chorionic plate arteries (CPAs) were obtained at term. CPA responses to Kv7 channel modulators was determined by wire myography. Presence of Kv7 channel mRNA (encoded by KCNQ1-5) and protein expression were assessed by RT-PCR and immunohistochemistry/immunofluorescence, respectively. Kv7 channel blockade with linopirdine increased CPA basal tone and AVP-induced contraction. Pre-contracted CPAs (AVP; 80 mM K(+) depolarization solution) exhibited significant relaxation to flupirtine, retigabine, the acrylamide (S)-1, and (S) BMS-204352, differential activators of Kv7.1 - Kv7.5 channels. All CPAs assessed expressed KCNQ1 and KCNQ3-5 mRNA; KCNQ2 was expressed only in a subset of CPAs. Kv7 protein expression was confirmed in intact CPAs and isolated VSMCs. Kv7 channels are present and active in fetoplacental vessels, contributing to vascular tone regulation in normal pregnancy. Targeting these channels may represent a therapeutic intervention in pregnancies complicated by increased vascular resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Inhibition of KV7 Channels Protects the Rat Heart against Myocardial Ischemia and Reperfusion Injury.

    PubMed

    Hedegaard, Elise R; Johnsen, Jacob; Povlsen, Jonas A; Jespersen, Nichlas R; Shanmuganathan, Jeffrey A; Laursen, Mia R; Kristiansen, Steen B; Simonsen, Ulf; Bøtker, Hans Erik

    2016-04-01

    The voltage-gated KV7 (KCNQ) potassium channels are activated by ischemia and involved in hypoxic vasodilatation. We investigated the effect of KV7 channel modulation on cardiac ischemia and reperfusion injury and its interaction with cardioprotection by ischemic preconditioning (IPC). Reverse-transcription polymerase chain reaction revealed expression of KV7.1, KV7.4, and KV7.5 in the left anterior descending rat coronary artery and all KV7 subtypes (KV7.1-KV7.5) in the left and right ventricles of the heart. Isolated hearts were subjected to no-flow global ischemia and reperfusion with and without IPC. Infarct size was quantified by 2,3,5-triphenyltetrazolium chloride staining. Two blockers of KV7 channels, XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone] (10 µM) and linopirdine (10 µM), reduced infarct size and exerted additive infarct reduction to IPC. An opener of KV7 channels, flupirtine (10 µM) abolished infarct size reduction by IPC. Hemodynamics were measured using a catheter inserted in the left ventricle and postischemic left ventricular recovery improved in accordance with reduction of infarct size and deteriorated with increased infarct size. XE991 (10 µM) reduced coronary flow in the reperfusion phase and inhibited vasodilatation in isolated small branches of the left anterior descending coronary artery during both simulated ischemia and reoxygenation. KV7 channels are expressed in rat coronary arteries and myocardium. Inhibition of KV7 channels exerts cardioprotection and opening of KV7 channels abrogates cardioprotection by IPC. Although safety issues should be further addressed, our findings suggest a potential role for KV7 blockers in the treatment of ischemia-reperfusion injury. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  14. Use-dependent activation of neuronal Kv1.2 channel complexes.

    PubMed

    Baronas, Victoria A; McGuinness, Brandon R; Brigidi, G Stefano; Gomm Kolisko, Rachel N; Vilin, Yury Y; Kim, Robin Y; Lynn, Francis C; Bamji, Shernaz X; Yang, Runying; Kurata, Harley T

    2015-02-25

    In excitable cells, ion channels are frequently challenged by repetitive stimuli, and their responses shape cellular behavior by regulating the duration and termination of bursts of action potentials. We have investigated the behavior of Shaker family voltage-gated potassium (Kv) channels subjected to repetitive stimuli, with a particular focus on Kv1.2. Genetic deletion of this subunit results in complete mortality within 2 weeks of birth in mice, highlighting a critical physiological role for Kv1.2. Kv1.2 channels exhibit a unique property described previously as "prepulse potentiation," in which activation by a depolarizing step facilitates activation in a subsequent pulse. In this study, we demonstrate that this property enables Kv1.2 channels to exhibit use-dependent activation during trains of very brief depolarizations. Also, Kv subunits usually assemble into heteromeric channels in the central nervous system, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that other Kv1 channel types do not exhibit use-dependent activation, but this property is conferred in heteromeric channel complexes containing even a single Kv1.2 subunit. This regulatory mechanism is observed in mammalian cell lines as well as primary cultures of hippocampal neurons. Our findings illustrate that use-dependent activation is a unique property of Kv1.2 that persists in heteromeric channel complexes and may influence function of hippocampal neurons. Copyright © 2015 the authors 0270-6474/15/353515-10$15.00/0.

  15. Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels.

    PubMed

    Sihn, Choong-Ryoul; Kim, Hyo Jeong; Woltz, Ryan L; Yarov-Yarovoy, Vladimir; Yang, Pei-Chi; Xu, Jun; Clancy, Colleen E; Zhang, Xiao-Dong; Chiamvimonvat, Nipavan; Yamoah, Ebenezer N

    2016-01-29

    Calmodulin (CaM), a Ca(2+)-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca(2+)-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca(2+)-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca(2+)/CaM. Ca(2+)/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca(2+)/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca(2+) on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca(2+)/CaM regulation of the Kv7.4 channel under physiological and pathological conditions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The importance of immunohistochemical analyses in evaluating the phenotype of Kv channel knockout mice.

    PubMed

    Menegola, Milena; Clark, Eliana; Trimmer, James S

    2012-06-01

    To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

  17. Heterogeneity in Kv7 channel function in the cerebral and coronary circulation.

    PubMed

    Lee, Sewon; Yang, Yan; Tanner, Miles A; Li, Min; Hill, Michael A

    2015-02-01

    Kv7 channels are considered important regulators of vascular smooth muscle contractility. The present study aimed to examine the hypotheses that (i) Kv7 channels are present in mouse cerebral and coronary arteries and regulate vascular reactivity and (ii) regional differences exist in the activity of these channels. PCR confirmed that basilar, Circle of Willis and LAD arteries express predominantly Kv7.1 and 7.4. Western blot analysis, however, showed greater Kv7.4 protein levels in the cerebral vessels. Relaxation to the Kv7 channel activator, retigabine (1-50 μM) was significantly greater in the basilar artery compared to the LAD artery. Similarly, the Kv7 channel inhibitor, linopirdine (10 μM) caused a stronger contraction of the basilar artery. Furthermore, pre-incubation with linopirdine reduced forskolin (cAMP activator)-induced vasorelaxation in basilar while not altering forskolin-induced vasorelaxation of the LAD, suggesting that Kv7 channels play a more prominent role in the cerebral than in the coronary circulation. Consistent with the vessel data, whole cell Kv7 currents in cerebral VSMCs were potentiated by retigabine and inhibited by linopirdine, while these responses were blunted in coronary VSMCs. This study provides evidence that mouse Kv7 channels may contribute differently to regulating the functional properties of cerebral and coronary arteries. Such heterogeneity has important implications for developing novel therapeutics for cardiovascular dysfunction. © 2014 John Wiley & Sons Ltd.

  18. Heterogeneity in Kv7 channel function in the Cerebral and Coronary Circulation

    PubMed Central

    Tanner, Miles A.; Li, Min; Hill, Michael A.

    2014-01-01

    Kv7 channels are considered important regulators of vascular smooth muscle contractility. The present study examined the hypotheses that 1. Kv7 channels are present in mouse cerebral and coronary arteries and regulate vascular reactivity, and 2. regional differences exist in the activity of these channels. PCR confirmed that basilar, Circle of Willis and left anterior descending (LAD) arteries express predominantly Kv7.1 and 7.4. Western blot analysis, however, showed greater Kv7.4 protein levels in the cerebral vessels. Relaxation to the Kv7 channel activator, retigabine (1-50μM) was significantly greater in basilar compared to LAD. Similarly, the Kv7 channel inhibitor, linopirdine (10μM) caused stronger contraction of the basilar artery. Furthermore, pre-incubation with linopirdine reduced forskolin (cAMP activator)-induced vasorelaxation in basilar while not altering forskolin-induced vasorelaxation of the LAD, suggesting that Kv7 channels play a more prominent role in the cerebral than coronary circulation. Consistent with the vessel data, whole cell Kv7 currents in cerebral VSMCs were potentiated by retigabine and inhibited by linopirdine, while these responses were blunted in coronary VSMCs. This study provides evidence that mouse Kv7 channels may contribute differently to regulating the functional properties of cerebral and coronary arteries. Such heterogeneity has important implications for developing novel therapeutics for cardiovascular dysfunction. PMID:25476662

  19. Developmental Expression of Kv Potassium Channels at the Axon Initial Segment of Cultured Hippocampal Neurons

    PubMed Central

    Sánchez-Ponce, Diana; DeFelipe, Javier; Garrido, Juan José; Muñoz, Alberto

    2012-01-01

    Axonal outgrowth and the formation of the axon initial segment (AIS) are early events in the acquisition of neuronal polarity. The AIS is characterized by a high concentration of voltage-dependent sodium and potassium channels. However, the specific ion channel subunits present and their precise localization in this axonal subdomain vary both during development and among the types of neurons, probably determining their firing characteristics in response to stimulation. Here, we characterize the developmental expression of different subfamilies of voltage-gated potassium channels in the AISs of cultured mouse hippocampal neurons, including subunits Kv1.2, Kv2.2 and Kv7.2. In contrast to the early appearance of voltage-gated sodium channels and the Kv7.2 subunit at the AIS, Kv1.2 and Kv2.2 subunits were tethered at the AIS only after 10 days in vitro. Interestingly, we observed different patterns of Kv1.2 and Kv2.2 subunit expression, with each confined to distinct neuronal populations. The accumulation of Kv1.2 and Kv2.2 subunits at the AIS was dependent on ankyrin G tethering, it was not affected by disruption of the actin cytoskeleton and it was resistant to detergent extraction, as described previously for other AIS proteins. This distribution of potassium channels in the AIS further emphasizes the heterogeneity of this structure in different neuronal populations, as proposed previously, and suggests corresponding differences in action potential regulation. PMID:23119056

  20. Somatodendritic surface expression of epitope-tagged and KChIP binding-deficient Kv4.2 channels in hippocampal neurons.

    PubMed

    Prechtel, Helena; Hartmann, Sven; Minge, Daniel; Bähring, Robert

    2018-01-01

    Kv4.2 channels mediate a subthreshold-activating somatodendritic A-type current (ISA) in hippocampal neurons. We examined the role of accessory Kv channel interacting protein (KChIP) binding in somatodendritic surface expression and activity-dependent decrease in the availability of Kv4.2 channels. For this purpose we transfected cultured hippocampal neurons with cDNA coding for Kv4.2 wild-type (wt) or KChIP binding-deficient Kv4.2 mutants. All channels were equipped with an externally accessible hemagglutinin (HA)-tag and an EGFP-tag, which was attached to the C-terminal end. Combined analyses of EGFP self-fluorescence, surface HA immunostaining and patch-clamp recordings demonstrated similar dendritic trafficking and functional surface expression for Kv4.2[wt]HA,EGFP and the KChIP binding-deficient Kv4.2[A14K]HA,EGFP. Coexpression of exogenous KChIP2 augmented the surface expression of Kv4.2[wt]HA,EGFP but not Kv4.2[A14K]HA,EGFP. Notably, activity-dependent decrease in availability was more pronounced in Kv4.2[wt]HA,EGFP + KChIP2 coexpressing than in Kv4.2[A14K]HA,EGFP + KChIP2 coexpressing neurons. Our results do not support the notion that accessory KChIP binding is a prerequisite for dendritic trafficking and functional surface expression of Kv4.2 channels, however, accessory KChIP binding may play a potential role in Kv4.2 modulation during intrinsic plasticity processes.

  1. Bladder contractility is modulated by Kv7 channels in pig detrusor.

    PubMed

    Svalø, Julie; Bille, Michala; Parameswaran Theepakaran, Neeraja; Sheykhzade, Majid; Nordling, Jørgen; Bouchelouche, Pierre

    2013-09-05

    Kv7 channels are involved in smooth muscle relaxation, and accordingly we believe that they constitute potential targets for the treatment of overactive bladder syndrome. We have therefore used myography to examine the function of Kv7 channels in detrusor, i.e. pig bladder, with a view to determining the effects of the following potassium channel activators: ML213 (Kv7.2/Kv7.4 channels) and retigabine (Kv7.2-7.5 channels). Retigabine produced a concentration-dependent relaxation of carbachol- and electric field-induced contractions. The potency was similar in magnitude to that of ML213-induced relaxation, suggesting that Kv7.2 and/or Kv7.4 channels constitute the subtypes that are relevant to bladder contractility. The effects of retigabine and ML213 were attenuated by pre-incubation with 10µM XE991 (Kv7.1-7.5 channel blocker) (P<0.05), which in turn confirmed Kv7 channel selectivity. Subtype-selective effects were further investigated by incubating the detrusor with 10µM chromanol 293B (Kv7.1 channel blocker). Regardless of the experimental protocol, this did not cause a further increase in the evoked contraction. In contrast, the addition of XE991 potentiated the KCl-induced contractions, but not those induced by carbachol or electric field, indicating the presence of a phosphatidyl-inositol-4,5-biphosphate-dependent mechanism amongst the Kv7 channels in detrusor. qRT-PCR studies of the mRNA transcript level of Kv7.3-7.5 channels displayed a higher level of Kv7.4 transcript in detrusor compared to that present in brain cortex and heart tissues. Thus, we have shown that Kv7.4 channels are expressed and functionally active in pig detrusor, and that the use of selective Kv7.4 channel modulators in the treatment of detrusor overactivity seems promising. © 2013 Elsevier B.V. All rights reserved.

  2. Kv4 Potassium Channels Modulate Hippocampal EPSP-Spike Potentiation and Spatial Memory in Rats

    ERIC Educational Resources Information Center

    Truchet, Bruno; Manrique, Christine; Sreng, Leam; Chaillan, Franck A.; Roman, Francois S.; Mourre, Christiane

    2012-01-01

    Kv4 channels regulate the backpropagation of action potentials (b-AP) and have been implicated in the modulation of long-term potentiation (LTP). Here we showed that blockade of Kv4 channels by the scorpion toxin AmmTX3 impaired reference memory in a radial maze task. In vivo, AmmTX3 intracerebroventricular (i.c.v.) infusion increased and…

  3. Multiprotein assembly of Kv4.2, KChIP3 and DPP10 produces ternary channel complexes with ISA-like properties.

    PubMed

    Jerng, Henry H; Kunjilwar, Kumud; Pfaffinger, Paul J

    2005-11-01

    Kv4 pore-forming subunits are the principal constituents of the voltage-gated K+ channel underlying somatodendritic subthreshold A-type currents (I(SA)) in neurones. Two structurally distinct types of Kv4 channel modulators, Kv channel-interacting proteins (KChIPs) and dipeptidyl-peptidase-like proteins (DPLs: DPP6 or DPPX, DPP10 or DPPY), enhance surface expression and modify functional properties. Since KChIP and DPL distributions overlap in the brain, we investigated the potential coassembly of Kv4.2, KChIP3 and DPL proteins, and the contribution of DPLs to ternary complex properties. Immunoprecipitation results show that KChIP3 and DPP10 associate simultaneously with Kv4.2 proteins in rat brain as well as heterologously expressing Xenopus oocytes, indicating Kv4.2 + KChIP3 + DPP10 multiprotein complexes. Consistent with ternary complex formation, coexpression of Kv4.2, KChIP3 and DPP10 in oocytes and CHO cells results in current waveforms distinct from the arithmetic sum of Kv4.2 + KChIP3 and Kv4.2 + DPP10 currents. Furthermore, the Kv4.2 + KChIP3 + DPP10 channels recover from inactivation very rapidly (tau(rec) approximately 18-26 ms), closely matching that of native I(SA) and significantly faster than the recovery of Kv4.2 + KChIP3 or Kv4.2 + DPP10 channels. For comparison, identical triple coexpression experiments were performed using DPP6 variants. While most results are similar, the Kv4.2 + KChIP3 + DPP6 channels exhibit inactivation that slows with increasing membrane potential, resulting in inactivation slower than that of Kv4.2 + KChIP3 + DPP10 channels at positive voltages. In conclusion, the native neuronal subthreshold A-type channel is probably a macromolecular complex formed from Kv4 and a combination of both KChIP and DPL proteins, with the precise composition of channel alpha and auxiliary subunits underlying tissue and regional variability in I(SA) properties.

  4. Molecular physiology and modulation of somatodendritic A-type potassium channels.

    PubMed

    Jerng, Henry H; Pfaffinger, Paul J; Covarrubias, Manuel

    2004-12-01

    The somatodendritic subthreshold A-type K+ current (ISA) in nerve cells is a critical component of the ensemble of voltage-gated ionic currents that determine somatodendritic signal integration. The underlying K+ channel belongs to the Shal subfamily of voltage-gated K+ channels. Most Shal channels across the animal kingdom share a high degree of structural conservation, operate in the subthreshold range of membrane potentials, and exhibit relatively fast inactivation and recovery from inactivation. Mammalian Shal K+ channels (Kv4) undergo preferential closed-state inactivation with features that are generally inconsistent with the classical mechanisms of inactivation typical of Shaker K+ channels. Here, we review (1) the physiological and genetic properties of ISA, 2 the molecular mechanisms of Kv4 inactivation and its remodeling by a family of soluble calcium-binding proteins (KChIPs) and a membrane-bound dipeptidase-like protein (DPPX), and (3) the modulation of Kv4 channels by protein phosphorylation.

  5. Expression and distribution of Kv4 potassium channel subunits and potassium channel interacting proteins in subpopulations of interneurons in the basolateral amygdala.

    PubMed

    Dabrowska, J; Rainnie, D G

    2010-12-15

    The Kv4 potassium channel α subunits, Kv4.1, Kv4.2, and Kv4.3, determine some of the fundamental physiological properties of neurons in the CNS. Kv4 subunits are associated with auxiliary β-subunits, such as the potassium channel interacting proteins (KChIP1 - 4), which are thought to regulate the trafficking and gating of native Kv4 potassium channels. Intriguingly, KChIP1 is thought to show cell type-selective expression in GABA-ergic inhibitory interneurons, while other β-subunits (KChIP2-4) are associated with principal glutamatergic neurons. However, nothing is known about the expression of Kv4 family α- and β-subunits in specific interneurons populations in the BLA. Here, we have used immunofluorescence, co-immunoprecipitation, and Western Blotting to determine the relative expression of KChIP1 in the different interneuron subtypes within the BLA, and its co-localization with one or more of the Kv4 α subunits. We show that all three α-subunits of Kv4 potassium channel are found in rat BLA neurons, and that the immunoreactivity of KChIP1 closely resembles that of Kv4.3. Indeed, Kv4.3 showed almost complete co-localization with KChIP1 in the soma and dendrites of a distinct subpopulation of BLA neurons. Dual-immunofluorescence studies revealed this to be in BLA interneurons immunoreactive for parvalbumin, cholecystokin-8, and somatostatin. Finally, co-immunoprecipitation studies showed that KChIP1 was associated with all three Kv4 α subunits. Together our results suggest that KChIP1 is selectively expressed in BLA interneurons where it may function to regulate the activity of A-type potassium channels. Hence, KChIP1 might be considered as a cell type-specific regulator of GABAergic inhibitory circuits in the BLA. Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Phosphatidic acid modulation of Kv channel voltage sensor function

    PubMed Central

    Hite, Richard K; Butterwick, Joel A; MacKinnon, Roderick

    2014-01-01

    Membrane phospholipids can function as potent regulators of ion channel function. This study uncovers and investigates the effect of phosphatidic acid on Kv channel gating. Using the method of reconstitution into planar lipid bilayers, in which protein and lipid components are defined and controlled, we characterize two effects of phosphatidic acid. The first is a non-specific electrostatic influence on activation mediated by electric charge density on the extracellular and intracellular membrane surfaces. The second is specific to the presence of a primary phosphate group, acts only through the intracellular membrane leaflet and depends on the presence of a particular arginine residue in the voltage sensor. Intracellular phosphatidic acid accounts for a nearly 50 mV shift in the midpoint of the activation curve in a direction consistent with stabilization of the voltage sensor's closed conformation. These findings support a novel mechanism of voltage sensor regulation by the signaling lipid phosphatidic acid. DOI: http://dx.doi.org/10.7554/eLife.04366.001 PMID:25285449

  7. Novel treatment strategies for smooth muscle disorders: Targeting Kv7 potassium channels.

    PubMed

    Haick, Jennifer M; Byron, Kenneth L

    2016-09-01

    Smooth muscle cells provide crucial contractile functions in visceral, vascular, and lung tissues. The contractile state of smooth muscle is largely determined by their electrical excitability, which is in turn influenced by the activity of potassium channels. The activity of potassium channels sustains smooth muscle cell membrane hyperpolarization, reducing cellular excitability and thereby promoting smooth muscle relaxation. Research over the past decade has indicated an important role for Kv7 (KCNQ) voltage-gated potassium channels in the regulation of the excitability of smooth muscle cells. Expression of multiple Kv7 channel subtypes has been demonstrated in smooth muscle cells from viscera (gastrointestinal, bladder, myometrial), from the systemic and pulmonary vasculature, and from the airways of the lung, from multiple species, including humans. A number of clinically used drugs, some of which were developed to target Kv7 channels in other tissues, have been found to exert robust effects on smooth muscle Kv7 channels. Functional studies have indicated that Kv7 channel activators and inhibitors have the ability to relax and contact smooth muscle preparations, respectively, suggesting a wide range of novel applications for the pharmacological tool set. This review summarizes recent findings regarding the physiological functions of Kv7 channels in smooth muscle, and highlights potential therapeutic applications based on pharmacological targeting of smooth muscle Kv7 channels throughout the body. Published by Elsevier Inc.

  8. β Subunits Control the Effects of Human Kv4.3 Potassium Channel Phosphorylation.

    PubMed

    Abbott, Geoffrey W

    2017-01-01

    The transient outward K + current, I to , activates early in the cardiac myocyte action potential, to begin repolarization. Human I to is generated primarily by two Kv4.3 potassium channel α subunit splice variants (Kv4.3L and Kv4.3S) that diverge only by a C-terminal, membrane-proximal, 19-residue stretch unique to Kv4.3L. Protein kinase C (PKC) phosphorylation of threonine 504 within the Kv4.3L-specific 19-residues mediates α-adrenergic inhibition of I to in human heart. Kv4.3 is regulated in human heart by various β subunits, including cytosolic KChIP2b and transmembrane KCNEs, yet their impact on the functional effects of human Kv4.3 phosphorylation has not been reported. Here, this gap in knowledge was addressed using human Kv4.3 splice variants, T504 mutants, and human β subunits. Subunits were co-expressed in Xenopus laevis oocytes and analyzed by two-electrode voltage-clamp, using phorbol 12-myristate 13-acetate (PMA) to stimulate PKC. Unexpectedly, KChIP2b removed the inhibitory effect of PKC on Kv4.3L (but not Kv4.3L threonine phosphorylation by PKC per-se ), while co-expression with KCNE2, but not KCNE4, restored PKC-dependent inhibition of Kv4.3L-KChIP2b to quantitatively resemble previously reported effects of α-adrenergic modulation of human ventricular I to . In addition, PKC accelerated recovery from inactivation of Kv4.3L-KChIP2b channels and, interestingly, of both Kv4.3L and Kv4.3S alone. Thus, β subunits regulate the response of human Kv4.3 to PKC phosphorylation and provide a potential mechanism for modifying the response of I to to α-adrenergic regulation in vivo .

  9. β Subunits Control the Effects of Human Kv4.3 Potassium Channel Phosphorylation

    PubMed Central

    Abbott, Geoffrey W.

    2017-01-01

    The transient outward K+ current, Ito, activates early in the cardiac myocyte action potential, to begin repolarization. Human Ito is generated primarily by two Kv4.3 potassium channel α subunit splice variants (Kv4.3L and Kv4.3S) that diverge only by a C-terminal, membrane-proximal, 19-residue stretch unique to Kv4.3L. Protein kinase C (PKC) phosphorylation of threonine 504 within the Kv4.3L-specific 19-residues mediates α-adrenergic inhibition of Ito in human heart. Kv4.3 is regulated in human heart by various β subunits, including cytosolic KChIP2b and transmembrane KCNEs, yet their impact on the functional effects of human Kv4.3 phosphorylation has not been reported. Here, this gap in knowledge was addressed using human Kv4.3 splice variants, T504 mutants, and human β subunits. Subunits were co-expressed in Xenopus laevis oocytes and analyzed by two-electrode voltage-clamp, using phorbol 12-myristate 13-acetate (PMA) to stimulate PKC. Unexpectedly, KChIP2b removed the inhibitory effect of PKC on Kv4.3L (but not Kv4.3L threonine phosphorylation by PKC per-se), while co-expression with KCNE2, but not KCNE4, restored PKC-dependent inhibition of Kv4.3L-KChIP2b to quantitatively resemble previously reported effects of α-adrenergic modulation of human ventricular Ito. In addition, PKC accelerated recovery from inactivation of Kv4.3L-KChIP2b channels and, interestingly, of both Kv4.3L and Kv4.3S alone. Thus, β subunits regulate the response of human Kv4.3 to PKC phosphorylation and provide a potential mechanism for modifying the response of Ito to α-adrenergic regulation in vivo. PMID:28919864

  10. Peripheral KV7 channels regulate visceral sensory function in mouse and human colon

    PubMed Central

    Hockley, James RF; Reed, David E; Smith, Ewan St. John; Bulmer, David C; Blackshaw, L Ashley

    2017-01-01

    Background Chronic visceral pain is a defining symptom of many gastrointestinal disorders. The KV7 family (KV7.1–KV7.5) of voltage-gated potassium channels mediates the M current that regulates excitability in peripheral sensory nociceptors and central pain pathways. Here, we use a combination of immunohistochemistry, gut-nerve electrophysiological recordings in both mouse and human tissues, and single-cell qualitative real-time polymerase chain reaction of gut-projecting sensory neurons, to investigate the contribution of peripheral KV7 channels to visceral nociception. Results Immunohistochemical staining of mouse colon revealed labelling of KV7 subtypes (KV7.3 and KV7.5) with CGRP around intrinsic enteric neurons of the myenteric plexuses and within extrinsic sensory fibres along mesenteric blood vessels. Treatment with the KV7 opener retigabine almost completely abolished visceral afferent firing evoked by the algogen bradykinin, in agreement with significant co-expression of mRNA transcripts by single-cell qualitative real-time polymerase chain reaction for KCNQ subtypes and the B2 bradykinin receptor in retrogradely labelled extrinsic sensory neurons from the colon. Retigabine also attenuated responses to mechanical stimulation of the bowel following noxious distension (0–80 mmHg) in a concentration-dependent manner, whereas the KV7 blocker XE991 potentiated such responses. In human bowel tissues, KV7.3 and KV7.5 were expressed in neuronal varicosities co-labelled with synaptophysin and CGRP, and retigabine inhibited bradykinin-induced afferent activation in afferent recordings from human colon. Conclusions We show that KV7 channels contribute to the sensitivity of visceral sensory neurons to noxious chemical and mechanical stimuli in both mouse and human gut tissues. As such, peripherally restricted KV7 openers may represent a viable therapeutic modality for the treatment of gastrointestinal pathologies. PMID:28566000

  11. Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

    PubMed

    Hristov, Kiril L; Chen, Muyan; Afeli, Serge A Y; Cheng, Qiuping; Rovner, Eric S; Petkov, Georgi V

    2012-06-01

    The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

  12. Mapping the Interaction Anatomy of BmP02 on Kv1.3 Channel

    NASA Astrophysics Data System (ADS)

    Wu, B.; Wu, B. F.; Feng, Y. J.; Tao, J.; Ji, Y. H.

    2016-07-01

    The potassium channel Kv 1.3 plays a vital part in the activation of T lymphocytes and is an attractive pharmacological target for autoimmune diseases. BmP02, a 28-residue peptide isolated from Chinese scorpion (Buthus martensi Karsch) venom, is a potent and selective Kv1.3 channel blocker. However, the mechanism through which BmP02 recognizes and inhibits the Kv1.3 channel is still unclear. In the present study, a complex molecular model of Kv1.3-BmP02 was developed by docking analysis and molecular dynamics simulations. From these simulations, it appears the large β-turn (residues 10-16) of BmP02 might be the binding interface with Kv 1.3. These results were confirmed by scanning alanine mutagenesis of BmP02, which identified His9, Lys11 and Lys13, which lie within BmP02’s β-turn, as key residues for interacting with Kv1.3. Based on these results and molecular modeling, two negatively charged residues of Kv1.3, D421 and D422, located in turret region, were predicted to act as the binding site for BmP02. Mutation of these residues reduced sensitivity of Kv 1.3 to BmP02 inhibition, suggesting that electrostatic interactions play a crucial role in Kv1.3-BmP02 interaction. This study revealed the molecular basis of Kv 1.3 recognition by BmP02 venom, and provides a novel interaction model for Kv channel-specific blocker complex, which may help guide future drug-design for Kv1.3-related channelopathies.

  13. A Kv7.2 mutation associated with early onset epileptic encephalopathy with suppression-burst enhances Kv7/M channel activity.

    PubMed

    Devaux, Jérôme; Abidi, Affef; Roubertie, Agathe; Molinari, Florence; Becq, Hélène; Lacoste, Caroline; Villard, Laurent; Milh, Mathieu; Aniksztejn, Laurent

    2016-05-01

    Mutations in the KCNQ2 gene encoding the voltage-gated potassium channel subunit Kv7.2 cause early onset epileptic encephalopathy (EOEE). Most mutations have been shown to induce a loss of function or to affect the subcellular distribution of Kv7 channels in neurons. Herein, we investigated functional consequences and subcellular distribution of the p.V175L mutation of Kv7.2 (Kv7.2(V175L) ) found in a patient presenting EOEE. We observed that the mutation produced a 25-40 mV hyperpolarizing shift of the conductance-voltage relationship of both the homomeric Kv7.2(V175L) and heteromeric Kv7.2(V175L) /Kv7.3 channels compared to wild-type channels and a 10 mV hyperpolarizing shift of Kv7.2(V175L) /Kv7.2/Kv7.3 channels in a 1:1:2 ratio mimicking the patient situation. Mutant channels also displayed faster activation kinetics and an increased current density that was prevented by 1 μm linopirdine. The p.V175L mutation did not affect the protein expression of Kv7 channels and its localization at the axon initial segment. We conclude that p.V175L is a gain of function mutation. This confirms previous observations showing that mutations having opposite consequences on M channels can produce EOEE. These findings alert us that drugs aiming to increase Kv7 channel activity might have adverse effects in EOEE in the case of gain-of-function variants. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

  14. Kv2 Channel Regulation of Action Potential Repolarization and Firing Patterns in Superior Cervical Ganglion Neurons and Hippocampal CA1 Pyramidal Neurons

    PubMed Central

    Liu, Pin W.

    2014-01-01

    Kv2 family “delayed-rectifier” potassium channels are widely expressed in mammalian neurons. Kv2 channels activate relatively slowly and their contribution to action potential repolarization under physiological conditions has been unclear. We explored the function of Kv2 channels using a Kv2-selective blocker, Guangxitoxin-1E (GxTX-1E). Using acutely isolated neurons, mixed voltage-clamp and current-clamp experiments were done at 37°C to study the physiological kinetics of channel gating and action potentials. In both rat superior cervical ganglion (SCG) neurons and mouse hippocampal CA1 pyramidal neurons, 100 nm GxTX-1E produced near-saturating block of a component of current typically constituting ∼60–80% of the total delayed-rectifier current. GxTX-1E also reduced A-type potassium current (IA), but much more weakly. In SCG neurons, 100 nm GxTX-1E broadened spikes and voltage clamp experiments using action potential waveforms showed that Kv2 channels carry ∼55% of the total outward current during action potential repolarization despite activating relatively late in the spike. In CA1 neurons, 100 nm GxTX-1E broadened spikes evoked from −70 mV, but not −80 mV, likely reflecting a greater role of Kv2 when other potassium channels were partially inactivated at −70 mV. In both CA1 and SCG neurons, inhibition of Kv2 channels produced dramatic depolarization of interspike voltages during repetitive firing. In CA1 neurons and some SCG neurons, this was associated with increased initial firing frequency. In all neurons, inhibition of Kv2 channels depressed maintained firing because neurons entered depolarization block more readily. Therefore, Kv2 channels can either decrease or increase neuronal excitability depending on the time scale of excitation. PMID:24695716

  15. A Calmodulin C-Lobe Ca2+-Dependent Switch Governs Kv7 Channel Function.

    PubMed

    Chang, Aram; Abderemane-Ali, Fayal; Hura, Greg L; Rossen, Nathan D; Gate, Rachel E; Minor, Daniel L

    2018-02-21

    Kv7 (KCNQ) voltage-gated potassium channels control excitability in the brain, heart, and ear. Calmodulin (CaM) is crucial for Kv7 function, but how this calcium sensor affects activity has remained unclear. Here, we present X-ray crystallographic analysis of CaM:Kv7.4 and CaM:Kv7.5 AB domain complexes that reveal an Apo/CaM clamp conformation and calcium binding preferences. These structures, combined with small-angle X-ray scattering, biochemical, and functional studies, establish a regulatory mechanism for Kv7 CaM modulation based on a common architecture in which a CaM C-lobe calcium-dependent switch releases a shared Apo/CaM clamp conformation. This C-lobe switch inhibits voltage-dependent activation of Kv7.4 and Kv7.5 but facilitates Kv7.1, demonstrating that mechanism is shared by Kv7 isoforms despite the different directions of CaM modulation. Our findings provide a unified framework for understanding how CaM controls different Kv7 isoforms and highlight the role of membrane proximal domains for controlling voltage-gated channel function. VIDEO ABSTRACT. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. KCNE1 constrains the voltage sensor of Kv7.1 K+ channels.

    PubMed

    Shamgar, Liora; Haitin, Yoni; Yisharel, Ilanit; Malka, Eti; Schottelndreier, Hella; Peretz, Asher; Paas, Yoav; Attali, Bernard

    2008-04-09

    Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K(+) channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs) and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K(+) channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions.

  17. KCNE1 Constrains the Voltage Sensor of Kv7.1 K+ Channels

    PubMed Central

    Yisharel, Ilanit; Malka, Eti; Schottelndreier, Hella; Peretz, Asher; Paas, Yoav; Attali, Bernard

    2008-01-01

    Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K+ channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs) and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K+ channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions. PMID:18398469

  18. Suppression of KV7/KCNQ potassium channel enhances neuronal differentiation of PC12 cells.

    PubMed

    Zhou, Najing; Huang, Sha; Li, Li; Huang, Dongyang; Yan, Yunli; Du, Xiaona; Zhang, Hailin

    2016-10-01

    Membrane potential shift driven by electrical activity is critical in determining the cell fate of proliferation or differentiation. As such, the ion channels that underlie the membrane electrical activity play an important role in cell proliferation/differentiation. KV7/KCNQ potassium channels are critical in determining the resting membrane potentials in many neuronal cells. However, the role of these channels in cell differentiation is not well studied. In the present study, we used PC12 cells as well as primary cultured rat cortical neurons to study the role and mechanism of KV7/KCNQ in neuronal differentiation. NGF induced PC12 cell differentiation into neuron-like cells with growth of neurites showing typical growth cone-like extensions. The Kv7/KCNQ blocker XE991 promoted NGF-induced neurite outgrowth, whereas Kv7/KCNQ opener retigabine (RTG) inhibited outgrowth. M-type Kv7 channels are likely involved in regulating neurite growth because overexpression of KCNQ2/Q3 inhibited neurite growth whereas suppression of KCNQ2/Q3 with shRNA promoted neurite growth. Membrane depolarization possibly underpins enhanced neurite growth induced by the suppression of Kv7/KCNQ. Additionally, high extracellular K(+) likely induced membrane depolarization and also promoted neurite growth. Finally, T-type Ca(2+) channels may be involved in membrane-depolarization-induced neurite growth. This study provides a new perspective for understanding neuronal differentiation as well as KV7/KCNQ channel function. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  19. Voltage-dependent gating and gating charge measurements in the Kv1.2 potassium channel

    PubMed Central

    Ishida, Itzel G.; Rangel-Yescas, Gisela E.; Carrasco-Zanini, Julia

    2015-01-01

    Much has been learned about the voltage sensors of ion channels since the x-ray structure of the mammalian voltage-gated potassium channel Kv1.2 was published in 2005. High resolution structural data of a Kv channel enabled the structural interpretation of numerous electrophysiological findings collected in various ion channels, most notably Shaker, and permitted the development of meticulous computational simulations of the activation mechanism. The fundamental premise for the structural interpretation of functional measurements from Shaker is that this channel and Kv1.2 have the same characteristics, such that correlation of data from both channels would be a trivial task. We tested these assumptions by measuring Kv1.2 voltage-dependent gating and charge per channel. We found that the Kv1.2 gating charge is near 10 elementary charges (eo), ∼25% less than the well-established 13–14 eo in Shaker. Next, we neutralized positive residues in the Kv1.2 S4 transmembrane segment to investigate the cause of the reduction of the gating charge and found that, whereas replacing R1 with glutamine decreased voltage sensitivity to ∼50% of the wild-type channel value, mutation of the subsequent arginines had a much smaller effect. These data are in marked contrast to the effects of charge neutralization in Shaker, where removal of the first four basic residues reduces the gating charge by roughly the same amount. In light of these differences, we propose that the voltage-sensing domains (VSDs) of Kv1.2 and Shaker might undergo the same physical movement, but the septum that separates the aqueous crevices in the VSD of Kv1.2 might be thicker than Shaker’s, accounting for the smaller Kv1.2 gating charge. PMID:25779871

  20. Retigabine holds KV7 channels open and stabilizes the resting potential

    PubMed Central

    Corbin-Leftwich, Aaron; Mossadeq, Sayeed M.; Ha, Junghoon; Ruchala, Iwona; Le, Audrey Han Ngoc

    2016-01-01

    The anticonvulsant Retigabine is a KV7 channel agonist used to treat hyperexcitability disorders in humans. Retigabine shifts the voltage dependence for activation of the heteromeric KV7.2/KV7.3 channel to more negative potentials, thus facilitating activation. Although the molecular mechanism underlying Retigabine’s action remains unknown, previous studies have identified the pore region of KV7 channels as the drug’s target. This suggested that the Retigabine-induced shift in voltage dependence likely derives from the stabilization of the pore domain in an open (conducting) conformation. Testing this idea, we show that the heteromeric KV7.2/KV7.3 channel has at least two open states, which we named O1 and O2, with O2 being more stable. The O1 state was reached after short membrane depolarizations, whereas O2 was reached after prolonged depolarization or during steady state at the typical neuronal resting potentials. We also found that activation and deactivation seem to follow distinct pathways, suggesting that the KV7.2/KV7.3 channel activity displays hysteresis. As for the action of Retigabine, we discovered that this agonist discriminates between open states, preferentially acting on the O2 state and further stabilizing it. Based on these findings, we proposed a novel mechanism for the therapeutic effect of Retigabine whereby this drug reduces excitability by enhancing the resting potential open state stability of KV7.2/KV7.3 channels. To address this hypothesis, we used a model for action potential (AP) in Xenopus laevis oocytes and found that the resting membrane potential became more negative as a function of Retigabine concentration, whereas the threshold potential for AP firing remained unaltered. PMID:26880756

  1. Retigabine holds KV7 channels open and stabilizes the resting potential.

    PubMed

    Corbin-Leftwich, Aaron; Mossadeq, Sayeed M; Ha, Junghoon; Ruchala, Iwona; Le, Audrey Han Ngoc; Villalba-Galea, Carlos A

    2016-03-01

    The anticonvulsant Retigabine is a KV7 channel agonist used to treat hyperexcitability disorders in humans. Retigabine shifts the voltage dependence for activation of the heteromeric KV7.2/KV7.3 channel to more negative potentials, thus facilitating activation. Although the molecular mechanism underlying Retigabine's action remains unknown, previous studies have identified the pore region of KV7 channels as the drug's target. This suggested that the Retigabine-induced shift in voltage dependence likely derives from the stabilization of the pore domain in an open (conducting) conformation. Testing this idea, we show that the heteromeric KV7.2/KV7.3 channel has at least two open states, which we named O1 and O2, with O2 being more stable. The O1 state was reached after short membrane depolarizations, whereas O2 was reached after prolonged depolarization or during steady state at the typical neuronal resting potentials. We also found that activation and deactivation seem to follow distinct pathways, suggesting that the KV7.2/KV7.3 channel activity displays hysteresis. As for the action of Retigabine, we discovered that this agonist discriminates between open states, preferentially acting on the O2 state and further stabilizing it. Based on these findings, we proposed a novel mechanism for the therapeutic effect of Retigabine whereby this drug reduces excitability by enhancing the resting potential open state stability of KV7.2/KV7.3 channels. To address this hypothesis, we used a model for action potential (AP) in Xenopus laevis oocytes and found that the resting membrane potential became more negative as a function of Retigabine concentration, whereas the threshold potential for AP firing remained unaltered. © 2016 Corbin-Leftwich et al.

  2. Chloride concentration affects Kv channel voltage-gating kinetics: Importance of experimental anion concentrations.

    PubMed

    Bekar, L K; Loewen, M E; Forsyth, G W; Walz, W

    2005-09-30

    Chloride concentration has been shown to have a dramatic impact on protein folding and subsequent tertiary conformation [K.D. Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300-311; I. Jelesarov, E. Durr, R.M. Thomas, H.R. Bosshard, Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper), Biochemistry 37 (1998) 7539-7550]. As it is known that Kv channel gating is linked to the stability of the cytoplasmic T1 multimerization domain conformation [D.L. Minor, Y.F. Lin, B.C. Mobley, A. Avelar, Y.N. Jan, L.Y. Jan, J.M. Berger, The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel, Cell 102 (2000) 657-670; B.A. Yi, D.L. Minor Jr., Y.F. Lin, Y.N. Jan, L.Y. Jan, Controlling potassium channel activities: interplay between the membrane and intracellular factors, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11016-11023] and that intracellular chloride concentration has been linked to Kv channel kinetics [L.K. Bekar, W. Walz, Intracellular chloride modulates A-type potassium currents in astrocytes, Glia 39 (2002) 207-216; W.B. Thoreson, S.L. Stella, Anion modulation of calcium current voltage dependence and amplitude in salamander rods, Biochim. Biophys. Acta 1464 (2000) 142-150], the objective of the present study was to address how chloride concentration changes affect Kv channel kinetics more closely in an isolated expression system. Initially, no significant chloride concentration-dependent effects on channel steady-state gating kinetics were observed. Only after disruption of the cytoskeleton with cytochalasin-D did we see significant chloride concentration-dependent shifts in gating kinetics. This suggests that the shift in gating kinetics is mediated through effects of intracellular chloride concentration on cytoplasmic domain tertiary

  3. β Subunits Functionally Differentiate Human Kv4.3 Potassium Channel Splice Variants

    PubMed Central

    Abbott, Geoffrey W.

    2017-01-01

    The human ventricular cardiomyocyte transient outward K+ current (Ito) mediates the initial phase of myocyte repolarization and its disruption is implicated in Brugada Syndrome and heart failure (HF). Human cardiac Ito is generated primarily by two Kv4.3 splice variants (Kv4.3L and Kv4.3S, diverging only by a C-terminal, S6-proximal, 19-residue stretch unique to Kv4.3L), which are differentially remodeled in HF, but considered functionally alike at baseline. Kv4.3 is regulated in human heart by β subunits including KChIP2b and KCNEs, but their effects were previously assumed to be Kv4.3 isoform-independent. Here, this assumption was tested experimentally using two-electrode voltage-clamp analysis of human subunits co-expressed in Xenopus laevis oocytes. Unexpectedly, Kv4.3L-KChIP2b channels exhibited up to 8-fold lower current augmentation, 40% slower inactivation, and 5 mV-shifted steady-state inactivation compared to Kv4.3S-KChIP2b. A synthetic peptide mimicking the 19-residue stretch diminished these differences, reinforcing the importance of this segment in mediating Kv4.3 regulation by KChIP2b. KCNE subunits induced further functional divergence, including a 7-fold increase in Kv4.3S-KCNE4-KChIP2b current compared to Kv4.3L-KCNE4-KChIP2b. The discovery of β-subunit-dependent functional divergence in human Kv4.3 splice variants suggests a C-terminal signaling hub is crucial to governing β-subunit effects upon Kv4.3, and demonstrates the potential significance of differential Kv4.3 gene-splicing and β subunit expression in myocyte physiology and pathobiology. PMID:28228734

  4. β Subunits Functionally Differentiate Human Kv4.3 Potassium Channel Splice Variants.

    PubMed

    Abbott, Geoffrey W

    2017-01-01

    The human ventricular cardiomyocyte transient outward K + current ( I to ) mediates the initial phase of myocyte repolarization and its disruption is implicated in Brugada Syndrome and heart failure (HF). Human cardiac I to is generated primarily by two Kv4.3 splice variants (Kv4.3L and Kv4.3S, diverging only by a C-terminal, S6-proximal, 19-residue stretch unique to Kv4.3L), which are differentially remodeled in HF, but considered functionally alike at baseline. Kv4.3 is regulated in human heart by β subunits including KChIP2b and KCNEs, but their effects were previously assumed to be Kv4.3 isoform-independent. Here, this assumption was tested experimentally using two-electrode voltage-clamp analysis of human subunits co-expressed in Xenopus laevis oocytes. Unexpectedly, Kv4.3L-KChIP2b channels exhibited up to 8-fold lower current augmentation, 40% slower inactivation, and 5 mV-shifted steady-state inactivation compared to Kv4.3S-KChIP2b. A synthetic peptide mimicking the 19-residue stretch diminished these differences, reinforcing the importance of this segment in mediating Kv4.3 regulation by KChIP2b. KCNE subunits induced further functional divergence, including a 7-fold increase in Kv4.3S-KCNE4-KChIP2b current compared to Kv4.3L-KCNE4-KChIP2b. The discovery of β-subunit-dependent functional divergence in human Kv4.3 splice variants suggests a C-terminal signaling hub is crucial to governing β-subunit effects upon Kv4.3, and demonstrates the potential significance of differential Kv4.3 gene-splicing and β subunit expression in myocyte physiology and pathobiology.

  5. Inhibitory effects of cortisone and hydrocortisone on human Kv1.5 channel currents.

    PubMed

    Yu, Jing; Park, Mi-Hyeong; Jo, Su-Hyun

    2015-01-05

    Glucocorticoids are the primary hormones that respond to stress and protect organisms from dangerous situations. The glucocorticoids hydrocortisone and its dormant form, cortisone, affect the cardiovascular system with changes such as increased blood pressure and cardioprotection. Kv1.5 channels play a critical role in the maintenance of cellular membrane potential and are widely expressed in pancreatic β-cells, neurons, myocytes, and smooth muscle cells of the pulmonary vasculature. We examined the electrophysiological effects of both cortisone and hydrocortisone on human Kv1.5 channels expressed in Xenopus oocytes using a two-microelectrode voltage clamp technique. Both cortisone and hydrocortisone rapidly and irreversibly suppressed the amplitude of Kv1.5 channel current with IC50 values of 50.2±4.2μM and 33.4±3.2μM, respectively, while sustained the current trace shape of Kv1.5 current. The inhibitory effect of cortisone on Kv1.5 decreased progressively from -10mV to +30mV, while hydrocortisone׳s inhibition of the channel did not change across the same voltage range. Both cortisone and hydrocortisone blocked Kv1.5 channel currents in a non-use-dependent manner and neither altered the channel׳s steady-state activation or inactivation curves. These results show that cortisone and hydrocortisone inhibited Kv1.5 channel currents differently, and that Kv1.5 channels were more sensitive to hydrocortisone than to cortisone. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Contribution of Kv7 channels to natriuretic peptide mediated vasodilation in normal and hypertensive rats.

    PubMed

    Stott, Jennifer B; Barrese, Vincenzo; Jepps, Thomas A; Leighton, Emma V; Greenwood, Iain A

    2015-03-01

    The Kv7 family of voltage-gated potassium channels are expressed within the vasculature where they are key regulators of vascular tone and mediate cAMP-linked endogenous vasodilator responses, a pathway that is compromised in hypertension. However, the role of Kv7 channels in non-cAMP-linked vasodilator pathways has not been investigated. Natriuretic peptides are potent vasodilators, which operate primarily through the activation of a cGMP-dependent signaling pathway. This study investigated the putative role of Kv7 channels in natriuretic peptide-dependent relaxations in the vasculature of normal and hypertensive animals. Relaxant responses of rat aorta to both atrial and C-type natriuretic peptides and the nitric oxide donor sodium nitroprusside were impaired by the Kv7 blocker linopirdine (10 μmol/L) but not by the Kv7.1-specific blocker HMR1556 (10 μmol/L) and other K(+) channel blockers. In contrast, only the atrial natriuretic peptide response was sensitive to linopirdine in the renal artery. These Kv7-mediated responses were attenuated in arteries from hypertensive rats. Quantitative polymerase chain reaction showed that A- and B-type natriuretic peptide receptors were expressed at high levels in the aorta and renal artery from normal and spontaneously hypertensive rats. This study provides the first evidence that natriuretic peptide responses are impaired in hypertension and that recruitment of Kv7 channels is a key component of natriuretic peptide-dependent vasodilations. © 2014 American Heart Association, Inc.

  7. Kv10.1 potassium channel: from the brain to the tumors.

    PubMed

    Cázares-Ordoñez, V; Pardo, L A

    2017-10-01

    The KCNH1 gene encodes the Kv10.1 (Eag1) ion channel, a member of the EAG (ether-à-go-go) family of voltage-gated potassium channels. Recent studies have demonstrated that KCHN1 mutations are implicated in Temple-Baraitser and Zimmermann-Laband syndromes and other forms of developmental deficits that all present with mental retardation and epilepsy, suggesting that Kv10.1 might be important for cognitive development in humans. Although the Kv10.1 channel is mainly expressed in the mammalian brain, its ectopic expression occurs in 70% of human cancers. Cancer cells and tumors expressing Kv10.1 acquire selective advantages that favor cancer progression through molecular mechanisms that involve several cellular pathways, indicating that protein-protein interactions may be important for Kv10.1 influence in cell proliferation and tumorigenesis. Several studies on transcriptional and post-transcriptional regulation of Kv10.1 expression have shown interesting mechanistic insights about Kv10.1 role in oncogenesis, increasing the importance of identifying the cellular factors that regulate Kv10.1 expression in tumors.

  8. From pan-reactive KV7 channel opener to subtype selective opener/inhibitor by addition of a methyl group.

    PubMed

    Blom, Sigrid Marie; Rottländer, Mario; Kehler, Jan; Bundgaard, Christoffer; Schmitt, Nicole; Jensen, Henrik Sindal

    2014-01-01

    The voltage-gated potassium channels of the KV7 family (KV7.1-5) play important roles in controlling neuronal excitability and are therefore attractive targets for treatment of CNS disorders linked to hyperexcitability. One of the main challenges in developing KV7 channel active drugs has been to identify compounds capable of discriminating between the neuronally expressed subtypes (KV7.2-5), aiding the identification of the subunit composition of KV7 currents in various tissues, and possessing better therapeutic potential for particular indications. By taking advantage of the structure-activity relationship of acrylamide KV7 channel openers and the effects of these compounds on mutant KV7 channels, we have designed and synthesized a novel KV7 channel modulator with a unique profile. The compound, named SMB-1, is an inhibitor of KV7.2 and an activator of KV7.4. SMB-1 inhibits KV7.2 by reducing the current amplitude and increasing the time constant for the slow component of the activation kinetics. The activation of KV7.4 is seen as an increase in the current amplitude and a slowing of the deactivation kinetics. Experiments studying mutant channels with a compromised binding site for the KV7.2-5 opener retigabine indicate that SMB-1 binds within the same pocket as retigabine for both inhibition of KV7.2 and activation of KV7.4. SMB-1 may serve as a valuable tool for KV7 channel research and may be used as a template for further design of better subtype selective KV7 channel modulators. A compound with this profile could hold novel therapeutic potential such as the treatment of both positive and cognitive symptoms in schizophrenia.

  9. Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions.

    PubMed

    Meisel, Eshcar; Tobelaim, William; Dvir, Meidan; Haitin, Yoni; Peretz, Asher; Attali, Bernard

    2018-01-01

    Inactivation is an intrinsic property of numerous voltage-gated K + (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.

  10. Role of N-terminal domain and accessory subunits in controlling deactivation-inactivation coupling of Kv4.2 channels.

    PubMed

    Barghaan, Jan; Tozakidou, Magdalini; Ehmke, Heimo; Bähring, Robert

    2008-02-15

    We examined the relationship between deactivation and inactivation in Kv4.2 channels. In particular, we were interested in the role of a Kv4.2 N-terminal domain and accessory subunits in controlling macroscopic gating kinetics and asked if the effects of N-terminal deletion and accessory subunit coexpression conform to a kinetic coupling of deactivation and inactivation. We expressed Kv4.2 wild-type channels and N-terminal deletion mutants in the absence and presence of Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) in human embryonic kidney 293 cells. Kv4.2-mediated A-type currents at positive and deactivation tail currents at negative membrane potentials were recorded under whole-cell voltage-clamp and analyzed by multi-exponential fitting. The observed changes in Kv4.2 macroscopic inactivation kinetics caused by N-terminal deletion, accessory subunit coexpression, or a combination of the two maneuvers were compared with respective changes in deactivation kinetics. Extensive correlation analyses indicated that modulatory effects on deactivation closely parallel respective effects on inactivation, including both onset and recovery kinetics. Searching for the structural determinants, which control deactivation and inactivation, we found that in a Kv4.2 Delta 2-10 N-terminal deletion mutant both the initial rapid phase of macroscopic inactivation and tail current deactivation were slowed. On the other hand, the intermediate and slow phase of A-type current decay, recovery from inactivation, and tail current decay kinetics were accelerated in Kv4.2 Delta 2-10 by KChIP2 and DPPX. Thus, a Kv4.2 N-terminal domain, which may control both inactivation and deactivation, is not necessary for active modulation of current kinetics by accessory subunits. Our results further suggest distinct mechanisms for Kv4.2 gating modulation by KChIPs and DPPs.

  11. Rhynchophylline from Uncaria rhynchophylla functionally turns delayed rectifiers into A-Type K+ channels.

    PubMed

    Chou, Chun-Hsiao; Gong, Chi-Li; Chao, Chia-Chia; Lin, Chia-Huei; Kwan, Chiu-Yin; Hsieh, Ching-Liang; Leung, Yuk-Man

    2009-05-22

    Rhynchophylline (1), a neuroprotective agent isolated from the traditional Chinese medicinal herb Uncaria rhynchophylla, was shown to affect voltage-gated K(+) (Kv) channel slow inactivation in mouse neuroblastoma N2A cells. Extracellular 1 (30 microM) accelerated the slow decay of Kv currents and shifted the steady-state inactivation curve to the left. Intracellular dialysis of 1 did not accelerate the slow current decay, suggesting that this compound acts extracellularly. In addition, the percent blockage of Kv currents by this substance was independent of the degree of depolarization and the intracellular K(+) concentration. Therefore, 1 did not appear to directly block the outer channel pore, with the results obtained suggesting that it drastically accelerated Kv channel slow inactivation. Interestingly, 1 also shifted the activation curve to the left. This alkaloid also strongly accelerated slow inactivation and caused a left shift of the activation curve of Kv1.2 channels heterologously expressed in HEK293 cells. Thus, this compound functionally turned delayed rectifiers into A-type K(+) channels.

  12. Kv7 potassium channel subunits and M currents in cultured hippocampal interneurons.

    PubMed

    Grigorov, Alexej; Moskalyuk, Anastasia; Kravchenko, Mykola; Veselovsky, Nikolai; Verkhratsky, Alexei; Fedulova, Svetlana

    2014-09-01

    Potassium channels of the Kv7 family that mediate the non-inactivating M current regulate the excitability of many types of neurons in the central nervous system, including some in the hippocampus. We report here that individual interneurons from newborn rat hippocampi in long-term culture strongly express messenger RNA specific for Kv7.2 and Kv7.3 and, to a lesser extent, Kv7.5 channel subunits but not for the Kv7.4 subunit. An M-like current was electrophysiologically identified in two subpopulations of interneurons distinct in their spiking behaviour (regular or fast spiking). The M-channel enhancer retigabine reduced interneuronal excitability by constraining the number of action potentials generated during imposed depolarisations; this effect was inhibited by specific the M-channel blocking drugs. In paired synaptically connected interneuron-target cell recordings, anatomically localised applications of retigabine indicated that M channels were present in both the interneuron soma and its GABA-ergic inhibitory axon. We conclude that M-channel subunits and functional M channels are broadly expressed in hippocampal interneurons and their axons and are potentially capable of strongly regulating their firing properties.

  13. IA Channels Encoded by Kv1.4 and Kv4.2 Regulate Circadian Period of PER2 Expression in the Suprachiasmatic Nucleus.

    PubMed

    Granados-Fuentes, Daniel; Hermanstyne, Tracey O; Carrasquillo, Yarimar; Nerbonne, Jeanne M; Herzog, Erik D

    2015-10-01

    Neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, display daily rhythms in electrical activity with more depolarized resting potentials and higher firing rates during the day than at night. Although these daily variations in the electrical properties of SCN neurons are required for circadian rhythms in physiology and behavior, the mechanisms linking changes in neuronal excitability to the molecular clock are not known. Recently, we reported that mice deficient for either Kcna4 (Kv1.4(-/-)) or Kcnd2 (Kv4.2(-/-); but not Kcnd3, Kv4.3(-/-)), voltage-gated K(+) (Kv) channel pore-forming subunits that encode subthreshold, rapidly activating, and inactivating K(+) currents (IA), have shortened (0.5 h) circadian periods in SCN firing and in locomotor activity compared with wild-type (WT) mice. In the experiments here, we used a mouse (Per2(Luc)) line engineered with a bioluminescent reporter construct, PERIOD2::LUCIFERASE (PER2::LUC), replacing the endogenous Per2 locus, to test the hypothesis that the loss of Kv1.4- or Kv4.2-encoded IA channels also modifies circadian rhythms in the expression of the clock protein PERIOD2 (PER2). We found that SCN explants from Kv1.4(-/-)Per2(Luc) and Kv4.2(-/-) Per2(Luc), but not Kv4.3(-/-)Per2(Luc), mice have significantly shorter (by approximately 0.5 h) circadian periods in PER2 rhythms, compared with explants from Per2(Luc) mice, revealing that the membrane properties of SCN neurons feedback to regulate clock (PER2) expression. The combined loss of both Kv1.4- and Kv4.2-encoded IA channels in Kv1.4(-/-)/Kv4.2(-/-)Per2(Luc) SCN explants did not result in any further alterations in PER2 rhythms. Interestingly, however, mice lacking both Kv1.4 and Kv4.2 show a striking (approximately 1.8 h) advance in their daily activity onset in a light cycle compared with WT mice, suggesting additional roles for Kv1.4- and Kv4.2-encoded IA channels in controlling the light-dependent responses of neurons within

  14. Molecular determinants of Kv7.1/KCNE1 channel inhibition by amitriptyline.

    PubMed

    Villatoro-Gómez, Kathya; Pacheco-Rojas, David O; Moreno-Galindo, Eloy G; Navarro-Polanco, Ricardo A; Tristani-Firouzi, Martin; Gazgalis, Dimitris; Cui, Meng; Sánchez-Chapula, José A; Ferrer, Tania

    2018-06-01

    Amitriptyline (AMIT) is a compound widely prescribed for psychiatric and non-psychiatric conditions including depression, migraine, chronic pain, and anorexia. However, AMIT has been associated with risks of cardiac arrhythmia and sudden death since it can induce prolongation of the QT interval on the surface electrocardiogram and torsade de pointes ventricular arrhythmia. These complications have been attributed to the inhibition of the rapid delayed rectifier potassium current (I Kr ). The slow delayed rectifier potassium current (I Ks ) is the main repolarizing cardiac current when I Kr is compromised and it has an important role in cardiac repolarization at fast heart rates induced by an elevated sympathetic tone. Therefore, we sought to characterize the effects of AMIT on Kv7.1/KCNE1 and homomeric Kv7.1 channels expressed in HEK-293H cells. Homomeric Kv7.1 and Kv7.1/KCNE1 channels were inhibited by AMIT in a concentration-dependent manner with IC50 values of 8.8 ± 2.1 μM and 2.5 ± 0.8 μM, respectively. This effect was voltage-independent for both homomeric Kv7.1 and Kv7.1/KCNE1 channels. Moreover, mutation of residues located on the P-loop and S6 domain along with molecular docking, suggest that T312, I337 and F340 are the most important molecular determinants for AMIT-Kv7.1 channel interaction. Our experimental findings and modeling suggest that AMIT preferentially blocks the open state of Kv7.1/KCNE1 channels by interacting with specific residues that were previously reported to be important for binding of other compounds, such as chromanol 293B and the benzodiazepine L7. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Physiological modulators of Kv3.1 channels adjust firing patterns of auditory brain stem neurons.

    PubMed

    Brown, Maile R; El-Hassar, Lynda; Zhang, Yalan; Alvaro, Giuseppe; Large, Charles H; Kaczmarek, Leonard K

    2016-07-01

    Many rapidly firing neurons, including those in the medial nucleus of the trapezoid body (MNTB) in the auditory brain stem, express "high threshold" voltage-gated Kv3.1 potassium channels that activate only at positive potentials and are required for stimuli to generate rapid trains of actions potentials. We now describe the actions of two imidazolidinedione derivatives, AUT1 and AUT2, which modulate Kv3.1 channels. Using Chinese hamster ovary cells stably expressing rat Kv3.1 channels, we found that lower concentrations of these compounds shift the voltage of activation of Kv3.1 currents toward negative potentials, increasing currents evoked by depolarization from typical neuronal resting potentials. Single-channel recordings also showed that AUT1 shifted the open probability of Kv3.1 to more negative potentials. Higher concentrations of AUT2 also shifted inactivation to negative potentials. The effects of lower and higher concentrations could be mimicked in numerical simulations by increasing rates of activation and inactivation respectively, with no change in intrinsic voltage dependence. In brain slice recordings of mouse MNTB neurons, both AUT1 and AUT2 modulated firing rate at high rates of stimulation, a result predicted by numerical simulations. Our results suggest that pharmaceutical modulation of Kv3.1 currents represents a novel avenue for manipulation of neuronal excitability and has the potential for therapeutic benefit in the treatment of hearing disorders. Copyright © 2016 the American Physiological Society.

  16. Physiological modulators of Kv3.1 channels adjust firing patterns of auditory brain stem neurons

    PubMed Central

    Brown, Maile R.; El-Hassar, Lynda; Zhang, Yalan; Alvaro, Giuseppe; Large, Charles H.

    2016-01-01

    Many rapidly firing neurons, including those in the medial nucleus of the trapezoid body (MNTB) in the auditory brain stem, express “high threshold” voltage-gated Kv3.1 potassium channels that activate only at positive potentials and are required for stimuli to generate rapid trains of actions potentials. We now describe the actions of two imidazolidinedione derivatives, AUT1 and AUT2, which modulate Kv3.1 channels. Using Chinese hamster ovary cells stably expressing rat Kv3.1 channels, we found that lower concentrations of these compounds shift the voltage of activation of Kv3.1 currents toward negative potentials, increasing currents evoked by depolarization from typical neuronal resting potentials. Single-channel recordings also showed that AUT1 shifted the open probability of Kv3.1 to more negative potentials. Higher concentrations of AUT2 also shifted inactivation to negative potentials. The effects of lower and higher concentrations could be mimicked in numerical simulations by increasing rates of activation and inactivation respectively, with no change in intrinsic voltage dependence. In brain slice recordings of mouse MNTB neurons, both AUT1 and AUT2 modulated firing rate at high rates of stimulation, a result predicted by numerical simulations. Our results suggest that pharmaceutical modulation of Kv3.1 currents represents a novel avenue for manipulation of neuronal excitability and has the potential for therapeutic benefit in the treatment of hearing disorders. PMID:27052580

  17. G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity.

    PubMed

    Stott, Jennifer B; Povstyan, Oleksandr V; Carr, Georgina; Barrese, Vincenzo; Greenwood, Iain A

    2015-05-19

    Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K(+) currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2-250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein-coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein-coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.

  18. G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity

    PubMed Central

    Stott, Jennifer B.; Povstyan, Oleksandr V.; Carr, Georgina; Barrese, Vincenzo; Greenwood, Iain A.

    2015-01-01

    Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K+ currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2–250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein–coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein–coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone. PMID:25941381

  19. KChIP1 modulation of Kv4.3-mediated A-type K(+) currents and repetitive firing in hippocampal interneurons.

    PubMed

    Bourdeau, M L; Laplante, I; Laurent, C E; Lacaille, J-C

    2011-03-10

    Neuronal A-type K(+) channels regulate action potential waveform, back-propagation and firing frequency. In hippocampal CA1 interneurons located at the stratum lacunosum-moleculare/radiatum junction (LM/RAD), Kv4.3 mediates A-type K(+) currents and a Kv4 β-subunit of the Kv channel interacting protein (KChIP) family, KChIP1, appears specifically expressed in these cells. However, the functional role of this accessory subunit in A-type K(+) currents and interneuron excitability remains largely unknown. Thus, first we studied KChIP1 and Kv4.3 channel interactions in human embryonic kidney 293 (HEK293) cells and determined that KChIP1 coexpression modulated the biophysical properties of Kv4.3 A-type currents (faster recovery from inactivation, leftward shift of activation curve, faster rise time and slower decay) and this modulation was selectively prevented by KChIP1 short interfering RNA (siRNA) knockdown. Next, we evaluated the effects of KChIP1 down-regulation by siRNA on A-type K(+) currents in LM/RAD interneurons in slice cultures. Recovery from inactivation of A-type K(+) currents was slower after KChIP1 down-regulation but other properties were unchanged. In addition, down-regulation of KChIP1 levels did not affect action potential waveform and firing, but increased firing frequency during suprathreshold depolarizations, indicating that KChIP1 regulates interneuron excitability. The effects of KChIP1 down-regulation were cell-specific since CA1 pyramidal cells that do not express KChIP1 were unaffected. Overall, our findings suggest that KChIP1 interacts with Kv4.3 in LM/RAD interneurons, enabling faster recovery from inactivation of A-type currents and thus promoting stronger inhibitory control of firing during sustained activity. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  20. Anti-voltage-gated potassium channel Kv1.4 antibodies in myasthenia gravis.

    PubMed

    Romi, Fredrik; Suzuki, Shigeaki; Suzuki, Norihiro; Petzold, Axel; Plant, Gordon T; Gilhus, Nils Erik

    2012-07-01

    Myasthenia gravis (MG) is an autoimmune disease characterized by skeletal muscle weakness mainly caused by acetylcholine receptor antibodies. MG can be divided into generalized and ocular, and into early-onset (<50 years of age) and late-onset (≥50 years of age). Anti-Kv1.4 antibodies targeting α-subunits (Kv1.4) of the voltage-gated potassium K(+) channel occurs frequently among patients with severe MG, accounting for 18% of a Japanese MG population. The aim of this study was to characterize the clinical features and serological associations of anti-Kv1.4 antibodies in a Caucasian MG population with mild and localized MG. Serum samples from 129 Caucasian MG patients with mainly ocular symptoms were tested for the presence of anti-Kv1.4 antibodies and compared to clinical and serological parameters. There were 22 (17%) anti-Kv1.4 antibody-positive patients, most of them women with late-onset MG, and all of them with mild MG. This contrasts to the Japanese anti-Kv1.4 antibody-positive patients who suffered from severe MG with bulbar symptoms, myasthenic crisis, thymoma, myocarditis and prolonged QT time on electrocardiography, despite equal anti-Kv1.4 antibody occurrence in both populations. No other clinical or serological parameters influenced anti-Kv1.4 antibody occurrence.

  1. Role of Kv7 channels in responses of the pulmonary circulation to hypoxia.

    PubMed

    Sedivy, Vojtech; Joshi, Shreena; Ghaly, Youssef; Mizera, Roman; Zaloudikova, Marie; Brennan, Sean; Novotna, Jana; Herget, Jan; Gurney, Alison M

    2015-01-01

    Hypoxic pulmonary vasoconstriction (HPV) is a beneficial mechanism that diverts blood from hypoxic alveoli to better ventilated areas of the lung, but breathing hypoxic air causes the pulmonary circulation to become hypertensive. Responses to airway hypoxia are associated with depolarization of smooth muscle cells in the pulmonary arteries and reduced activity of K(+) channels. As Kv7 channels have been proposed to play a key role in regulating the smooth muscle membrane potential, we investigated their involvement in the development of HPV and hypoxia-induced pulmonary hypertension. Vascular effects of the selective Kv7 blocker, linopirdine, and Kv7 activator, flupirtine, were investigated in isolated, saline-perfused lungs from rats maintained for 3-5 days in an isobaric hypoxic chamber (FiO2 = 0.1) or room air. Linopirdine increased vascular resistance in lungs from normoxic, but not hypoxic rats. This effect was associated with reduced mRNA expression of the Kv7.4 channel α-subunit in hypoxic arteries, whereas Kv7.1 and Kv7.5 were unaffected. Flupirtine had no effect in normoxic lungs but reduced vascular resistance in hypoxic lungs. Moreover, oral dosing with flupirtine (30 mg/kg/day) prevented short-term in vivo hypoxia from increasing pulmonary vascular resistance and sensitizing the arteries to acute hypoxia. These findings suggest a protective role for Kv7.4 channels in the pulmonary circulation, limiting its reactivity to pressor agents and preventing hypoxia-induced pulmonary hypertension. They also provide further support for the therapeutic potential of Kv7 activators in pulmonary vascular disease. Copyright © 2015 the American Physiological Society.

  2. Modulatory mechanisms and multiple functions of somatodendritic A-type K+ channel auxiliary subunits

    PubMed Central

    Jerng, Henry H.; Pfaffinger, Paul J.

    2014-01-01

    Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders. PMID:24723849

  3. Modulatory mechanisms and multiple functions of somatodendritic A-type K (+) channel auxiliary subunits.

    PubMed

    Jerng, Henry H; Pfaffinger, Paul J

    2014-01-01

    Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders.

  4. Tracking single Kv2.1 channels in live cells reveals anomalous subdiffusion and ergodicity breaking

    NASA Astrophysics Data System (ADS)

    Weigel, Aubrey; Simon, Blair; Tamkun, Michael; Krapf, Diego

    2011-03-01

    The dynamic organization of the plasma membrane is responsible for essential cellular processes, such as receptor trafficking and signaling. By studying the dynamics of transmembrane proteins a greater understanding of these processes as a whole can be achieved. It is broadly observed that the diffusion pattern of membrane protein displays anomalous subdiffusion. However, the mechanisms responsible for this behavior are not yet established. We explore the dynamics of the voltage gated potassium channel Kv2.1 by using single-particle tracking. We analyze Kv2.1 channel trajectories in terms of the time and ensemble distributions of square displacements. Our results reveal that all Kv2.1 channels experience anomalous subdiffusion and we observe that the Kv2.1 diffusion pattern is non-ergodic. We further investigated the role of the actin cytoskeleton in these channel dynamics by applying actin depolymerizing drugs. It is seen that with the breakdown of the actin cytoskeleton the Kv2.1 channel trajectories recover ergodicity.

  5. Polarized targeting of a shaker-like (A-type) K(+)-channel in the polarized epithelial cell line MDCK.

    PubMed

    Le Maout, S; Sewing, S; Coudrier, E; Elalouf, J M; Pongs, O; Merot, J

    1996-01-01

    Functional Kv 1-4 channels were stably expressed in filter-grown MDCK cells which form a polarized epithelium with two distinct plasma membrane domains: a basolateral and an apical cell surface. The Shaker-related Kv 1-4 channels mediated in MDCK cells fast transient (A-type) voltage-activated outward currents having similar properties to the ones reported for Kv 1-4 in the Xenopus oocytes expression system. Immunoblot analysis with specific anti-Kv 1-4 antibodies showed that two Kv 1-4 protein forms are expressed in MDCK cells which most likely represent the glycosylated and non-glycosylated Kv 1-4 protein, respectively. Using immunocytochemistry and confocal microscopy we showed that the Kv 1-4 channels are specifically localized in the basolateral membranes of MDCK cells. Thus, the MDCK cells may provide an important model system to analyse the polarized transport of ion channels such as Kv 1-4, which are distinctly expressed in the mammalian central nervous system.

  6. Suppression of Inflammatory Demyelinaton and Axon Degeneration through Inhibiting Kv3 Channels

    PubMed Central

    Jukkola, Peter; Gu, Yuanzheng; Lovett-Racke, Amy E.; Gu, Chen

    2017-01-01

    The development of neuroprotective and repair strategies for treating progressive multiple sclerosis (MS) requires new insights into axonal injury. 4-aminopyridine (4-AP), a blocker of voltage-gated K+ (Kv) channels, is used in symptomatic treatment of progressive MS, but the underlying mechanism remains unclear. Here we report that deleting Kv3.1—the channel with the highest 4-AP sensitivity—reduces clinical signs in experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. In Kv3.1 knockout (KO) mice, EAE lesions in sensory and motor tracts of spinal cord were markedly reduced, and radial astroglia were activated with increased expression of brain derived neurotrophic factor (BDNF). Kv3.3/Kv3.1 and activated BDNF receptors were upregulated in demyelinating axons in EAE and MS lesions. In spinal cord myelin coculture, BDNF treatment promoted myelination, and neuronal firing via altering channel expression. Therefore, suppressing Kv3.1 alters neural circuit activity, which may enhance BNDF signaling and hence protect axons from inflammatory insults. PMID:29123469

  7. Functional and Molecular Evidence for Kv7 Channel Subtypes in Human Detrusor from Patients with and without Bladder Outflow Obstruction

    PubMed Central

    Svalø, Julie; Sheykhzade, Majid; Nordling, Jørgen; Matras, Christina; Bouchelouche, Pierre

    2015-01-01

    The aim of the study was to investigate whether Kv7 channels and their ancillary β-subunits, KCNE, are functionally expressed in the human urinary bladder. Kv7 channels were examined at the molecular level and by functional studies using RT-qPCR and myography, respectively. We found mRNA expression of KCNQ1, KCNQ3-KCNQ5 and KCNE1-5 in the human urinary bladder from patients with normal bladder function (n = 7) and in patients with bladder outflow obstruction (n = 3). Interestingly, a 3.4-fold up-regulation of KCNQ1 was observed in the latter. The Kv7 channel subtype selective modulators, ML277 (activator of Kv7.1 channels, 10 μM) and ML213 (activator of Kv7.2, Kv7.4, Kv7.4/7.5 and Kv7.5 channels, 10 μM), reduced the tone of 1 μM carbachol pre-constricted bladder strips. XE991 (blocker of Kv7.1–7.5 channels, 10 μM) had opposing effects as it increased contractions achieved with 20 mM KPSS. Furthermore, we investigated if there is interplay between Kv7 channels and β-adrenoceptors. Using cumulative additions of isoprenaline (β-adrenoceptor agonist) and forskolin (adenylyl cyclase activator) in combination with the Kv7 channel activator and blocker, retigabine and XE991, we did not find interplay between Kv7 channels and β-adrenoceptors in the human urinary bladder. The performed gene expression analysis combined with the organ bath studies imply that compounds that activate Kv7 channels could be useful for treatment of overactive bladder syndrome. PMID:25692982

  8. Functional and molecular evidence for Kv7 channel subtypes in human detrusor from patients with and without bladder outflow obstruction.

    PubMed

    Svalø, Julie; Sheykhzade, Majid; Nordling, Jørgen; Matras, Christina; Bouchelouche, Pierre

    2015-01-01

    The aim of the study was to investigate whether Kv7 channels and their ancillary β-subunits, KCNE, are functionally expressed in the human urinary bladder. Kv7 channels were examined at the molecular level and by functional studies using RT-qPCR and myography, respectively. We found mRNA expression of KCNQ1, KCNQ3-KCNQ5 and KCNE1-5 in the human urinary bladder from patients with normal bladder function (n = 7) and in patients with bladder outflow obstruction (n = 3). Interestingly, a 3.4-fold up-regulation of KCNQ1 was observed in the latter. The Kv7 channel subtype selective modulators, ML277 (activator of Kv7.1 channels, 10 μM) and ML213 (activator of Kv7.2, Kv7.4, Kv7.4/7.5 and Kv7.5 channels, 10 μM), reduced the tone of 1 μM carbachol pre-constricted bladder strips. XE991 (blocker of Kv7.1-7.5 channels, 10 μM) had opposing effects as it increased contractions achieved with 20 mM KPSS. Furthermore, we investigated if there is interplay between Kv7 channels and β-adrenoceptors. Using cumulative additions of isoprenaline (β-adrenoceptor agonist) and forskolin (adenylyl cyclase activator) in combination with the Kv7 channel activator and blocker, retigabine and XE991, we did not find interplay between Kv7 channels and β-adrenoceptors in the human urinary bladder. The performed gene expression analysis combined with the organ bath studies imply that compounds that activate Kv7 channels could be useful for treatment of overactive bladder syndrome.

  9. Coexpression of high-voltage-activated ion channels Kv3.4 and Cav1.2 in pioneer axons during pathfinding in the developing rat forebrain.

    PubMed

    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. Copyright © 2012 Wiley Periodicals, Inc.

  10. KCNE1 remodels the voltage sensor of Kv7.1 to modulate channel function.

    PubMed

    Wu, Dick; Pan, Hua; Delaloye, Kelli; Cui, Jianmin

    2010-12-01

    The KCNE1 auxiliary subunit coassembles with the Kv7.1 channel and modulates its properties to generate the cardiac I(Ks) current. Recent biophysical evidence suggests that KCNE1 interacts with the voltage-sensing domain (VSD) of Kv7.1. To investigate the mechanism of how KCNE1 affects the VSD to alter the voltage dependence of channel activation, we perturbed the VSD of Kv7.1 by mutagenesis and chemical modification in the absence and presence of KCNE1. Mutagenesis of S4 in Kv7.1 indicates that basic residues in the N-terminal half (S4-N) and C-terminal half (S4-C) of S4 are important for stabilizing the resting and activated states of the channel, respectively. KCNE1 disrupts electrostatic interactions involving S4-C, specifically with the lower conserved glutamate in S2 (Glu(170) or E2). Likewise, Trp scanning of S4 shows that mutations to a cluster of residues in S4-C eliminate current in the presence of KCNE1. In addition, KCNE1 affects S4-N by enhancing MTS accessibility to the top of the VSD. Consistent with the structure of Kv channels and previous studies on the KCNE1-Kv7.1 interaction, these results suggest that KCNE1 alters the interactions of S4 residues with the surrounding protein environment, possibly by changing the protein packing around S4, thereby affecting the voltage dependence of Kv7.1. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  11. Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability

    PubMed Central

    Speca, David J.; Ogata, Genki; Mandikian, Danielle; Bishop, Hannah I.; Wiler, Steve W.; Eum, Kenneth; Wenzel, H. Jürgen; Doisy, Emily T.; Matt, Lucas; Campi, Katharine L.; Golub, Mari S.; Nerbonne, Jeanne M.; Hell, Johannes W.; Trainor, Brian C.; Sack, Jon T.; Schwartzkroin, Philip A.; Trimmer, James S.

    2014-01-01

    The Kv2.1 delayed rectifier potassium channel exhibits high-level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity-dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1−/−) mice lacking this channel. Kv2.1−/− mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1−/− mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1−/− mice appears unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1−/− animals. Field recordings from hippocampal slices of Kv2.1−/− mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1−/− mice, long-term potentiation at the Schaffer collateral – CA1 synapse is decreased. Kv2.1−/− mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1−/− mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1−/− mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function. PMID:24494598

  12. Induction of stable ER–plasma-membrane junctions by Kv2.1 potassium channels

    PubMed Central

    Fox, Philip D.; Haberkorn, Christopher J.; Akin, Elizabeth J.; Seel, Peter J.; Krapf, Diego; Tamkun, Michael M.

    2015-01-01

    ABSTRACT Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER–plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K+ channel in the mammalian brain, induces the formation of ER–plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER–plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER–plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca2+ signaling. PMID:25908859

  13. Molecular basis for the toxin insensitivity of scorpion voltage-gated potassium channel MmKv1.

    PubMed

    Zhang, Chuangeng; Xie, Zili; Li, Xinxin; Chen, Jing; Feng, Jing; Lang, Yange; Yang, Weishan; Li, Wenxin; Chen, Zongyun; Yao, Jing; Cao, Zhijian; Wu, Yingliang

    2016-05-01

    Scorpions are insensitive to their own venoms, which contain various neurotoxins specific for mammalian or insect ion channels, whose molecular mechanism remains unsolved. Using MmKv1, a potassium channel identified from the genome of the scorpion Mesobuthus martensii, channel kinetic experiments showed that MmKv1 was a classical voltage-gated potassium channel with a voltage-dependent fast activation and slow inactivation. Compared with the human Kv1.3 channel (hKv1.3), the MmKv1 channel exhibited a remarkable insensitivity to both scorpion venom and toxin. The chimaeric channels of MmKv1 and hKv1.3 revealed that both turret and filter regions of the MmKv1 channel were critical for the toxin insensitivity of MmKv1. Furthermore, mutagenesis of the chimaeric channel indicated that two basic residues (Arg(399) and Lys(403)) in the MmKv1 turret region and Arg(425) in the MmKv1 filter region significantly affected its toxin insensitivity. Moreover, when these three basic residues of MmKv1 were simultaneously substituted with the corresponding residues from hKv1.3, the MmKv1-R399T/K403S/R425H mutant channels exhibited similar sensitivity to both scorpion venom and toxin to hKv1.3, which revealed the determining role of these three basic residues in the toxin insensitivity of the MmKv1 channel. More strikingly, a similar triad sequence structure is present in all Shaker-like channels from venomous invertebrates, which suggested a possible convergent functional evolution of these channels to enable them to resist their own venoms. Together, these findings first illustrate the mechanism by which scorpions are insensitive to their own venoms at the ion channel receptor level and enrich our knowledge of the insensitivity of scorpions and other venomous animals to their own venoms. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

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

    PubMed

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

    2010-05-19

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

  15. A Quantitative Analysis of Neurons with Kv3 Potassium Channel Subunits–Kv3.1b and Kv3.2–in Macaque Primary Visual Cortex

    PubMed Central

    Constantinople, Christine M.; Disney, Anita A; Maffie, Jonathan; Rudy, Bernardo; Hawken, Michael J

    2010-01-01

    Voltage-gated potassium channels that are composed of Kv3 subunits exhibit distinct electrophysiological properties: activation at more depolarized potentials than other voltage-gated K+ channels and fast kinetics. These channels have been shown to contribute to the high-frequency firing of fast-spiking (FS) GABAergic interneurons in the rat and mouse brain. In the rodent neocortex, there are distinct patterns of expression for the Kv3.1b and Kv3.2 channel subunits and of co-expression of these subunits with neurochemical markers, such as the calcium-binding proteins parvalbumin (PV) and calbindin D-28K (CB). The distribution of Kv3 channels and interrelationship with calcium-binding protein expression has not been investigated in primate cortex. We used immunoperoxidase and immunofluorescent labeling and stereological counting techniques to characterize the laminar and cell-type distributions of Kv3-ir neurons in macaque V1. We found that across the cortical layers ~25% of both Kv3.1b- and Kv3.2-ir neurons are non-GABAergic. In contrast all Kv3-ir neurons in rodent cortex are GABAergic (Chow et al., 1999). The putatively excitatory Kv3-ir neurons were mostly located in layers 2, 3 and 4b. Further, the proportion of Kv3-ir neurons that express PV or CB also differs between macaque V1 and rodent cortex. These data indicate that, within the population of cortical neurons, a broader population of neurons, encompassing cells of a wider range of morphological classes may be capable of sustaining high-frequency firing in macaque V1. PMID:19634181

  16. The pan-Kv7 (KCNQ) Channel Opener Retigabine Inhibits Striatal Excitability by Direct Action on Striatal Neurons In Vivo.

    PubMed

    Hansen, Henrik H; Weikop, Pia; Mikkelsen, Maria D; Rode, Frederik; Mikkelsen, Jens D

    2017-01-01

    Central Kv7 (KCNQ) channels are voltage-dependent potassium channels composed of different combinations of four Kv7 subunits, being differently expressed in the brain. Notably, striatal dopaminergic neurotransmission is strongly suppressed by systemic administration of the pan-Kv7 channel opener retigabine. The effect of retigabine likely involves the inhibition of the activity in mesencephalic dopaminergic neurons projecting to the striatum, but whether Kv7 channels expressed in the striatum may also play a role is not resolved. We therefore assessed the effect of intrastriatal retigabine administration on striatal neuronal excitability in the rat determined by c-Fos immunoreactivity, a marker of neuronal activation. When retigabine was applied locally in the striatum, this resulted in a marked reduction in the number of c-Fos-positive neurons after a strong excitatory striatal stimulus induced by acute systemic haloperidol administration in the rat. The relative mRNA levels of Kv7 subunits in the rat striatum were found to be Kv7.2 = Kv7.3 = Kv7.5 > >Kv7.4. These data suggest that intrastriatal Kv7 channels play a direct role in regulating striatal excitability in vivo. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  17. Hydrophobic interactions between the voltage sensor and pore mediate inactivation in Kv11.1 channels

    PubMed Central

    Perry, Matthew D.; Wong, Sophia; Ng, Chai Ann

    2013-01-01

    Kv11.1 channels are critical for the maintenance of a normal heart rhythm. The flow of potassium ions through these channels is controlled by two voltage-regulated gates, termed “activation” and “inactivation,” located at opposite ends of the pore. Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating. Although it is clear that the fourth transmembrane segments (S4), within each subunit of the tetrameric channel, are important for controlling the opening and closing of the activation gate, their role during inactivation gating is much less clear. Here, we use rate equilibrium free energy relationship (REFER) analysis to probe the contribution of the S4 “voltage-sensor” helix during inactivation of Kv11.1 channels. Contrary to the important role that charged residues play during activation gating, it is the hydrophobic residues (Leu529, Leu530, Leu532, and Val535) that are the key molecular determinants of inactivation gating. Within the context of an interconnected multi-domain model of Kv11.1 inactivation gating, our REFER analysis indicates that the S4 helix and the S4–S5 linker undergo a conformational rearrangement shortly after that of the S5 helix and S5P linker, but before the S6 helix. Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices. Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating. PMID:23980196

  18. Kv1 channels control spike threshold dynamics and spike timing in cortical pyramidal neurones

    PubMed Central

    Higgs, Matthew H; Spain, William J

    2011-01-01

    Abstract Previous studies showed that cortical pyramidal neurones (PNs) have a dynamic spike threshold that functions as a high-pass filter, enhancing spike timing in response to high-frequency input. While it is commonly assumed that Na+ channel inactivation is the primary mechanism of threshold accommodation, the possible role of K+ channel activation in fast threshold changes has not been well characterized. The present study tested the hypothesis that low-voltage activated Kv1 channels affect threshold dynamics in layer 2–3 PNs, using α-dendrotoxin (DTX) or 4-aminopyridine (4-AP) to block these conductances. We found that Kv1 blockade reduced the dynamic changes of spike threshold in response to a variety of stimuli, including stimulus-evoked synaptic input, current steps and ramps of varied duration, and noise. Analysis of the responses to noise showed that Kv1 channels increased the coherence of spike output with high-frequency components of the stimulus. A simple model demonstrates that a dynamic spike threshold can account for this effect. Our results show that the Kv1 conductance is a major mechanism that contributes to the dynamic spike threshold and precise spike timing of cortical PNs. PMID:21911608

  19. Restoration of Kv7 Channel-Mediated Inhibition Reduces Cued-Reinstatement of Cocaine Seeking.

    PubMed

    Parrilla-Carrero, Jeffrey; Buchta, William C; Goswamee, Priyodarshan; Culver, Oliver; McKendrick, Greer; Harlan, Benjamin; Moutal, Aubin; Penrod, Rachel; Lauer, Abigail; Ramakrishnan, Viswanathan; Khanna, Rajesh; Kalivas, Peter; Riegel, Arthur C

    2018-04-25

    Cocaine addicts display increased sensitivity to drug-associated cues, due in part to changes in the prelimbic prefrontal cortex (PL-PFC). The cellular mechanisms underlying cue-induced reinstatement of cocaine seeking remain unknown. Reinforcement learning for addictive drugs may produce persistent maladaptations in intrinsic excitability within sparse subsets of PFC pyramidal neurons. Using a model of relapse in male rats, we sampled >600 neurons to examine spike frequency adaptation (SFA) and afterhyperpolarizations (AHPs), two systems that attenuate low-frequency inputs to regulate neuronal synchronization. We observed that training to self-administer cocaine or nondrug (sucrose) reinforcers decreased SFA and AHPs in a subpopulation of PL-PFC neurons. Only with cocaine did the resulting hyperexcitability persist through extinction training and increase during reinstatement. In neurons with intact SFA, dopamine enhanced excitability by inhibiting Kv7 potassium channels that mediate SFA. However, dopamine effects were occluded in neurons from cocaine-experienced rats, where SFA and AHPs were reduced. Pharmacological stabilization of Kv7 channels with retigabine restored SFA and Kv7 channel function in neuroadapted cells. When microinjected bilaterally into the PL-PFC 10 min before reinstatement testing, retigabine reduced cue-induced reinstatement of cocaine seeking. Last, using cFos-GFP transgenic rats, we found that the loss of SFA correlated with the expression of cFos-GFP following both extinction and re-exposure to drug-associated cues. Together, these data suggest that cocaine self-administration desensitizes inhibitory Kv7 channels in a subpopulation of PL-PFC neurons. This subpopulation of neurons may represent a persistent neural ensemble responsible for driving drug seeking in response to cues. SIGNIFICANCE STATEMENT Long after the cessation of drug use, cues associated with cocaine still elicit drug-seeking behavior, in part by activation of the

  20. PKC-dependent regulation of Kv7.5 channels by the bronchoconstrictor histamine in human airway smooth muscle cells.

    PubMed

    Haick, Jennifer M; Brueggemann, Lioubov I; Cribbs, Leanne L; Denning, Mitchell F; Schwartz, Jeffrey; Byron, Kenneth L

    2017-06-01

    Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca 2+ ([Ca 2+ ] cyt ). The latter response was inhibited by verapamil, a blocker of L-type voltage-sensitive Ca 2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, although the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca 2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction. Copyright © 2017 the American Physiological Society.

  1. Kv7 Channel Activation Underpins EPAC-Dependent Relaxations of Rat Arteries.

    PubMed

    Stott, Jennifer B; Barrese, Vincenzo; Greenwood, Iain A

    2016-12-01

    To establish the role of Kv7 channels in EPAC (exchange protein directly activated by cAMP)-dependent relaxations of the rat vasculature and to investigate whether this contributes to β-adrenoceptor-mediated vasorelaxations. Isolated rat renal and mesenteric arteries (RA and MA, respectively) were used for isometric tension recording to study the relaxant effects of a specific EPAC activator and the β-adrenoceptor agonist isoproterenol in the presence of potassium channel inhibitors and cell signaling modulators. Isolated myocytes were used in proximity ligation assay studies to detect localization of signaling intermediaries with Kv7.4 before and after cell stimulation. Our studies showed that the EPAC activator (8-pCPT-2Me-cAMP-AM) produced relaxations and enhanced currents of MA and RA that were sensitive to linopirdine (Kv7 inhibitor). Linopirdine also inhibited isoproterenol-mediated relaxations in both RA and MA. In the MA, isoproterenol relaxations were sensitive to EPAC inhibition, but not protein kinase A inhibition. In contrast, isoproterenol relaxations in RA were attenuated by protein kinase A but not by EPAC inhibition. Proximity ligation assay showed a localization of Kv7.4 with A-kinase anchoring protein in both vessels in the basal state, which increased only in the RA with isoproterenol stimulation. In the MA, but not the RA, a localization of Kv7.4 with both Rap1a and Rap2 (downstream of EPAC) increased with isoproterenol stimulation. EPAC-dependent vasorelaxations occur in part via activation of Kv7 channels. This contributes to the isoproterenol-mediated relaxation in mesenteric, but not renal, arteries. © 2016 American Heart Association, Inc.

  2. Kv7 channels critically determine coronary artery reactivity: left-right differences and down-regulation by hyperglycaemia.

    PubMed

    Morales-Cano, Daniel; Moreno, Laura; Barreira, Bianca; Pandolfi, Rachele; Chamorro, Virginia; Jimenez, Rosario; Villamor, Eduardo; Duarte, Juan; Perez-Vizcaino, Francisco; Cogolludo, Angel

    2015-04-01

    Voltage-gated potassium channels encoded by KCNQ genes (Kv7 channels) are emerging as important regulators of vascular tone. In this study, we analysed the contribution of Kv7 channels to the vasodilation induced by hypoxia and the cyclic AMP pathway in the coronary circulation. We also assessed their regional distribution and possible impairment by diabetes. We examined the effects of Kv7 channel modulators on K+ currents and vascular reactivity in rat left and right coronary arteries (LCAs and RCAs, respectively). Currents from LCA were more sensitive to Kv7 channel inhibitors (XE991, linopirdine) and activators (flupirtine, retigabine) than those from RCA. Accordingly, LCAs were more sensitive than RCAs to the relaxation induced by Kv7 channel enhancers. Likewise, relaxation induced by the adenylyl cyclase activator forskolin and hypoxia, which were mediated through Kv7 channel activation, were greater in LCA than in RCA. KCNQ1 and KCNQ5 expression was markedly higher in LCA than in RCA. After incubation with high glucose (HG, 30 mmol/L), myocytes from LCA, but not from RCA, were more depolarized and showed reduced Kv7 currents. In HG-incubated LCA, the effects of Kv7 channel modulators and forskolin were diminished, and the expression of KCNQ1 and KCNQ5 was reduced. Finally, vascular responses induced by Kv7 channel modulators were impaired in LCA, but not in RCA, from type 1 diabetic rats. Our results reveal that the high expression and function of Kv7 channels in the LCA and their down-regulation by diabetes critically determine the sensitivity to key regulators of coronary tone. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

  3. Alternative splicing modulates Kv channel clustering through a molecular ball and chain mechanism

    NASA Astrophysics Data System (ADS)

    Zandany, Nitzan; Marciano, Shir; Magidovich, Elhanan; Frimerman, Teddy; Yehezkel, Rinat; Shem-Ad, Tzilhav; Lewin, Limor; Abdu, Uri; Orr, Irit; Yifrach, Ofer

    2015-03-01

    Ion channel clustering at the post-synaptic density serves a fundamental role in action potential generation and transmission. Here, we show that interaction between the Shaker Kv channel and the PSD-95 scaffold protein underlying channel clustering is modulated by the length of the intrinsically disordered C terminal channel tail. We further show that this tail functions as an entropic clock that times PSD-95 binding. We thus propose a ‘ball and chain’ mechanism to explain Kv channel binding to scaffold proteins, analogous to the mechanism describing channel fast inactivation. The physiological relevance of this mechanism is demonstrated in that alternative splicing of the Shaker channel gene to produce variants of distinct tail lengths resulted in differential channel cell surface expression levels and clustering metrics that correlate with differences in affinity of the variants for PSD-95. We suggest that modulating channel clustering by specific spatial-temporal spliced variant targeting serves a fundamental role in nervous system development and tuning.

  4. KV7 channels contribute to paracrine, but not metabolic or ischemic, regulation of coronary vascular reactivity in swine

    PubMed Central

    Goodwill, Adam G.; Fu, Lijuan; Noblet, Jillian N.; Casalini, Eli D.; Berwick, Zachary C.; Kassab, Ghassan S.; Tune, Johnathan D.

    2016-01-01

    Hydrogen peroxide (H2O2) and voltage-dependent K+ (KV) channels play key roles in regulating coronary blood flow in response to metabolic, ischemic, and paracrine stimuli. The KV channels responsible have not been identified, but KV7 channels are possible candidates. Existing data regarding KV7 channel function in the coronary circulation (limited to ex vivo assessments) are mixed. Thus we examined the hypothesis that KV7 channels are present in cells of the coronary vascular wall and regulate vasodilation in swine. We performed a variety of molecular, biochemical, and functional (in vivo and ex vivo) studies. Coronary arteries expressed KCNQ genes (quantitative PCR) and KV7.4 protein (Western blot). Immunostaining demonstrated KV7.4 expression in conduit and resistance vessels, perhaps most prominently in the endothelial and adventitial layers. Flupirtine, a KV7 opener, relaxed coronary artery rings, and this was attenuated by linopirdine, a KV7 blocker. Endothelial denudation inhibited the flupirtine-induced and linopirdine-sensitive relaxation of coronary artery rings. Moreover, linopirdine diminished bradykinin-induced endothelial-dependent relaxation of coronary artery rings. There was no effect of intracoronary flupirtine or linopirdine on coronary blood flow at the resting heart rate in vivo. Linopirdine had no effect on coronary vasodilation in vivo elicited by ischemia, H2O2, or tachycardia. However, bradykinin increased coronary blood flow in vivo, and this was attenuated by linopirdine. These data indicate that KV7 channels are expressed in some coronary cell type(s) and influence endothelial function. Other physiological functions of coronary vascular KV7 channels remain unclear, but they do appear to contribute to endothelium-dependent responses to paracrine stimuli. PMID:26825518

  5. KV7 channels contribute to paracrine, but not metabolic or ischemic, regulation of coronary vascular reactivity in swine.

    PubMed

    Goodwill, Adam G; Fu, Lijuan; Noblet, Jillian N; Casalini, Eli D; Sassoon, Daniel; Berwick, Zachary C; Kassab, Ghassan S; Tune, Johnathan D; Dick, Gregory M

    2016-03-15

    Hydrogen peroxide (H2O2) and voltage-dependent K(+) (KV) channels play key roles in regulating coronary blood flow in response to metabolic, ischemic, and paracrine stimuli. The KV channels responsible have not been identified, but KV7 channels are possible candidates. Existing data regarding KV7 channel function in the coronary circulation (limited to ex vivo assessments) are mixed. Thus we examined the hypothesis that KV7 channels are present in cells of the coronary vascular wall and regulate vasodilation in swine. We performed a variety of molecular, biochemical, and functional (in vivo and ex vivo) studies. Coronary arteries expressed KCNQ genes (quantitative PCR) and KV7.4 protein (Western blot). Immunostaining demonstrated KV7.4 expression in conduit and resistance vessels, perhaps most prominently in the endothelial and adventitial layers. Flupirtine, a KV7 opener, relaxed coronary artery rings, and this was attenuated by linopirdine, a KV7 blocker. Endothelial denudation inhibited the flupirtine-induced and linopirdine-sensitive relaxation of coronary artery rings. Moreover, linopirdine diminished bradykinin-induced endothelial-dependent relaxation of coronary artery rings. There was no effect of intracoronary flupirtine or linopirdine on coronary blood flow at the resting heart rate in vivo. Linopirdine had no effect on coronary vasodilation in vivo elicited by ischemia, H2O2, or tachycardia. However, bradykinin increased coronary blood flow in vivo, and this was attenuated by linopirdine. These data indicate that KV7 channels are expressed in some coronary cell type(s) and influence endothelial function. Other physiological functions of coronary vascular KV7 channels remain unclear, but they do appear to contribute to endothelium-dependent responses to paracrine stimuli. Copyright © 2016 the American Physiological Society.

  6. Heteromeric Kv7.2/7.3 channels differentially regulate action potential initiation and conduction in neocortical myelinated axons.

    PubMed

    Battefeld, Arne; Tran, Baouyen T; Gavrilis, Jason; Cooper, Edward C; Kole, Maarten H P

    2014-03-05

    Rapid energy-efficient signaling along vertebrate axons is achieved through intricate subcellular arrangements of voltage-gated ion channels and myelination. One recently appreciated example is the tight colocalization of K(v)7 potassium channels and voltage-gated sodium (Na(v)) channels in the axonal initial segment and nodes of Ranvier. The local biophysical properties of these K(v)7 channels and the functional impact of colocalization with Na(v) channels remain poorly understood. Here, we quantitatively examined K(v)7 channels in myelinated axons of rat neocortical pyramidal neurons using high-resolution confocal imaging and patch-clamp recording. K(v)7.2 and 7.3 immunoreactivity steeply increased within the distal two-thirds of the axon initial segment and was mirrored by the conductance density estimates, which increased from ~12 (proximal) to 150 pS μm(-2) (distal). The axonal initial segment and nodal M-currents were similar in voltage dependence and kinetics, carried by K(v)7.2/7.3 heterotetramers, 4% activated at the resting membrane potential and rapidly activated with single-exponential time constants (~15 ms at 28 mV). Experiments and computational modeling showed that while somatodendritic K(v)7 channels are strongly activated by the backpropagating action potential to attenuate the afterdepolarization and repetitive firing, axonal K(v)7 channels are minimally recruited by the forward-propagating action potential. Instead, in nodal domains K(v)7.2/7.3 channels were found to increase Na(v) channel availability and action potential amplitude by stabilizing the resting membrane potential. Thus, K(v)7 clustering near axonal Na(v) channels serves specific and context-dependent roles, both restraining initiation and enhancing conduction of the action potential.

  7. Heteromeric Kv7.2/7.3 Channels Differentially Regulate Action Potential Initiation and Conduction in Neocortical Myelinated Axons

    PubMed Central

    Battefeld, Arne; Tran, Baouyen T.; Gavrilis, Jason; Cooper, Edward C.

    2014-01-01

    Rapid energy-efficient signaling along vertebrate axons is achieved through intricate subcellular arrangements of voltage-gated ion channels and myelination. One recently appreciated example is the tight colocalization of Kv7 potassium channels and voltage-gated sodium (Nav) channels in the axonal initial segment and nodes of Ranvier. The local biophysical properties of these Kv7 channels and the functional impact of colocalization with Nav channels remain poorly understood. Here, we quantitatively examined Kv7 channels in myelinated axons of rat neocortical pyramidal neurons using high-resolution confocal imaging and patch-clamp recording. Kv7.2 and 7.3 immunoreactivity steeply increased within the distal two-thirds of the axon initial segment and was mirrored by the conductance density estimates, which increased from ∼12 (proximal) to 150 pS μm−2 (distal). The axonal initial segment and nodal M-currents were similar in voltage dependence and kinetics, carried by Kv7.2/7.3 heterotetramers, 4% activated at the resting membrane potential and rapidly activated with single-exponential time constants (∼15 ms at 28 mV). Experiments and computational modeling showed that while somatodendritic Kv7 channels are strongly activated by the backpropagating action potential to attenuate the afterdepolarization and repetitive firing, axonal Kv7 channels are minimally recruited by the forward-propagating action potential. Instead, in nodal domains Kv7.2/7.3 channels were found to increase Nav channel availability and action potential amplitude by stabilizing the resting membrane potential. Thus, Kv7 clustering near axonal Nav channels serves specific and context-dependent roles, both restraining initiation and enhancing conduction of the action potential. PMID:24599470

  8. Nonreciprocal mechanisms in up- and downregulation of spinal motoneuron excitability by modulators of KCNQ/Kv7 channels

    PubMed Central

    Lombardo, Joseph

    2016-01-01

    KCNQ/Kv7 channels form a slow noninactivating K+ current, also known as the M current. They activate in the subthreshold range of membrane potentials and regulate different aspects of excitability in neurons of the central nervous system. In spinal motoneurons (MNs), KCNQ/Kv7 channels have been identified in the somata, axonal initial segment, and nodes of Ranvier, where they generate a slow, noninactivating, K+ current sensitive to both muscarinic receptor-mediated inhibition and KCNQ/Kv7 channel blockers. In this study, we thoroughly reevaluated the function of up- and downregulation of KCNQ/Kv7 channels in mouse immature spinal MNs. Using electrophysiological techniques together with specific pharmacological modulators of the activity of KCNQ/Kv7 channels, we show that enhancement of the activity of these channels decreases the excitability of spinal MNs in mouse neonates. This action on MNs results from a combination of hyperpolarization of the resting membrane potential, a decrease in the input resistance, and depolarization of the voltage threshold. On the other hand, the effect of inhibition of KCNQ/Kv7 channels suggested that these channels play a limited role in regulating basal excitability. Computer simulations confirmed that pharmacological enhancement of KCNQ/Kv7 channel activity decreases excitability and also suggested that the effects of inhibition of KCNQ/Kv7 channels on the excitability of spinal MNs do not depend on a direct effect in these neurons but likely on spinal cord synaptic partners. These results indicate that KCNQ/Kv7 channels have a fundamental role in the modulation of the excitability of spinal MNs acting both in these neurons and in their local presynaptic partners. PMID:27512022

  9. Voltage-Gated K+ Channel, Kv3.3 Is Involved in Hemin-Induced K562 Differentiation

    PubMed Central

    Song, Min Seok; Choi, Seon Young; Ryu, Pan Dong; Lee, So Yeong

    2016-01-01

    Voltage-gated K+ (Kv) channels are well known to be involved in cell proliferation. However, even though cell proliferation is closely related to cell differentiation, the relationship between Kv channels and cell differentiation remains poorly investigated. This study demonstrates that Kv3.3 is involved in K562 cell erythroid differentiation. Down-regulation of Kv3.3 using siRNA-Kv3.3 increased hemin-induced K562 erythroid differentiation through decreased activation of signal molecules such as p38, cAMP response element-binding protein, and c-fos. Down-regulation of Kv3.3 also enhanced cell adhesion by increasing integrin β3 and this effect was amplified when the cells were cultured with fibronectin. The Kv channels, or at least Kv3.3, appear to be associated with cell differentiation; therefore, understanding the mechanisms of Kv channel regulation of cell differentiation would provide important information regarding vital cellular processes. PMID:26849432

  10. Selective Modulation of K+ Channel Kv7.4 Significantly Affects the Excitability of DRN 5-HT Neurons.

    PubMed

    Zhao, Chen; Su, Min; Wang, Yingzi; Li, Xinmeng; Zhang, Yongxue; Du, Xiaona; Zhang, Hailin

    2017-01-01

    The serotonin (5-HT) system originating in the dorsal raphe nucleus (DRN) is implicated in various mood- and emotion-related disorders, such as anxiety, fear and stress. Abnormal activity of DRN 5-HT neurons is the key factor in the development of these disorders. Here, we describe a crucial role for the Kv7.4 potassium channel in modulating DRN 5-HT neuronal excitability. We demonstrate that Kv7.4 is selectively expressed in 5-HT neurons of the DRN. Using selective Kv7.4 opener fasudil and Kv7.4 knock-out mice, we demonstrate that Kv7.4 is a potent modulator of DRN 5-HT neuronal excitability. Furthermore, we demonstrate that the cellular redox signaling mechanism is involved in this 5-HT activation of Kv7.4. The current study suggests a new strategy for treating psychiatric disorders related to altered activity of DRN 5-HT neurons using K + channel modulators.

  11. Altered expression and localization of hippocampal A-type potassium channel subunits in the pilocarpine-induced model of temporal lobe epilepsy.

    PubMed

    Monaghan, M M; Menegola, M; Vacher, H; Rhodes, K J; Trimmer, J S

    2008-10-15

    Altered ion channel expression and/or function may contribute to the development of certain human epilepsies. In rats, systemic administration of pilocarpine induces a model of human temporal lobe epilepsy, wherein a brief period of status epilepticus (SE) triggers development of spontaneous recurrent seizures that appear after a latency of 2-3 weeks. Here we investigate changes in expression of A-type voltage-gated potassium (Kv) channels, which control neuronal excitability and regulate action potential propagation and neurotransmitter release, in the pilocarpine model of epilepsy. Using immunohistochemistry, we examined the expression of component subunits of somatodendritic (Kv4.2, Kv4.3, KChIPl and KChIP2) and axonal (Kv1.4) A-type Kv channels in hippocampi of pilocarpine-treated rats that entered SE. We found that Kv4.2, Kv4.3 and KChIP2 staining in the molecular layer of the dentate gyrus changes from being uniformly distributed across the molecular layer to concentrated in just the outer two-thirds. We also observed a loss of KChIP1 immunoreactive interneurons, and a reduction of Kv4.2 and KChIP2 staining in stratum radiatum of CA1. These changes begin to appear 1 week after pilocarpine treatment and persist or are enhanced at 4 and 12 weeks. As such, these changes in Kv channel distribution parallel the acquisition of recurrent spontaneous seizures as observed in this model. We also found temporal changes in Kv1.4 immunoreactivity matching those in Timm's stain, being expanded in stratum lucidum of CA3 and in the inner third of the dentate molecular layer. Among pilocarpine-treated rats, changes were only observed in those that entered SE. These changes in A-type Kv channel expression may contribute to hyperexcitability of dendrites in the associated hippocampal circuits as observed in previous studies of the effects of pilocarpine-induced SE.

  12. Zn2+-dependent redox switch in the intracellular T1-T1 interface of a Kv channel.

    PubMed

    Wang, Guangyu; Strang, Candace; Pfaffinger, Paul J; Covarrubias, Manuel

    2007-05-04

    The thiol-based redox regulation of proteins plays a central role in cellular signaling. Here, we investigated the redox regulation at the Zn(2+) binding site (HX(5)CX(20)CC) in the intracellular T1-T1 inter-subunit interface of a Kv4 channel. This site undergoes conformational changes coupled to voltage-dependent gating, which may be sensitive to oxidative stress. The main results show that internally applied nitric oxide (NO) inhibits channel activity profoundly. This inhibition is reversed by reduced glutathione and suppressed by intracellular Zn(2+), and at least two Zn(2+) site cysteines are required to observe the NO-induced inhibition (Cys-110 from one subunit and Cys-132 from the neighboring subunit). Biochemical evidence suggests strongly that NO induces a disulfide bridge between Cys-110 and Cys-132 in intact cells. Finally, further mutational studies suggest that intra-subunit Zn(2+) coordination involving His-104, Cys-131, and Cys-132 protects against the formation of the inhibitory disulfide bond. We propose that the interfacial T1 Zn(2+) site of Kv4 channels acts as a Zn(2+)-dependent redox switch that may regulate the activity of neuronal and cardiac A-type K(+) currents under physiological and pathological conditions.

  13. A functional Kv1.2-hERG chimaeric channel expressed in Pichia pastoris

    PubMed Central

    Dhillon, Mandeep S.; Cockcroft, Christopher J.; Munsey, Tim; Smith, Kathrine J.; Powell, Andrew J.; Carter, Paul; Wrighton, David C.; Rong, Hong-lin; Yusaf, Shahnaz P.; Sivaprasadarao, Asipu

    2014-01-01

    Members of the six-transmembrane segment family of ion channels share a common structural design. However, there are sequence differences between the members that confer distinct biophysical properties on individual channels. Currently, we do not have 3D structures for all members of the family to help explain the molecular basis for the differences in their biophysical properties and pharmacology. This is due to low-level expression of many members in native or heterologous systems. One exception is rat Kv1.2 which has been overexpressed in Pichia pastoris and crystallised. Here, we tested chimaeras of rat Kv1.2 with the hERG channel for function in Xenopus oocytes and for overexpression in Pichia. Chimaera containing the S1–S6 transmembrane region of HERG showed functional and pharmacological properties similar to hERG and could be overexpressed and purified from Pichia. Our results demonstrate that rat Kv1.2 could serve as a surrogate to express difficult-to-overexpress members of the six-transmembrane segment channel family. PMID:24569544

  14. Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase.

    PubMed

    Gomes, Pedro; Saito, Tomoaki; Del Corsso, Cris; Alioua, Abderrahmane; Eghbali, Mansoureh; Toro, Ligia; Stefani, Enrico

    2008-10-01

    Voltage-gated K(+) (Kv) channels are key determinants of cardiac and neuronal excitability. A substantial body of evidence has accumulated in support of a role for Src family tyrosine kinases in the regulation of Kv channels. In this study, we examined the possibility that c-Src tyrosine kinase participates in the modulation of the transient voltage-dependent K(+) channel Kv4.3. Supporting a mechanistic link between Kv4.3 and c-Src, confocal microscopy analysis of HEK293 cells stably transfected with Kv4.3 showed high degree of co-localization of the two proteins at the plasma membrane. Our results further demonstrate an association between Kv4.3 and c-Src by co-immunoprecipitation and GST pull-down assays, this interaction being mediated by the SH2 and SH3 domains of c-Src. Furthermore, we show that Kv4.3 is tyrosine phosphorylated under basal conditions. The functional relevance of the observed interaction between Kv4.3 and c-Src was established in patch-clamp experiments, where application of the Src inhibitor PP2 caused a decrease in Kv4.3 peak current amplitude, but not the inactive structural analogue PP3. Conversely, intracellular application of recombinant c-Src kinase or the protein tyrosine phosphatase inhibitor bpV(phen) increased Kv4.3 peak current amplitude. In conclusion, our findings provide evidence that c-Src-induced Kv4.3 channel activation involves their association in a macromolecular complex and suggest a role for c-Src-Kv4.3 pathway in regulating cardiac and neuronal excitability.

  15. How does KCNE1 regulate the Kv7.1 potassium channel? Model-structure, mutations, and dynamics of the Kv7.1-KCNE1 complex.

    PubMed

    Gofman, Yana; Shats, Simona; Attali, Bernard; Haliloglu, Turkan; Ben-Tal, Nir

    2012-08-08

    The voltage-gated potassium channel Kv7.1 and its auxiliary subunit KCNE1 are expressed in the heart and give rise to the major repolarization current. The interaction of Kv7.1 with the single transmembrane helix of KCNE1 considerably slows channel activation and deactivation, raises single-channel conductance, and prevents slow voltage-dependent inactivation. We built a Kv7.1-KCNE1 model-structure. The model-structure agrees with previous disulfide mapping studies and enables us to derive molecular interpretations of electrophysiological recordings that we obtained for two KCNE1 mutations. An elastic network analysis of Kv7.1 fluctuations in the presence and absence of KCNE1 suggests a mechanistic perspective on the known effects of KCNE1 on Kv7.1 function: slow deactivation is attributed to the low mobility of the voltage-sensor domains upon KCNE1 binding, abolishment of voltage-dependent inactivation could result from decreased fluctuations in the external vestibule, and amalgamation of the fluctuations in the pore region is associated with enhanced ion conductivity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Identification of a key residue in Kv7.1 potassium channel essential for sensing external potassium ions.

    PubMed

    Wang, Wenying; Flores, Maria Cristina Perez; Sihn, Choong-Ryoul; Kim, Hyo Jeong; Zhang, Yinuo; Doyle, Karen J; Chiamvimonvat, Nipavan; Zhang, Xiao-Dong; Yamoah, Ebenezer N

    2015-03-01

    Kv7.1 voltage-gated K(+) (Kv) channels are present in the apical membranes of marginal cells of the stria vascularis of the inner ear, where they mediate K(+) efflux into the scala media (cochlear duct) of the cochlea. As such, they are exposed to the K(+)-rich (∼ 150 mM of external K(+) (K(+) e)) environment of the endolymph. Previous studies have shown that Kv7.1 currents are substantially suppressed by high K(+) e (independent of the effects of altering the electrochemical gradient). However, the molecular basis for this inhibition, which is believed to involve stabilization of an inactivated state, remains unclear. Using sequence alignment of S5-pore linkers of several Kv channels, we identified a key residue, E290, found in only a few Kv channels including Kv7.1. We used substituted cysteine accessibility methods and patch-clamp analysis to provide evidence that the ability of Kv7.1 to sense K(+) e depends on E290, and that the charge at this position is essential for Kv7.1's K(+) e sensitivity. We propose that Kv7.1 may use this feedback mechanism to maintain the magnitude of the endocochlear potential, which boosts the driving force to generate the receptor potential of hair cells. The implications of our findings transcend the auditory system; mutations at this position also result in long QT syndrome in the heart. © 2015 Wang et al.

  17. Cellular Mechanism of Inner Ear Genetic Disease, roles of Kv7.1 (KCNQ1) Channel

    NASA Astrophysics Data System (ADS)

    Mousavi Nik, Atefeh

    Potassium channels are the most diverse and widely distributed membrane protein in all living organisms. They have various roles in the body such as controlling membrane potential, cell volume, and cell migration. Many studies have shown that mutation in these channels is associated with different diseases for example: Hearing Defect, Cardiac Arrhythmia, Episodic Ataxia, Seizure and Neuromyotonia. One of the most important diseases associated with K+ channel mutations is called Jervell and Lange-Nielsen syndrome (JLNS). This disease causes bilateral congenital deafness and the patients also suffer from Long QT and they usually experience syncopal episodes in their life and eventually die as a result of cardiac arrest. The gene KCNQ1 encodes the Kv7.1 voltage gated potassium channel. This channel expresses in apical membrane of marginal cell in stria vasularis of cochlea and secret K+ ion to endolymp to keep the endocochlear potential stable, which is necessary for the inner ear to function properly. Kv7.1 channel also expresses in cardiac myocytes and mutation in this gene is associated with another syndrome called Romano-Ward syndrome (RWS). Although Romano-Ward patients have mutation in KCNQ1, similar to Jervell and Lange-Nielsen patients, they only suffer from cardiac defect, and their hearing is completely normal. Several studies identified that mutations in Kv7.1 gene is associated with JLNS and RWS, but the biophysical and cellular mechanisms of these mutations are still unknown. To determine the cellular mechanisms of JLNS and RWS, and to provide mechanistic insight on the functional outputs of JLNS versus RWS mutations, we generated several mutant forms of the human Kv7.1 ( KCNQ1) clone, using site-directed mutagenesis to define their sub-cellular localization and examined their electrophysiological properties. We identified JLNS and RWS mutations at the S4-S5-linker, the pore loop (P-loop) and the C-terminus of hKv7.1 which have been found to control

  18. miR-1 is increased in pulmonary hypertension and downregulates Kv1.5 channels in rat pulmonary arteries.

    PubMed

    Mondejar-Parreño, Gema; Callejo, María; Barreira, Bianca; Morales-Cano, Daniel; Esquivel-Ruiz, Sergio; Moreno, Laura; Cogolludo, Angel; Perez-Vizcaino, Francisco

    2018-05-02

    ■The expression of miR-1 is increased in lungs from the Hyp/Su5416 PAH rat model. ■PASMC from this animal model are more depolarised and show decreased expression and activity of Kv1.5. ■miR-1 directly targets Kv1.5 channels, reduces Kv1.5 activity and induces membrane depolarization. ■Antagomir-1 prevents Kv1.5 channel downregulation and the depolarization induced by hypoxia/Su5416 exposition. Impairment of voltage-dependent potassium channel (Kv) plays a central role in the development of cardiovascular diseases, including pulmonary arterial hypertension (PAH). MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the 3'-UTR region of specific mRNAs. The aim of this study was to analyze the effects of miR-1 on Kv channel function in pulmonary arteries (PA). Kv channel activity was studied in PA from healthy animals transfected with miR-1 or scrambled-miR. Kv currents were studied using the whole-cell configuration of patch-clamp technique. The characterization of the Kv1.5 currents was performed with the selective inhibitor DPO-1. miR-1 expression was increased and Kv1.5 channels were decreased in lungs from a rat model of PAH induced by hypoxia and Su5416. miR-1 transfection increased cell capacitance, reduced Kv1.5 currents and induced membrane depolarization in isolated pulmonary artery smooth muscle cells (PASMCs). Luciferase reporter assay indicated that KCNA5, which encodes Kv1.5 channels, is a direct target gene of miR-1. Incubation of PA with Su5416 and hypoxia (3% O 2 ) increased miR-1 and induced a decline in Kv1.5 currents, which was prevented by antagomiR-1. In conclusion, these data indicate that miR-1 induces PASMC hypertrophy and reduces the activity and expression of Kv channels, suggesting a pathophysiological role in PAH. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  19. Discovery of a novel Kv7 channel opener as a treatment for epilepsy.

    PubMed

    Davoren, Jennifer E; Claffey, Michelle M; Snow, Sheri L; Reese, Matthew R; Arora, Gaurav; Butler, Christopher R; Boscoe, Brian P; Chenard, Lois; DeNinno, Shari L; Drozda, Susan E; Duplantier, Allen J; Moine, Ludivine; Rogers, Bruce N; Rong, SuoBao; Schuyten, Katherine; Wright, Ann S; Zhang, Lei; Serpa, Kevin A; Weber, Mark L; Stolyar, Polina; Whisman, Tammy L; Baker, Karen; Tse, Karen; Clark, Alan J; Rong, Haojing; Mather, Robert J; Lowe, John A

    2015-11-01

    Facilitating activation, or delaying inactivation, of the native Kv7 channel reduces neuronal excitability, which may be beneficial in controlling spontaneous electrical activity during epileptic seizures. In an effort to identify a compound with such properties, the structure-activity relationship (SAR) and in vitro ADME for a series of heterocyclic Kv7.2-7.5 channel openers was explored. PF-05020182 (2) demonstrated suitable properties for further testing in vivo where it dose-dependently decreased the number of animals exhibiting full tonic extension convulsions in response to corneal stimulation in the maximal electroshock (MES) assay. In addition, PF-05020182 (2) significantly inhibited convulsions in the MES assay at doses tested, consistent with in vitro activity measure. The physiochemical properties, in vitro and in vivo activities of PF-05020182 (2) support further development as an adjunctive treatment of refractory epilepsy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Gating currents from Kv7 channels carrying neuronal hyperexcitability mutations in the voltage-sensing domain.

    PubMed

    Miceli, Francesco; Vargas, Ernesto; Bezanilla, Francisco; Taglialatela, Maurizio

    2012-03-21

    Changes in voltage-dependent gating represent a common pathogenetic mechanism for genetically inherited channelopathies, such as benign familial neonatal seizures or peripheral nerve hyperexcitability caused by mutations in neuronal K(v)7.2 channels. Mutation-induced changes in channel voltage dependence are most often inferred from macroscopic current measurements, a technique unable to provide a detailed assessment of the structural rearrangements underlying channel gating behavior; by contrast, gating currents directly measure voltage-sensor displacement during voltage-dependent gating. In this work, we describe macroscopic and gating current measurements, together with molecular modeling and molecular-dynamics simulations, from channels carrying mutations responsible for benign familial neonatal seizures and/or peripheral nerve hyperexcitability; K(v)7.4 channels, highly related to K(v)7.2 channels both functionally and structurally, were used for these experiments. The data obtained showed that mutations affecting charged residues located in the more distal portion of S(4) decrease the stability of the open state and the active voltage-sensing domain configuration but do not directly participate in voltage sensing, whereas mutations affecting a residue (R4) located more proximally in S(4) caused activation of gating-pore currents at depolarized potentials. These results reveal that distinct molecular mechanisms underlie the altered gating behavior of channels carrying disease-causing mutations at different voltage-sensing domain locations, thereby expanding our current view of the pathogenesis of neuronal hyperexcitability diseases. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Pharmacological Targeting Of Neuronal Kv7.2/3 Channels: A Focus On Chemotypes And Receptor Sites.

    PubMed

    Miceli, Francesco; Soldovieri, Maria Virginia; Ambrosino, Paolo; Manocchio, Laura; Medoro, Alessandro; Mosca, Ilaria; Taglialatela, Maurizio

    2017-10-12

    The Kv7 (KCNQ) subfamily of voltage-gated potassium channels consists of 5 members (Kv7.1-5) each showing a characteristic tissue distribution and physiological roles. Given their functional heterogeneity, Kv7 channels represent important pharmacological targets for development of new drugs for neuronal, cardiac and metabolic diseases. In the present manuscript, we focus on describing the pharmacological relevance and the potential therapeutic applications of drugs acting on neuronally-expressed Kv7.2/3 channels, placing particular emphasis on the different modulator chemotypes, and highlighting their pharmacodynamic and, whenever possible, pharmacokinetic peculiarities. The present work is based on an in-depth search of the currently available scientific literature, and on our own experience and knowledge in the field of neuronal Kv7 channel pharmacology. Space limitations impeded to describe the full pharmacological potential of Kv7 channels; thus, we have chosen to focus on neuronal channels composed of Kv7.2 and Kv7.3 subunits, and to mainly concentrate on their involvement in epilepsy. An astonishing heterogeneity in the molecular scaffolds exploitable to develop Kv7.2/3 modulators is evident, with important structural/functional peculiarities of distinct compound classes. In the present work we have attempted to show the current status and growing potential of the Kv7 pharmacology field. We anticipate a bright future for the field, and we express our hopes that the efforts herein reviewed will result in an improved treatment of hyperexcitability (or any other) diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. The role of PSD-95 in the rearrangement of Kv1.3 channels to the immunological synapse.

    PubMed

    Szilágyi, Orsolya; Boratkó, Anita; Panyi, György; Hajdu, Péter

    2013-09-01

    Establishment of the immunological synapse (IS) between T lymphocytes and antigen-presenting cells is a key step in the adaptive immune response. Several proteins accumulate in the IS, such as the Kv1.3 potassium channel; however, the mechanism of this translocation is unknown. PSD-95 and SAP97 are adaptor proteins that regulate the polarized cell surface expression and localization of Kv1 channels in neurons. We investigated whether these proteins affect the redistribution of Kv1.3 into the IS in non-excitable human T cells. We show here that PSD-95 and SAP97 are expressed in Jurkat and interact with the C terminus of Kv1.3. Disruption of the interaction between PSD-95 or SAP97 and Kv1.3 in Jurkat was realized by the expression of a C-terminal truncated Kv1.3, which lacks the binding domain for these proteins, or by the knockdown of the expression of PSD-95 or SAP97 using specific shRNA. Expression of the truncated Kv1.3 or knockdown of PSD-95, but not the knockdown of SAP97, inhibited the recruitment of Kv1.3 into the IS; the fraction of cells showing polarized Kv1.3 expression upon engagement in an IS was significantly lower than in control cells expressing the full-length Kv1.3, and the rearrangement of Kv1.3 did not show time dependence. In contrast, Jurkat cells expressing the full-length channel showed marked time dependence in the recruitment into the IS peaking at 1 min after the conjugation of the cells. These results demonstrate that PSD-95 participates in the targeting of Kv1.3 into the IS, implying its important role in human T-cell activation.

  3. Kv4 channels underlie A-currents with highly variable inactivation time courses but homogeneous other gating properties in the nucleus tractus solitarii.

    PubMed

    Strube, Caroline; Saliba, Layal; Moubarak, Estelle; Penalba, Virginie; Martin-Eauclaire, Marie-France; Tell, Fabien; Clerc, Nadine

    2015-04-01

    In the nucleus of the tractus solitarii (NTS), a large proportion of neurones express transient A-type potassium currents (I KA) having deep influence on the fidelity of the synaptic transmission of the visceral primary afferent inputs to second-order neurones. Up to now, the strong impact of I KA within the NTS was considered to result exclusively from its variation in amplitude, and its molecular correlate(s) remained unknown. In order to identify which Kv channels underlie I KA in NTS neurones, the gating properties and the pharmacology of this current were determined using whole cell patch clamp recordings in slices. Complementary information was brought by immunohistochemistry. Strikingly, two neurone subpopulations characterized by fast or slow inactivation time courses (respectively about 50 and 200 ms) were discriminated. Both characteristics matched those of the Kv4 channel subfamily. The other gating properties, also matching the Kv4 channel ones, were homogeneous through the NTS. The activation and inactivation occurred at membrane potentials around the threshold for generating action potentials, and the time course of recovery from inactivation was rapid. Pharmacologically, I KA in NTS neurones was found to be resistant to tetraethylammonium (TEA), sea anemone toxin blood-depressing substance (BDS) and dendrotoxin (DTX), whereas Androctonus mauretanicus mauretanicus toxin 3 (AmmTX3), a scorpion toxin of the α-KTX 15 family that has been shown to block all the members of the Kv4 family, inhibited 80 % of I KA irrespectively of its inactivation time course. Finally, immunohistochemistry data suggested that, among the Kv4 channel subfamily, Kv4.3 is the prevalent subunit expressed in the NTS.

  4. Expression and function of Kv1.1 potassium channels in human atria from patients with atrial fibrillation.

    PubMed

    Glasscock, Edward; Voigt, Niels; McCauley, Mark D; Sun, Qiang; Li, Na; Chiang, David Y; Zhou, Xiao-Bo; Molina, Cristina E; Thomas, Dierk; Schmidt, Constanze; Skapura, Darlene G; Noebels, Jeffrey L; Dobrev, Dobromir; Wehrens, Xander H T

    2015-09-01

    Voltage-gated Kv1.1 channels encoded by the Kcna1 gene are traditionally regarded as being neural-specific with no known expression or intrinsic functional role in the heart. However, recent studies in mice reveal low-level Kv1.1 expression in heart and cardiac abnormalities associated with Kv1.1-deficiency suggesting that the channel may have a previously unrecognized cardiac role. Therefore, this study tests the hypothesis that Kv1.1 channels are associated with arrhythmogenesis and contribute to intrinsic cardiac function. In intra-atrial burst pacing experiments, Kcna1-null mice exhibited increased susceptibility to atrial fibrillation (AF). The atria of Kcna1-null mice showed minimal Kv1 family ion channel remodeling and fibrosis as measured by qRT-PCR and Masson's trichrome histology, respectively. Using RT-PCR, immunocytochemistry, and immunoblotting, KCNA1 mRNA and protein were detected in isolated mouse cardiomyocytes and human atria for the first time. Patients with chronic AF (cAF) showed no changes in KCNA1 mRNA levels relative to controls; however, they exhibited increases in atrial Kv1.1 protein levels, not seen in paroxysmal AF patients. Patch-clamp recordings of isolated human atrial myocytes revealed significant dendrotoxin-K (DTX-K)-sensitive outward current components that were significantly increased in cAF patients, reflecting a contribution by Kv1.1 channels. The concomitant increases in Kv1.1 protein and DTX-K-sensitive currents in atria of cAF patients suggest that the channel contributes to the pathological mechanisms of persistent AF. These findings provide evidence of an intrinsic cardiac role of Kv1.1 channels and indicate that they may contribute to atrial repolarization and AF susceptibility.

  5. Expression and function of Kv1.1 potassium channels in human atria from patients with atrial fibrillation

    PubMed Central

    Glasscock, Edward; Voigt, Niels; McCauley, Mark D.; Sun, Qiang; Li, Na; Chiang, David Y.; Zhou, Xiao-Bo; Molina, Cristina E.; Thomas, Dierk; Schmidt, Constanze; Skapura, Darlene G.; Noebels, Jeffrey L.; Dobrev, Dobromir; Wehrens, Xander H. T.

    2016-01-01

    Voltage-gated Kv1.1 channels encoded by the Kcna1 gene are traditionally regarded as being neural-specific with no known expression or intrinsic functional role in the heart. However, recent studies in mice reveal low-level Kv1.1 expression in heart and cardiac abnormalities associated with Kv1.1-deficiency suggesting that the channel may have a previously unrecognized cardiac role. Therefore, this study tests the hypothesis that Kv1.1 channels are associated with arrhythmogenesis and contribute to intrinsic cardiac function. In intra-atrial burst pacing experiments, Kcna1-null mice exhibited increased susceptibility to atrial fibrillation (AF). The atria of Kcna1-null mice showed minimal Kv1 family ion channel remodeling and fibrosis as measured by qRT-PCR and Masson’s trichrome histology, respectively. Using RT-PCR, immunocytochemistry, and immunoblotting, KCNA1 mRNA and protein were detected in isolated mouse cardiomyocytes and human atria for the first time. Patients with chronic AF (cAF) showed no changes in KCNA1 mRNA levels relative to controls; however, they exhibited increases in atrial Kv1.1 protein levels, not seen in paroxysmal AF patients. Patch-clamp recordings of isolated human atrial myocytes revealed significant dendrotoxin-K (DTX-K)-sensitive outward current components that were significantly increased in cAF patients, reflecting a contribution by Kv1.1 channels. The concomitant increases in Kv1.1 protein and DTX-K-sensitive currents in atria of cAF patients suggest that the channel contributes to the pathological mechanisms of persistent AF. These findings provide evidence of an intrinsic cardiac role of Kv1.1 channels and indicate that they may contribute to atrial repolarization and AF susceptibility. PMID:26162324

  6. Kv7 channels are upregulated during striatal neuron development and promote maturation of human iPSC-derived neurons.

    PubMed

    Telezhkin, Vsevolod; Straccia, Marco; Yarova, Polina; Pardo, Monica; Yung, Sun; Vinh, Ngoc-Nga; Hancock, Jane M; Barriga, Gerardo Garcia-Diaz; Brown, David A; Rosser, Anne E; Brown, Jonathan T; Canals, Josep M; Randall, Andrew D; Allen, Nicholas D; Kemp, Paul J

    2018-05-24

    Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.

  7. Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels*

    PubMed Central

    Ng, Chai Ann; Phan, Kevin; Hill, Adam P.; Vandenberg, Jamie I.; Perry, Matthew D.

    2014-01-01

    The intracellular domains of many ion channels are important for fine-tuning their gating kinetics. In Kv11.1 channels, the slow kinetics of channel deactivation, which are critical for their function in the heart, are largely regulated by the N-terminal N-Cap and Per-Arnt-Sim (PAS) domains, as well as the C-terminal cyclic nucleotide-binding homology (cNBH) domain. Here, we use mutant cycle analysis to probe for functional interactions between the N-Cap/PAS domains and the cNBH domain. We identified a specific and stable charge-charge interaction between Arg56 of the PAS domain and Asp803 of the cNBH domain, as well an additional interaction between the cNBH domain and the N-Cap, both of which are critical for maintaining slow deactivation kinetics. Furthermore, we found that positively charged arginine residues within the disordered region of the N-Cap interact with negatively charged residues of the C-linker domain. Although this interaction is likely more transient than the PAS-cNBD interaction, it is strong enough to stabilize the open conformation of the channel and thus slow deactivation. These findings provide novel insights into the slow deactivation mechanism of Kv11.1 channels. PMID:25074935

  8. Ca2+-Calmodulin and PIP2 interactions at the proximal C-terminus of Kv7 channels.

    PubMed

    Tobelaim, William S; Dvir, Meidan; Lebel, Guy; Cui, Meng; Buki, Tal; Peretz, Asher; Marom, Milit; Haitin, Yoni; Logothetis, Diomedes E; Hirsch, Joel A; Attali, Bernard

    2017-11-02

    In the heart, co-assembly of Kv7.1 with KCNE1 produces the slow I KS potassium current, which repolarizes the cardiac action potential and mutations in human Kv7.1 and KCNE1 genes cause cardiac arrhythmias. The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ) and recently we revealed the competition of PIP 2 with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation. Data indicated that PIP 2 and Ca 2+ -CaM perform the same function on I KS channel gating to stabilize the channel open state. Here we show that similar features were observed for Kv7.1 currents expressed alone. We also find that conservation of homologous residues in helix B of other Kv7 subtypes confer similar competition of Ca 2+ -CaM with PIP2 binding to their proximal C-termini and suggest that PIP2-CaM interactions converge to Kv7 helix B to modulates channel activity in a Kv7 subtype-dependent manner.

  9. Modulation of Kv7 potassium channels by a novel opener pyrazolo[1,5-a]pyrimidin-7(4H)-one compound QO-58

    PubMed Central

    Zhang, F; Mi, Y; Qi, JL; Li, JW; Si, M; Guan, BC; Du, XN; An, HL; Zhang, HL

    2013-01-01

    Background and Purpose Modulation of Kv7/M channel function represents a relatively new strategy to treat neuronal excitability disorders such as epilepsy and neuropathic pain. We designed and synthesized a novel series of pyrazolo[1,5-a] pyrimidin-7(4H)-one compounds, which activate Kv7 channels. Here, we characterized the effects of the lead compound, QO-58, on Kv7 channels and investigated its mechanism of action. Experimental Approach A perforated whole-cell patch technique was used to record Kv7 currents expressed in mammalian cell lines and M-type currents from rat dorsal root ganglion neurons. The effects of QO-58 in a rat model of neuropathic pain, chronic constriction injury (CCI) of the sciatic nerve, were also examined. Key Results QO-58 increased the current amplitudes, shifted the voltage-dependent activation curve in a more negative direction and slowed the deactivation of Kv7.2/Kv7.3 currents. QO-58 activated Kv7.1, Kv7.2, Kv7.4 and Kv7.3/Kv7.5 channels with a more selective effect on Kv7.2 and Kv7.4, but little effect on Kv7.3. The mechanism of QO-58's activation of Kv7 channels was clearly distinct from that used by retigabine. A chain of amino acids, Val224Val225Tyr226, in Kv7.2 was important for QO-58 activation of this channel. QO-58 enhanced native neuronal M currents, resulting in depression of evoked action potentials. QO-58 also elevated the pain threshold of neuropathic pain in the sciatic nerve CCI model. Conclusions and Implications The results indicate that QO-58 is a potent modulator of Kv7 channels with a mechanism of action different from those of known Kv7 openers. Hence, QO-58 shows potential as a treatment for diseases associated with neuronal hyperexcitability. PMID:23013484

  10. Decrease of a Current Mediated by Kv1.3 Channels Causes Striatal Cholinergic Interneuron Hyperexcitability in Experimental Parkinsonism.

    PubMed

    Tubert, Cecilia; Taravini, Irene R E; Flores-Barrera, Eden; Sánchez, Gonzalo M; Prost, María Alejandra; Avale, María Elena; Tseng, Kuei Y; Rela, Lorena; Murer, Mario Gustavo

    2016-09-06

    The mechanism underlying a hypercholinergic state in Parkinson's disease (PD) remains uncertain. Here, we show that disruption of the Kv1 channel-mediated function causes hyperexcitability of striatal cholinergic interneurons in a mouse model of PD. Specifically, our data reveal that Kv1 channels containing Kv1.3 subunits contribute significantly to the orphan potassium current known as IsAHP in striatal cholinergic interneurons. Typically, this Kv1 current provides negative feedback to depolarization that limits burst firing and slows the tonic activity of cholinergic interneurons. However, such inhibitory control of cholinergic interneuron excitability by Kv1.3-mediated current is markedly diminished in the parkinsonian striatum, suggesting that targeting Kv1.3 subunits and their regulatory pathways may have therapeutic potential in PD therapy. These studies reveal unexpected roles of Kv1.3 subunit-containing channels in the regulation of firing patterns of striatal cholinergic interneurons, which were thought to be largely dependent on KCa channels. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Synergistic interplay of Gβγ and phosphatidylinositol 4,5-bisphosphate dictates Kv7.4 channel activity.

    PubMed

    Povstyan, Oleksandr V; Barrese, Vincenzo; Stott, Jennifer B; Greenwood, Iain A

    2017-02-01

    Kv7.4 channels are key determinants of arterial contractility and cochlear mechanosensation that, like all Kv7 channels, have an obligatory requirement for phosphatidylinositol 4,5-bisphosphate (PIP 2 ). βγ G proteins (Gβγ) have been identified as novel positive regulators of Kv7.4. The present study ascertained whether Gβγ increased Kv7.4 open probability through an increased sensitivity to PIP 2 . In HEK cells stably expressing Kv7.4, PIP 2 or Gβγ increased open probability in a concentration dependent manner. Depleting PIP 2 prevented any Gβγ-mediated stimulation whilst an array of Gβγ inhibitors prohibited any PIP 2 -induced current enhancement. A combination of PIP 2 and Gβγ at sub-efficacious concentrations increased channel open probability considerably. The stimulatory effects of three Kv7.2-7.5 channel activators were also lost by PIP 2 depletion or Gβγ inhibitors. This study alters substantially our understanding of the fundamental processes that dictate Kv7.4 activity, revealing a more complex and subtle paradigm where the reliance on local phosphoinositide is dictated by interaction with Gβγ.

  12. Functional analysis of potassium channels in Kv7.2 G271V mutant causing early onset familial epilepsy.

    PubMed

    Wang, Juanjuan; Li, Yuan; Hui, Zhiyan; Cao, Min; Shi, Ruiming; Zhang, Wei; Geng, Limeng; Zhou, Xihui

    2015-08-07

    Kv7 (KCNQ) channels underlying a class of voltage-gated K+ current are best known for regulating neuronal excitability. The first glycine (G) residue in the pore helix of Kv7.2 (KCNQ2) subunit is highly conserved among different classes of Kv7 channel family. A missense mutation causing the replacement of the corresponding G residues with a valine (p.G271V) in Kv7.2 was found in a large, four-generation pedigree. Here, we set out to examine the molecular pathomechanism of G271V mutants using patch clamp technology combined with biochemical and immunocytochemical techniques in transiently transfected human embryonic kidney (HEK) 293 cells. The expression of Kv7.2 protein had the same intensity for both wild type (WT) and G271V. In transfected HEK cells, G271V mutants induced large depolarizing shifts of the conductance-voltage relationships and marked slowing of current activation kinetics compared to WT. In addition, G271V mutants abolished currents in homomeric channels, and resulted in about 50% reduction of current in Kv7.2/G271V/Kv7.3 heteromultimeric condition, indicating a more severe functional defect. To test for G271V mutant channel expression in surface membrane, we performed fluorescence confocal microscopy imaging, which revealed no differences between the mutant and WT, suggesting that G271V channels fail to open in response to depolarization even though they are present in the membrane. Furthermore, pharmacologic intervention experiments revealed that upon specific incubation of transfected HEK 293 cells expressing G271V heteromultimeric channels in presence of Kv7 channel enhancer retigabine (ezogabine), the potassium currents increased significantly, suggesting the potential of retigabine as gene-specific therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Taurine activates delayed rectifier KV channels via a metabotropic pathway in retinal neurons

    PubMed Central

    Bulley, Simon; Liu, Yufei; Ripps, Harris; Shen, Wen

    2013-01-01

    Taurine is one of the most abundant amino acids in the retina, throughout the CNS, and in heart and muscle cells. In keeping with its broad tissue distribution, taurine serves as a modulator of numerous basic processes, such as enzyme activity, cell development, myocardial function and cytoprotection. Despite this multitude of functional roles, the precise mechanism underlying taurine's actions has not yet been identified. In this study we report findings that indicate a novel role for taurine in the regulation of voltage-gated delayed rectifier potassium (KV) channels in retinal neurons by means of a metabotropic receptor pathway. The metabotropic taurine response was insensitive to the Cl− channel blockers, picrotoxin and strychnine, but it was inhibited by a specific serotonin 5-HT2A receptor antagonist, MDL11939. Moreover, we found that taurine enhanced KV channels via intracellular protein kinase C-mediated pathways. When 5-HT2A receptors were expressed in human embryonic kidney cells, taurine and AL34662, a non-specific 5-HT2 receptor activator, produced a similar regulation of KIR channels. In sum, this study provides new evidence that taurine activates a serotonin system, apparently via 5-HT2A receptors and related intracellular pathways. PMID:23045337

  14. Molecular Expression and Pharmacological Evidence for a Functional Role of Kv7 Channel Subtypes in Guinea Pig Urinary Bladder Smooth Muscle

    PubMed Central

    Afeli, Serge A. Y.; Malysz, John; Petkov, Georgi V.

    2013-01-01

    Voltage-gated Kv7 (KCNQ) channels are emerging as essential regulators of smooth muscle excitability and contractility. However, their physiological role in detrusor smooth muscle (DSM) remains to be elucidated. Here, we explored the molecular expression and function of Kv7 channel subtypes in guinea pig DSM by RT-PCR, qRT-PCR, immunohistochemistry, electrophysiology, and isometric tension recordings. In whole DSM tissue, mRNAs for all Kv7 channel subtypes were detected in a rank order: Kv7.1~Kv7.2Kv7.3~Kv7.5Kv7.4. In contrast, freshly-isolated DSM cells showed mRNA expression of: Kv7.1~Kv7.2Kv7.5Kv7.3~Kv7.4. Immunohistochemical confocal microscopy analyses of DSM, conducted by using co-labeling of Kv7 channel subtype-specific antibodies and α-smooth muscle actin, detected protein expression for all Kv7 channel subtypes, except for the Kv7.4, in DSM cells. L-364373 (R-L3), a Kv7.1 channel activator, and retigabine, a Kv7.2-7.5 channel activator, inhibited spontaneous phasic contractions and the 10-Hz electrical field stimulation (EFS)-induced contractions of DSM isolated strips. Linopiridine and XE991, two pan-Kv7 (effective at Kv7.1-Kv7.5 subtypes) channel inhibitors, had opposite effects increasing DSM spontaneous phasic and 10 Hz EFS-induced contractions. EFS-induced DSM contractions generated by a wide range of stimulation frequencies were decreased by L-364373 (10 µM) or retigabine (10 µM), and increased by XE991 (10 µM). Retigabine (10 µM) induced hyperpolarization and inhibited spontaneous action potentials in freshly-isolated DSM cells. In summary, Kv7 channel subtypes are expressed at mRNA and protein levels in guinea pig DSM cells. Their pharmacological modulation can control DSM contractility and excitability; therefore, Kv7 channel subtypes provide potential novel therapeutic targets for urinary bladder dysfunction. PMID:24073284

  15. Molecular expression and pharmacological evidence for a functional role of kv7 channel subtypes in Guinea pig urinary bladder smooth muscle.

    PubMed

    Afeli, Serge A Y; Malysz, John; Petkov, Georgi V

    2013-01-01

    Voltage-gated Kv7 (KCNQ) channels are emerging as essential regulators of smooth muscle excitability and contractility. However, their physiological role in detrusor smooth muscle (DSM) remains to be elucidated. Here, we explored the molecular expression and function of Kv7 channel subtypes in guinea pig DSM by RT-PCR, qRT-PCR, immunohistochemistry, electrophysiology, and isometric tension recordings. In whole DSM tissue, mRNAs for all Kv7 channel subtypes were detected in a rank order: Kv7.1~Kv7.2Kv7.3~Kv7.5Kv7.4. In contrast, freshly-isolated DSM cells showed mRNA expression of: Kv7.1~Kv7.2Kv7.5Kv7.3~Kv7.4. Immunohistochemical confocal microscopy analyses of DSM, conducted by using co-labeling of Kv7 channel subtype-specific antibodies and α-smooth muscle actin, detected protein expression for all Kv7 channel subtypes, except for the Kv7.4, in DSM cells. L-364373 (R-L3), a Kv7.1 channel activator, and retigabine, a Kv7.2-7.5 channel activator, inhibited spontaneous phasic contractions and the 10-Hz electrical field stimulation (EFS)-induced contractions of DSM isolated strips. Linopiridine and XE991, two pan-Kv7 (effective at Kv7.1-Kv7.5 subtypes) channel inhibitors, had opposite effects increasing DSM spontaneous phasic and 10 Hz EFS-induced contractions. EFS-induced DSM contractions generated by a wide range of stimulation frequencies were decreased by L-364373 (10 µM) or retigabine (10 µM), and increased by XE991 (10 µM). Retigabine (10 µM) induced hyperpolarization and inhibited spontaneous action potentials in freshly-isolated DSM cells. In summary, Kv7 channel subtypes are expressed at mRNA and protein levels in guinea pig DSM cells. Their pharmacological modulation can control DSM contractility and excitability; therefore, Kv7 channel subtypes provide potential novel therapeutic targets for urinary bladder dysfunction.

  16. Spectroscopic classification of SN 2018kv as a type Ia supernova

    NASA Astrophysics Data System (ADS)

    Zhang, Jujia; Lu, Kaixing; Wang, Xiaofeng; Xu, Zhijian; Li, Wenxiong; Zhang, Tianmeng; Tan, Hanjie; Li, Zhitong; Song, Hao; University, Jun Mo (Tsinghua

    2018-01-01

    We obtained an optical spectrum (range 350-900 nm) of SN 2018kv, discovered by the Tsinghua-NAOC Transient Survey, on UT 2018 Jan. 26.9 with the Li-Jiang 2.4 m telescope (LJT+YFOSC) at Li-Jiang Observatory of Yunnan Observatories.

  17. Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

    SciTech Connect

    Jang, Soo Hwa; Choi, Changsun; Hong, Seong-Geun

    2009-06-26

    Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a rolemore » in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.« less

  18. Reconstitution of a Kv channel into lipid membranes for structural and functional studies.

    PubMed

    Lee, Sungsoo; Zheng, Hui; Shi, Liang; Jiang, Qiu-Xing

    2013-07-13

    To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

  19. XE991 and Linopirdine Are State-Dependent Inhibitors for Kv7/KCNQ Channels that Favor Activated Single Subunits.

    PubMed

    Greene, Derek L; Kang, Seungwoo; Hoshi, Naoto

    2017-07-01

    M-channel inhibitors, especially XE991, are being used increasingly in animal experiments; however, insufficient characterization of XE991 at times confounds the interpretation of results when using this compound. Here, we demonstrate that XE991 and linopirdine are state-dependent inhibitors that favor the activated-subunit of neuronal Kv7/KCNQ channels. We performed patch-clamp experiments on homomeric Kv7.2 or heteromeric Kv7.2/3 channels expressed in Chinese hamster ovary cells to characterize XE991 and linopirdine. Neither inhibitor was efficacious around the resting membrane potential of cells in physiologic conditions. Inhibition of Kv7.2 and Kv7.2/3 channels by XE991 was closely related with channel activation. When the voltage dependence of activation was left-shifted by retigabine or right-shifted by the mutation, Kv7.2(R214D), the shift in half-activation voltage proportionally coincided with the shift in the half-effective potential for XE991 inhibition. Inhibition kinetics during XE991 wash-in was facilitated at depolarized potentials. Ten-minute washout of XE991 resulted in ∼30% current recovery, most of which was attributed to surface transport of Kv7.2 channels. Linopirdine also exhibited similar inhibition characteristics, with the exception of near- complete current recovery after washout at depolarized potentials. Inhibition kinetics of both XE991 and linopirdine was not as sensitive to changes in voltage as would be predicted by open- channel inhibition. Instead, they were well explained by binding to a single activated subunit. The characteristics of XE991 and linopirdine should be taken into account when these M-channel inhibitors are used in experiments. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  20. Expression and function of Kv7.4 channels in rat cardiac mitochondria: possible targets for cardioprotection.

    PubMed

    Testai, Lara; Barrese, Vincenzo; Soldovieri, Maria Virginia; Ambrosino, Paolo; Martelli, Alma; Vinciguerra, Iolanda; Miceli, Francesco; Greenwood, Iain Andrew; Curtis, Michael John; Breschi, Maria Cristina; Sisalli, Maria Josè; Scorziello, Antonella; Canduela, Miren Josune; Grandes, Pedro; Calderone, Vincenzo; Taglialatela, Maurizio

    2016-05-01

    Plasmalemmal Kv7.1 (KCNQ1) channels are critical players in cardiac excitability; however, little is known on the functional role of additional Kv7 family members (Kv7.2-5) in cardiac cells. In this work, the expression, function, cellular and subcellular localization, and potential cardioprotective role against anoxic-ischaemic cardiac injury of Kv7.4 channels have been investigated. Expression of Kv7.1 and Kv7.4 transcripts was found in rat heart tissue by quantitative polymerase chain reaction. Western blots detected Kv7.4 subunits in mitochondria from Kv7.4-transfected cells, H9c2 cardiomyoblasts, freshly isolated adult cardiomyocytes, and whole hearts. Immunofluorescence experiments revealed that Kv7.4 subunits co-localized with mitochondrial markers in cardiac cells, with ∼ 30-40% of cardiac mitochondria being labelled by Kv7.4 antibodies, a result also confirmed by immunogold electron microscopy experiments. In isolated cardiac (but not liver) mitochondria, retigabine (1-30 µM) and flupirtine (30 µM), two selective Kv7 activators, increased Tl(+) influx, depolarized the membrane potential, and inhibited calcium uptake; all these effects were antagonized by the Kv7 blocker XE991. In intact H9c2 cells, reducing Kv7.4 expression by RNA interference blunted retigabine-induced mitochondrial membrane depolarization; in these cells, retigabine decreased mitochondrial Ca(2+) levels and increased radical oxygen species production, both effects prevented by XE991. Finally, retigabine reduced cellular damage in H9c2 cells exposed to anoxia/re-oxygenation and largely prevented the functional and morphological changes triggered by global ischaemia/reperfusion (I/R) in Langendorff-perfused rat hearts. Kv7.4 channels are present and functional in cardiac mitochondria; their activation exerts a significant cardioprotective role, making them potential therapeutic targets against I/R-induced cardiac injury. Published on behalf of the European Society of Cardiology. All

  1. Inhalational anaesthetics and n-alcohols share a site of action in the neuronal Shaw2 Kv channel

    PubMed Central

    Bhattacharji, Aditya; Klett, Nathan; Go, Ramon Christopher V; Covarrubias, Manuel

    2010-01-01

    Background and purpose: Neuronal ion channels are key targets of general anaesthetics and alcohol, and binding of these drugs to pre-existing and relatively specific sites is thought to alter channel gating. However, the underlying molecular mechanisms of this action are still poorly understood. Here, we investigated the neuronal Shaw2 voltage-gated K+ (Kv) channel to ask whether the inhalational anaesthetic halothane and n-alcohols share a binding site near the activation gate of the channel. Experimental approach: Focusing on activation gate mutations that affect channel modulation by n-alcohols, we investigated n-alcohol-sensitive and n-alcohol-resistant Kv channels heterologously expressed in Xenopus oocytes to probe the functional modulation by externally applied halothane using two-electrode voltage clamping and a gas-tight perfusion system. Key results: Shaw2 Kv channels are reversibly inhibited by halothane in a dose-dependent and saturable manner (K0.5= 400 µM; nH= 1.2). Also, discrete mutations in the channel's S4S5 linker are sufficient to reduce or confer inhibition by halothane (Shaw2-T330L and Kv3.4-G371I/T378A respectively). Furthermore, a point mutation in the S6 segment of Shaw2 (P410A) converted the halothane-induced inhibition into halothane-induced potentiation. Lastly, the inhibition resulting from the co-application of n-butanol and halothane is consistent with the presence of overlapping binding sites for these drugs and weak binding cooperativity. Conclusions and implications: These observations strongly support a molecular model of a general anaesthetic binding site in the Shaw2 Kv channel. This site may involve the amphiphilic interface between the S4S5 linker and the S6 segment, which plays a pivotal role in Kv channel activation. PMID:20136839

  2. Fe2O3 nanoparticles suppress Kv1.3 channels via affecting the redox activity of Kvβ2 subunit in Jurkat T cells

    NASA Astrophysics Data System (ADS)

    Yan, Li; Liu, Xiao; Liu, Wei-Xia; Tan, Xiao-Qiu; Xiong, Fei; Gu, Ning; Hao, Wei; Gao, Xue; Cao, Ji-Min

    2015-12-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) are promising nanomaterials in medical practice due to their special magnetic characteristics and nanoscale size. However, their potential impacts on immune cells are not well documented. This study aims to investigate the effects of Fe2O3 nanoparticles (Fe2O3-NPs) on the electrophysiology of Kv1.3 channels in Jurkat T cells. Using the whole-cell patch-clamp technique, we demonstrate that incubation of Jurkat cells with Fe2O3-NPs dose- and time-dependently decreased the current density and shifted the steady-state inactivation curve and the recovery curve of Kv1.3 channels to a rightward direction. Fe2O3-NPs increased the NADP level but decreased the NADPH level of Jurkat cells. Direct induction of NADPH into the cytosole of Jurkat cells via the pipette abolished the rightward shift of the inactivation curve. In addition, transmission electron microscopy showed that Fe2O3-NPs could be endocytosed by Jurkat cells with relatively low speed and capacity. Fe2O3-NPs did not significantly affect the viability of Jurkat cells, but suppressed the expressions of certain cytokines (TNFα, IFNγ and IL-2) and interferon responsive genes (IRF-1 and PIM-1), and the time courses of Fe2O3-NPs endocytosis and effects on the expressions of cytokines and interferon responsive genes were compatible. We conclude that Fe2O3-NPs can be endocytosed by Jurkat cells and act intracellularly. Fe2O3-NPs decrease the current density and delay the inactivation and recovery kinetics of Kv1.3 channels in Jurkat cells by oxidizing NADPH and therefore disrupting the redox activity of the Kvβ2 auxiliary subunit, and as a result, lead to changes of the Kv1.3 channel function. These results suggest that iron oxide nanoparticles may affect T cell function by disturbing the activity of Kv1.3 channels. Further, the suppressing effects of Fe2O3-NPs on the expressions of certain inflammatory cytokines and interferon responsive genes suggest that iron

  3. LGI1 tunes intrinsic excitability by regulating the density of axonal Kv1 channels.

    PubMed

    Seagar, Michael; Russier, Michael; Caillard, Olivier; Maulet, Yves; Fronzaroli-Molinieres, Laure; De San Feliciano, Marina; Boumedine-Guignon, Norah; Rodriguez, Léa; Zbili, Mickael; Usseglio, Fabrice; Formisano-Tréziny, Christine; Youssouf, Fahamoe; Sangiardi, Marion; Boillot, Morgane; Baulac, Stéphanie; Benitez, María José; Garrido, Juan-José; Debanne, Dominique; El Far, Oussama

    2017-07-18

    Autosomal dominant epilepsy with auditory features results from mutations in leucine-rich glioma-inactivated 1 (LGI1), a soluble glycoprotein secreted by neurons. Animal models of LGI1 depletion display spontaneous seizures, however, the function of LGI1 and the mechanisms by which deficiency leads to epilepsy are unknown. We investigated the effects of pure recombinant LGI1 and genetic depletion on intrinsic excitability, in the absence of synaptic input, in hippocampal CA3 neurons, a classical focus for epileptogenesis. Our data indicate that LGI1 is expressed at the axonal initial segment and regulates action potential firing by setting the density of the axonal Kv1.1 channels that underlie dendrotoxin-sensitive D-type potassium current. LGI1 deficiency incurs a >50% down-regulation of the expression of Kv1.1 and Kv1.2 via a posttranscriptional mechanism, resulting in a reduction in the capacity of axonal D-type current to limit glutamate release, thus contributing to epileptogenesis.

  4. Retigabine, a Kv7.2/Kv7.3-Channel Opener, Attenuates Drug-Induced Seizures in Knock-In Mice Harboring Kcnq2 Mutations.

    PubMed

    Ihara, Yukiko; Tomonoh, Yuko; Deshimaru, Masanobu; Zhang, Bo; Uchida, Taku; Ishii, Atsushi; Hirose, Shinichi

    2016-01-01

    The hetero-tetrameric voltage-gated potassium channel Kv7.2/Kv7.3, which is encoded by KCNQ2 and KCNQ3, plays an important role in limiting network excitability in the neonatal brain. Kv7.2/Kv7.3 dysfunction resulting from KCNQ2 mutations predominantly causes self-limited or benign epilepsy in neonates, but also causes early onset epileptic encephalopathy. Retigabine (RTG), a Kv7.2/ Kv7.3-channel opener, seems to be a rational antiepileptic drug for epilepsies caused by KCNQ2 mutations. We therefore evaluated the effects of RTG on seizures in two strains of knock-in mice harboring different Kcnq2 mutations, in comparison to the effects of phenobarbital (PB), which is the first-line antiepileptic drug for seizures in neonates. The subjects were heterozygous knock-in mice (Kcnq2Y284C/+ and Kcnq2A306T/+) bearing the Y284C or A306T Kcnq2 mutation, respectively, and their wild-type (WT) littermates, at 63-100 days of age. Seizures induced by intraperitoneal injection of kainic acid (KA, 12mg/kg) were recorded using a video-electroencephalography (EEG) monitoring system. Effects of RTG on KA-induced seizures of both strains of knock-in mice were assessed using seizure scores from a modified Racine's scale and compared with those of PB. The number and total duration of spike bursts on EEG and behaviors monitored by video recording were also used to evaluate the effects of RTG and PB. Both Kcnq2Y284C/+ and Kcnq2A306T/+ mice showed significantly more KA-induced seizures than WT mice. RTG significantly attenuated KA-induced seizure activities in both Kcnq2Y284C/+ and Kcnq2A306T/+ mice, and more markedly than PB. This is the first reported evidence of RTG ameliorating KA-induced seizures in knock-in mice bearing mutations of Kcnq2, with more marked effects than those observed with PB. RTG or other Kv7.2-channel openers may be considered as first-line antiepileptic treatments for epilepsies resulting from KCNQ2 mutations.

  5. Quantitative Confocal Microscopy Analysis as a Basis for Search and Study of Potassium Kv1.x Channel Blockers

    NASA Astrophysics Data System (ADS)

    Feofanov, Alexey V.; Kudryashova, Kseniya S.; Nekrasova, Oksana V.; Vassilevski, Alexander A.; Kuzmenkov, Alexey I.; Korolkova, Yuliya V.; Grishin, Eugene V.; Kirpichnikov, Mikhail P.

    Artificial KcsA-Kv1.x (x = 1, 3) receptors were recently designed by transferring the ligand-binding site from human Kv1.x voltage-gated potassium channels into corresponding domain of the bacterial KscA channel. We found that KcsA-Kv1.x receptors expressed in E. coli cells are embedded into cell membrane and bind ligands when the cells are transformed to spheroplasts. We supposed that E. coli spheroplasts with membrane-embedded KcsA-Kv1.x and fluorescently labeled ligand agitoxin-2 (R-AgTx2) can be used as elements of an advanced analytical system for search and study of Kv1-channel blockers. To realize this idea, special procedures were developed for measurement and quantitative treatment of fluorescence signals obtained from spheroplast membrane using confocal laser scanning microscopy (CLSM). The worked out analytical "mix and read" systems supported by quantitative CLSM analysis were demonstrated to be reliable alternative to radioligand and electrophysiology techniques in the search and study of selective Kv1.x channel blockers of high scientific and medical importance.

  6. Convergent phosphomodulation of the major neuronal dendritic potassium channel Kv4.2 by pituitary adenylate cyclase-activating polypeptide.

    PubMed

    Gupte, Raeesa P; Kadunganattil, Suraj; Shepherd, Andrew J; Merrill, Ronald; Planer, William; Bruchas, Michael R; Strack, Stefan; Mohapatra, Durga P

    2016-02-01

    The endogenous neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is secreted by both neuronal and non-neuronal cells in the brain and spinal cord, in response to pathological conditions such as stroke, seizures, chronic inflammatory and neuropathic pain. PACAP has been shown to exert various neuromodulatory and neuroprotective effects. However, direct influence of PACAP on the function of intrinsically excitable ion channels that are critical to both hyperexcitation as well as cell death, remain largely unexplored. The major dendritic K(+) channel Kv4.2 is a critical regulator of neuronal excitability, back-propagating action potentials in the dendrites, and modulation of synaptic inputs. We identified, cloned and characterized the downstream signaling originating from the activation of three PACAP receptor (PAC1) isoforms that are expressed in rodent hippocampal neurons that also exhibit abundant expression of Kv4.2 protein. Activation of PAC1 by PACAP leads to phosphorylation of Kv4.2 and downregulation of channel currents, which can be attenuated by inhibition of either PKA or ERK1/2 activity. Mechanistically, this dynamic downregulation of Kv4.2 function is a consequence of reduction in the density of surface channels, without any influence on the voltage-dependence of channel activation. Interestingly, PKA-induced effects on Kv4.2 were mediated by ERK1/2 phosphorylation of the channel at two critical residues, but not by direct channel phosphorylation by PKA, suggesting a convergent phosphomodulatory signaling cascade. Altogether, our findings suggest a novel GPCR-channel signaling crosstalk between PACAP/PAC1 and Kv4.2 channel in a manner that could lead to neuronal hyperexcitability. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. The Novel KV7.2/KV7.3 Channel Opener ICA-069673 Reveals Subtype-Specific Functional Roles in Guinea Pig Detrusor Smooth Muscle Excitability and Contractility

    PubMed Central

    Provence, Aaron; Malysz, John

    2015-01-01

    The physiologic roles of voltage-gated KV7 channel subtypes (KV7.1–KV7.5) in detrusor smooth muscle (DSM) are poorly understood. Here, we sought to elucidate the functional roles of KV7.2/KV7.3 channels in guinea pig DSM excitability and contractility using the novel KV7.2/KV7.3 channel activator ICA-069673 [N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide]. We employed a multilevel experimental approach using Western blot analysis, immunocytochemistry, isometric DSM tension recordings, fluorescence Ca2+ imaging, and perforated whole-cell patch-clamp electrophysiology. Western blot experiments revealed the protein expression of KV7.2 and KV7.3 channel subunits in DSM tissue. In isolated DSM cells, immunocytochemistry with confocal microscopy further confirmed protein expression for KV7.2 and KV7.3 channel subunits, where they localize within the vicinity of the cell membrane. ICA-069673 inhibited spontaneous phasic, pharmacologically induced, and nerve-evoked contractions in DSM isolated strips in a concentration-dependent manner. The inhibitory effects of ICA-069673 on DSM spontaneous phasic and tonic contractions were abolished in the presence of the KV7 channel inhibitor XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride]. Under conditions of elevated extracellular K+ (60 mM), the effects of ICA-069673 on DSM tonic contractions were significantly attenuated. ICA-069673 decreased the global intracellular Ca2+ concentration in DSM cells, an effect blocked by the L-type Ca2+ channel inhibitor nifedipine. ICA-069673 hyperpolarized the membrane potential and inhibited spontaneous action potentials of isolated DSM cells, effects that were blocked in the presence of XE991. In conclusion, using the novel KV7.2/KV7.3 channel activator ICA-069673, this study provides strong evidence for a critical role for the KV7.2- and KV7.3-containing channels in DSM function at both cellular and tissue levels. PMID:26087697

  8. The Novel KV7.2/KV7.3 Channel Opener ICA-069673 Reveals Subtype-Specific Functional Roles in Guinea Pig Detrusor Smooth Muscle Excitability and Contractility.

    PubMed

    Provence, Aaron; Malysz, John; Petkov, Georgi V

    2015-09-01

    The physiologic roles of voltage-gated KV7 channel subtypes (KV7.1-KV7.5) in detrusor smooth muscle (DSM) are poorly understood. Here, we sought to elucidate the functional roles of KV7.2/KV7.3 channels in guinea pig DSM excitability and contractility using the novel KV7.2/KV7.3 channel activator ICA-069673 [N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide]. We employed a multilevel experimental approach using Western blot analysis, immunocytochemistry, isometric DSM tension recordings, fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp electrophysiology. Western blot experiments revealed the protein expression of KV7.2 and KV7.3 channel subunits in DSM tissue. In isolated DSM cells, immunocytochemistry with confocal microscopy further confirmed protein expression for KV7.2 and KV7.3 channel subunits, where they localize within the vicinity of the cell membrane. ICA-069673 inhibited spontaneous phasic, pharmacologically induced, and nerve-evoked contractions in DSM isolated strips in a concentration-dependent manner. The inhibitory effects of ICA-069673 on DSM spontaneous phasic and tonic contractions were abolished in the presence of the KV7 channel inhibitor XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride]. Under conditions of elevated extracellular K(+) (60 mM), the effects of ICA-069673 on DSM tonic contractions were significantly attenuated. ICA-069673 decreased the global intracellular Ca(2+) concentration in DSM cells, an effect blocked by the L-type Ca(2+) channel inhibitor nifedipine. ICA-069673 hyperpolarized the membrane potential and inhibited spontaneous action potentials of isolated DSM cells, effects that were blocked in the presence of XE991. In conclusion, using the novel KV7.2/KV7.3 channel activator ICA-069673, this study provides strong evidence for a critical role for the KV7.2- and KV7.3-containing channels in DSM function at both cellular and tissue levels. Copyright © 2015 by The American Society for

  9. Phosphorylation regulates the sensitivity of voltage-gated Kv7.2 channels towards phosphatidylinositol-4,5-bisphosphate.

    PubMed

    Salzer, Isabella; Erdem, Fatma Asli; Chen, Wei-Qiang; Heo, Seok; Koenig, Xaver; Schicker, Klaus W; Kubista, Helmut; Lubec, Gert; Boehm, Stefan; Yang, Jae-Won

    2017-02-01

    Phosphatidylinositol-4,5-bisphosphate (PIP 2 ) is a key regulator of many membrane proteins, including voltage-gated Kv7.2 channels. In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP 2 -binding domains in Kv7.2. Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP 2 . Dephosphorylation of Kv7.2 affected channel inhibition via M 1 muscarinic receptors, but not via bradykinin receptors. Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP 2 , thereby ensuring the tight regulation of the channel via G protein-coupled receptors. The function of numerous ion channels is tightly controlled by G protein-coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol-4,5-bisphosphate (PIP 2 ). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP 2 and through phosphorylation. Using liquid chromatography-coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2-binding domains. To evaluate the effect of phosphorylation on PIP 2 -mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A 5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP 2 depletion via the voltage-sensitive phosphatase Dr-VSP than were wild-type channels. In vitro phosphorylation assays with the purified C-terminus of Kv7

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

    PubMed

    Teisseyre, Andrzej; Gąsiorowska, Justyna; Michalak, Krystyna

    2015-01-01

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

  11. Ubiquitin ligase Nedd4-2 modulates Kv1.3 current amplitude and ion channel protein targeting

    PubMed Central

    Velez, Patricio; Schwartz, Austin B.; Iyer, Subashini R.; Warrington, Anthony

    2016-01-01

    Voltage-dependent potassium channels (Kv) go beyond the stabilization of the resting potential and regulate biochemical pathways, regulate intracellular signaling, and detect energy homeostasis. Because targeted deletion and pharmacological block of the Kv1.3 channel protein produce marked changes in metabolism, resistance to diet-induced obesity, and changes in olfactory structure and function, this investigation explored Nedd4-2-mediated ubiquitination and degradation to regulate Kv1.3 channel density. Heterologous coexpression of Nedd4-2 ligase and Kv1.3 in HEK 293 cells reduced Kv1.3 current density without modulation of kinetic properties as measured by patch-clamp electrophysiology. Modulation of current density was dependent on ligase activity and was lost through point mutation of cysteine 938 in the catalytic site of the ligase (Nedd4-2CS). Incorporation of adaptor protein Grb10 relieved Nedd4-2-induced current suppression as did application of the proteasome inhibitor Mg-132. SDS-PAGE and immunoprecipitation strategies demonstrated a channel/adaptor/ligase signalplex. Pixel immunodensity was reduced for Kv1.3 in the presence of Nedd4-2, which was eliminated upon additional incorporation of Grb10. We confirmed Nedd4-2/Grb10 coimmunoprecipitation and observed an increased immunodensity for Nedd4-2 in the presence of Kv1.3 plus Grb10, regardless of whether the catalytic site was active. Kv1.3/Nedd4-2 were reciprocally coimmunoprecipated, whereby mutation of the COOH-terminal, SH3-recognition (493–498), or ubiquitination sites on Kv1.3 (lysines 467, 476, 498) retained coimmunoprecipitation, while the latter prevented the reduction in channel density. A model is presented for which an atypical interaction outside the canonical PY motif may permit channel/ligase interaction to lead to protein degradation and reduced current density, which can involve Nedd4-2/Grb10 interactions to disrupt Kv1.3 loss of current density. PMID:27146988

  12. Remodeling of Kv1.5 channel in right atria from Han Chinese patients with atrial fibrillation.

    PubMed

    Ou, Xian-hong; Li, Miao-ling; Liu, Rui; Fan, Xin-rong; Mao, Liang; Fan, Xue-hui; Yang, Yan; Zeng, Xiao-rong

    2015-04-28

    The incidence of atrial fibrillation (AF) in rheumatic heart diseases (RHD) is very high and increases with age. Occurrence and maintenance of AF are very complicated process accompanied by many different mechanisms. Ion-channel remodeling, including the voltage-gated potassium channel Kv1.5, plays an important role in the pathophysiology of AF. However, the changes of Kv1.5 channel expression in Han Chinese patients with RHD and AF remain poorly understood. The aim of the present study was to investigate whether the Kv1.5 channels of the right atria may be altered with RHD, age, and sex to contribute to AF. Right atrial appendages were obtained from 20 patients with normal cardiac functions who had undergone surgery, and 26 patients with AF. Subjects were picked from 4 groups: adult and aged patients in normal sinus rhythm (SR) and AF. Patients were divided into non-RHD and RHD groups or men and women groups in normal SR and AF, respectively. The expression of Kv1.5 protein and messenger RNA (mRNA) were measured using Western blotting and polymerase chain reaction (PCR) method, respectively. Compared with the SR group, the expression of Kv1.5 protein decreased significantly in the AF group. However, neither Kv1.5 protein nor KCNA5 mRNA had significant differences in adult and aged groups, non-RHD and RHD group, and men and women group of AF. The expression of Kv1.5 channel protein changes with AF but not with age, RHD, and sex in AF.

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

    PubMed

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

    2013-08-06

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

  14. Catalytic mechanism and substrate specificity of the β-subunit of the voltage-gated potassium (Kv) channel

    PubMed Central

    Tipparaju, Srinivas M.; Barski, Oleg A.; Srivastava, Sanjay; Bhatnagar, Aruni

    2008-01-01

    The β-subunits of voltage-gated potassium (Kv) channels are members of aldo-keto reductase (AKR) superfamily. These proteins regulate inactivation and membrane localization of Kv1 and Kv4 channels. The Kvβ proteins bind to pyridine nucleotides with high affinity; however, their catalytic properties remain unclear. Here we report that recombinant rat Kvβ2 catalyzes the reduction of a wide range of aldehydes and ketones. The rate of catalysis was slower (0.06 to 0.2 min−1) than that of other AKRs, but displayed the expected hyperbolic dependence on substrate concentration, with no evidence of allosteric cooperativity. Catalysis was prevented by site-directed substitution of Tyr-90 with phenylalanine, indicating that the acid-base catalytic residue, identified in other AKRs, has a conserved function in Kvβ2. The protein catalyzed the reduction of a broad range of carbonyls including aromatic carbonyls, electrophilic aldehydes and prostaglandins, phospholipid and sugar aldehydes. Little or no activity was detected with carbonyl steroids. Initial velocity profiles were consistent with an ordered bi-bi rapid-equilibrium mechanism in which NADPH binding precedes carbonyl binding. Significant primary kinetic isotope effects (2.0 – 3.1) were observed under single and multiple turnover conditions, indicating that the bond-breaking chemical step is rate-limiting. Structure-activity relationships with a series of para-substituted benzaldehydes indicated that the electronic interactions predominate during substrate binding and that no significant charge develops during the transition state. These data strengthen the view that Kvβ proteins are catalytically-active AKRs that impart redox-sensitivity to Kv channels. PMID:18672894

  15. KV7 channels in the human detrusor: channel modulator effects and gene and protein expression.

    PubMed

    Bientinesi, Riccardo; Mancuso, Cesare; Martire, Maria; Bassi, Pier Francesco; Sacco, Emilio; Currò, Diego

    2017-02-01

    Voltage-gated type 7 K + (K V 7 or KCNQ) channels regulate the contractility of various smooth muscles. With this study, we aimed to assess the role of K V 7 channels in the regulation of human detrusor contractility, as well as the gene and protein expression of K V 7 channels in this tissue. For these purposes, the isolated organ technique, RT-qPCR, and Western blot were used, respectively. XE-991, a selective K V 7 channel blocker, concentration-dependently contracted the human detrusor; mean EC 50 and E max of XE-991-induced concentration-response curve were 14.1 μM and 28.8 % of the maximal bethanechol-induced contraction, respectively. Flupirtine and retigabine, selective K V 7.2-7.5 channel activators, induced concentration-dependent relaxations of bethanechol-precontracted strips, with maximal relaxations of 51.6 and 51.8 % of the precontraction, respectively. XE-991 blocked the relaxations induced by flupirtine and retigabine. All five KCNQ genes were found to be expressed in the detrusor with KCNQ4 being the most expressed among them. Different bands, having sizes similar to some of reported K V 7.1, 7.4, and 7.5 channel subunit isoforms, were detected in the detrusor by Western blot with the K V 7.4 band being the most intense among them. In conclusion, K V 7 channels contribute to set the basal tone of the human detrusor. In addition, K V 7 channel activators significantly relax the detrusor. The K V 7.4 channels are probably the most important K V 7 channels expressed in the human detrusor. These data suggest that selective K V 7.4 channel activators might represent new pharmacological tools for inducing therapeutic relaxation of the detrusor.

  16. Rearrangement of potassium ions and Kv1.1/Kv1.2 potassium channels in regenerating axons following end-to-end neurorrhaphy: ionic images from TOF-SIMS.

    PubMed

    Liu, Chiung-Hui; Chang, Hung-Ming; Wu, Tsung-Huan; Chen, Li-You; Yang, Yin-Shuo; Tseng, To-Jung; Liao, Wen-Chieh

    2017-10-01

    The voltage-gated potassium channels Kv1.1 and Kv1.2 that cluster at juxtaparanodal (JXP) regions are essential in the regulation of nerve excitability and play a critical role in axonal conduction. When demyelination occurs, Kv1.1/Kv1.2 activity increases, suppressing the membrane potential nearly to the equilibrium potential of K + , which results in an axonal conduction blockade. The recovery of K + -dependent communication signals and proper clustering of Kv1.1/Kv1.2 channels at JXP regions may directly reflect nerve regeneration following peripheral nerve injury. However, little is known about potassium channel expression and its relationship with the dynamic potassium ion distribution at the node of Ranvier during the regenerative process of peripheral nerve injury (PNI). In the present study, end-to-end neurorrhaphy (EEN) was performed using an in vivo model of PNI. The distribution of K + at regenerating axons following EEN was detected by time-of-flight secondary-ion mass spectrometry. The specific localization and expression of Kv1.1/Kv1.2 channels were examined by confocal microscopy and western blotting. Our data showed that the re-establishment of K + distribution and intensity was correlated with the functional recovery of compound muscle action potential morphology in EEN rats. Furthermore, the re-clustering of Kv1.1/1.2 channels 1 and 3 months after EEN at the nodal region of the regenerating nerve corresponded to changes in the K + distribution. This study provided direct evidence of K + distribution in regenerating axons for the first time. We proposed that the Kv1.1/Kv1.2 channels re-clustered at the JXP regions of regenerating axons are essential for modulating the proper patterns of K + distribution in axons for maintaining membrane potential stability after EEN.

  17. Updating In Vivo and In Vitro Phosphorylation and Methylation Sites of Voltage-Gated Kv7.2 Potassium Channels.

    PubMed

    Erdem, Fatma Asli; Salzer, Isabella; Heo, Seok; Chen, Wei-Qiang; Jung, Gangsoo; Lubec, Gert; Boehm, Stefan; Yang, Jae-Won

    2017-10-01

    Voltage-gated Kv7.2 potassium channels regulate neuronal excitability. The gating of these channels is tightly controlled by various mediators and neurotransmitters acting via G protein-coupled receptors; the underlying signaling cascades involve phosphatidylinositol-4,5-bisphosphate (PIP 2 ), Ca 2+ /calmodulin, and phosphorylation. Recent studies found that the PIP 2 sensitivity of Kv7.2 channels is affected by two posttranslational modifications, phosphorylation and methylation, harboured within putative PIP 2 -binding domains. In this study, we updated phosphorylation and methylation sites in Kv7.2 either heterologously expressed in mammalian cells or as GST-fusion proteins exposed to recombinant protein kinases by using LC-MS/MS. In vitro kinase assays revealed that CDK5, protein kinase C (PKC) alpha, PKA, p38 MAPK, CamKIIα, and GSK3β could mediate phosphorylation. Taken together, we provided a comprehensive map of phosphorylation and methylation in Kv7.2 within protein-protein and protein-lipid interaction domains. This may help to interpret the functional roles of individual PTM sites in Kv7.2 channels. All MS data are available via ProteomeXchange with the identifier PXD005567. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. DPP10 splice variants are localized in distinct neuronal populations and act to differentially regulate the inactivation properties of Kv4-based ion channels.

    PubMed

    Jerng, Henry H; Lauver, Aaron D; Pfaffinger, Paul J

    2007-08-01

    Dipeptidyl peptidase-like proteins (DPLs) and Kv-channel-interacting proteins (KChIPs) join Kv4 pore-forming subunits to form multi-protein complexes that underlie subthreshold A-type currents (I(SA)) in neuronal somatodendritic compartments. Here, we characterize the functional effects and brain distributions of N-terminal variants belonging to the DPL dipeptidyl peptidase 10 (DPP10). In the Kv4.2+KChIP3+DPP10 channel complex, all DPP10 variants accelerate channel gating kinetics; however, the splice variant DPP10a produces uniquely fast inactivation kinetics that accelerates with increasing depolarization. This DPP10a-specific inactivation dominates in co-expression studies with KChIP4a and other DPP10 isoforms. Real-time qRT-PCR and in situ hybridization analyses reveal differential expression of DPP10 variants in rat brain. DPP10a transcripts are prominently expressed in the cortex, whereas DPP10c and DPP10d mRNAs exhibit more diffuse distributions. Our results suggest that DPP10a underlies rapid inactivation of cortical I(SA), and the regulation of isoform expression may contribute to the variable inactivation properties of I(SA) across different brain regions.

  19. Kv7 channels in the nucleus accumbens are altered by chronic drinking and are targets for reducing alcohol consumption.

    PubMed

    McGuier, Natalie S; Griffin, William C; Gass, Justin T; Padula, Audrey E; Chesler, Elissa J; Mulholland, Patrick J

    2016-11-01

    Alcohol use disorders (AUDs) are a major public health issue and produce enormous societal and economic burdens. Current Food and Drug Administration (FDA)-approved pharmacotherapies for treating AUDs suffer from deleterious side effects and are only effective in a subset of individuals. It is therefore essential to find improved medications for the management of AUDs. Emerging evidence suggests that anticonvulsants are a promising class of drugs for treating individuals with AUDs. In these studies, we used integrative functional genomics to demonstrate that genes that encode Kv7 channels (i.e. Kcnq2/3) are related to alcohol (ethanol) consumption, preference and acceptance in rodents. We then tested the ability of the FDA-approved anticonvulsant retigabine, a Kv7 channel opener, to reduce voluntary ethanol consumption of Wistar rats in a two-bottle choice intermittent alcohol access paradigm. Systemic administration and microinjections of retigabine into the nucleus accumbens significantly reduced alcohol drinking, and retigabine was more effective at reducing intake in high- versus low-drinking populations of Wistar rats. Prolonged voluntary drinking increased the sensitivity to the proconvulsant effects of pharmacological blockade of Kv7 channels and altered surface trafficking and SUMOylation patterns of Kv7.2 channels in the nucleus accumbens. These data implicate Kcnq2/3 in the regulation of ethanol drinking and demonstrate that long-term drinking produces neuroadaptations in Kv7 channels. In addition, these results have identified retigabine as a potential pharmacotherapy for treating AUDs and Kv7 channels as a novel therapeutic target for reducing heavy drinking. © 2015 Society for the Study of Addiction.

  20. Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K+ channel

    PubMed Central

    Villarroel, Álvaro; Millet, Oscar

    2018-01-01

    The Kv7.2 (KCNQ2) channel is the principal molecular component of the slow voltage-gated, noninactivating K+ M-current, a key controller of neuronal excitability. To investigate the calmodulin (CaM)-mediated Ca2+ gating of the channel, we used NMR spectroscopy to structurally and dynamically describe the association of helices hA and hB of Kv7.2 with CaM, as a function of Ca2+ concentration. The structures of the CaM/Kv7.2-hAB complex at two different calcification states are reported here. In the presence of a basal cytosolic Ca2+ concentration (10–100 nM), only the N-lobe of CaM is Ca2+-loaded and the complex (representative of the open channel) exhibits collective dynamics on the millisecond time scale toward a low-populated excited state (1.5%) that corresponds to the inactive state of the channel. In response to a chemical or electrical signal, intracellular Ca2+ levels rise up to 1–10 μM, triggering Ca2+ association with the C-lobe. The associated conformational rearrangement is the key biological signal that shifts populations to the closed/inactive channel. This reorientation affects the C-lobe of CaM and both helices in Kv7.2, allosterically transducing the information from the Ca2+-binding site to the transmembrane region of the channel. PMID:29463698

  1. Protective role of Kv7 channels in oxygen and glucose deprivation-induced damage in rat caudate brain slices

    PubMed Central

    Barrese, Vincenzo; Taglialatela, Maurizio; Greenwood, Iain A; Davidson, Colin

    2015-01-01

    Ischemic stroke can cause striatal dopamine efflux that contributes to cell death. Since Kv7 potassium channels regulate dopamine release, we investigated the effects of their pharmacological modulation on dopamine efflux, measured by fast cyclic voltammetry (FCV), and neurotoxicity, in Wistar rat caudate brain slices undergoing oxygen and glucose deprivation (OGD). The Kv7 activators retigabine and ICA27243 delayed the onset, and decreased the peak level of dopamine efflux induced by OGD; and also decreased OGD-induced damage measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Retigabine also reduced OGD-induced necrotic cell death evaluated by lactate dehydrogenase activity assay. The Kv7 blocker linopirdine increased OGD-evoked dopamine efflux and OGD-induced damage, and attenuated the effects of retigabine. Quantitative-PCR experiments showed that OGD caused an ~6-fold decrease in Kv7.2 transcript, while levels of mRNAs encoding for other Kv7 subunits were unaffected; western blot experiments showed a parallel reduction in Kv7.2 protein levels. Retigabine also decreased the peak level of dopamine efflux induced by L-glutamate, and attenuated the loss of TTC staining induced by the excitotoxin. These results suggest a role for Kv7.2 in modulating ischemia-evoked caudate damage. PMID:25966943

  2. Selective Modulation of K+ Channel Kv7.4 Significantly Affects the Excitability of DRN 5-HT Neurons

    PubMed Central

    Zhao, Chen; Su, Min; Wang, Yingzi; Li, Xinmeng; Zhang, Yongxue; Du, Xiaona; Zhang, Hailin

    2017-01-01

    The serotonin (5-HT) system originating in the dorsal raphe nucleus (DRN) is implicated in various mood- and emotion-related disorders, such as anxiety, fear and stress. Abnormal activity of DRN 5-HT neurons is the key factor in the development of these disorders. Here, we describe a crucial role for the Kv7.4 potassium channel in modulating DRN 5-HT neuronal excitability. We demonstrate that Kv7.4 is selectively expressed in 5-HT neurons of the DRN. Using selective Kv7.4 opener fasudil and Kv7.4 knock-out mice, we demonstrate that Kv7.4 is a potent modulator of DRN 5-HT neuronal excitability. Furthermore, we demonstrate that the cellular redox signaling mechanism is involved in this 5-HT activation of Kv7.4. The current study suggests a new strategy for treating psychiatric disorders related to altered activity of DRN 5-HT neurons using K+ channel modulators. PMID:29311835

  3. Margatoxin-bound quantum dots as a novel inhibitor of the voltage-gated ion channel Kv1.3.

    PubMed

    Schwartz, Austin B; Kapur, Anshika; Wang, Wentao; Huang, Zhenbo; Fardone, Erminia; Palui, Goutam; Mattoussi, Hedi; Fadool, Debra Ann

    2017-02-01

    Venom-derived ion channel inhibitors have strong channel selectivity, potency, and stability; however, tracking delivery to their target can be challenging. Herein, we utilized luminescent quantum dots (QDs) conjugated to margatoxin (MgTx) as a traceable vehicle to target a voltage-dependent potassium channel, Kv1.3, which has a select distribution and well-characterized role in immunity, glucose metabolism, and sensory ability. We screened both unconjugated (MgTx) and conjugated MgTx (QD-MgTx) for their ability to inhibit Shaker channels Kv1.1 to Kv1.7 using patch-clamp electrophysiology in HEK293 cells. Our data indicate that MgTx inhibits 79% of the outward current in Kv1.3-transfected cells and that the QD-MgTx conjugate is able to achieve a similar level of block, albeit a slightly reduced efficacy (66%) and at a slower time course (50% block by 10.9 ± 1.1 min, MgTx; vs. 15.3 ± 1.2 min, QD-MgTx). Like the unbound peptide, the QD-MgTx conjugate inhibits both Kv1.3 and Kv1.2 at a 1 nM concentration, whereas it does not inhibit other screened Shaker channels. We tested the ability of QD-MgTx to inhibit native Kv1.3 expressed in the mouse olfactory bulb (OB). In brain slices of the OB, the conjugate acted similarly to MgTx to inhibit Kv1.3, causing an increased action potential firing frequency attributed to decreased intraburst duration rather than interspike interval. Our data demonstrate a retention of known biophysical properties associated with block of the vestibule of Kv1.3 by QD-MgTx conjugate compared to that of MgTx, inferring QDs could provide a useful tool to deliver ion channel inhibitors to targeted tissues in vivo. © 2016 International Society for Neurochemistry.

  4. Kv7 potassium channel activation with ICA-105665 reduces photoparoxysmal EEG responses in patients with epilepsy.

    PubMed

    Kasteleijn-Nolst Trenité, Dorotheé G A; Biton, Victor; French, Jacqueline A; Abou-Khalil, Bassel; Rosenfeld, William E; Diventura, Bree; Moore, Elizabeth L; Hetherington, Seth V; Rigdon, Greg C

    2013-08-01

    To assess the effects of ICA-105665, an agonist of neuronal Kv7 potassium channels, on epileptiform EEG discharges, evoked by intermittent photic stimulation (IPS), the so-called photoparoxysmal responses (PPRs) in patients with epilepsy. Male and female patients aged 18-60 years with reproducible PPRs were eligible for enrollment. The study was conducted as a single-blind, single-dose, multiple-cohort study. Four patients were enrolled in each of the first three cohorts. Six patients were enrolled in the fourth cohort and one patient was enrolled in the fifth cohort. PPR responses to 14 IPS frequencies (steps) were used to determine the standard photosensitivity range (SPR) following placebo on day 1 and ICA-105665 on day 2. The SPR was quantified for three eye conditions (eyes closing, eyes closed, and eyes open), and the most sensitive condition was used for assessment of efficacy. A partial response was defined as a reduction in the SPR of at least three units at three separate time points following ICA-105665 compared to the same time points following placebo with no time points with more than three units of increase. Complete suppression was defined by no PPRs in any eye condition at one or more time points. Six individual patients participated in the first three cohorts (100, 200, and 400 mg). Six patients participated in the fourth cohort (500 mg), and one patient participated in the fifth cohort (600 mg). Decreases in SPR occurred in one patient at 100 mg, two patients receiving 400 mg ICA-105665 (complete abolishment of SPR occurred in one patient at 400 mg), and in four of six patients receiving 500 mg. The most common adverse events (AEs) were those related to the nervous system, and dizziness appeared to be the first emerging AE. The single patient in the 600 mg cohort developed a brief generalized seizure within 1 h of dosing, leading to the discontinuation of additional patients at this dose, per the predefined protocol stopping rules. ICA-105665

  5. A novel DPP6 isoform (DPP6-E) can account for differences between neuronal and reconstituted A-type K(+) channels.

    PubMed

    Maffie, Jonathon; Blenkinsop, Timothy; Rudy, Bernardo

    2009-01-16

    The channels mediating most of the somatodendritic A-type K(+) current in neurons are thought to be ternary complexes of Kv4 pore-forming subunits and two types of auxiliary subunits, the K(+) channel interacting proteins (KChIPs) and dipeptidyl-peptidase-like (DPPL) proteins. The channels expressed in heterologous expression systems by mixtures of Kv4.2, KChIP1 and DPP6-S resemble in many properties the A-type current in hippocampal CA1 pyramidal neurons and cerebellar granule cells, neurons with prominent A-type K(+) currents. However, the native currents have faster kinetics. Moreover, the A-type currents in neurons in intermediary layers of the superior colliculus have even faster inactivating rates. We have characterized a new DPP6 spliced isoform, DPP6-E, that produces in heterologous cells ternary Kv4 channels with very fast kinetics. DPP6-E is selectively expressed in a few neuronal populations in brain including cerebellar granule neurons, hippocampal pyramidal cells and neurons in intermediary layers of the superior colliculus. The effects of DPP6-E explain past discrepancies between reconstituted and native Kv4 channels in some neurons, and contributes to the diversity of A-type K(+) currents in neurons.

  6. Characterization of the Kv channels of mouse carotid body chemoreceptor cells and their role in oxygen sensing

    PubMed Central

    Pérez-García, M Teresa; Colinas, Olaia; Miguel-Velado, Eduardo; Moreno-Domínguez, Alejandro; López-López, José Ramón

    2004-01-01

    As there are wide interspecies variations in the molecular nature of the O2-sensitive Kv channels in arterial chemoreceptors, we have characterized the expression of these channels and their hypoxic sensitivity in the mouse carotid body (CB). CB chemoreceptor cells were obtained from a transgenic mouse expressing green fluorescent protein (GFP) under the control of tyrosine hydroxylase (TH) promoter. Immunocytochemical identification of TH in CB cell cultures reveals a good match with GFP-positive cells. Furthermore, these cells show an increase in [Ca2+]i in response to low PO2, demonstrating their ability to engender a physiological response. Whole-cell experiments demonstrated slow-inactivating K+ currents with activation threshold around −30 mV and a bi-exponential kinetic of deactivation (τ of 6.24 ± 0.52 and 32.85 ± 4.14 ms). TEA sensitivity of the currents identified also two different components (IC50 of 17.8 ± 2.8 and 940.0 ± 14.7 μm). Current amplitude decreased reversibly in response to hypoxia, which selectively affected the fast deactivating component. Hypoxic inhibition was also abolished in the presence of low (10–50 μm) concentrations of TEA, suggesting that O2 interacts with the component of the current most sensitive to TEA. The kinetic and pharmacological profile of the currents suggested the presence of Kv2 and Kv3 channels as their molecular correlates, and we have identified several members of these two subfamilies by single-cell PCR and immunocytochemistry. This report represents the first functional and molecular characterization of Kv channels in mouse CB chemoreceptor cells, and strongly suggests that O2-sensitive Kv channels in this preparation belong to the Kv3 subfamily. PMID:15034123

  7. Complex oligosaccharides are N-linked to Kv3 voltage-gated K+ channels in rat brain.

    PubMed

    Cartwright, Tara A; Corey, Melissa J; Schwalbe, Ruth A

    2007-04-01

    Neuronal Kv3 voltage-gated K(+) channels have two absolutely conserved N-glycosylation sites. Here, it is shown that Kv3.1, 3.3, and 3.4 channels are N-glycosylated in rat brain. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced faster migrating immunobands than those of undigested membranes. Additionally, partial PNGase F digests showed that both sites are occupied by oligosaccharides. Neuraminidase treatment produced a smaller immunoband shift relative to PNGase F treatment. These results indicate that both sites are highly available and occupied by N-linked oligosaccharides for Kv3.1, 3.3, and 3.4 in rat brain, and furthermore that at least one oligosaccharide is of complex type. Additionally, these results point to an extracytoplasmic S1-S2 linker in Kv3 proteins expressed in native membranes. We suggest that N-glycosylation processing of Kv3 channels is critical for the expression of K(+) currents at the surface of neurons, and perhaps contributes to the pathophysiology of congenital disorders of glycosylation.

  8. The C-terminus SH3-binding domain of Kv1.3 is required for the actin-mediated immobilization of the channel via cortactin

    PubMed Central

    Hajdu, Peter; Martin, Geoffrey V.; Chimote, Ameet A.; Szilagyi, Orsolya; Takimoto, Koichi; Conforti, Laura

    2015-01-01

    Kv1.3 channels play a pivotal role in the activation and migration of T-lymphocytes. These functions are accompanied by the channels' polarization, which is essential for associated downstream events. However, the mechanisms that govern the membrane movement of Kv1.3 channels remain unclear. F-actin polymerization occurs concomitantly to channel polarization, implicating the actin cytoskeleton in this process. Here we show that cortactin, a factor initiating the actin network, controls the membrane mobilization of Kv1.3 channels. FRAP with EGFP-tagged Kv1.3 channels demonstrates that knocking down cortactin decreases the actin-based immobilization of the channels. Using various deletion and mutation constructs, we show that the SH3 motif of Kv1.3 mediates the channel immobilization. Proximity ligation assays indicate that deletion or mutation of the SH3 motif also disrupts interaction of the channel with cortactin. In T-lymphocytes, the interaction between HS1 (the cortactin homologue) and Kv1.3 occurs at the immune synapse and requires the channel's C-terminal domain. These results show that actin dynamics regulates the membrane motility of Kv1.3 channels. They also provide evidence that the SH3 motif of the channel and cortactin plays key roles in this process. PMID:25739456

  9. Dynamics of internal pore opening in KV channels probed by a fluorescent unnatural amino acid

    PubMed Central

    Kalstrup, Tanja; Blunck, Rikard

    2013-01-01

    Atomic-scale models on the gating mechanism of voltage-gated potassium channels (Kv) are based on linear interpolations between static structures of their initial and final state derived from crystallography and molecular dynamics simulations, and, thus, lack dynamic structural information. The lack of information on dynamics and intermediate states makes it difficult to associate the structural with the dynamic functional data obtained with electrophysiology. Although voltage-clamp fluorometry fills this gap, it is limited to sites extracellularly accessible, when the key region for gating is located at the cytosolic side of the channels. Here, we solved this problem by performing voltage-clamp fluorometry with a fluorescent unnatural amino acid. By using an orthogonal tRNA-synthetase pair, the fluorescent unnatural amino acid was incorporated in the Shaker voltage-gated potassium channel at key regions that were previously inaccessible. Thus, we defined which parts act independently and which parts act cooperatively and found pore opening to occur in two sequential transitions. PMID:23630265

  10. Roles of specific Kv channel types in repolarization of the action potential in genetically identified subclasses of pyramidal neurons in mouse neocortex

    PubMed Central

    Pathak, Dhruba; Guan, Dongxu

    2016-01-01

    The action potential (AP) is a fundamental feature of excitable cells that serves as the basis for long-distance signaling in the nervous system. There is considerable diversity in the appearance of APs and the underlying repolarization mechanisms in different neuronal types (reviewed in Bean BP. Nat Rev Neurosci 8: 451–465, 2007), including among pyramidal cell subtypes. In the present work, we used specific pharmacological blockers to test for contributions of Kv1, Kv2, or Kv4 channels to repolarization of single APs in two genetically defined subpopulations of pyramidal cells in layer 5 of mouse somatosensory cortex (etv1 and glt) as well as pyramidal cells from layer 2/3. These three subtypes differ in AP properties (Groh A, Meyer HS, Schmidt EF, Heintz N, Sakmann B, Krieger P. Cereb Cortex 20: 826–836, 2010; Guan D, Armstrong WE, Foehring RC. J Neurophysiol 113: 2014–2032, 2015) as well as laminar position, morphology, and projection targets. We asked what the roles of Kv1, Kv2, and Kv4 channels are in AP repolarization and whether the underlying mechanisms are pyramidal cell subtype dependent. We found that Kv4 channels are critically involved in repolarizing neocortical pyramidal cells. There are also pyramidal cell subtype-specific differences in the role for Kv1 channels. Only Kv4 channels were involved in repolarizing the narrow APs of glt cells. In contrast, in etv1 cells and layer 2/3 cells, the broader APs are partially repolarized by Kv1 channels in addition to Kv4 channels. Consistent with their activation in the subthreshold range, Kv1 channels also regulate AP voltage threshold in all pyramidal cell subtypes. PMID:26864770

  11. A novel N-terminal motif of dipeptidyl peptidase-like proteins produces rapid inactivation of KV4.2 channels by a pore-blocking mechanism.

    PubMed

    Jerng, Henry H; Dougherty, Kevin; Covarrubias, Manuel; Pfaffinger, Paul J

    2009-11-01

    The somatodendritic subthreshold A-type K(+) current in neurons (I(SA)) depends on its kinetic and voltage-dependent properties to regulate membrane excitability, action potential repetitive firing, and signal integration. Key functional properties of the K(V)4 channel complex underlying I(SA) are determined by dipeptidyl peptidase-like proteins known as dipeptidyl peptidase 6 (DPP6) and dipeptidyl peptidase 10 (DPP10). Among the multiple known DPP10 isoforms with alternative N-terminal sequences, DPP10a confers exceptionally fast inactivation to K(V)4.2 channels. To elucidate the molecular basis of this fast inactivation, we investigated the structure-function relationship of the DPP10a N-terminal region and its interaction with the K(V)4.2 channel. Here, we show that DPP10a shares a conserved N-terminal sequence (MNQTA) with DPP6a (aka DPP6-E), which also induces fast inactivation. Deletion of the NQTA sequence in DPP10a eliminates this dramatic fast inactivation, and perfusion of MNQTA peptide to the cytoplasmic face of inside-out patches inhibits the K(V)4.2 current. DPP10a-induced fast inactivation exhibits competitive interactions with internally applied tetraethylammonium (TEA), and elevating the external K(+) concentration accelerates recovery from DPP10a-mediated fast inactivation. These results suggest that fast inactivation induced by DPP10a or DPP6a is mediated by a common N-terminal inactivation motif via a pore-blocking mechanism. This mechanism may offer an attractive target for novel pharmacological interventions directed at impairing I(SA) inactivation and reducing neuronal excitability.

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

    PubMed

    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.

  13. Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel

    PubMed Central

    Tobelaim, William Sam; Dvir, Meidan; Lebel, Guy; Cui, Meng; Buki, Tal; Peretz, Asher; Marom, Milit; Haitin, Yoni; Logothetis, Diomedes E.; Hirsch, Joel Alan; Attali, Bernard

    2017-01-01

    Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current IKS that repolarizes the cardiac action potential. The physiological importance of the IKS channel is underscored by the existence of mutations in human Kv7.1 and KCNE1 genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2), but the role of CaM in channel function is still unclear, and its possible interaction with PIP2 is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP2 and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1 helix B form a critical site where CaM competes with PIP2 to stabilize the channel open state. Data indicate that both PIP2 and Ca2+-CaM perform the same function on IKS channel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca2+-CaM. The results suggest that, after receptor-mediated PIP2 depletion and increased cytosolic Ca2+, calcified CaM N lobe interacts with helix B in place of PIP2 to limit excessive IKS current inhibition. PMID:28096388

  14. Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel.

    PubMed

    Tobelaim, William Sam; Dvir, Meidan; Lebel, Guy; Cui, Meng; Buki, Tal; Peretz, Asher; Marom, Milit; Haitin, Yoni; Logothetis, Diomedes E; Hirsch, Joel Alan; Attali, Bernard

    2017-01-31

    Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current I KS that repolarizes the cardiac action potential. The physiological importance of the I KS channel is underscored by the existence of mutations in human Kv7.1 and KCNE1 genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ), but the role of CaM in channel function is still unclear, and its possible interaction with PIP 2 is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP 2 and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1 helix B form a critical site where CaM competes with PIP 2 to stabilize the channel open state. Data indicate that both PIP 2 and Ca 2+ -CaM perform the same function on I KS channel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca 2+ -CaM. The results suggest that, after receptor-mediated PIP 2 depletion and increased cytosolic Ca 2+ , calcified CaM N lobe interacts with helix B in place of PIP 2 to limit excessive I KS current inhibition.

  15. Altered Expression and Localization of Hippocampal A-Type Potassium Channel Subunits in the Pilocarpine-Induced Model of Temporal Lobe Epilepsy

    PubMed Central

    Monaghan, Michael M.; Menegola, Milena; Vacher, Helene; Rhodes, Kenneth J.; Trimmer, James S.

    2010-01-01

    Summary Altered ion channel expression and/or function may contribute to the development of certain human epilepsies. In rats, systemic administration of pilocarpine induces a model of human temporal lobe epilepsy, wherein a brief period of status epilepticus (SE) triggers development of spontaneous recurrent seizures that appear after a latency of two-three weeks. Here we investigate changes in expression of A-type voltage-gated potassium (Kv) channels, which control neuronal excitability and regulate action potential propagation and neurotransmitter release, in the pilocarpine model of epilepsy. Using immunohistochemistry, we examined the expression of component subunits of somatodendritic (Kv4.2, Kv4.3, KChIPl and KChIP2) and axonal (Kv1.4) A-type Kv channels in hippocampi of pilocarpine-treated rats that entered SE. We found that Kv4.2, Kv4.3 and KChIP2 staining in the molecular layer of the dentate gyrus changes from being uniformly distributed across the molecular layer to concentrated in just the outer two-thirds. We also observed a loss of KChIP1 immunoreactive interneurons, and a reduction of Kv4.2 and KChIP2 staining in stratum radiatum of CA1. These changes begin to appear 1 week after pilocarpine treatment and persist or are enhanced at 4 and 12 weeks. As such, these changes in Kv channel distribution parallel the acquisition of recurrent spontaneous seizures as observed in this model. We also found temporal changes in Kv1.4 immunoreactivity matching those in Timm's stain, being expanded in stratum lucidum of CA3 and in the inner third of the dentate molecular layer. Among pilocarpine-treated rats, changes were only observed in those that entered SE. These changes in A-type Kv channel expression may contribute to hyperexcitability of dendrites in the associated hippocampal circuits as observed in previous studies of the effects of pilocarpine-induced SE. PMID:18727953

  16. Kv1.3 channel blocker (ImKTx88) maintains blood-brain barrier in experimental autoimmune encephalomyelitis.

    PubMed

    Huang, Jie; Han, Song; Sun, Qi; Zhao, Yipeng; Liu, Junchen; Yuan, Xiaolu; Mao, Wenqian; Peng, Biwen; Liu, Wanhong; Yin, Jun; He, Xiaohua

    2017-01-01

    Disruption of blood-brain barrier (BBB) and subsequent infiltration of auto-reactive T lymphocytes are major characteristics of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Kv1.3 channel blockers are demonstrated potential therapeutic effects on MS patients and EAE models, maybe via reducing activation of T cells. However, it remains to be explored whether Kv1.3 channel blockers maintain integrity of BBB in MS model. In this study, ImKTx88, a highly selective Kv1.3 channel blocker, was used to determine the role of Kv1.3 channel in the pathogenesis of EAE, particularly in the maintenance of BBB. ImKTx88 ameliorated pathological severity in the EAE rats, and reduced extravasation into CNS. ImKTx88 also ameliorated the severity of loss or redistribution of tight junction proteins, and inhibited over-expression of ICAM-1 and VCAM-1 in the brain from EAE rats. Furthermore ImKTx88 protection was associated with activation of Ang-1/Tie-2 axis, and might be due to decreased IL-17 production. ImKTx88 may be a novel therapeutic agent for MS treatment by stabilizing the BBB.

  17. Molecular characterization of voltage-gated potassium channel (Kv) and its importance in functional dynamics in bull spermatozoa.

    PubMed

    Gupta, Rishi Kumar; Swain, Dilip Kumar; Singh, Vijay; Anand, Mukul; Choudhury, Soumen; Yadav, Sarvajeet; Saxena, Atul; Garg, Satish Kumar

    2018-07-01

    Present study was undertaken to characterize the voltage gated potassium channel (K v 1.1) in bull spermatozoa using sixty four ejaculates collected from four Hariana bulls. Functional characterization was undertaken using a selective blocker of Kv channel, 4-Aminopyridine (4-AP) while molecular presence of Kv on bull spermatozoa by immunoblotting and indirect immunofluorescence. Three sets of 100 μL diluted sperm samples namely-negative control (100 μL of sperm dilution medium (SDM) containing 10 × 10 6  cells), vehicle control (99 μL of SDM containing 10 × 10 6  cells, and DMSO- 1  μL) and 4-AP treatment group (99 μL of SDM containing 10 × 10 6  cells, and drug 1 μL 4-AP) were used in the study. Immunoblotting identified a single band of 56 kDa corresponding to Kv1.1 in Hariana bull spermatozoa. Immunolocalization showed the positive immunoreactivity at head, middle piece and principal piece of the spermatozoa for Kv 1.1. Blocking of Kv using 4-AP resulted in significant (p < 0.05) reduction in sperm progressive motility, per cent capacitated spermatozoa (B-pattern) and acrosome reacted (AR-pattern) spermatozoa, while significant (P < 0.05) increase in per cent swollen spermatozoa. Blocking of Kv channels resulted in significantly (P < 0.05) increased percentage of spermatozoa with lower mitochondrial transmembrane potential. Computer assisted semen analysis (CASA) of motion and kinematic parameters in 4-AP treated spermatozoa indicated reduction in sperm motion parameters like LIN, STR, VSL and VAP and higher ALH, VCL, and BCF indicating hyperactivity of spermatozoa. Based on our findings, it may be concluded that voltage-gated potassium channel (Kv) are present on bull spermatozoa and these are associated with functional dynamics of spermatozoa. However, based on our limited study, it is not possible to deduce that how these channels are associated with induction of hyperactivity. Therefore, further studies

  18. Contribution of Kv2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra.

    PubMed

    Kyle, B; Bradley, E; Ohya, S; Sergeant, G P; McHale, N G; Thornbury, K D; Hollywood, M A

    2011-11-01

    We have characterized the native voltage-dependent K(+) (K(v)) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with K(v)2.1 and K(v)2.2 channels cloned from the rabbit urethra and stably expressed in human embryonic kidney (HEK)-293 cells (HEK(Kv2.1) and HEK(Kv2.2)). RUSMC were perfused with Hanks' solution at 37°C and studied using the patch-clamp technique with K(+)-rich pipette solutions. Cells were bathed in 100 nM Penitrem A (Pen A) to block large-conductance Ca(2+)-activated K(+) (BK) currents and depolarized to +40 mV for 500 ms to evoke K(v) currents. These were unaffected by margatoxin, κ-dendrotoxin, or α-dendrotoxin (100 nM, n = 3-5) but were blocked by stromatoxin-1 (ScTx, IC(50) ∼130 nM), consistent with the idea that the currents were carried through K(v)2 channels. RNA was detected for K(v)2.1, K(v)2.2, and the silent subunit K(v)9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both K(v)2 subtypes and K(v)9.3 in isolated RUSMC. HEK(Kv2.1) and HEK(Kv2.2) currents were blocked in a concentration-dependent manner by ScTx, with estimated IC(50) values of ∼150 nM (K(v)2.1, n = 5) and 70 nM (K(v)2.2, n = 6). The mean half-maximal voltage (V(1/2)) of inactivation of the USMC K(v) current was -56 ± 3 mV (n = 9). This was similar to the HEK(Kv2.1) current (-55 ± 3 mV, n = 13) but significantly different from the HEK(Kv2.2) currents (-30 ± 3 mV, n = 11). Action potentials (AP) evoked from RUSMC studied under current-clamp mode were unaffected by ScTx. However, when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that K(v)2.1 channels contribute significantly to the K(v) current in RUSMC.

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

    PubMed

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

    2006-11-22

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

  20. Co-expression of the voltage-gated potassium channel Kv1.4 with transient receptor potential channels (TRPV1 and TRPV2) and the cannabinoid receptor CB1 in rat dorsal root ganglion neurons.

    PubMed

    Binzen, U; Greffrath, W; Hennessy, S; Bausen, M; Saaler-Reinhardt, S; Treede, R-D

    2006-10-13

    Potassium channels contribute to basic neuronal excitability and modulation. Here, we examined expression patterns of the voltage-gated potassium channel Kv1.4, the nociceptive transduction channels TRPV1 and TRPV2 as well as the putative anti-nociceptive cannabinoid receptor CB1 by immunofluorescence double-labelings in sections of rat dorsal root ganglia (DRGs). Kv1.4, TRPV1 and CB1 were each detected in about one third of neurons (35.7+/-0.5%, 29.4+/-1.1% and 36.4+/-0.5%, respectively, mean diameter 19.1+/-0.3 microm). TRPV2 was present in 4.4+/-0.4% of all neurons that were significantly larger in diameter (27.4+/-0.7 microm; P < 0.001). Antibody double-labeling revealed that the majority of Kv1.4-positive neurons co-expressed TRPV1 (73.9+/-1.5%) whereas none expressed TRPV2. The largest overlap was found with CB1 (93.1+/-0.1%). CB1 expression resembled that seen for Kv1.4 since the majority of neurons expressing CB1-protein also expressed TRPV1 (69.4+/-6.5%) but not TRPV2 (0.6+/-0.3%). When CB1-mRNA was detected using in situ hybridizations an additional subset of larger neurons was labeled including 82.4+/-17.7% of the TRPV2 expressing neurons. However, co-localization of Kv1.4 with CB1-mRNA (92%, mean diameter: 18.5 microm) was essentially the same as with CB1-protein. The almost complete overlap of CB1 and Kv1.4 in nociceptive DRG neurons suggests a functional synergistic action between Kv1.4 and CB1. The potassium channel may have two important roles in nociception. As the molecular basis of A-type current it could be involved in the control of repetitive discharges at peripheral terminals and as a downstream signal transduction site of CB1 in the control of presynaptic transmitter release at central terminals.

  1. Closed-state inactivation involving an internal gate in Kv4.1 channels modulates pore blockade by intracellular quaternary ammonium ions

    PubMed Central

    Fineberg, Jeffrey D.; Szanto, Tibor G.; Panyi, Gyorgy; Covarrubias, Manuel

    2016-01-01

    Voltage-gated K+ (Kv) channel activation depends on interactions between voltage sensors and an intracellular activation gate that controls access to a central pore cavity. Here, we hypothesize that this gate is additionally responsible for closed-state inactivation (CSI) in Kv4.x channels. These Kv channels undergo CSI by a mechanism that is still poorly understood. To test the hypothesis, we deduced the state of the Kv4.1 channel intracellular gate by exploiting the trap-door paradigm of pore blockade by internally applied quaternary ammonium (QA) ions exhibiting slow blocking kinetics and high-affinity for a blocking site. We found that inactivation gating seemingly traps benzyl-tributylammonium (bTBuA) when it enters the central pore cavity in the open state. However, bTBuA fails to block inactivated Kv4.1 channels, suggesting gated access involving an internal gate. In contrast, bTBuA blockade of a Shaker Kv channel that undergoes open-state P/C-type inactivation exhibits fast onset and recovery inconsistent with bTBuA trapping. Furthermore, the inactivated Shaker Kv channel is readily blocked by bTBuA. We conclude that Kv4.1 closed-state inactivation modulates pore blockade by QA ions in a manner that depends on the state of the internal activation gate. PMID:27502553

  2. Inhibition of Voltage-Gated K+ Channel Kv1.5 by Antiarrhythmic Drugs.

    PubMed

    Chen, Rong; Chung, Shin-Ho

    2018-05-08

    Molecular dynamics simulations are employed to determine the inhibitory mechanisms of three drugs, 5-(4-phenoxybutoxy)psoralen (PAP-1), vernakalant, and flecainide, on the voltage-gated K + channel Kv1.5, a target for the treatment of cardiac arrhythmia. At neutral pH, PAP-1 is neutral, whereas the other two molecules carry one positive charge. We show that PAP-1 forms stable dimers in water, primarily through hydrophobic interactions between aromatic rings. All three molecules bind to the cavity between the Ile508 and Val512 residues from the four subunits of the channel. Once bound, the drug molecules are flexible, with the average root-mean-square fluctuation being between 2 and 3 Å, which is larger than the radius of gyration of a bulky amino acid. The presence of a monomeric PAP-1 causes the permeating K + ion to dehydrate, thereby creating a significant energy barrier. In contrast, vernakalant blocks the ion permeation primarily via an electrostatic mechanism and, therefore, must be in the protonated and charged form to be effective.

  3. AKAP79/150 impacts intrinsic excitability of hippocampal neurons through phospho-regulation of A-type K+ channel trafficking.

    PubMed

    Lin, Lin; Sun, Wei; Kung, Faith; Dell'Acqua, Mark L; Hoffman, Dax A

    2011-01-26

    Kv4.2, as the primary α-subunit of rapidly inactivating, A-type voltage-gated K(+) (Kv) channels expressed in hippocampal CA1 pyramidal dendrites, plays a critical role in regulating their excitability. Activity-dependent trafficking of Kv4.2 relies on C-terminal protein kinase A (PKA) phosphorylation. A-kinase-anchoring proteins (AKAPs) target PKA to glutamate receptor and ion channel complexes to allow for discrete, local signaling. As part of a previous study, we showed that AKAP79/150 interacts with Kv4.2 complexes and that the two proteins colocalize in hippocampal neurons. However, the nature and functional consequence of their interaction has not been previously explored. Here, we report that the C-terminal domain of Kv4.2 interacts with an internal region of AKAP79/150 that overlaps with its MAGUK (membrane-associated guanylate kinase)-binding domain. We show that AKAP79/150-anchored PKA activity controls Kv4.2 surface expression in heterologous cells and hippocampal neurons. Consistent with these findings, disrupting PKA anchoring led to a decrease in neuronal excitability, while preventing dephosphorylation by the phosphatase calcineurin resulted in increased excitability. These results demonstrate that AKAP79/150 provides a platform for dynamic PKA regulation of Kv4.2 expression, fundamentally impacting CA1 excitability.

  4. Pathogenic plasticity of Kv7.2/3 channel activity is essential for the induction of tinnitus.

    PubMed

    Li, Shuang; Choi, Veronica; Tzounopoulos, Thanos

    2013-06-11

    Tinnitus, the perception of phantom sound, is often a debilitating condition that affects many millions of people. Little is known, however, about the molecules that participate in the induction of tinnitus. In brain slices containing the dorsal cochlear nucleus, we reveal a tinnitus-specific increase in the spontaneous firing rate of principal neurons (hyperactivity). This hyperactivity is observed only in noise-exposed mice that develop tinnitus and only in the dorsal cochlear nucleus regions that are sensitive to high frequency sounds. We show that a reduction in Kv7.2/3 channel activity is essential for tinnitus induction and for the tinnitus-specific hyperactivity. This reduction is due to a shift in the voltage dependence of Kv7 channel activation to more positive voltages. Our in vivo studies demonstrate that a pharmacological manipulation that shifts the voltage dependence of Kv7 to more negative voltages prevents the development of tinnitus. Together, our studies provide an important link between the biophysical properties of the Kv7 channel and the generation of tinnitus. Moreover, our findings point to previously unknown biological targets for designing therapeutic drugs that may prevent the development of tinnitus in humans.

  5. Pseudomonas fluorescens lipopolysaccharide inhibits both delayed rectifier and transient A-type K+ channels of cultured rat cerebellar granule neurons.

    PubMed

    Mezghani-Abdelmoula, Sana; Chevalier, Sylvie; Lesouhaitier, Olivier; Orange, Nicole; Feuilloley, Marc G J; Cazin, Lionel

    2003-09-05

    Pseudomonas fluorescens is a Gram-negative bacillus closely related to the pathogen P. aeruginosa known to provoke infectious disorders in the central nervous system (CNS). The endotoxin lipopolysaccharide (LPS) expressed by the bacteria is the first infectious factor that can interact with the plasma membrane of host cells. In the present study, LPS extracted from P. fluorescens MF37 was examined for its actions on delayed rectifier and A-type K(+) channels, two of the main types of voltage-activated K(+) channels involved in the action potential firing. Current recordings were performed in cultured rat cerebellar granule neurons at days 7 or 8, using the whole-cell patch-clamp technique. A 3-h incubation with LPS (200 ng/ml) markedly depressed both the delayed rectifier (I(KV)) and transient A-type (I(A)) K(+) currents evoked by depolarizations above 0 and -40 mV, respectively. The percent decrease of I(KV) and I(A) ( approximately 30%) did not vary with membrane potential, suggesting that inhibition of both types of K(+) channels by LPS was voltage-insensitive. The endotoxin did neither modify the steady-state voltage-dependent activation properties of I(KV) and I(A) nor the steady-state inactivation of I(A). The present results suggest that, by inhibiting I(KV) and I(A), LPS applied extracellulary increases the action potential firing in cerebellar granule neurons. It is concluded that P. fluorescens MF37 may provoke in the CNS disorders associated with sever alterations of membrane ionic channel functions.

  6. Physiological roles of Kv2 channels in entorhinal cortex layer II stellate cells revealed by Guangxitoxin‐1E

    PubMed Central

    Hönigsperger, Christoph; Nigro, Maximiliano J.

    2016-01-01

    Key points Kv2 channels underlie delayed‐rectifier potassium currents in various neurons, although their physiological roles often remain elusive. Almost nothing is known about Kv2 channel functions in medial entorhinal cortex (mEC) neurons, which are involved in representing space, memory formation, epilepsy and dementia.Stellate cells in layer II of the mEC project to the hippocampus and are considered to be space‐representing grid cells. We used the new Kv2 blocker Guangxitoxin‐1E (GTx) to study Kv2 functions in these neurons.Voltage clamp recordings from mEC stellate cells in rat brain slices showed that GTx inhibited delayed‐rectifier K+ current but not transient A‐type current.In current clamp, GTx had multiple effects: (i) increasing excitability and bursting at moderate spike rates but reducing firing at high rates; (ii) enhancing after‐depolarizations; (iii) reducing the fast and medium after‐hyperpolarizations; (iv) broadening action potentials; and (v) reducing spike clustering.GTx is a useful tool for studying Kv2 channels and their functions in neurons. Abstract The medial entorhinal cortex (mEC) is strongly involved in spatial navigation, memory, dementia and epilepsy. Although potassium channels shape neuronal activity, their roles in mEC are largely unknown. We used the new Kv2 blocker Guangxitoxin‐1E (GTx; 10–100 nm) in rat brain slices to investigate Kv2 channel functions in mEC layer II stellate cells (SCs). These neurons project to the hippocampus and are considered to be grid cells representing space. Voltage clamp recordings from SCs nucleated patches showed that GTx inhibited a delayed rectifier K+ current activating beyond –30 mV but not transient A‐type current. In current clamp, GTx (i) had almost no effect on the first action potential but markedly slowed repolarization of late spikes during repetitive firing; (ii) enhanced the after‐depolarization (ADP); (iii) reduced fast and medium after

  7. KV1 channels identified in rodent myelinated axons, linked to Cx29 in innermost myelin: support for electrically active myelin in mammalian saltatory conduction

    PubMed Central

    Vanderpool, Kimberly G.; Yasumura, Thomas; Hickman, Jordan; Beatty, Jonathan T.; Nagy, James I.

    2016-01-01

    Saltatory conduction in mammalian myelinated axons was thought to be well understood before recent discoveries revealed unexpected subcellular distributions and molecular identities of the K+-conductance pathways that provide for rapid axonal repolarization. In this study, we visualize, identify, localize, quantify, and ultrastructurally characterize axonal KV1.1/KV1.2 channels in sciatic nerves of rodents. With the use of light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling electron microscopy, KV1.1/KV1.2 channels are localized to three anatomically and compositionally distinct domains in the internodal axolemmas of large myelinated axons, where they form densely packed “rosettes” of 9-nm intramembrane particles. These axolemmal KV1.1/KV1.2 rosettes are precisely aligned with and ultrastructurally coupled to connexin29 (Cx29) channels, also in matching rosettes, in the surrounding juxtaparanodal myelin collars and along the inner mesaxon. As >98% of transmembrane proteins large enough to represent ion channels in these specialized domains, ∼500,000 KV1.1/KV1.2 channels define the paired juxtaparanodal regions as exclusive membrane domains for the voltage-gated K+ conductance that underlies rapid axonal repolarization in mammals. The 1:1 molecular linkage of KV1 channels to Cx29 channels in the apposed juxtaparanodal collars, plus their linkage to an additional 250,000–400,000 Cx29 channels along each inner mesaxon in every large-diameter myelinated axon examined, supports previously proposed K+ conductance directly from juxtaparanodal axoplasm into juxtaparanodal myeloplasm in mammalian axons. With neither Cx29 protein nor myelin rosettes detectable in frog myelinated axons, these data showing axon-to-myelin linkage by abundant KV1/Cx29 channels in rodent axons support renewed consideration of an electrically active role for myelin in increasing both saltatory conduction velocity and maximum propagation frequency in

  8. Serum starvation-induced voltage-gated potassium channel Kv7.5 expression and its regulation by Sp1 in canine osteosarcoma cells.

    PubMed

    Lee, Bo Hyung; Ryu, Pan Dong; Lee, So Yeong

    2014-01-10

    The KCNQ gene family, whose members encode Kv7 channels, belongs to the voltage-gated potassium (Kv) channel group. The roles of this gene family have been widely investigated in nerve and muscle cells. In the present study, we investigated several characteristics of Kv7.5, which is strongly expressed in the canine osteosarcoma cell line, CCL-183. Serum starvation upregulated Kv7.5 expression, and the Kv7 channel opener, flupirtine, attenuated cell proliferation by arresting cells in the G0/G1 phase. We also showed that Kv7.5 knockdown helps CCL-183 cells to proliferate. In an effort to find an endogenous regulator of Kv7.5, we used mithramycin A to reduce the level of the transcription factor Sp1, and it strongly inhibited the induction of Kv7.5 in CCL-183 cells. These results suggest that the activation of Kv7.5 by flupirtine may exert an anti-proliferative effect in canine osteosarcoma. Therefore, Kv7.5 is a possible molecular target for canine osteosarcoma therapy.

  9. Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

    PubMed Central

    Kuzmenkov, Alexey I.; Vassilevski, Alexander A.; Kudryashova, Kseniya S.; Nekrasova, Oksana V.; Peigneur, Steve; Tytgat, Jan; Feofanov, Alexey V.; Kirpichnikov, Mikhail P.; Grishin, Eugene V.

    2015-01-01

    The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus, and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here, we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands, and we compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis, we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β and inhibitor cystine knot types of fold. M. eupeus venom was then separated to individual components by multistage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore binding activity during venom separation. As a result, eight high affinity Kv1.1 channel blockers were identified, including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1–780 nm) by the two-electrode voltage clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs. PMID:25792741

  10. Dendritic Kv3.3 potassium channels in cerebellar purkinje cells regulate generation and spatial dynamics of dendritic Ca2+ spikes.

    PubMed

    Zagha, Edward; Manita, Satoshi; Ross, William N; Rudy, Bernardo

    2010-06-01

    Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

  11. Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K+ channel.

    PubMed

    Bernardo-Seisdedos, Ganeko; Nuñez, Eider; Gomis-Perez, Carolina; Malo, Covadonga; Villarroel, Álvaro; Millet, Oscar

    2018-03-06

    The Kv7.2 (KCNQ2) channel is the principal molecular component of the slow voltage-gated, noninactivating K + M-current, a key controller of neuronal excitability. To investigate the calmodulin (CaM)-mediated Ca 2+ gating of the channel, we used NMR spectroscopy to structurally and dynamically describe the association of helices h A and h B of Kv7.2 with CaM, as a function of Ca 2+ concentration. The structures of the CaM/Kv7.2-hAB complex at two different calcification states are reported here. In the presence of a basal cytosolic Ca 2+ concentration (10-100 nM), only the N-lobe of CaM is Ca 2+ -loaded and the complex (representative of the open channel) exhibits collective dynamics on the millisecond time scale toward a low-populated excited state (1.5%) that corresponds to the inactive state of the channel. In response to a chemical or electrical signal, intracellular Ca 2+ levels rise up to 1-10 μM, triggering Ca 2+ association with the C-lobe. The associated conformational rearrangement is the key biological signal that shifts populations to the closed/inactive channel. This reorientation affects the C-lobe of CaM and both helices in Kv7.2, allosterically transducing the information from the Ca 2+ -binding site to the transmembrane region of the channel. Copyright © 2018 the Author(s). Published by PNAS.

  12. Differential Regulation of Action Potential Shape and Burst-Frequency Firing by BK and Kv2 Channels in Substantia Nigra Dopaminergic Neurons

    PubMed Central

    Kimm, Tilia; Khaliq, Zayd M.

    2015-01-01

    Little is known about the voltage-dependent potassium currents underlying spike repolarization in midbrain dopaminergic neurons. Studying mouse substantia nigra pars compacta dopaminergic neurons both in brain slice and after acute dissociation, we found that BK calcium-activated potassium channels and Kv2 channels both make major contributions to the depolarization-activated potassium current. Inhibiting Kv2 or BK channels had very different effects on spike shape and evoked firing. Inhibiting Kv2 channels increased spike width and decreased the afterhyperpolarization, as expected for loss of an action potential-activated potassium conductance. BK inhibition also increased spike width but paradoxically increased the afterhyperpolarization. Kv2 channel inhibition steeply increased the slope of the frequency–current (f–I) relationship, whereas BK channel inhibition had little effect on the f–I slope or decreased it, sometimes resulting in slowed firing. Action potential clamp experiments showed that both BK and Kv2 current flow during spike repolarization but with very different kinetics, with Kv2 current activating later and deactivating more slowly. Further experiments revealed that inhibiting either BK or Kv2 alone leads to recruitment of additional current through the other channel type during the action potential as a consequence of changes in spike shape. Enhancement of slowly deactivating Kv2 current can account for the increased afterhyperpolarization produced by BK inhibition and likely underlies the very different effects on the f–I relationship. The cross-regulation of BK and Kv2 activation illustrates that the functional role of a channel cannot be defined in isolation but depends critically on the context of the other conductances in the cell. SIGNIFICANCE STATEMENT This work shows that BK calcium-activated potassium channels and Kv2 voltage-activated potassium channels both regulate action potentials in dopamine neurons of the substantia nigra

  13. Differential Regulation of Action Potential Shape and Burst-Frequency Firing by BK and Kv2 Channels in Substantia Nigra Dopaminergic Neurons.

    PubMed

    Kimm, Tilia; Khaliq, Zayd M; Bean, Bruce P

    2015-12-16

    Little is known about the voltage-dependent potassium currents underlying spike repolarization in midbrain dopaminergic neurons. Studying mouse substantia nigra pars compacta dopaminergic neurons both in brain slice and after acute dissociation, we found that BK calcium-activated potassium channels and Kv2 channels both make major contributions to the depolarization-activated potassium current. Inhibiting Kv2 or BK channels had very different effects on spike shape and evoked firing. Inhibiting Kv2 channels increased spike width and decreased the afterhyperpolarization, as expected for loss of an action potential-activated potassium conductance. BK inhibition also increased spike width but paradoxically increased the afterhyperpolarization. Kv2 channel inhibition steeply increased the slope of the frequency-current (f-I) relationship, whereas BK channel inhibition had little effect on the f-I slope or decreased it, sometimes resulting in slowed firing. Action potential clamp experiments showed that both BK and Kv2 current flow during spike repolarization but with very different kinetics, with Kv2 current activating later and deactivating more slowly. Further experiments revealed that inhibiting either BK or Kv2 alone leads to recruitment of additional current through the other channel type during the action potential as a consequence of changes in spike shape. Enhancement of slowly deactivating Kv2 current can account for the increased afterhyperpolarization produced by BK inhibition and likely underlies the very different effects on the f-I relationship. The cross-regulation of BK and Kv2 activation illustrates that the functional role of a channel cannot be defined in isolation but depends critically on the context of the other conductances in the cell. This work shows that BK calcium-activated potassium channels and Kv2 voltage-activated potassium channels both regulate action potentials in dopamine neurons of the substantia nigra pars compacta. Although both

  14. Spatial distributions of Kv4 channels and KChip2 isoforms in the murine heart based on laser capture microdissection.

    PubMed

    Teutsch, Christine; Kondo, Richard P; Dederko, Dorothy A; Chrast, Jacqueline; Chien, Kenneth R; Giles, Wayne R

    2007-03-01

    Regional differences in repolarizing K(+) current densities and expression levels of their molecular components are important for coordinating the pattern of electrical excitation and repolarization of the heart. The small size of hearts from mice may obscure these interventricular and/or transmural expression differences of K(+) channels. We have examined this possibility in adult mouse ventricle using a technology that provides very high spatial resolution of tissue collection. Conventional manual dissection and laser capture microdissection (LCM) were utilized to dissect tissue from distinct ventricular regions. RNA was isolated from epicardial, mid-myocardial and endocardial layers of both the right and left ventricles. Real-time RT-PCR was used to quantify the transcript expression in these different regions. LCM revealed significant interventricular and transmural gradients for both Kv4.2 and the alpha-subunit of KChIP2. The expression profile of a second K(+) channel transcript, Kir2.1, which is responsible for the inwardly rectifying K(+) current I(k1), showed no interventricular or transmural gradients and therefore served as a negative control. Our findings are in contrast to previous reports of a relatively uniform left ventricular transmural pattern of expression of Kv4.2, Kv4.3 and KChIP2 in adult mouse heart, which appear to be different than that in larger mammals. Specifically, our results demonstrate significant epi- to endocardial differences in the patterns of expression of both Kv4.2 and KChIP2.

  15. Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation.

    PubMed

    Ng, Chai-Ann; Gravel, Andrée E; Perry, Matthew D; Arnold, Alexandre A; Marcotte, Isabelle; Vandenberg, Jamie I

    2016-08-12

    Slow deactivation of Kv11.1 channels is critical for its function in the heart. The S4-S5 linker, which joins the voltage sensor and pore domains, plays a critical role in this slow deactivation gating. Here, we use NMR spectroscopy to identify the membrane-bound surface of the S4S5 linker, and we show that two highly conserved tyrosine residues within the KCNH subfamily of channels are membrane-associated. Site-directed mutagenesis and electrophysiological analysis indicates that Tyr-542 interacts with both the pore domain and voltage sensor residues to stabilize activated conformations of the channel, whereas Tyr-545 contributes to the slow kinetics of deactivation by primarily stabilizing the transition state between the activated and closed states. Thus, the two tyrosine residues in the Kv11.1 S4S5 linker play critical but distinct roles in the slow deactivation phenotype, which is a hallmark of Kv11.1 channels. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. K+ channels expression in hypertension after arterial injury, and effect of selective Kv1.3 blockade with PAP-1 on intimal hyperplasia formation.

    PubMed

    Cidad, P; Novensà, L; Garabito, M; Batlle, M; Dantas, A P; Heras, M; López-López, J R; Pérez-García, M T; Roqué, M

    2014-12-01

    K(+) channels are central to vascular pathophysiology. Previous results demonstrated that phenotypic modulation associates with a change in Kv1.3 to Kv1.5 expression, and that Kv1.3 blockade inhibits proliferation of VSMCs cultures. To explore whether the Kv1.3 to Kv1.5 switch could be a marker of the increased risk of intimal hyperplasia in essential hypertension and whether systemic treatment with Kv1.3 blockers can prevent intimal hyperplasia after endoluminal lesion . Morphometric and immunohistochemical analysis were performed in arterial segments following arterial injury and constant infusion of the Kv1.3 blocker PAP-1 during 28 days. Differential expression of K(+) channel genes was studied in VSMC from hypertensive (BPH) and normotensive (BPN) mice, both in control and after endoluminal lesion. Finally, the migration and proliferation rate of BPN and BPH VSMCs was explored in vitro. Changes in mRNA expression led to an increased Kv1.3/Kv1.5 ratio in BPH VSMC. Consistent with this, arterial injury in BPH mice induced a higher degree of luminal stenosis, (84 ± 4% vs. 70 ± 5% in BPN, p < 0.01), although no differences in migration and proliferation rate were observed in cultured VSMCs. The in vivo proliferative lesions were significantly decreased upon PAP-1 systemic infusion (18 ± 6% vs. 58 ± 20% with vehicle, p < 0.05). Hypertension leads to a higher degree of luminal stenosis in our arterial injury model, that correlates with a decreased expression of Kv1.5 channels. Kv1.3 blockers decreased in vitro VSMCs proliferation, migration, and in vivo intimal hyperplasia formation, pointing to Kv1.3 channels as promising therapeutical targets against restenosis.

  17. K(v) channel interacting protein 3 expression and regulation by haloperidol in midbrain dopaminergic neurons.

    PubMed

    Duncan, Carlotta E; Schofield, Peter R; Weickert, Cynthia Shannon

    2009-12-22

    Antipsychotic drugs are the main treatment for schizophrenia, despite their adverse side effects and uncertain mode of action. Gene expression studies in the brains of rodents treated with antipsychotic drugs aim to uncover this mechanism and elucidate more specific targets for schizophrenia treatment. Previous expression profiling analyses showed that K(v) channel interacting protein 3 (KChIP3) was down-regulated in the mouse brain following treatment with multiple antipsychotic drugs. In this study, we used in situ hybridization to anatomically define the expression of KChIP3 mRNA in the mouse brain and to quantify its regulation by 7-day haloperidol treatment. We used immunohistochemistry to localize KChIP3 protein expression in the midbrain, dorsal and ventral striatum and the prefrontal cortex. We found KChIP3 mRNA throughout the grey matter of the brain, with high expression in the hippocampus, specific thalamic nuclei, deeper cortical layers and in the midbrain. KChIP3 mRNA was significantly down-regulated in the dorsal striatum and the ventral tegmental area following haloperidol treatment. KChIP3 protein is expressed in the neuropil in the cortex and striatum, as well as in the soma of deeper layer cortical and striatal neurons. This study, for the first time, also localized KChIP3 protein in the cell bodies and processes of dopaminergic neurons in the midbrain. These findings indicate that regulation of KChIP3, particularly in mesocortical dopamine neurons, may be part of the action of antipsychotic drugs and that prolonged and more specific targeting of ion channel subunits may enhance the therapeutic effects of antipsychotic drugs.

  18. A recurrent KCNQ2 pore mutation causing early onset epileptic encephalopathy has a moderate effect on M current but alters subcellular localization of Kv7 channels.

    PubMed

    Abidi, Affef; Devaux, Jérôme J; Molinari, Florence; Alcaraz, Gisèle; Michon, François-Xavier; Sutera-Sardo, Julie; Becq, Hélène; Lacoste, Caroline; Altuzarra, Cécilia; Afenjar, Alexandra; Mignot, Cyril; Doummar, Diane; Isidor, Bertrand; Guyen, Sylvie N; Colin, Estelle; De La Vaissière, Sabine; Haye, Damien; Trauffler, Adeline; Badens, Catherine; Prieur, Fabienne; Lesca, Gaetan; Villard, Laurent; Milh, Mathieu; Aniksztejn, Laurent

    2015-08-01

    Mutations in the KCNQ2 gene encoding the voltage-dependent potassium M channel Kv7.2 subunit cause either benign epilepsy or early onset epileptic encephalopathy (EOEE). It has been proposed that the disease severity rests on the inhibitory impact of mutations on M current density. Here, we have analyzed the phenotype of 7 patients carrying the p.A294V mutation located on the S6 segment of the Kv7.2 pore domain (Kv7.2(A294V)). We investigated the functional and subcellular consequences of this mutation and compared it to another mutation (Kv7.2(A294G)) associated with a benign epilepsy and affecting the same residue. We report that all the patients carrying the p.A294V mutation presented the clinical and EEG characteristics of EOEE. In CHO cells, the total expression of Kv7.2(A294V) alone, assessed by western blotting, was only 20% compared to wild-type. No measurable current was recorded in CHO cells expressing Kv7.2(A294V) channel alone. Although the total Kv7.2(A294V) expression was rescued to wild-type levels in cells co-expressing the Kv7.3 subunit, the global current density was still reduced by 83% compared to wild-type heteromeric channel. In a configuration mimicking the patients' heterozygous genotype i.e., Kv7.2(A294V)/Kv7.2/Kv7.3, the global current density was reduced by 30%. In contrast to Kv7.2(A294V), the current density of homomeric Kv7.2(A294G) was not significantly changed compared to wild-type Kv7.2. However, the current density of Kv7.2(A294G)/Kv7.2/Kv7.3 and Kv7.2(A294G)/Kv7.3 channels were reduced by 30% and 50% respectively, compared to wild-type Kv7.2/Kv7.3. In neurons, the p.A294V mutation induced a mislocalization of heteromeric mutant channels to the somato-dendritic compartment, while the p.A294G mutation did not affect the localization of the heteromeric channels to the axon initial segment. We conclude that this position is a hotspot of mutation that can give rise to a severe or a benign epilepsy. The p.A294V mutation does not exert a

  19. Tannic acid activates the Kv7.4 and Kv7.3/7.5 K(+) channels expressed in HEK293 cells and reduces tension in the rat mesenteric arteries.

    PubMed

    Zhang, Yuanyuan; Chu, Xi; Liu, Ling; Zhang, Nan; Guo, Hui; Yang, Fan; Liu, Zhenyi; Dong, Yongsheng; Bao, Yifan; Zhang, Xuan; Zhang, Jianping

    2016-04-01

    This study investigated the effect of tannic acid (TA), a plant-derived hydrolyzable polyphenol, on Kv7.4 and Kv7.5 K(+) channels and rat mesenteric artery. Whole-cell patch clamp experiments were used to record the Kv7.4 and Kv7.3/7.5 K(+) currents expressed in HEK293 cells; and the tension changes of mesenteric arteries isolated from rats were recorded using small vessel myography apparatus. Tannic acid increases the Kv7.4 and Kv7.3/7.5 K(+) currents in a concentration-dependent manner (median effective concentration (EC50 ) = 27.3 ± 3.6 μm and EC50 = 23.1 ± 3.9 μm, respectively). In addition, 30 μm TA shifts the G-V curve of Kv7.4 and Kv7.3/7.5 K(+) currents to the left by 14.18 and 25.24 mV, respectively, and prolongs the deactivation time constants by 184.44 and 154.77 ms, respectively. Moreover, TA relaxes the vascular tension of rat mesenteric arteries in a concentration-dependent manner (half inhibitory concentration (IC50 ) = 148.7 ± 13.4 μm). These results confirms the vasodilatory effects of TA on rat mesenteric artery and the activating effects on the Kv7.4 and Kv7.3/7.5 K(+) channels, which may be a mechanism to explain the vasodilatory effect and this mechanism can be used in the research of antihypertension. © 2016 Royal Pharmaceutical Society.

  20. Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

    PubMed Central

    Soldovieri, Maria Virginia; Ambrosino, Paolo; Mosca, Ilaria; De Maria, Michela; Moretto, Edoardo; Miceli, Francesco; Alaimo, Alessandro; Iraci, Nunzio; Manocchio, Laura; Medoro, Alessandro; Passafaro, Maria; Taglialatela, Maurizio

    2016-01-01

    Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K+ current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP2 levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP2 is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies. PMID:27905566

  1. Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate.

    PubMed

    Soldovieri, Maria Virginia; Ambrosino, Paolo; Mosca, Ilaria; De Maria, Michela; Moretto, Edoardo; Miceli, Francesco; Alaimo, Alessandro; Iraci, Nunzio; Manocchio, Laura; Medoro, Alessandro; Passafaro, Maria; Taglialatela, Maurizio

    2016-12-01

    Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K + current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP 2 levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP 2 is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies.

  2. Kv7.3 Compound Heterozygous Variants in Early Onset Encephalopathy Reveal Additive Contribution of C-Terminal Residues to PIP2-Dependent K+ Channel Gating.

    PubMed

    Ambrosino, Paolo; Freri, Elena; Castellotti, Barbara; Soldovieri, Maria Virginia; Mosca, Ilaria; Manocchio, Laura; Gellera, Cinzia; Canafoglia, Laura; Franceschetti, Silvana; Salis, Barbara; Iraci, Nunzio; Miceli, Francesco; Ragona, Francesca; Granata, Tiziana; DiFrancesco, Jacopo C; Taglialatela, Maurizio

    2018-01-30

    Over one hundred mutations in the Kv7.2 (KCNQ2) gene encoding for phosphatidylinositol 4,5-bisphosphate (PIP 2 )-sensitive voltage-gated K + channel subunits have been identified in early-onset epilepsies with wide phenotypic variability. By contrast, only few mutations in the closely related Kv7.3 (KCNQ3) gene have been reported, mostly associated with typical benign familial neonatal seizures (BFNS). We herein describe a patient affected by early onset epileptic encephalopathy (EOEE) carrying two Kv7.3 missense mutations (p.Val359Leu/V359L and p.Asp542Asn/D542N) in compound heterozygosis, each inherited from an asymptomatic parent. Patch-clamp recordings from transiently transfected CHO cells showed that, when incorporated in physiologically relevant Kv7.2 + Kv7.3 heteromeric channels, expression of Kv7.3 V359L or Kv7.3 D542N subunits failed to affect current density, whereas a significant decrease was instead observed when these mutant subunits were both simultaneously present. Modeling and functional experiments revealed that each variant decreased PIP 2 -dependent current regulation, with additive effects when the two were co-expressed. Moreover, expression of Kv7.2 subunits carrying the D535N variant previously described in three sporadic EOEE cases prompted functional changes more dramatic when compared to those of the corresponding D542N variant in Kv7.3, but similar to those observed when both Kv7.3 V359L and Kv7.3 D542N subunits were expressed together. Finally, the Kv7 activator retigabine restored channel dysfunction induced by each Kv7.2 or Kv7.3 variant(s). These results provide a plausible molecular explanation for the apparent recessive inheritance of the phenotype in the family investigated, and a rational basis for personalized therapy with Kv7 channel activators in EOEE patients carrying loss-of-function mutations in Kv7.2 or Kv7.3.

  3. Neuroplasticity of A-type potassium channel complexes induced by chronic alcohol exposure enhances dendritic calcium transients in hippocampus.

    PubMed

    Mulholland, Patrick J; Spencer, Kathryn B; Hu, Wei; Kroener, Sven; Chandler, L Judson

    2015-06-01

    Chronic alcohol-induced cognitive impairments and maladaptive plasticity of glutamatergic synapses are well-documented. However, it is unknown if prolonged alcohol exposure affects dendritic signaling that may underlie hippocampal dysfunction in alcoholics. Back-propagation of action potentials (bAPs) into apical dendrites of hippocampal neurons provides distance-dependent signals that modulate dendritic and synaptic plasticity. The amplitude of bAPs decreases with distance from the soma that is thought to reflect an increase in the density of Kv4.2 channels toward distal dendrites. The aim of this study was to quantify changes in hippocampal Kv4.2 channel function and expression using electrophysiology, Ca(2+) imaging, and western blot analyses in a well-characterized in vitro model of chronic alcohol exposure. Chronic alcohol exposure significantly decreased expression of Kv4.2 channels and KChIP3 in hippocampus. This reduction was associated with an attenuation of macroscopic A-type K(+) currents in CA1 neurons. Chronic alcohol exposure increased bAP-evoked Ca(2+) transients in the distal apical dendrites of CA1 pyramidal neurons. The enhanced bAP-evoked Ca(2+) transients induced by chronic alcohol exposure were not related to synaptic targeting of N-methyl-D-aspartate (NMDA) receptors or morphological adaptations in apical dendritic arborization. These data suggest that chronic alcohol-induced decreases in Kv4.2 channel function possibly mediated by a downregulation of KChIP3 drive the elevated bAP-associated Ca(2+) transients in distal apical dendrites. Alcohol-induced enhancement of bAPs may affect metaplasticity and signal integration in apical dendrites of hippocampal neurons leading to alterations in hippocampal function.

  4. Novel Roles for Kv7 Channels in Shaping Histamine-Induced Contractions and Bradykinin-Dependent Relaxations in Pig Coronary Arteries

    PubMed Central

    Chen, Xingjuan; Li, Wennan; Hiett, S. Christopher; Obukhov, Alexander G.

    2016-01-01

    Voltage-gated Kv7 channels are inhibited by agonists of Gq-protein-coupled receptors, such as histamine. Recent works have provided evidence that inhibition of vascular Kv7 channels may trigger vessel contractions. In this study, we investigated how Kv7 activity modulates the histamine-induced contractions in “healthy” and metabolic syndrome (MetS) pig right coronary arteries (CAs). We performed isometric tension and immunohistochemical studies with domestic, lean Ossabaw, and MetS Ossabaw pig CAs. We found that neither the Kv7.2/Kv7.4/Kv7.5 activator ML213 nor the general Kv7 inhibitor XE991 altered the tension of CA rings under preload, indicating that vascular Kv7 channels are likely inactive in the preloaded rings. Conversely, ML213 potently dilated histamine-pre-contracted CAs, suggesting that Kv7 channels are activated during histamine applications and yet partially inhibited by histamine. Immunohistochemistry analysis revealed strong Kv7.4 immunostaining in the medial and intimal layers of the CA wall, whereas Kv7.5 immunostaining intensity was strong in the intimal but weak in the medial layers. The medial Kv7 immunostaining was significantly weaker in MetS Ossabaw CAs as compared to lean Ossabaw or domestic CAs. Consistently, histamine-pre-contracted MetS Ossabaw CAs exhibited attenuated ML213-dependent dilations. In domestic pig CAs, where medial Kv7 immunostaining intensity was stronger, histamine-induced contractions spontaneously decayed to ~31% of the peak amplitude within 4 minutes. Oppositely, in Ossabaw CAs, where Kv7 immunostaining intensity was weaker, the histamine-induced contractions were more sustained. XE991 pretreatment significantly slowed the decay rate of histamine-induced contractions in domestic CAs, supporting the hypothesis that increased Kv7 activity correlates with a faster rate of histamine-induced contraction decay. Alternatively, XE991 significantly decreased the amplitude of bradykinin-dependent dilations in pre

  5. Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly destabilizing epileptogenic mutation.

    PubMed

    Alberdi, Araitz; Gomis-Perez, Carolina; Bernardo-Seisdedos, Ganeko; Alaimo, Alessandro; Malo, Covadonga; Aldaregia, Juncal; Lopez-Robles, Carlos; Areso, Pilar; Butz, Elisabeth; Wahl-Schott, Christian; Villarroel, Alvaro

    2015-11-01

    We show that the combination of an intracellular bi-partite calmodulin (CaM)-binding site and a distant assembly region affect how an ion channel is regulated by a membrane lipid. Our data reveal that regulation by phosphatidylinositol(4,5)bisphosphate (PIP2) and stabilization of assembled Kv7.2 subunits by intracellular coiled-coil regions far from the membrane are coupled molecular processes. Live-cell fluorescence energy transfer measurements and direct binding studies indicate that remote coiled-coil formation creates conditions for different CaM interaction modes, each conferring different PIP2 dependency to Kv7.2 channels. Disruption of coiled-coil formation by epilepsy-causing mutation decreases apparent CaM-binding affinity and interrupts CaM influence on PIP2 sensitivity. © 2015. Published by The Company of Biologists Ltd.

  6. Overlapping Binding Sites of Structurally Different Antiarrhythmics Flecainide and Propafenone in the Subunit Interface of Potassium Channel Kv2.1*

    PubMed Central

    Madeja, Michael; Steffen, Wibke; Mesic, Ivana; Garic, Bojan; Zhorov, Boris S.

    2010-01-01

    Kv2.1 channels, which are expressed in brain, heart, pancreas, and other organs and tissues, are important targets for drug design. Flecainide and propafenone are known to block Kv2.1 channels more potently than other Kv channels. Here, we sought to explore structural determinants of this selectivity. We demonstrated that flecainide reduced the K+ currents through Kv2.1 channels expressed in Xenopus laevis oocytes in a voltage- and time-dependent manner. By systematically exchanging various segments of Kv2.1 with those from Kv1.2, we determined flecainide-sensing residues in the P-helix and inner helix S6. These residues are not exposed to the inner pore, a conventional binding region of open channel blockers. The flecainide-sensing residues also contribute to propafenone binding, suggesting overlapping receptors for the drugs. Indeed, propafenone and flecainide compete for binding in Kv2.1. We further used Monte Carlo-energy minimizations to map the receptors of the drugs. Flecainide docking in the Kv1.2-based homology model of Kv2.1 predicts the ligand ammonium group in the central cavity and the benzamide moiety in a niche between S6 and the P-helix. Propafenone also binds in the niche. Its carbonyl group accepts an H-bond from the P-helix, the amino group donates an H-bond to the P-loop turn, whereas the propyl group protrudes in the pore and blocks the access to the selectivity filter. Thus, besides the binding region in the central cavity, certain K+ channel ligands can expand in the subunit interface whose residues are less conserved between K+ channels and hence may be targets for design of highly desirable subtype-specific K+ channel drugs. PMID:20709754

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

    PubMed

    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.

  8. Functional characterization of Kv11.1 (hERG) potassium channels split in the voltage-sensing domain.

    PubMed

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

    2018-03-23

    Voltage-dependent KCNH family potassium channel functionality can be reconstructed using non-covalently linked voltage-sensing domain (VSD) and pore modules (split channels). However, the necessity of a covalent continuity for channel function has not been evaluated at other points within the two functionally independent channel modules. We find here that by cutting Kv11.1 (hERG, KCNH2) channels at the different loops linking the transmembrane spans of the channel core, not only channels split at the S4-S5 linker level, but also those split at the intracellular S2-S3 and the extracellular S3-S4 loops, yield fully functional channel proteins. Our data indicate that albeit less markedly, channels split after residue 482 in the S2-S3 linker resemble the uncoupled gating phenotype of those split at the C-terminal end of the VSD S4 transmembrane segment. Channels split after residues 514 and 518 in the S3-S4 linker show gating characteristics similar to those of the continuous wild-type channel. However, breaking the covalent link at this level strongly accelerates the voltage-dependent accessibility of a membrane impermeable methanethiosulfonate reagent to an engineered cysteine at the N-terminal region of the S4 transmembrane helix. Thus, besides that of the S4-S5 linker, structural integrity of the intracellular S2-S3 linker seems to constitute an important factor for proper transduction of VSD rearrangements to opening and closing the cytoplasmic gate. Furthermore, our data suggest that the short and probably rigid characteristics of the extracellular S3-S4 linker are not an essential component of the Kv11.1 voltage sensing machinery.

  9. Kv7 voltage-activated potassium channel inhibitors reduce fluid resuscitation requirements after hemorrhagic shock in rats.

    PubMed

    Nassoiy, Sean P; Byron, Kenneth L; Majetschak, Matthias

    2017-01-17

    Recent evidence suggests that drugs targeting Kv7 channels could be used to modulate vascular function and blood pressure. Here, we studied whether Kv7 channel inhibitors can be utilized to stabilize hemodynamics and reduce resuscitation fluid requirements after hemorrhagic shock. Anesthetized male Sprague-Dawley rats were instrumented with arterial and venous catheters for blood pressure monitoring, hemorrhage and fluid resuscitation. Series 1: Linopirdine (Kv7 channel blocker, 0.1-6 mg/kg) or retigabine (Kv7 channel activator, 0.1-12 mg/kg) were administered to normal animals. Series 2: Animals were hemorrhaged to a MAP of 25 mmHg for 30 min, followed by fluid resuscitation with normal saline (NS) to a MAP of 70 mmHg until t = 75 min. Animals were treated with single bolus injections of vehicle, linopirdine (1-6 mg/kg), XE-991 (structural analogue of linopirdine with higher potency for channel blockade, 1 mg/kg) prior to fluid resuscitation. Series 3: Animals were resuscitated with NS alone or NS supplemented with linopirdine (1.25-200 μg/mL). Data were analyzed with 2-way ANOVA/Bonferroni post-hoc testing. Series 1: Linopirdine transiently (10-15 min) and dose-dependently increased MAP by up to 15%. Retigabine dose-dependently reduced MAP by up to 60%, which could be reverted with linopirdine. Series 2: Fluid requirements to maintain MAP at 70 mmHg were 65 ± 34 mL/kg with vehicle, and 57 ± 13 mL/kg, 22 ± 8 mL/kg and 22 ± 11 mL/kg with intravenous bolus injection of 1, 3 and 6 mg/kg linopirdine, respectively. XE-991 (1 mg/kg), reduced resuscitation requirements comparable to 3 mg/kg linopirdine. Series 3: When resuscitation was performed with linopirdine-supplemented normal saline (NS), fluid requirements to stabilize MAP were 73 ± 12 mL/kg with NS alone and 72 ± 24, 61 ± 20, 36 ± 9 and 31 ± 9 mL/kg with NS supplemented with 1.25, 6.25, 12.5 and 200 μg/mL linopirdine, respectively. Our data suggest that Kv7

  10. Contribution of KV1.5 Channel to H2O2-Induced Human Arteriolar Dilation and its Modulation by Coronary Artery Disease

    PubMed Central

    Nishijima, Yoshinori; Cao, Sheng; Chabowski, Dawid S.; Korishettar, Ankush; Ge, Alyce; Zheng, Xiaodong; Sparapani, Rodney; Gutterman, David D.; Zhang, David X.

    2016-01-01

    Rationale Hydrogen peroxide (H2O2) regulates vascular tone in the human microcirculation under physiological and pathophysiological conditions. It dilates arterioles by activating BKCa channels in subjects with coronary artery disease (CAD), but its mechanisms of action in subjects without CAD (non-CAD) as compared to those with CAD remain unknown. Objective We hypothesize that H2O2-elicited dilation involves different K+ channels in non-CAD versus CAD, resulting in an altered capacity for vasodilation during disease. Methods and Results H2O2 induced endothelium-independent vasodilation in non-CAD adipose arterioles, which was reduced by paxilline, a BKCa channel blocker, and by 4-AP, a KV channel blocker. Assays of mRNA transcripts, protein expression and subcellular localization revealed that KV1.5 is the major KV1 channel expressed in vascular smooth muscle cells (VSMCs) and is abundantly localized on the plasma membrane. The selective KV1.5 blocker DPO-1 and the KV1.3/1.5 blocker Psora-4 reduced H2O2-elicited dilation to a similar extent as 4-AP, but the selective KV1.3 blocker PAP-1 was without effect. In arterioles from CAD subjects, H2O2-induced dilation was significantly reduced and this dilation was inhibited by paxilline but not by 4-AP, DPO-1 or Psora-4. KV1.5 cell membrane localization and DPO-1-sensitive K+ currents were markedly reduced in isolated VSMCs from CAD arterioles, although mRNA or total cellular protein expression were largely unchanged. Conclusions In human arterioles, H2O2-induced dilation is impaired in CAD, which is associated with a transition from a combined BKCa- and KV (KV1.5)-mediated vasodilation toward a BKCa-predominant mechanism of dilation. Loss of KV1.5 vasomotor function may play an important role in microvascular dysfunction in CAD or other vascular diseases. PMID:27872049

  11. Initial segment Kv2.2 channels mediate a slow delayed rectifier and maintain high frequency action potential firing in medial nucleus of the trapezoid body neurons

    PubMed Central

    Johnston, Jamie; Griffin, Sarah J; Baker, Claire; Skrzypiec, Anna; Chernova, Tatanya; Forsythe, Ian D

    2008-01-01

    The medial nucleus of the trapezoid body (MNTB) is specialized for high frequency firing by expression of Kv3 channels, which minimize action potential (AP) duration, and Kv1 channels, which suppress multiple AP firing, during each calyceal giant EPSC. However, the outward K+ current in MNTB neurons is dominated by another unidentified delayed rectifier. It has slow kinetics and a peak conductance of ∼37 nS; it is half-activated at −9.2 ± 2.1 mV and half-inactivated at −35.9 ± 1.5 mV. It is blocked by several non-specific potassium channel antagonists including quinine (100 μm) and high concentrations of extracellular tetraethylammonium (TEA; IC50 = 11.8 mm), but no specific antagonists were found. These characteristics are similar to recombinant Kv2-mediated currents. Quantitative RT-PCR showed that Kv2.2 mRNA was much more prevalent than Kv2.1 in the MNTB. A Kv2.2 antibody showed specific staining and Western blots confirmed that it recognized a protein ∼110 kDa which was absent in brainstem tissue from a Kv2.2 knockout mouse. Confocal imaging showed that Kv2.2 was highly expressed in axon initial segments of MNTB neurons. In the absence of a specific antagonist, Hodgkin–Huxley modelling of voltage-gated conductances showed that Kv2.2 has a minor role during single APs (due to its slow activation) but assists recovery of voltage-gated sodium channels (Nav) from inactivation by hyperpolarizing interspike potentials during repetitive AP firing. Current-clamp recordings during high frequency firing and characterization of Nav inactivation confirmed this hypothesis. We conclude that Kv2.2-containing channels have a distinctive initial segment location and crucial function in maintaining AP amplitude by regulating the interspike potential during high frequency firing. PMID:18511484

  12. Specification of skeletal muscle differentiation by repressor element-1 silencing transcription factor (REST)-regulated Kv7.4 potassium channels

    PubMed Central

    Iannotti, Fabio Arturo; Barrese, Vincenzo; Formisano, Luigi; Miceli, Francesco; Taglialatela, Maurizio

    2013-01-01

    Changes in the expression of potassium (K+) channels is a pivotal event during skeletal muscle differentiation. In mouse C2C12 cells, similarly to human skeletal muscle cells, myotube formation increased the expression of Kv7.1, Kv7.3, and Kv7.4, the last showing the highest degree of regulation. In C2C12 cells, Kv7.4 silencing by RNA interference reduced the expression levels of differentiation markers (myogenin, myosin heavy chain, troponinT-1, and Pax3) and impaired myotube formation and multinucleation. In Kv7.4-silenced cells, the differentiation-promoting effect of the Kv7 activator N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester (retigabine) was abrogated. Expression levels for the repressor element-1 silencing transcription factor (REST) declined during myotube formation. Transcript levels for Kv7.4, as well as for myogenin, troponinT-1, and Pax3, were reduced by REST overexpression and enhanced upon REST suppression by RNA interference. Four regions containing potential REST-binding sites in the 5′ untranslated region and in the first intron of the Kv7.4 gene were identified by bioinformatic analysis. Chromatin immunoprecipitation assays showed that REST binds to these regions, exhibiting a higher efficiency in myoblasts than in myotubes. These data suggest that Kv7.4 plays a permissive role in skeletal muscle differentiation and highlight REST as a crucial transcriptional regulator for this K+ channel subunit. PMID:23242999

  13. Kv7(KCNQ)-K+-Channels Influence Total Peripheral Resistance in Female but Not Male Rats, and Hamper Catecholamine Release in Hypertensive Rats of Both Sexes

    PubMed Central

    Berg, Torill

    2018-01-01

    K+-channels of the Kv7/KCNQ-family hyperpolarize and stabilize excitable cells such as autonomic neurons and vascular smooth muscle cells (VSMC). Kv7 may therefore play a role in blood pressure (BP) homeostasis, and prevent a high total peripheral vascular resistance (TPR), a hallmark of hypertensive disease. The present study analyzed if Kv7 channels influence catecholamine release and TPR in normotensive (WKY) and spontaneously hypertensive rats (SHR), and if they may contribute to the antihypertensive protection seen in young, female SHR. Tyramine-stimulated norepinephrine release evokes an adrenergic cardiovascular response, and also allows modulation of release to be reflected in the overflow to plasma. The experiment itself activated some secretion of epinephrine. The results show: (1) XE-991 (Kv7.1-7.4-inhibitor), but not chromanol 293B (Kv7.1-inhibitor), increased tyramine-stimulated norepinephrine overflow and epinephrine secretion in both sexes in SHR, but not WKY. (2) Surprisingly, the Kv7-openers retigabine (Kv7.2-7.5) and ICA-27243 (Kv7.2-7.3-preferring) increased catecholamine release in female SHR. (3) The rise in TPR following tyramine-stimulated norepinephrine release was increased by XE-991 but not chromanol in the female WKY only. (4) Retigabine and ICA-27243 reduced the TPR-response to tyramine in the female SHR only. These results suggested: (1) Up-regulation of Kv7.2-7.3 function in sympathetic neurons and chromaffin cells hampered catecholamine release in SHR of both sexes. (2) The increase catecholamine release observed after channel openers in the female SHR may possibly involve reduced transmission in cholinergic neurons which hamper catecholamine release. These two mechanisms may serve to counter-act the hyperadrenergic state in SHR. (3) Kv7.4, most likely in the vasculature, opposed the tension-response to norepinephrine in the female WKY. (4) Vascular Kv7.4-7.5 could be stimulated and then opposed norepinephrine-induced vasoconstriction

  14. Kv7(KCNQ)-K+-Channels Influence Total Peripheral Resistance in Female but Not Male Rats, and Hamper Catecholamine Release in Hypertensive Rats of Both Sexes.

    PubMed

    Berg, Torill

    2018-01-01

    K + -channels of the Kv7/KCNQ-family hyperpolarize and stabilize excitable cells such as autonomic neurons and vascular smooth muscle cells (VSMC). Kv7 may therefore play a role in blood pressure (BP) homeostasis, and prevent a high total peripheral vascular resistance (TPR), a hallmark of hypertensive disease. The present study analyzed if Kv7 channels influence catecholamine release and TPR in normotensive (WKY) and spontaneously hypertensive rats (SHR), and if they may contribute to the antihypertensive protection seen in young, female SHR. Tyramine-stimulated norepinephrine release evokes an adrenergic cardiovascular response, and also allows modulation of release to be reflected in the overflow to plasma. The experiment itself activated some secretion of epinephrine. The results show: (1) XE-991 (Kv7.1-7.4-inhibitor), but not chromanol 293B (Kv7.1-inhibitor), increased tyramine-stimulated norepinephrine overflow and epinephrine secretion in both sexes in SHR, but not WKY. (2) Surprisingly, the Kv7-openers retigabine (Kv7.2-7.5) and ICA-27243 (Kv7.2-7.3-preferring) increased catecholamine release in female SHR. (3) The rise in TPR following tyramine-stimulated norepinephrine release was increased by XE-991 but not chromanol in the female WKY only. (4) Retigabine and ICA-27243 reduced the TPR-response to tyramine in the female SHR only. These results suggested: (1) Up-regulation of Kv7.2-7.3 function in sympathetic neurons and chromaffin cells hampered catecholamine release in SHR of both sexes. (2) The increase catecholamine release observed after channel openers in the female SHR may possibly involve reduced transmission in cholinergic neurons which hamper catecholamine release. These two mechanisms may serve to counter-act the hyperadrenergic state in SHR. (3) Kv7.4, most likely in the vasculature, opposed the tension-response to norepinephrine in the female WKY. (4) Vascular Kv7.4-7.5 could be stimulated and then opposed norepinephrine

  15. The Effects of 4-Hydroxybenzoic Acid Identified from Bamboo (Dendrocalamus asper) Shoots on Kv1.4 Channel

    PubMed Central

    Mohamad, Fatin H.; Wong, Jia Hui; Mohamad, Habsah; Ismail, Abdul Hadi; Mohamed Yusoff, Abdul Aziz; Osman, Hasnah; Wong, Kok Tong; Idris, Zamzuri; Abdullah, Jafri Malin

    2018-01-01

    Background Bamboo shoot has been used as a treatment for epilepsy in traditional Chinese medicine for generations to treat neuronal disorders such as convulsive, dizziness and headaches. 4-hydroxybenzoic acid (4-hba) is a non-flavonoid phenol found abundantly in Dendrocalamus asper shoots (bamboo), fruits (strawberries and apples) and flowers. Kv1.4 is a rapidly inactivating Shaker-related member of the voltage-gated potassium channels with two inactivation mechanisms; the fast N-type and slow C-type. It plays vital roles in repolarisation, hyperpolarisation and signaling the restoration of resting membrane potential through the regulation of the movement of K+ across the cellular membrane. Methods Chemical compounds from Dendrocalamus asper bamboo shoots were purified and identified as major palmitic acids mixed with other minor fatty acids, palmitic acid, 4-hydroxybenzaldehyde, lauric acid, 4-hydroxybenzoic acid and cholest-4-ene-3-one. The response of synthetic 4-hydroxybenzoic acid was tested on Kv1.4 potassium channel which was injected into viable oocytes that was extracted from Xenopus laevis. The current were detected by the two-microelectrode voltage clamp, holding potential starting from −80 mV with 20 mV step-up until +80 mV. Readings of treatments with 0.1% DMSO, 4-hba concentrations and K channel blockers were taken at +60 mV. The ratio of tail/peak amplitude is the index of the activity of the Kv1.4 channels with n ≥ 6 (number of oocytes tested). The decreases of the ratios of five different concentrations (1 μM, 10 μM, 100 μM, 1 mM and 2.5 mM) were compared with 0.1% DMSO as the control. Results All concentration showed statistically significant results with P < 0.05 except for 100 μM. The normalised current of the 4-hba concentrations were compared with potassium channel blockers (TEA and 4-AP) and all groups showed statistically significant results. This study also showed that time taken for each concentration to affect Kv1.4 does not play

  16. 4-aminopyridine, a Kv channel antagonist, prevents apoptosis of rat cerebellar granule neurons.

    PubMed

    Hu, Chang-Long; Liu, Zheng; Zeng, Xi-Min; Liu, Zi-Qiang; Chen, Xian-Hua; Zhang, Zhi-Hong; Mei, Yan-Ai

    2006-09-01

    Compelling evidence indicates that excessive potassium (K+) efflux and intracellular K+ depletion are the key early steps in apoptosis. Previously, we reported that apoptosis of cerebellar granule neurons induced by incubation in low-K+ (5 mM) and serum-free medium was associated with an increase in A-type transient inactivation of K+ channel current (IA) amplitude and modulation of channels' gating properties. Here, we showed that a classic K+ channel blocker, 4-aminopyradine (4-AP), significantly inhibited IA amplitude in a concentration-dependent manner (reduction of current by 10 microM and 10 mM 4-AP was 11.4+/-1.3% and 72.2+/-3.3%, respectively). Moreover, 4-AP modified the steady-state activation and inactivation kinetics of IA channels, such that the activation and inactivation curves were shifted to the right about 20 mV and 17 mV, respectively. Fluorescence staining showed that 4-AP dramatically increased the viability of cells undergoing apoptosis in a dose-dependent manner. That is, while 5 mM 4-AP was present, cell viability was 84.9+/-5.2%. Consistent with the cell viability analysis, internucleosomal DNA fragmentation by gel electrophoresis analysis showed that 5 mM 4-AP also protected against neuronal apoptosis. Furthermore, 4-AP significantly inhibited cytochrome c release and caspase-3 activity induced by low-K+/serum-free incubation. Finally, current-clamp analysis indicated that 5 mM 4-AP did not significantly depolarize the membrane potential. These results suggest that 4-AP has robust neuroprotective effects on apoptotic granule cells. The neuroprotective effect of 4-AP is likely not due to membrane depolarization, but rather that 4-AP may modulate the gating properties of IA channels in an anti-apoptotic manner.

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  19. Molecular Dynamics Simulation Reveals Specific Interaction Sites between Scorpion Toxins and Kv1.2 Channel: Implications for Design of Highly Selective Drugs

    PubMed Central

    Yuan, Shouli; Gao, Bin

    2017-01-01

    The Kv1.2 channel plays an important role in the maintenance of resting membrane potential and the regulation of the cellular excitability of neurons, whose silencing or mutations can elicit neuropathic pain or neurological diseases (e.g., epilepsy and ataxia). Scorpion venom contains a variety of peptide toxins targeting the pore region of this channel. Despite a large amount of structural and functional data currently available, their detailed interaction modes are poorly understood. In this work, we choose four Kv1.2-targeted scorpion toxins (Margatoxin, Agitoxin-2, OsK-1, and Mesomartoxin) to construct their complexes with Kv1.2 based on the experimental structure of ChTx-Kv1.2. Molecular dynamics simulation of these complexes lead to the identification of hydrophobic patches, hydrogen-bonds, and salt bridges as three essential forces mediating the interactions between this channel and the toxins, in which four Kv1.2-specific interacting amino acids (D353, Q358, V381, and T383) are identified for the first time. This discovery might help design highly selective Kv1.2-channel inhibitors by altering amino acids of these toxins binding to the four channel residues. Finally, our results provide new evidence in favor of an induced fit model between scorpion toxins and K+ channel interactions. PMID:29104247

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

    NASA Astrophysics Data System (ADS)

    Wawrzkiewicz-Jałowiecka, Agata; Grzywna, Zbigniew J.

    2018-03-01

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

  1. The Prion Protein Modulates A-type K+ Currents Mediated by Kv4.2 Complexes through Dipeptidyl Aminopeptidase-like Protein 6*

    PubMed Central

    Mercer, Robert C. C.; Ma, Li; Watts, Joel C.; Strome, Robert; Wohlgemuth, Serene; Yang, Jing; Cashman, Neil R.; Coulthart, Michael B.; Schmitt-Ulms, Gerold; Jhamandas, Jack H.; Westaway, David

    2013-01-01

    Widely expressed in the adult central nervous system, the cellular prion protein (PrPC) is implicated in a variety of processes, including neuronal excitability. Dipeptidyl aminopeptidase-like protein 6 (DPP6) was first identified as a PrPC interactor using in vivo formaldehyde cross-linking of wild type (WT) mouse brain. This finding was confirmed in three cell lines and, because DPP6 directs the functional assembly of K+ channels, we assessed the impact of WT and mutant PrPC upon Kv4.2-based cell surface macromolecular complexes. Whereas a Gerstmann-Sträussler-Scheinker disease version of PrP with eight extra octarepeats was a loss of function both for complex formation and for modulation of Kv4.2 channels, WT PrPC, in a DPP6-dependent manner, modulated Kv4.2 channel properties, causing an increase in peak amplitude, a rightward shift of the voltage-dependent steady-state inactivation curve, a slower inactivation, and a faster recovery from steady-state inactivation. Thus, the net impact of wt PrPC was one of enhancement, which plays a critical role in the down-regulation of neuronal membrane excitability and is associated with a decreased susceptibility to seizures. Insofar as previous work has established a requirement for WT PrPC in the Aβ-dependent modulation of excitability in cholinergic basal forebrain neurons, our findings implicate PrPC regulation of Kv4.2 channels as a mechanism contributing to the effects of oligomeric Aβ upon neuronal excitability and viability. PMID:24225951

  2. Contribution of Kv2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra

    PubMed Central

    Kyle, B.; Bradley, E.; Ohya, S.; Sergeant, G. P.; McHale, N. G.; Thornbury, K. D.

    2011-01-01

    We have characterized the native voltage-dependent K+ (Kv) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with Kv2.1 and Kv2.2 channels cloned from the rabbit urethra and stably expressed in human embryonic kidney (HEK)-293 cells (HEKKv2.1 and HEKKv2.2). RUSMC were perfused with Hanks′ solution at 37°C and studied using the patch-clamp technique with K+-rich pipette solutions. Cells were bathed in 100 nM Penitrem A (Pen A) to block large-conductance Ca2+-activated K+ (BK) currents and depolarized to +40 mV for 500 ms to evoke Kv currents. These were unaffected by margatoxin, κ-dendrotoxin, or α-dendrotoxin (100 nM, n = 3–5) but were blocked by stromatoxin-1 (ScTx, IC50 ∼130 nM), consistent with the idea that the currents were carried through Kv2 channels. RNA was detected for Kv2.1, Kv2.2, and the silent subunit Kv9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both Kv2 subtypes and Kv9.3 in isolated RUSMC. HEKKv2.1 and HEKKv2.2 currents were blocked in a concentration-dependent manner by ScTx, with estimated IC50 values of ∼150 nM (Kv2.1, n = 5) and 70 nM (Kv2.2, n = 6). The mean half-maximal voltage (V1/2) of inactivation of the USMC Kv current was −56 ± 3 mV (n = 9). This was similar to the HEKKv2.1 current (−55 ± 3 mV, n = 13) but significantly different from the HEKKv2.2 currents (−30 ± 3 mV, n = 11). Action potentials (AP) evoked from RUSMC studied under current-clamp mode were unaffected by ScTx. However, when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that Kv2.1 channels contribute significantly to the Kv current in RUSMC. PMID:21813710

  3. BmP02 Atypically Delays Kv4.2 Inactivation: Implication for a Unique Interaction between Scorpion Toxin and Potassium Channel

    PubMed Central

    Wu, Bin; Zhu, Yan; Shi, Jian; Tao, Jie; Ji, Yonghua

    2016-01-01

    BmP02, a short-chain peptide with 28 residues from the venom of Chinese scorpion Buthus martensi Karsch, has been reported to inhibit the transient outward potassium currents (Ito) in rat ventricular muscle cells. However, it remains unclear whether BmP02 modulates the Kv4.2 channel, one of the main contributors to Ito. The present study investigated the effects of BmP02 on Kv4.2 kinetics and its underlying molecular mechanism. The electrophysiological recordings showed that the inactivation of Kv4.2 expressed in HEK293T cells was significantly delayed by BmP02 in a dose-response manner with EC50 of ~850 nM while the peak current, activation and voltage-dependent inactivation of Kv4.2 were not affected. Meanwhile, the recovery from inactivation of Kv4.2 was accelerated and the deactivation was slowed after the application of BmP02. The site-directed mutagenesis combined with computational modelling identified that K347 and K353, located in the turret motif of the Kv4.2, and E4/E5, D20/D21 in BmP02 are key residues to interact with BmP02 through electrostatic force. These findings not only reveal a novel interaction between Kv4.2 channel and its peptidyl modulator, but also provide valuable information for design of highly-selective Kv4.2 modulators. PMID:27690098

  4. Kv4.2 Knockout Mice Have Hippocampal-Dependent Learning and Memory Deficits

    ERIC Educational Resources Information Center

    Lugo, Joaquin N.; Brewster, Amy L.; Spencer, Corinne M.; Anderson, Anne E.

    2012-01-01

    Kv4.2 channels contribute to the transient, outward K[superscript +] current (A-type current) in hippocampal dendrites, and modulation of this current substantially alters dendritic excitability. Using Kv4.2 knockout (KO) mice, we examined the role of Kv4.2 in hippocampal-dependent learning and memory. We found that Kv4.2 KO mice showed a deficit…

  5. KCNE1 induces fenestration in the Kv7.1/KCNE1 channel complex that allows for highly specific pharmacological targeting

    NASA Astrophysics Data System (ADS)

    Wrobel, Eva; Rothenberg, Ina; Krisp, Christoph; Hundt, Franziska; Fraenzel, Benjamin; Eckey, Karina; Linders, Joannes T. M.; Gallacher, David J.; Towart, Rob; Pott, Lutz; Pusch, Michael; Yang, Tao; Roden, Dan M.; Kurata, Harley T.; Schulze-Bahr, Eric; Strutz-Seebohm, Nathalie; Wolters, Dirk; Seebohm, Guiscard

    2016-10-01

    Most small-molecule inhibitors of voltage-gated ion channels display poor subtype specificity because they bind to highly conserved residues located in the channel's central cavity. Using a combined approach of scanning mutagenesis, electrophysiology, chemical ligand modification, chemical cross-linking, MS/MS-analyses and molecular modelling, we provide evidence for the binding site for adamantane derivatives and their putative access pathway in Kv7.1/KCNE1 channels. The adamantane compounds, exemplified by JNJ303, are highly potent gating modifiers that bind to fenestrations that become available when KCNE1 accessory subunits are bound to Kv7.1 channels. This mode of regulation by auxiliary subunits may facilitate the future development of potent and highly subtype-specific Kv channel inhibitors.

  6. Inhibitory effects of retigabine, a Kv7 channel activator, on mechanosensitive primary bladder afferent activities and nociceptive behaviors in rats.

    PubMed

    Aizawa, Naoki; Wakamatsu, Daisuke; Kida, Jun; Otsuki, Takeya; Saito, Yasuho; Matsuya, Hidekazu; Homma, Yukio; Igawa, Yasuhiko

    2017-02-01

    Kv7 voltage-gated potassium channels have been suggested to modulate mechano-afferent transduction and nociception in the bladder. We investigated the effects of retigabine, a Kv7 channel activator, on rhythmic bladder contractions (RBCs), and single-unit afferent activities (SAAs) of the primary bladder mechanosensitive afferent nerve fibers in urethane-anesthetized rats. In addition, the effects of pretreatment with retigabine on the nociceptive behaviors provoked by an intravesical instillation of resiniferatoxin (RTX) were evaluated in the conscious condition. Female Sprague-Dawley rats were used. Under urethane anesthesia, saline was instilled into the bladder until RBCs were induced reproducibly. Then, the effects of intravenous, cumulative administrations of retigabine (0.1-3 mg/kg) or vehicle (saline) on RBCs were assessed. In separate animals, SAAs of Aδ- and C-fibers were identified by electrical stimulation of the pelvic nerve and by bladder distention with saline. After baseline recording, vehicle or retigabine (0.01-1 mg/kg) was administered intravenously and further recordings were performed. Under pretreatment with vehicle or retigabine (3 mg/kg intraperitoneally), the frequencies of lower abdominal licking and freezing were counted and scored as the bladder nociceptive behaviors induced by intravesical RTX instillation (3 µM, 0.3 ml). Retigabine dose-dependently decreased both the frequency and the amplitude of RBCs and SAAs of both Aδ- and C-fibers. The effect on RBCs was more potent on the frequency than the amplitude. Retigabine inhibited the RTX-induced abdominal licking, but not freezing. Kv7 channels are likely to be implicated in inhibition of bladder mechano- and nociceptive sensory transduction. Neurourol. Urodynam. 36:280-285, 2017. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  7. C-Terminal β9-Strand of the Cyclic Nucleotide-Binding Homology Domain Stabilizes Activated States of Kv11.1 Channels

    PubMed Central

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

    2013-01-01

    Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K+ channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH) domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels. PMID:24204727

  8. Comparative Study of the Energetics of Ion Permeation in Kv1.2 and KcsA Potassium Channels

    PubMed Central

    Baştuğ, Turgut; Kuyucak, Serdar

    2011-01-01

    Biological ion channels rely on a multi-ion transport mechanism for fast yet selective permeation of ions. The crystal structure of the KcsA potassium channel provided the first microscopic picture of this process. A similar mechanism is assumed to operate in all potassium channels, but the validity of this assumption has not been well investigated. Here, we examine the energetics of ion permeation in Shaker Kv1.2 and KcsA channels, which exemplify the six-transmembrane voltage-gated and two-transmembrane inward-rectifier channels. We study the feasibility of binding a third ion to the filter and the concerted motion of ions in the channel by constructing the potential of mean force for K+ ions in various configurations. For both channels, we find that a pair of K+ ions can move almost freely within the filter, but a relatively large free-energy barrier hinders the K+ ion from stepping outside the filter. We discuss the effect of the CMAP dihedral energy correction that was recently incorporated into the CHARMM force field on ion permeation dynamics. PMID:21281577

  9. Clinical-pathologic correlations in voltage-gated Kv1 potassium channel complex-subtyped autoimmune painful polyneuropathy.

    PubMed

    Lahoria, Rajat; Pittock, Sean J; Gadoth, Avi; Engelstad, Janean K; Lennon, Vanda A; Klein, Christopher J

    2017-04-01

    Voltage-gated Kv1 potassium channel complex (VGKC) autoantibodies subtyped for leucine-rich glioma-inactivated 1 (LGI1), contactin-associated-proteinlike 2 (CASPR2), and Kv IgGs have a spectrum of neurological presentations. Painful polyneuropathy is seen in some patients, but nerve pathology descriptions are lacking. Clinicopathologic features were studied in subtyped VGKC-autoantibody-seropositive patients who had undergone nerve biopsies. Five patients were identified, 1 LGI1 IgG positive and 1 CASPR2 IgG positive, but all negative for Kv1.1-, 1.2-, 1.6-subtyped IgG autoantibodies. Median symptom duration was 17 months. Pain was the predominant symptom; 3 had mild sensory loss and/or weakness. Histopathological abnormalities were limited to axonal loss in 3. None had mononuclear cellular infiltrates. Electron micrographs revealed no interstitial abnormalities. Three patients reported marked improvement in pain with immunotherapy. The nerve biopsy histopathology of patients subtyped for LGI1 and CASPR2 IgGs within the VGKC-complex spectrum disorders shows either normal density or axonal fiber loss without inflammatory infiltrates. A reversible neural hyperexcitable mechanism is considered to be the cause of this painful polyneuropathy. Muscle Nerve 55: 520-525, 2017. © 2016 Wiley Periodicals, Inc.

  10. The facilitating effect of systemic administration of Kv7/M channel blocker XE991 on LTP induction in the hippocampal CA1 area independent of muscarinic activation.

    PubMed

    Song, Ming-Ke; Cui, Yong-Yao; Zhang, Wei-Wei; Zhu, Liang; Lu, Yang; Chen, Hong-Zhuan

    2009-09-11

    A large amount of in vitro studies demonstrate suppression of M-current in hippocampal neurons by Kv7/M channel blocker results in depolarization of membrane potential and release of neurotransmitters, such as acetylcholine and glutamate, suggesting that Kv7/M channel may play important roles in regulating synaptic plasticity. In the present study, we examined the in vivo effect of Kv7/M channel inhibition on the long-term potentiation (LTP) induction at basal dendrites in hippocampal CA1 area of urethane-anaesthetized rats. The Kv7/M channel was inhibited by intraperitoneal injection of XE991 (10mg/kg) and the LTP of field excitatory postsynaptic potential (fEPSP) was induced by supra-threshold high frequency stimulation (S1 HFS). A weak protocol which was just below the threshold for evoking LTP was used as sub-threshold high frequency stimulation (S2 HFS). XE991 did not significantly alter the slope of fEPSP and the magnitude of LTP induced by S1 HFS, suggesting that Kv7/M channel inhibition had little or no effect on glutamatergic transmission under basal conditions. However, XE991 could make S2 HFS evoke LTP even after the application of the muscarinic cholinergic (mACh) receptor antagonist scopolamine, suggesting that Kv7/M channel inhibition lowered the threshold for LTP induction and the effect was independent of muscarinic activation. Based on the above findings, we concluded that the facilitating effect of XE991 on LTP induction is not mediated by its ability to enhance the release of acetylcholine; therefore, Kv7/M channel blockers may provide a therapeutic benefit to cholinergic deficiency-related cognitive impairment, e.g., Alzheimer's disease.

  11. Heterogeneity in Kv2 Channel Expression Shapes Action Potential Characteristics and Firing Patterns in CA1 versus CA2 Hippocampal Pyramidal Neurons

    PubMed Central

    Chevaleyre, Vivien; Murray, Karl D.; Piskorowski, Rebecca A.

    2017-01-01

    Abstract The CA1 region of the hippocampus plays a critical role in spatial and contextual memory, and has well-established circuitry, function and plasticity. In contrast, the properties of the flanking CA2 pyramidal neurons (PNs), important for social memory, and lacking CA1-like plasticity, remain relatively understudied. In particular, little is known regarding the expression of voltage-gated K+ (Kv) channels and the contribution of these channels to the distinct properties of intrinsic excitability, action potential (AP) waveform, firing patterns and neurotransmission between CA1 and CA2 PNs. In the present study, we used multiplex fluorescence immunolabeling of mouse brain sections, and whole-cell recordings in acute mouse brain slices, to define the role of heterogeneous expression of Kv2 family Kv channels in CA1 versus CA2 pyramidal cell excitability. Our results show that the somatodendritic delayed rectifier Kv channel subunits Kv2.1, Kv2.2, and their auxiliary subunit AMIGO-1 have region-specific differences in expression in PNs, with the highest expression levels in CA1, a sharp decrease at the CA1-CA2 boundary, and significantly reduced levels in CA2 neurons. PNs in CA1 exhibit a robust contribution of Guangxitoxin-1E-sensitive Kv2-based delayed rectifier current to AP shape and after-hyperpolarization potential (AHP) relative to that seen in CA2 PNs. Our results indicate that robust Kv2 channel expression confers a distinct pattern of intrinsic excitability to CA1 PNs, potentially contributing to their different roles in hippocampal network function. PMID:28856240

  12. Genotype–phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor of Kv7.2 potassium channel subunits

    PubMed Central

    Miceli, Francesco; Soldovieri, Maria Virginia; Ambrosino, Paolo; Barrese, Vincenzo; Migliore, Michele; Cilio, Maria Roberta; Taglialatela, Maurizio

    2013-01-01

    Mutations in the KV7.2 gene encoding for voltage-dependent K+ channel subunits cause neonatal epilepsies with wide phenotypic heterogeneity. Two mutations affecting the same positively charged residue in the S4 domain of KV7.2 have been found in children affected with benign familial neonatal seizures (R213W mutation) or with neonatal epileptic encephalopathy with severe pharmacoresistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct neuroradiological features (R213Q mutation). To examine the molecular basis for this strikingly different phenotype, we studied the functional characteristics of mutant channels by using electrophysiological techniques, computational modeling, and homology modeling. Functional studies revealed that, in homomeric or heteromeric configuration with KV7.2 and/or KV7.3 subunits, both mutations markedly destabilized the open state, causing a dramatic decrease in channel voltage sensitivity. These functional changes were (i) more pronounced for channels incorporating R213Q- than R213W-carrying KV7.2 subunits; (ii) proportional to the number of mutant subunits incorporated; and (iii) fully restored by the neuronal Kv7 activator retigabine. Homology modeling confirmed a critical role for the R213 residue in stabilizing the activated voltage sensor configuration. Modeling experiments in CA1 hippocampal pyramidal cells revealed that both mutations increased cell firing frequency, with the R213Q mutation prompting more dramatic functional changes compared with the R213W mutation. These results suggest that the clinical disease severity may be related to the extent of the mutation-induced functional K+ channel impairment, and set the preclinical basis for the potential use of Kv7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates with KV7.2 encephalopathy. PMID:23440208

  13. Activity-dependent downregulation of M-Type (Kv7) K⁺ channels surface expression requires the activation of iGluRs/Ca²⁺/PKC signaling pathway in hippocampal neuron.

    PubMed

    Li, Cai; Lu, Qing; Huang, Pengcheng; Fu, Tianli; Li, Changjun; Guo, Lianjun; Xu, Xulin

    2015-08-01

    M-type (Kv7) K(+) channels, encoded by KCNQ2-KCNQ5 genes, play a pivotal role in controlling neuronal excitability. However, precisely how neuronal activity regulates Kv7 channel translocation has not yet been fully defined. Here we reported activity-dependent changes in Kv7 channel subunits Kv7.2 and Kv7.3 surface expression by glutamate (glu). In the present study, we found that treatment with glutamate rapidly caused a specific decrease in M-current as well as Kv7 channel surface expression in primary cultured hippocampal neurons. The glutamate effects were mimicked by NMDA and AMPA. The glutamate effects on Kv7 channels were partially attenuated by pre-treatment of NMDA receptors antagonist d,l-APV or AMPA-KA receptors antagonist CNQX. The signal required Ca(2+) influx through L-type Ca(2+) channel and intracellular Ca(2+) elevations. PKC activation was involved in the glutamate-induced reduction of Kv7 channel surface expression. Moreover, a significant reduction of Kv7 channel surface expression occurred following glycine-induced "chem"-LTP in vitro and hippocampus-dependent behavioral learning training in vivo. These results demonstrated that activity-dependent reduction of Kv7 channel surface expression through activation of ionotropic glutamate receptors (iGluRs)/Ca(2+)/PKC signaling pathway might be an important molecular mechanism for regulation of neuronal excitability and synaptic plasticity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Voltage-gated potassium channel (K(v) 1) autoantibodies in patients with chagasic gut dysmotility and distribution of K(v) 1 channels in human enteric neuromusculature (autoantibodies in GI dysmotility).

    PubMed

    Hubball, A W; Lang, B; Souza, M A N; Curran, O D; Martin, J E; Knowles, C H

    2012-08-01

    Autoantibodies directed against specific neuronal antigens are found in a significant number of patients with gastrointestinal neuromuscular diseases (GINMDs) secondary to neoplasia. This study examined the presence of antineuronal antibodies in idiopathic GINMD and GINMD secondary to South American Trypanosomiasis. The GI distribution of voltage-gated potassium channels (VGKCs) was also investigated. Seventy-three patients were included in the study with diagnoses of primary achalasia, enteric dysmotility, chronic intestinal pseudo-obstruction, esophageal or colonic dysmotility secondary to Chagas' disease. Sera were screened for specific antibodies to glutamic acid decarboxylase, voltage-gated calcium channels (VGCCs; P/Q subtype), nicotinic acetylcholine receptors (nAChRs; α3 subtype), and voltage-gated potassium channels (VGKCs, K(V) 1 subtype) using validated immunoprecipitation assays. The distribution of six VGKC subunits (K(V) 1.1-1.6), including those known to be antigenic targets of anti-VGKC antibodies was immunohistochemically investigated in all main human GI tract regions. Three patients (14%) with chagasic GI dysmotility were found to have positive anti-VGKC antibody titers. No antibodies were detected in patients with idiopathic GINMD. The VGKCs were found in enteric neurons at every level of the gut in unique yet overlapping distributions. The VGKC expression in GI smooth muscle was found to be limited to the esophagus. A small proportion of patients with GI dysfunction secondary to Chagas' disease have antibodies against VGKCs. The presence of these channels in the human enteric nervous system may have pathological relevance to the growing number of GINMDs with which anti-VGKC antibodies have been associated. © 2012 Blackwell Publishing Ltd.

  15. Early-onset epileptic encephalopathy caused by gain-of-function mutations in the voltage sensor of Kv7.2 and Kv7.3 potassium channel subunits.

    PubMed

    Miceli, Francesco; Soldovieri, Maria Virginia; Ambrosino, Paolo; De Maria, Michela; Migliore, Michele; Migliore, Rosanna; Taglialatela, Maurizio

    2015-03-04

    Mutations in Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3) genes, encoding for voltage-gated K(+) channel subunits underlying the neuronal M-current, have been associated with a wide spectrum of early-onset epileptic disorders ranging from benign familial neonatal seizures to severe epileptic encephalopathies. The aim of the present work has been to investigate the molecular mechanisms of channel dysfunction caused by voltage-sensing domain mutations in Kv7.2 (R144Q, R201C, and R201H) or Kv7.3 (R230C) recently found in patients with epileptic encephalopathies and/or intellectual disability. Electrophysiological studies in mammalian cells transfected with human Kv7.2 and/or Kv7.3 cDNAs revealed that each of these four mutations stabilized the activated state of the channel, thereby producing gain-of-function effects, which are opposite to the loss-of-function effects produced by previously found mutations. Multistate structural modeling revealed that the R201 residue in Kv7.2, corresponding to R230 in Kv7.3, stabilized the resting and nearby voltage-sensing domain states by forming an intricate network of electrostatic interactions with neighboring negatively charged residues, a result also confirmed by disulfide trapping experiments. Using a realistic model of a feedforward inhibitory microcircuit in the hippocampal CA1 region, an increased excitability of pyramidal neurons was found upon incorporation of the experimentally defined parameters for mutant M-current, suggesting that changes in network interactions rather than in intrinsic cell properties may be responsible for the neuronal hyperexcitability by these gain-of-function mutations. Together, the present results suggest that gain-of-function mutations in Kv7.2/3 currents may cause human epilepsy with a severe clinical course, thus revealing a previously unexplored level of complexity in disease pathogenetic mechanisms. Copyright © 2015 the authors 0270-6474/15/353782-12$15.00/0.

  16. Expression and high glucose-mediated regulation of K+ channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells.

    PubMed

    Chavira-Suárez, Erika; Sandoval, Alejandro; Felix, Ricardo; Lamas, Mónica

    2011-01-14

    Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K(+) channels K(V)4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K(V)4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K(+) current and an alteration in the inactivation of the macroscopic outward K(+) currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K(V)4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy. Copyright © 2010 Elsevier Inc. All rights reserved.

  17. Chronic deficit in the expression of voltage-gated potassium channel Kv3.4 subunit in the hippocampus of pilocarpine-treated epileptic rats

    PubMed Central

    Pacheco Otalora, Luis F.; Skinner, Frank; Oliveira, Mauro S.; Dotson, Bianca Farrel; Arshadmansab, Massoud F.; Pandari, Tarun; Garcia, Ileana; Robles, Leslie; Rosas, Gerardo; Mello, Carlos F.; Ermolinsky, Boris S.; Garrido-Sanabria, Emilio R.

    2010-01-01

    Voltage gated K+ channels (Kv) are a highly diverse group of channels critical in determining neuronal excitability. Deficits of Kv channel subunit expression and function have been implicated in the pathogenesis of epilepsy. In this study, we investigate whether the expression of the specific subunit Kv3.4 is affected during epileptogenesis following pilocarpine-induced status epilepticus. For this purpose, we used immunohistochemistry, Western blotting assays and comparative analysis of gene expression using TaqMan-based probes and delta-delta cycle threshold (Δ ΔCT) method of quantitative real-time polymerase chain reaction (qPCR) technique in samples obtained from age-matched control and epileptic rats. A marked down-regulation of Kv3.4 immunoreactivity was detected in the stratum lucidum and hilus of dentate gyrus in areas corresponding to the mossy fiber system of chronically epileptic rats. Correspondingly, a 20% reduction of Kv3.4 protein levels was detected in the hippocampus of chronic epileptic rats. Real-time quantitative PCR analysis of gene expression revealed that a significant 33% reduction of transcripts for Kv3.4 (gene Kcnc4) occurred after 1 month of pilocarpine-induced status epilepticus and persisted during the chronic phase of the model. These data indicate a reduced expression of Kv3.4 channels at protein and transcript levels in the epileptic hippocampus. Down-regulation of Kv3.4 in mossy fibers may contribute to enhanced presynaptic excitability leading to recurrent seizures in the pilocarpine model of temporal lobe epilepsy. PMID:20971086

  18. The ladder-shaped polyether toxin gambierol anchors the gating machinery of Kv3.1 channels in the resting state

    PubMed Central

    Kopljar, Ivan; Labro, Alain J.; de Block, Tessa; Rainier, Jon D.; Tytgat, Jan

    2013-01-01

    Voltage-gated potassium (Kv) and sodium (Nav) channels are key determinants of cellular excitability and serve as targets of neurotoxins. Most marine ciguatoxins potentiate Nav channels and cause ciguatera seafood poisoning. Several ciguatoxins have also been shown to affect Kv channels, and we showed previously that the ladder-shaped polyether toxin gambierol is a potent Kv channel inhibitor. Most likely, gambierol acts via a lipid-exposed binding site, located outside the K+ permeation pathway. However, the mechanism by which gambierol inhibits Kv channels remained unknown. Using gating and ionic current analysis to investigate how gambierol affected S6 gate opening and voltage-sensing domain (VSD) movements, we show that the resting (closed) channel conformation forms the high-affinity state for gambierol. The voltage dependence of activation was shifted by >120 mV in the depolarizing direction, precluding channel opening in the physiological voltage range. The (early) transitions between the resting and the open state were monitored with gating currents, and provided evidence that strong depolarizations allowed VSD movement up to the activated-not-open state. However, for transition to the fully open (ion-conducting) state, the toxin first needed to dissociate. These dissociation kinetics were markedly accelerated in the activated-not-open state, presumably because this state displayed a much lower affinity for gambierol. A tetrameric concatemer with only one high-affinity binding site still displayed high toxin sensitivity, suggesting that interaction with a single binding site prevented the concerted step required for channel opening. We propose a mechanism whereby gambierol anchors the channel’s gating machinery in the resting state, requiring more work from the VSD to open the channel. This mechanism is quite different from the action of classical gating modifier peptides (e.g., hanatoxin). Therefore, polyether toxins open new opportunities in structure

  19. Phosphorylation regulates the sensitivity of voltage‐gated Kv7.2 channels towards phosphatidylinositol‐4,5‐bisphosphate

    PubMed Central

    Salzer, Isabella; Erdem, Fatma Asli; Chen, Wei‐Qiang; Heo, Seok; Koenig, Xaver; Schicker, Klaus W.; Kubista, Helmut; Lubec, Gert; Boehm, Stefan

    2016-01-01

    Key points Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key regulator of many membrane proteins, including voltage‐gated Kv7.2 channels.In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2‐binding domains in Kv7.2.Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2.Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors.Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channel via G protein‐coupled receptors. Abstract The function of numerous ion channels is tightly controlled by G protein‐coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol‐4,5‐bisphosphate (PIP2). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP2 and through phosphorylation. Using liquid chromatography‐coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2‐binding domains. To evaluate the effect of phosphorylation on PIP2‐mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP2 depletion via the voltage‐sensitive phosphatase Dr‐VSP than were wild‐type channels. In vitro phosphorylation assays with the

  20. Clicked bis-PEG-peptide conjugates for studying calmodulin-Kv7.2 channel binding.

    PubMed

    Bonache, M Angeles; Alaimo, Alessandro; Malo, Covadonga; Millet, Oscar; Villarroel, Alvaro; González-Muñiz, Rosario

    2014-11-28

    The recombinant Kv7.2 calmodulin (CaM) binding site (Q2AB CaMBD) shows a high tendency to aggregate, thus complicating biochemical and structural studies. To facilitate these studies we have conceived bis-PEG-peptide CaMBD-mimetics linking helices A and B in single, easy to handle molecules. Short PEG chains were selected as spacers between the two peptide molecules, and a Cu(i)-catalyzed cycloaddition (CuAAC) protocol was used to assemble the final bis-PEG-peptide conjugate, by the convenient functionalization of PEG arms with azide and alkyne groups. The resulting conjugates, with a certain helical character in TFE solutions (CD), showed nanomolar affinity in a fluorescence CaM binding in vitro assay, higher than just the sum of the precursor PEG-peptide affinities, thus validating our design. The approach to these first described examples of Kv7.2 CaMBD-mimetics could pave the way to chimeric conjugates merging helices A and B from different Kv7 subunits.

  1. Conformational Changes and Slow Dynamics through Microsecond Polarized Atomistic Molecular Simulation of an Integral Kv1.2 Ion Channel

    PubMed Central

    Bjelkmar, Pär; Niemelä, Perttu S.; Vattulainen, Ilpo; Lindahl, Erik

    2009-01-01

    Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how transitions occur in membrane proteins—not to mention numerous applications in drug design. Here, we present a full 1 µs atomic-detail molecular dynamics simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements, including up to 120° rotation of the S4 segment, changes in hydrogen-bonding patterns, but only low amounts of translation. A smaller rotation (∼35°) of the extracellular end of all S4 segments is present also in a reference 0.5 µs simulation without applied field, which indicates that the crystal structure might be slightly different from the natural state of the voltage sensor. The conformation change upon hyperpolarization is closely coupled to an increase in 310 helix contents in S4, starting from the intracellular side. This could support a model for transition from the crystal structure where the hyperpolarization destabilizes S4–lipid hydrogen bonds, which leads to the helix rotating to keep the arginine side chains away from the hydrophobic phase, and the driving force for final relaxation by downward translation is partly entropic, which would explain the slow process. The coordinates of the transmembrane part of the simulated channel actually stay closer to the recently determined higher-resolution Kv1.2 chimera channel than the starting structure for the entire second half of the simulation (0.5–1 µs). Together with lipids binding in matching positions and significant thinning of the membrane also observed in experiments, this provides additional support for the predictive power of microsecond-scale membrane protein simulations. PMID:19229308

  2. Residues within the myristoylation motif determine intracellular targeting of the neuronal Ca2+ sensor protein KChIP1 to post-ER transport vesicles and traffic of Kv4 K+ channels.

    PubMed

    O'Callaghan, Dermott W; Hasdemir, Burcu; Leighton, Mark; Burgoyne, Robert D

    2003-12-01

    KChIPs (K+ channel interacting proteins) regulate the function of A-type Kv4 potassium channels by modifying channel properties and by increasing their cell surface expression. We have explored factors affecting the localisation of Kv4.2 and the targeting of KChIP1 and other NCS proteins by using GFP-variant fusion proteins expressed in HeLa cells. ECFP-Kv4.2 expressed alone was not retained in the ER but reached the Golgi complex. In cells co-expressing ECFP-Kv4.2 and KChIP1-EYFP, the two proteins were co-localised and were mainly present on the plasma membrane. When KChIP1-EYFP was expressed alone it was instead targeted to punctate structures. This was distinct from the localisation of the NCS proteins NCS-1 and hippocalcin, which were targeted to the trans-Golgi network (TGN) and plasma membrane. The membrane localisation of each NCS protein required myristoylation and minimal myristoylation motifs of hippocalcin or KChIP1 were sufficient to target fusion proteins to either TGN/plasma membrane or to punctate structures. The existence of targeting information within the N-terminal motifs was confirmed by mutagenesis of residues corresponding to three conserved basic amino acids in hippocalcin and NCS-1 at positions 3, 7 and 9. Residues at these positions determined intracellular targeting to the different organelles. Myristoylation and correct targeting of KChIP1 was required for the efficient traffic of ECFP-Kv4.2 to the plasma membrane. Expression of KChIP1(1-11)-EYFP resulted in the formation of enlarged structures that were positive for ERGIC-53 and beta-COP. ECFP-Kv4.2 was also accumulated in these structures suggesting that KChIP1(1-11)-EYFP inhibited traffic out of the ERGIC. We suggest that KChIP1 is targeted by its myristoylation motif to post-ER transport vesicles where it could interact with and regulate the traffic of Kv4 channels to the plasma membrane under the influence of localised Ca2+ signals.

  3. Modulation of slow inactivation in human cardiac Kv1.5 channels by extra- and intracellular permeant cations

    PubMed Central

    Fedida, David; Maruoka, Neil D; Lin, Shunping

    1999-01-01

    The properties and regulation of slow inactivation by intracellular and extracellular cations in the human heart K+ channel hKv1.5 have been investigated. Extensive NH2- and COOH-terminal deletions outside the central core of transmembrane domains did not affect the degree of inactivation. The voltage dependence of steady-state inactivation curves of hKv1.5 channels was unchanged in Rb+ and Cs+, compared with K+, but biexponential inactivation over 10 s was reduced from ∼100% of peak current in Na+ to ∼65% in K+, ∼50% in Rb+ and ∼30% in Cs+. This occurred as a result of a decrease in both fast and slow components of inactivation, with little change in inactivation time constants. Changes in extracellular cation species and concentration (5-300 mM) had only small effects on the rates of inactivation and recovery from inactivation (τrecovery∼1 s). Mutation of residues at a putative regulatory site at R487 in the outer pore mouth did not affect slow inactivation or recovery from inactivation of hKv1.5, although sensitivity to extracellular TEA was conferred. Symmetrical reduction of both intra- and extracellular cation concentrations accelerated and augmented both components of inactivation of K+ (Kd = 34.7 mM) and Cs+ (Kd = 20.5 mM) currents. These effects could be quantitatively accounted for by unilateral reduction of intracellular K+ (Ki+) (Kd = 43.4 mM) or Csi+ with constant 135 mM external ion concentrations. We conclude that inactivation and recovery from inactivation in hKv1.5 were not typically C-type in nature. However, the ion species dependence of inactivation was still closely coupled to ion permeation through the pore. Intracellular ion modulatory actions were more potent than extracellular actions, although still of relatively low affinity. These results suggest the presence of ion binding sites capable of regulating inactivation located on both intracellular and extracellular sides of the pore selectivity filter. PMID:10050000

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

    USDA-ARS?s Scientific Manuscript database

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

  5. Functional Invalidation of Putative Sudden Infant Death Syndrome-Associated Variants in the KCNH2-Encoded Kv11.1 Channel.

    PubMed

    Smith, Jennifer L; Tester, David J; Hall, Allison R; Burgess, Don E; Hsu, Chun-Chun; Claude Elayi, Samy; Anderson, Corey L; January, Craig T; Luo, Jonathan Z; Hartzel, Dustin N; Mirshahi, Uyenlinh L; Murray, Michael F; Mirshahi, Tooraj; Ackerman, Michael J; Delisle, Brian P

    2018-05-01

    Heterologous functional validation studies of putative long-QT syndrome subtype 2-associated variants clarify their pathological potential and identify disease mechanism(s) for most variants studied. The purpose of this study is to clarify the pathological potential for rare nonsynonymous KCNH2 variants seemingly associated with sudden infant death syndrome. Genetic testing of 292 sudden infant death syndrome cases identified 9 KCNH2 variants: E90K, R181Q, A190T, G294V, R791W, P967L, R1005W, R1047L, and Q1068R. Previous studies show R181Q-, P967L-, and R1047L-Kv11.1 channels function similar to wild-type Kv11.1 channels, whereas Q1068R-Kv11.1 channels accelerate inactivation gating. We studied the biochemical and biophysical properties for E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels expressed in human embryonic kidney 293 cells; examined the electronic health records of patients who were genotype positive for the sudden infant death syndrome-linked KCNH2 variants; and simulated their functional impact using computational models of the human ventricular action potential. Western blot and voltage-clamping analyses of cells expressing E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels demonstrated these variants express and generate peak Kv11.1 current levels similar to cells expressing wild-type-Kv11.1 channels, but R791W- and R1005W-Kv11.1 channels accelerated deactivation and activation gating, respectively. Electronic health records of patients with the sudden infant death syndrome-linked KCNH2 variants showed that the patients had median heart rate-corrected QT intervals <480 ms and none had been diagnosed with long-QT syndrome or experienced cardiac arrest. Simulating the impact of dysfunctional gating variants predicted that they have little impact on ventricular action potential duration. We conclude that these rare Kv11.1 missense variants are not long-QT syndrome subtype 2-causative variants and therefore do not represent the pathogenic substrate for

  6. KCNQ/Kv7 channel activator flupirtine protects against acute stress-induced impairments of spatial memory retrieval and hippocampal LTP in rats.

    PubMed

    Li, C; Huang, P; Lu, Q; Zhou, M; Guo, L; Xu, X

    2014-11-07

    Spatial memory retrieval and hippocampal long-term potentiation (LTP) are impaired by stress. KCNQ/Kv7 channels are closely associated with memory and the KCNQ/Kv7 channel activator flupirtine represents neuroprotective effects. This study aims to test whether KCNQ/Kv7 channel activation prevents acute stress-induced impairments of spatial memory retrieval and hippocampal LTP. Rats were placed on an elevated platform in the middle of a bright room for 30 min to evoke acute stress. The expression of KCNQ/Kv7 subunits was analyzed at 1, 3 and 12 h after stress by Western blotting. Spatial memory was examined by the Morris water maze (MWM) and the field excitatory postsynaptic potential (fEPSP) in the hippocampal CA1 area was recorded in vivo. Acute stress transiently decreased the expression of KCNQ2 and KCNQ3 in the hippocampus. Acute stress impaired the spatial memory retrieval and hippocampal LTP, the KCNQ/Kv7 channel activator flupirtine prevented the impairments, and the protective effects of flupirtine were blocked by XE-991 (10,10-bis(4-Pyridinylmethyl)-9(10H)-anthracenone), a selective KCNQ channel blocker. Furthermore, acute stress decreased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9 in the hippocampus, and flupirtine inhibited the reduction. These results suggest that the KCNQ/Kv7 channels may be a potential target for protecting both hippocampal synaptic plasticity and spatial memory retrieval from acute stress influences. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. Pore Helices Play a Dynamic Role as Integrators of Domain Motion during Kv11.1 Channel Inactivation Gating*

    PubMed Central

    Perry, Matthew D.; Ng, Chai Ann; Vandenberg, Jamie I.

    2013-01-01

    Proteins that form ion-selective pores in the membrane of cells are integral to many rapid signaling processes, including regulating the rhythm of the heartbeat. In potassium channels, the selectivity filter is critical for both endowing an exquisite selectivity for potassium ions, as well as for controlling the flow of ions through the pore. Subtle rearrangements in the complex hydrogen-bond network that link the selectivity filter to the surrounding pore helices differentiate conducting (open) from nonconducting (inactivated) conformations of the channel. Recent studies suggest that beyond the selectivity filter, inactivation involves widespread rearrangements of the channel protein. Here, we use rate equilibrium free energy relationship analysis to probe the structural changes that occur during selectivity filter gating in Kv11.1 channels, at near atomic resolution. We show that the pore helix plays a crucial dynamic role as a bidirectional interface during selectivity filter gating. We also define the molecular bases of the energetic coupling between the pore helix and outer helix of the pore domain that occurs early in the transition from open to inactivated states, as well as the coupling between the pore helix and inner helix late in the transition. Our data demonstrate that the pore helices are more than just static structural elements supporting the integrity of the selectivity filter; instead they play a crucial dynamic role during selectivity filter gating. PMID:23471968

  8. New Molecular Targets for Antiepileptic Drugs: α2δ, SV2A, and Kv7/KCNQ/M Potassium Channels

    PubMed Central

    Rogawski, Michael A.; Bazil, Carl W.

    2008-01-01

    Many currently prescribed antiepileptic drugs (AEDs) act via voltage-gated sodium channels, through effects on γ-aminobutyric acid–mediated inhibition, or via voltage-gated calcium channels. Some newer AEDs do not act via these traditional mechanisms. The molecular targets for several of these nontraditional AEDs have been defined using cellular electrophysiology and molecular approaches. Here, we describe three of these targets: α2δ, auxiliary subunits of voltage-gated calcium channels through which the gabapentinoids gabapentin and pregabalin exert their anticonvulsant and analgesic actions; SV2A, a ubiquitous synaptic vesicle glycoprotein that may prepare vesicles for fusion and serves as the target for levetiracetam and its analog brivaracetam (which is currently in late-stage clinical development); and Kv7/KCNQ/M potassium channels that mediate the M-current, which acts a brake on repetitive firing and burst generation and serves as the target for the investigational AEDs retigabine and ICA-105665. Functionally, all of the new targets modulate neurotransmitter output at synapses, focusing attention on presynaptic terminals as critical sites of action for AEDs. PMID:18590620

  9. Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

    PubMed

    Shem-Ad, Tzilhav; Irit, Orr; Yifrach, Ofer

    2013-01-01

    The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

  10. Blockade of the Kv1.3 K+ Channel Enhances BCG Vaccine Efficacy by Expanding Central Memory T Lymphocytes.

    PubMed

    Singh, Dhiraj Kumar; Dwivedi, Ved Prakash; Ranganathan, Anand; Bishai, William R; Van Kaer, Luc; Das, Gobardhan

    2016-11-01

    Tuberculosis is the oldest known infectious disease, yet there is no effective vaccine against adult pulmonary tuberculosis. Emerging evidence indicates that T-helper 1 and T-helper 17 cells play important roles in host protection against tuberculosis. However, tuberculosis vaccine efficacy in mice is critically dependent on the balance between antigen-specific central memory T (Tcm) and effector memory T (Tem) cells. Specifically, a high Tcm/Tem cell ratio is essential for optimal vaccine efficacy. Here, we show that inhibition of Kv1.3, a potassium channel preferentially expressed by Tem cells, by Clofazimine selectively expands Tcm cells during BCG vaccination. Furthermore, mice that received clofazimine after BCG vaccination exhibited significantly enhanced resistance against tuberculosis. This superior activity against tuberculosis could be adoptively transferred to naive, syngeneic mice by CD4 + T cells. Therefore, clofazimine enhances Tcm cell expansion, which in turn provides improved vaccine efficacy. Thus, Kv1.3 blockade is a promising approach for enhancing the efficacy of the BCG vaccine in humans. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  11. Down-regulation of delayed rectifier K+ channels in the hippocampus of seizure sensitive gerbils.

    PubMed

    Lee, Sang-Moo; Kim, Ji-Eun; Sohn, Jong-Hee; Choi, Hui-Chul; Lee, Ju-Sang; Kim, Sung-Hun; Kim, Min-Ju; Choi, Ihn-Geun; Kang, Tae-Cheon

    2009-12-16

    In order to confirm the species-specific distribution of voltage-gated K(+) (Kv) channels and the definitive relationship between their immunoreactivities and seizure activity, we investigated Kv2.x, Kv3.x and Kv4.x channel immunoreactivities in the hippocampi of seizure-resistant (SR) and seizure-sensitive (SS) gerbils. There was no difference in Kv2.1, Kv3.4, Kv4.2 and Kv4.3 immunoreactivity in the hippocampus between SR and SS gerbils. In comparison to SR gerbils, Kv3.1b immunoreactivity in neurons was significantly lower in SS gerbils instead Kv3.1b-immunoreactive astrocytes were clearly observed in SS gerbils (p<0.05). Kv3.2 immunoreactivity was also significantly lower in neurons of SS gerbils than in those of SR gerbils (p<0.05). Considering the findings of our previous study, these findings suggest that delayed rectifier K(+) channels (Kv1.1, Kv1.2, Kv1.5, Kv1.6, Kv2.1 and Kv3.1-2), not A-type K(+) channels (Kv1.4, Kv3.4 and Kv4.x), may be down-regulated in the SS gerbil hippocampus, as compared to SR gerbils.

  12. Expression of Kv3.1b potassium channel is widespread in macaque motor cortex pyramidal cells: A histological comparison between rat and macaque

    PubMed Central

    Soares, David; Goldrick, Isabelle; Lemon, Roger N.; Kraskov, Alexander; Greensmith, Linda

    2017-01-01

    Abstract There are substantial differences across species in the organization and function of the motor pathways. These differences extend to basic electrophysiological properties. Thus, in rat motor cortex, pyramidal cells have long duration action potentials, while in the macaque, some pyramidal neurons exhibit short duration “thin” spikes. These differences may be related to the expression of the fast potassium channel Kv3.1b, which in rat interneurons is associated with generation of thin spikes. Rat pyramidal cells typically lack these channels, while there are reports that they are present in macaque pyramids. Here we made a systematic, quantitative comparison of the Kv3.1b expression in sections from macaque and rat motor cortex, using two different antibodies (NeuroMab, Millipore). As our standard reference, we examined, in the same sections, Kv3.1b staining in parvalbumin‐positive interneurons, which show strong Kv3.1b immunoreactivity. In macaque motor cortex, a large sample of pyramidal neurons were nearly all found to express Kv3.1b in their soma membranes. These labeled neurons were identified as pyramidal based either by expression of SMI32 (a pyramidal marker), or by their shape and size, and lack of expression of parvalbumin (a marker for some classes of interneuron). Large (Betz cells), medium, and small pyramidal neurons all expressed Kv3.1b. In rat motor cortex, SMI32‐postive pyramidal neurons expressing Kv3.1b were very rare and weakly stained. Thus, there is a marked species difference in the immunoreactivity of Kv3.1b in pyramidal neurons, and this may be one of the factors explaining the pronounced electrophysiological differences between rat and macaque pyramidal neurons. PMID:28213922

  13. Expression of Kv3.1b potassium channel is widespread in macaque motor cortex pyramidal cells: A histological comparison between rat and macaque.

    PubMed

    Soares, David; Goldrick, Isabelle; Lemon, Roger N; Kraskov, Alexander; Greensmith, Linda; Kalmar, Bernadett

    2017-06-15

    There are substantial differences across species in the organization and function of the motor pathways. These differences extend to basic electrophysiological properties. Thus, in rat motor cortex, pyramidal cells have long duration action potentials, while in the macaque, some pyramidal neurons exhibit short duration "thin" spikes. These differences may be related to the expression of the fast potassium channel Kv3.1b, which in rat interneurons is associated with generation of thin spikes. Rat pyramidal cells typically lack these channels, while there are reports that they are present in macaque pyramids. Here we made a systematic, quantitative comparison of the Kv3.1b expression in sections from macaque and rat motor cortex, using two different antibodies (NeuroMab, Millipore). As our standard reference, we examined, in the same sections, Kv3.1b staining in parvalbumin-positive interneurons, which show strong Kv3.1b immunoreactivity. In macaque motor cortex, a large sample of pyramidal neurons were nearly all found to express Kv3.1b in their soma membranes. These labeled neurons were identified as pyramidal based either by expression of SMI32 (a pyramidal marker), or by their shape and size, and lack of expression of parvalbumin (a marker for some classes of interneuron). Large (Betz cells), medium, and small pyramidal neurons all expressed Kv3.1b. In rat motor cortex, SMI32-postive pyramidal neurons expressing Kv3.1b were very rare and weakly stained. Thus, there is a marked species difference in the immunoreactivity of Kv3.1b in pyramidal neurons, and this may be one of the factors explaining the pronounced electrophysiological differences between rat and macaque pyramidal neurons. © 2017 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

  14. KV7 Channel Pharmacological Activation by the Novel Activator ML213: Role for Heteromeric KV7.4/KV7.5 Channels in Guinea Pig Detrusor Smooth Muscle Function.

    PubMed

    Provence, Aaron; Angoli, Damiano; Petkov, Georgi V

    2018-01-01

    Voltage-gated K V 7 channels (K V 7.1 to K V 7.5) are important regulators of the cell membrane potential in detrusor smooth muscle (DSM) of the urinary bladder. This study sought to further the current knowledge of K V 7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca 2+ imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N -(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of K V 7.2, K V 7.4, and K V 7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 µ M) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the K V 7 channel inhibitor XE991 (10 µ M). ML213 (0.1-30 µ M) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 µ M) decreased global intracellular Ca 2+ concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 µ M) caused an increase in the amplitude of whole-cell K V 7 currents. Further, in current-clamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 µ M), consistent with ML213 activation of K V 7 channel currents. Preapplication of XE991 (10 µ M) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for K V 7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric K V 7.4/K V 7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive K V 7.4- and K V 7.5-containing channels are essential regulators of DSM excitability and contractility. Copyright © 2017 by The American

  15. Complementary functions of SK and Kv7/M potassium channels in excitability control and synaptic integration in rat hippocampal dentate granule cells

    PubMed Central

    Mateos-Aparicio, Pedro; Murphy, Ricardo; Storm, Johan F

    2014-01-01

    The dentate granule cells (DGCs) form the most numerous neuron population of the hippocampal memory system, and its gateway for cortical input. Yet, we have only limited knowledge of the intrinsic membrane properties that shape their responses. Since SK and Kv7/M potassium channels are key mechanisms of neuronal spiking and excitability control, afterhyperpolarizations (AHPs) and synaptic integration, we studied their functions in DGCs. The specific SK channel blockers apamin or scyllatoxin increased spike frequency (excitability), reduced early spike frequency adaptation, fully blocked the medium-duration AHP (mAHP) after a single spike or spike train, and increased postsynaptic EPSP summation after spiking, but had no effect on input resistance (Rinput) or spike threshold. In contrast, blockade of Kv7/M channels by XE991 increased Rinput, lowered the spike threshold, and increased excitability, postsynaptic EPSP summation, and EPSP–spike coupling, but only slightly reduced mAHP after spike trains (and not after single spikes). The SK and Kv7/M channel openers 1-EBIO and retigabine, respectively, had effects opposite to the blockers. Computational modelling reproduced many of these effects. We conclude that SK and Kv7/M channels have complementary roles in DGCs. These mechanisms may be important for the dentate network function, as CA3 neurons can be activated or inhibition recruited depending on DGC firing rate. PMID:24366266

  16. Discovery of three toxin peptides with Kv1.3 channel and IL-2 cytokine-inhibiting activities from Non-Buthidae scorpions, Chaerilus tricostatus and Chaerilus tryznai.

    PubMed

    Ding, Li; Chen, Jing; Hao, Jinbo; Zhang, Jiahui; Huang, Xuejun; Hu, Fangfang; Wu, Zheng; Liu, Yaru; Li, Wenxin; Cao, Zhijian; Wu, Yingliang; Li, Jian; Li, Shan; Liu, Hongyan; Wu, Wenlong; Chen, Zongyun

    2017-05-01

    Non-Buthidae venomous scorpions are huge natural sources of toxin peptides; however, only a few studies have been done to understand their toxin peptides. Herein, we describe three new potential immunomodulating toxin peptides, Ctri18, Ctry68 and Ctry2908, from two non-Buthidae scorpions, Chaerilus tricostatus and Chaerilus tryznai. Sequence alignment analyses showed that Ctri18, Ctry68 and Ctry2908 are three new members of the scorpion toxin α-KTx15 subfamily. Electrophysiological experiments showed that Ctri18, Ctry68 and Ctry2908 blocked the Kv1.3 channel at micromole to nanomole levels, but had weak effects on potassium channel KCNQ1 and sodium channel Nav1.4, which indicated that Ctri18, Ctry68 and Ctry2908 might have specific inhibiting effects on the Kv1.3 channel. ELISA experiments showed that Ctri18, Ctry68 and Ctry2908 inhibited IL-2 cytokine secretions of activated T lymphocyte in human PBMCs. Excitingly, consistent with the good Kv1.3 channel inhibitory activity, Ctry2908 inhibited cytokine IL-2 secretion in nanomole level, which indicated that Ctry2908 might be a new lead drug template toward Kv1.3 channels. Together, these studies discovered three new toxin peptides, Ctri18, Ctry68 and Ctry2908, with Kv1.3 channel and IL-2 cytokine-inhibiting activities from two scorpions, C. tricostatus and C. tryznai, and highlighted that non-Buthidae venomous scorpions are new natural toxin peptide sources. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Effects of the Kv7 voltage-activated potassium channel inhibitor linopirdine in rat models of haemorrhagic shock.

    PubMed

    Nassoiy, Sean P; Babu, Favin S; LaPorte, Heather M; Byron, Kenneth L; Majetschak, Matthias

    2018-04-27

    Recently, we demonstrated that Kv7 voltage-activated potassium channel inhibitors reduce fluid resuscitation requirements in short-term rat models of haemorrhagic shock. The aim of the present study was to further delineate the therapeutic potential and side effect profile of the Kv7 channel blocker linopirdine in various rat models of severe haemorrhagic shock over clinically relevant time periods. Intravenous administration of linopirdine, either before (1 or 3 mg/kg) or after (3 mg/kg) a 40% blood volume haemorrhage, did not affect blood pressure and survival in lethal haemorrhage models without fluid resuscitation. A single bolus of linopirdine (3 mg/kg) at the beginning of fluid resuscitation after haemorrhagic shock transiently reduced early fluid requirements in spontaneously breathing animals that were resuscitated for 3.5 hours. When mechanically ventilated rats were resuscitated after haemorrhagic shock with normal saline (NS) or with linopirdine-supplemented (10, 25 or 50 μg/mL) NS for 4.5 hours, linopirdine significantly and dose-dependently reduced fluid requirements by 14%, 45% and 55%, respectively. Lung and colon wet/dry weight ratios were reduced with linopirdine (25/50 μg/mL). There was no evidence for toxicity or adverse effects based on measurements of routine laboratory parameters and inflammation markers in plasma and tissue homogenates. Our findings support the concept that linopirdine-supplementation of resuscitation fluids is a safe and effective approach to reduce fluid requirements and tissue oedema formation during resuscitation from haemorrhagic shock. © 2018 John Wiley & Sons Australia, Ltd.

  18. Unanticipated region- and cell-specific downregulation of individual KChIP auxiliary subunit isotypes in Kv4.2 knock-out mouse brain.

    PubMed

    Menegola, Milena; Trimmer, James S

    2006-11-22

    Kv4 family voltage-gated potassium channel alpha subunits and Kv channel-interacting protein (KChIP) and dipeptidyl aminopeptidase-like protein subunits comprise somatodendritic A-type channels in mammalian neurons. Recently, a mouse was generated with a targeted deletion of Kv4.2, a Kv4 alpha subunit expressed in many but not all mammalian brain neurons. Kv4.2-/- mice are grossly indistinguishable from wild-type (WT) littermates. Here we used immunohistochemistry to analyze expression of component Kv4 and KChIP subunits of A-type channels in WT and Kv4.2-/- brains. We found that the expression level, and cellular and subcellular distribution of the other prominent brain Kv4 family member Kv4.3, was indistinguishable between WT and Kv4.2-/- samples. However, we found unanticipated regional and cell-specific decreases in expression of KChIPs. The degree of altered expression of individual KChIP isoforms in different regions and neurons precisely follows the level of Kv4.2 normally found at those sites and presumably their extent of association of these KChIPs with Kv4.2. The dramatic effects of Kv4.2 deletion on KChIP expression suggest that, in addition to previously characterized effects of KChIPs on the functional properties, trafficking, and turnover rate of Kv4 channels, Kv4:KChIP association may confer reciprocal Kv4.2-dependent effects on KChIPs. The impact of Kv4.2 deletion on KChIP expression also supports the major role of KChIPs as auxiliary subunits of Kv4 channels.

  19. The open gate of the K(V)1.2 channel: quantum calculations show the key role of hydration.

    PubMed

    Kariev, Alisher M; Njau, Philipa; Green, Michael E

    2014-02-04

    The open gate of the Kv1.2 voltage-gated potassium channel can just hold a hydrated K(+) ion. Quantum calculations starting from the x-ray coordinates of the channel confirm this, showing little change from the x-ray coordinates for the protein. Water molecules not in the x-ray coordinates, and the ion itself, are placed by the calculation. The water molecules, including their orientation and hydrogen bonding, with and without an ion, are critical for the path of the ion, from the solution to the gate. A sequence of steps is postulated in which the potential experienced by the ion in the pore is influenced by the position of the ion. The gate structure, with and without the ion, has been optimized. The charges on the atoms and bond lengths have been calculated using natural bond orbital calculations, giving K(+) ~0.77 charges, rather than 1.0. The PVPV hinge sequence has been mutated in silico to PVVV (P407V in the 2A79 numbering). The water structure around the ion becomes discontinuous, separated into two sections, above and below the ion. PVPV conservation closely relates to maintaining the water structure. Finally, these results have implications concerning gating. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  20. The Open Gate of the KV1.2 Channel: Quantum Calculations Show the Key Role of Hydration

    PubMed Central

    Kariev, Alisher M.; Njau, Philipa; Green, Michael E.

    2014-01-01

    The open gate of the Kv1.2 voltage-gated potassium channel can just hold a hydrated K+ ion. Quantum calculations starting from the x-ray coordinates of the channel confirm this, showing little change from the x-ray coordinates for the protein. Water molecules not in the x-ray coordinates, and the ion itself, are placed by the calculation. The water molecules, including their orientation and hydrogen bonding, with and without an ion, are critical for the path of the ion, from the solution to the gate. A sequence of steps is postulated in which the potential experienced by the ion in the pore is influenced by the position of the ion. The gate structure, with and without the ion, has been optimized. The charges on the atoms and bond lengths have been calculated using natural bond orbital calculations, giving K+ ∼0.77 charges, rather than 1.0. The PVPV hinge sequence has been mutated in silico to PVVV (P407V in the 2A79 numbering). The water structure around the ion becomes discontinuous, separated into two sections, above and below the ion. PVPV conservation closely relates to maintaining the water structure. Finally, these results have implications concerning gating. PMID:24507595

  1. A basic residue in the proximal C-terminus is necessary for efficient activation of the M-channel subunit Kv7.2 by PI(4,5)P₂.

    PubMed

    Telezhkin, Vsevolod; Thomas, Alison M; Harmer, Stephen C; Tinker, Andrew; Brown, David A

    2013-07-01

    All Kv7 potassium channels require membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) for their normal function and hence can be physiologically regulated by neurotransmitters and hormones that stimulate phosphoinositide hydrolysis. Recent mutational analysis indicates that a cluster of basic residues in the proximal C-terminus (K354/K358/R360/K362) is crucial for PI(4,5)P2 activation of cardiac Kv7.1 channels. Since this cluster is largely conserved in all Kv7 subunits, we tested whether homologous residues are also required for activation of Kv7.2 (a subunit of neuronal M-channels). We found that the mutation Kv7.2 (R325A) (corresponding to R360 in Kv7.1) reduced Kv7.2 current amplitude by ∼60 % (P < 0.02) without change in voltage sensitivity and reduced the sensitivity of Kv7.2 channels to dioctanoyl-phosphatidylinositol-4,5-bisphosphate by ∼eightfold (P < 0.001). Taking into account previous experiments (Zhang et al., Neuron 37:963-75, 2003) implicating Kv7.2 (H328), and since R325 and H328 are conserved in homologous positions in all other Kv7 channels, we suggest that this proximal C-terminal domain adjacent to the last transmembrane domain that contains R325 and H328 (in Kv7.2) might play a major role in the activation of all members of the Kv7 channel family by PI(4,5)P2.

  2. Modulation by clamping: Kv4 and KChIP interactions.

    PubMed

    Wang, Kewei

    2008-10-01

    The rapidly inactivating (A-type) potassium channels regulate membrane excitability that defines the fundamental mechanism of neuronal functions such as pain signaling. Cytosolic Kv channel-interacting proteins KChIPs that belong to neuronal calcium sensor (NCS) family of calcium binding EF-hand proteins co-assemble with Kv4 (Shal) alpha subunits to form a native complex that encodes major components of neuronal somatodendritic A-type K+ current, I(SA), in neurons and transient outward current, I(TO), in cardiac myocytes. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Here, I attempt to emphasize the interaction between KChIPs and Kv4 based on recent progress made in understanding the structure complex in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner. Greater insights into molecular mechanism between KChIPs and Kv4 interaction may provide therapeutic potentials of designing compounds aimed at disrupting the protein-protein interaction for treatment of membrane excitability-related disorders.

  3. Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells.

    PubMed

    Yuan, X J; Wang, J; Juhaszova, M; Golovina, V A; Rubin, L J

    1998-04-01

    K(+)-channel activity-mediated alteration of the membrane potential and cytoplasmic free Ca2+ concentration ([Ca2+]cyt) is a pivotal mechanism in controlling pulmonary vasomotor tone. By using combined approaches of patch clamp, imaging fluorescent microscopy, and molecular biology, we examined the electrophysiological properties of K+ channels and the role of different K+ currents in regulating [Ca2+]cyt and explored the molecular identification of voltage-gated K+ (KV)- and Ca(2+)-activated K+ (KCa)-channel genes expressed in pulmonary arterial smooth muscle cells (PASMC). Two kinetically distinct KV currents [IK(V)], a rapidly inactivating (A-type) and a noninactivating delayed rectifier, as well as a slowly activated KCa current [IK(Ca)] were identified. IK(V) was reversibly inhibited by 4-aminopyridine (5 mM), whereas IK(Ca) was significantly inhibited by charybdotoxin (10-20 nM). K+ channels are composed of pore-forming alpha-subunits and auxiliary beta-subunits. Five KV-channel alpha-subunit genes from the Shaker subfamily (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), a KV-channel alpha-subunit gene from the Shab subfamily (KV2.1), a KV-channel modulatory alpha-subunit (KV9.3), and a KCa-channel alpha-subunit gene (rSlo), as well as three KV-channel beta-subunit genes (KV beta 1.1, KV beta 2, and KV beta 3) are expressed in PASMC. The data suggest that 1) native K+ channels in PASMC are encoded by multiple genes; 2) the delayed rectifier IK(V) may be generated by the KV1.1, KV1.2, KV1.5, KV1.6, KV2.1, and/or KV2.1/KV9.3 channels; 3) the A-type IK(V) may be generated by the KV1.4 channel and/or the delayed rectifier KV channels (KV1 subfamily) associated with beta-subunits; and 4) the IK(Ca) may be generated by the rSlo gene product. The function of the KV channels plays an important role in the regulation of membrane potential and [Ca2+]cyt in PASMC.

  4. Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1: AN INTEGRATED TRANSCRIPTOMIC AND PROTEOMIC STUDY.

    PubMed

    Kuzmenkov, Alexey I; Vassilevski, Alexander A; Kudryashova, Kseniya S; Nekrasova, Oksana V; Peigneur, Steve; Tytgat, Jan; Feofanov, Alexey V; Kirpichnikov, Mikhail P; Grishin, Eugene V

    2015-05-08

    The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus, and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here, we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands, and we compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis, we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β and inhibitor cystine knot types of fold. M. eupeus venom was then separated to individual components by multistage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore binding activity during venom separation. As a result, eight high affinity Kv1.1 channel blockers were identified, including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1-780 nm) by the two-electrode voltage clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Peripheral KV7 channels regulate visceral sensory function in mouse and human colon.

    PubMed

    Peiris, Madusha; Hockley, James Rf; Reed, David E; Smith, Ewan St John; Bulmer, David C; Blackshaw, L Ashley

    2017-01-01

    Background Chronic visceral pain is a defining symptom of many gastrointestinal disorders. The K V 7 family (K V 7.1-K V 7.5) of voltage-gated potassium channels mediates the M current that regulates excitability in peripheral sensory nociceptors and central pain pathways. Here, we use a combination of immunohistochemistry, gut-nerve electrophysiological recordings in both mouse and human tissues, and single-cell qualitative real-time polymerase chain reaction of gut-projecting sensory neurons, to investigate the contribution of peripheral K V 7 channels to visceral nociception. Results Immunohistochemical staining of mouse colon revealed labelling of K V 7 subtypes (K V 7.3 and K V 7.5) with CGRP around intrinsic enteric neurons of the myenteric plexuses and within extrinsic sensory fibres along mesenteric blood vessels. Treatment with the K V 7 opener retigabine almost completely abolished visceral afferent firing evoked by the algogen bradykinin, in agreement with significant co-expression of mRNA transcripts by single-cell qualitative real-time polymerase chain reaction for KCNQ subtypes and the B 2 bradykinin receptor in retrogradely labelled extrinsic sensory neurons from the colon. Retigabine also attenuated responses to mechanical stimulation of the bowel following noxious distension (0-80 mmHg) in a concentration-dependent manner, whereas the K V 7 blocker XE991 potentiated such responses. In human bowel tissues, K V 7.3 and K V 7.5 were expressed in neuronal varicosities co-labelled with synaptophysin and CGRP, and retigabine inhibited bradykinin-induced afferent activation in afferent recordings from human colon. Conclusions We show that K V 7 channels contribute to the sensitivity of visceral sensory neurons to noxious chemical and mechanical stimuli in both mouse and human gut tissues. As such, peripherally restricted K V 7 openers may represent a viable therapeutic modality for the treatment of gastrointestinal pathologies.

  6. Activation of neuronal Kv7/KCNQ/M-channels by the opener QO58-lysine and its anti-nociceptive effects on inflammatory pain in rodents.

    PubMed

    Teng, Bo-Chuan; Song, Yan; Zhang, Fan; Ma, Tian-Yang; Qi, Jin-Long; Zhang, Hai-Lin; Li, Gang; Wang, KeWei

    2016-08-01

    The aim of this study was to examine the activation of neuronal Kv7/KCNQ channels by a novel modified Kv7 opener QO58-lysine and to test the anti-nociceptive effects of QO58-lysine on inflammatory pain in rodent models. Assays including whole-cell patch clamp recordings, HPLC, and in vivo pain behavioral evaluations were employed. QO58-lysine caused instant activation of Kv7.2/7.3 currents, and increasing the dose of QO58-lysine resulted in a dose-dependent activation of Kv7.2/Kv7.3 currents with an EC50 of 1.2±0.2 μmol/L. QO58-lysine caused a leftward shift of the voltage-dependent activation of Kv7.2/Kv7.3 to a hyperpolarized potential at V1/2=-54.4±2.5 mV from V1/2=-26.0±0.6 mV. The half-life in plasma (t1/2) was derived as 2.9, 2.7, and 3.0 h for doses of 12.5, 25, and 50 mg/kg, respectively. The absolute bioavailabilities for the three doses (12.5, 25, and 50 mg/kg) of QO58-lysine (po) were determined as 13.7%, 24.3%, and 39.3%, respectively. QO58-lysine caused a concentration-dependent reduction in the licking times during phase II pain induced by the injection of formalin into the mouse hindpaw. In the Complete Freund's adjuvant (CFA)-induced inflammatory pain model in rats, oral or intraperitoneal administration of QO58-lysine resulted in a dose-dependent increase in the paw withdrawal threshold, and the anti-nociceptive effect on mechanical allodynia could be reversed by the channel-specific blocker XE991 (3 mg/kg). Taken together, our findings show that a modified QO58 compound (QO58-lysine) can specifically activate Kv7.2/7.3/M-channels. Oral or intraperitoneal administration of QO58-lysine, which has improved bioavailability and a half-life of approximately 3 h in plasma, can reverse inflammatory pain in rodent animal models.

  7. A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide–induced proarrhythmic early afterdepolarizations

    PubMed Central

    Al-Owais, Moza M.; Hettiarachchi, Nishani T.; Kirton, Hannah M.; Hardy, Matthew E.; Boyle, John P.; Scragg, Jason L.; Steele, Derek S.; Peers, Chris

    2017-01-01

    Exposure to CO causes early afterdepolarization arrhythmias. Previous studies in rats have indicated that arrhythmias arose as a result of augmentation of the late Na+ current. The purpose of the present study was to examine the basis for CO-induced arrhythmias in guinea pig myocytes in which action potentials (APs) more closely resemble those of human myocytes. Whole-cell current- and voltage-clamp recordings were made from isolated guinea pig myocytes as well as from human embryonic kidney 293 (HEK293) cells that express wild-type or a C723S mutant form of ether-a-go-go–related gene (ERG; Kv11.1). We also monitored the formation of peroxynitrite (ONOO−) in HEK293 cells fluorimetrically. CO—applied as the CO-releasing molecule, CORM-2—prolonged the APs and induced early afterdepolarizations in guinea pig myocytes. In HEK293 cells, CO inhibited wild-type, but not C723S mutant, Kv11.1 K+ currents. Inhibition was prevented by an antioxidant, mitochondrial inhibitors, or inhibition of NO formation. CO also raised ONOO− levels, an effect that was reversed by the ONOO− scavenger, FeTPPS [5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato-iron(III)], which also prevented the CO inhibition of Kv11.1 currents and abolished the effects of CO on Kv11.1 tail currents and APs in guinea pig myocytes. Our data suggest that CO induces arrhythmias in guinea pig cardiac myocytes via the ONOO−-mediated inhibition of Kv11.1 K+ channels.—Al-Owais, M. M., Hettiarachchi, N. T., Kirton, H. M., Hardy, M. E., Boyle, J. P., Scragg, J. L., Steele, D. S., Peers, C. A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide–induced proarrhythmic early afterdepolarizations. PMID:28743763

  8. Schizophrenia-Associated hERG channel Kv11.1-3.1 Exhibits a Unique Trafficking Deficit that is Rescued Through Proteasome Inhibition for High Throughput Screening.

    PubMed

    Calcaterra, Nicholas E; Hoeppner, Daniel J; Wei, Huijun; Jaffe, Andrew E; Maher, Brady J; Barrow, James C

    2016-02-16

    The primate-specific brain voltage-gated potassium channel isoform Kv11.1-3.1 has been identified as a novel therapeutic target for the treatment of schizophrenia. While this ether-a-go-go related K(+)channel has shown clinical relevance, drug discovery efforts have been hampered due to low and inconsistent activity in cell-based assays. This poor activity is hypothesized to result from poor trafficking via the lack of an intact channel-stabilizing Per-Ant-Sim (PAS) domain. Here we characterize Kv11.1-3.1 cellular localization and show decreased channel expression and cell surface trafficking relative to the PAS-domain containing major isoform, Kv11.1-1A. Using small molecule inhibition of proteasome degradation, cellular expression and plasma membrane trafficking are rescued. These findings implicate the importance of the unfolded-protein response and endoplasmic reticulum associated degradation pathways in the expression and regulation of this schizophrenia risk factor. Utilizing this identified phenomenon, an electrophysiological and high throughput in-vitro fluorescent assay platform has been developed for drug discovery in order to explore a potentially new class of cognitive therapeutics.

  9. Conformational changes of an ion-channel during gating and emerging electrophysiologic properties: Application of a computational approach to cardiac Kv7.1.

    PubMed

    Nekouzadeh, Ali; Rudy, Yoram

    2016-01-01

    Ion channels are the "building blocks" of the excitation process in excitable tissues. Despite advances in determining their molecular structure, understanding the relationship between channel protein structure and electrical excitation remains a challenge. The Kv7.1 potassium channel is an important determinant of the cardiac action potential and its adaptation to rate changes. It is subject to beta adrenergic regulation, and many mutations in the channel protein are associated with the arrhythmic long QT syndrome. In this theoretical study, we use a novel computational approach to simulate the conformational changes that Kv7.1 undergoes during activation gating and compute the resulting electrophysiologic function in terms of single-channel and macroscopic currents. We generated all possible conformations of the S4-S5 linker that couples the S3-S4 complex (voltage sensor domain, VSD) to the pore, and all associated conformations of VSD and the pore (S6). Analysis of these conformations revealed that VSD-to-pore mechanical coupling during activation gating involves outward translation of the voltage sensor, accompanied by a translation away from the pore and clockwise twist. These motions cause pore opening by moving the S4-S5 linker upward and away from the pore, providing space for the S6 tails to move away from each other. Single channel records, computed from the simulated motion trajectories during gating, have stochastic properties similar to experimentally recorded traces. Macroscopic current through an ensemble of channels displays two key properties of Kv7.1: an initial delay of activation and fast inactivation. The simulations suggest a molecular mechanism for fast inactivation; a large twist of the VSD following its outward translation results in movement of the base of the S4-S5 linker toward the pore, eliminating open pore conformations to cause inactivation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Rapid activity-dependent modulation of the intrinsic excitability through up-regulation of KCNQ/Kv7 channel function in neonatal spinal motoneurons.

    PubMed

    Lombardo, Joseph; Sun, Jianli; Harrington, Melissa A

    2018-01-01

    Activity-dependent changes in the properties of the motor system underlie the necessary adjustments in its responsiveness on the basis of the environmental and developmental demands of the organism. Although plastic changes in the properties of the spinal cord have historically been neglected because of the archaic belief that the spinal cord is constituted by a hardwired network that simply relays information to muscles, plenty of evidence has been accumulated showing that synapses impinging on spinal motoneurons undergo short- and long-term plasticity. In the brain, brief changes in the activity level of the network have been shown to be paralleled by changes in the intrinsic excitability of the neurons and are suggested to either reinforce or stabilize the changes at the synaptic level. However, rapid activity-dependent changes in the intrinsic properties of spinal motoneurons have never been reported. In this study, we show that in neonatal mice the intrinsic excitability of spinal motoneurons is depressed after relatively brief but sustained changes in the spinal cord network activity. Using electrophysiological techniques together with specific pharmacological blockers of KCNQ/Kv7 channels, we demonstrate their involvement in the reduction of the intrinsic excitability of spinal motoneurons. This action results from an increased M-current, the product of the activation of KCNQ/Kv7 channels, which leads to a hyperpolarization of the resting membrane potential and a decrease in the input resistance of spinal motoneurons. Computer simulations showed that specific up-regulations in KCNQ/Kv7 channels functions lead to a modulation of the intrinsic excitability of spinal motoneurons as observed experimentally. These results indicate that KCNQ/Kv7 channels play a fundamental role in the activity-dependent modulation of the excitability of spinal motoneurons.

  11. The Kv7/KCNQ channel blocker XE991 protects nigral dopaminergic neurons in the 6-hydroxydopamine rat model of Parkinson's disease.

    PubMed

    Liu, Haixia; Jia, Lu; Chen, Xiaoyan; Shi, Limin; Xie, Junxia

    2018-03-01

    The excitability of dopaminergic neurons in the substantia nigra pars compacta (SNc) that supply the striatum with dopamine (DA) determines the function of the nigrostriatal system for motor coordination. We previously showed that 4-pyridinylmethyl-9(10H)-anthracenone (XE991), a specific blocker of Kv7/KCNQ channels, enhanced the excitability of nigral DA neurons and resulted in attenuation of haloperidol-induced catalepsy in a Parkinson's disease (PD) rat model. However, whether XE991 exhibits neuroprotective effects towards DA neuron degeneration remains unknown. The aim of this study was to investigate the effects of Kv7/KCNQ channel blocker, XE991, on 6-hydroxydopamine (6-OHDA)-induced nigral DA neuron degeneration and motor dysfunction. Using immunofluorescence staining and western blotting, we showed that intracerebroventricular administration of XE991 prevented the 6-OHDA-induced decrease in tyrosine hydroxylase (TH)-positive neurons and TH protein expression in the SNc. High-performance liquid chromatography with electrochemical detection (HPLC-ECD) also revealed that XE991 partly restored the levels of DA and its metabolites in the striatum. Moreover, XE991 decreased apomorphine (APO)-induced contralateral rotations, enhanced balance and coordination, and attenuated muscle rigidity in 6-OHDA-treated rats. Importantly, all neuroprotective effects by XE991 were abolished by co-application of Kv7/KCNQ channel opener retigabine and XE991. Thus, Kv7/KCNQ channel inhibition by XE991 can exert neuroprotective effects against 6-OHDA-induced degeneration of the nigrostriatal DA system and motor dysfunction. Copyright © 2017. Published by Elsevier Inc.

  12. A limited 4 Å radial displacement of the S4-S5 linker is sufficient for internal gate closing in Kv channels.

    PubMed

    Faure, Élise; Starek, Greg; McGuire, Hugo; Bernèche, Simon; Blunck, Rikard

    2012-11-16

    Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3-4 Å is sufficient to prevent ion permeation through the pore.

  13. Immunomodulation by memantine in therapy of Alzheimer's disease is mediated through inhibition of Kv1.3 channels and T cell responsiveness

    PubMed Central

    Lowinus, Theresa; Bose, Tanima; Busse, Stefan; Busse, Mandy; Reinhold, Dirk; Schraven, Burkhart; Bommhardt, Ursula H.H.

    2016-01-01

    Memantine is approved for the treatment of advanced Alzheimer's disease (AD) and reduces glutamate-mediated neuronal excitotoxicity by antagonism of N-methyl-D-aspartate receptors. In the pathophysiology of AD immune responses deviate and infectious side effects are observed during memantine therapy. However, the particular effects of memantine on human T lymphocytes are unresolved. Here, we provide evidence that memantine blocks Kv1.3 potassium channels, inhibits CD3-antibody- and alloantigen-induced proliferation and suppresses chemokine-induced migration of peripheral blood T cells of healthy donors. Concurrent with the in vitro data, CD4+ T cells from AD patients receiving therapeutic doses of memantine show a transient decline of Kv1.3 channel activity and a long-lasting reduced proliferative response to alloantigens in mixed lymphocyte reactions. Furthermore, memantine treatment provokes a profound depletion of peripheral blood memory CD45RO+ CD4+ T cells. Thus, standard doses of memantine profoundly reduce T cell responses in treated patients through blockade of Kv1.3 channels. This may normalize deviant immunopathology in AD and contribute to the beneficial effects of memantine, but may also account for the enhanced infection rate. PMID:27462773

  14. Immunomodulation by memantine in therapy of Alzheimer's disease is mediated through inhibition of Kv1.3 channels and T cell responsiveness.

    PubMed

    Lowinus, Theresa; Bose, Tanima; Busse, Stefan; Busse, Mandy; Reinhold, Dirk; Schraven, Burkhart; Bommhardt, Ursula H H

    2016-08-16

    Memantine is approved for the treatment of advanced Alzheimer´s disease (AD) and reduces glutamate-mediated neuronal excitotoxicity by antagonism of N-methyl-D-aspartate receptors. In the pathophysiology of AD immune responses deviate and infectious side effects are observed during memantine therapy. However, the particular effects of memantine on human T lymphocytes are unresolved. Here, we provide evidence that memantine blocks Kv1.3 potassium channels, inhibits CD3-antibody- and alloantigen-induced proliferation and suppresses chemokine-induced migration of peripheral blood T cells of healthy donors. Concurrent with the in vitro data, CD4+ T cells from AD patients receiving therapeutic doses of memantine show a transient decline of Kv1.3 channel activity and a long-lasting reduced proliferative response to alloantigens in mixed lymphocyte reactions. Furthermore, memantine treatment provokes a profound depletion of peripheral blood memory CD45RO+ CD4+ T cells. Thus, standard doses of memantine profoundly reduce T cell responses in treated patients through blockade of Kv1.3 channels. This may normalize deviant immunopathology in AD and contribute to the beneficial effects of memantine, but may also account for the enhanced infection rate.

  15. Effects of natural and synthetic isothiocyanate-based H2S-releasers against chemotherapy-induced neuropathic pain: Role of Kv7 potassium channels.

    PubMed

    Di Cesare Mannelli, Lorenzo; Lucarini, Elena; Micheli, Laura; Mosca, Ilaria; Ambrosino, Paolo; Soldovieri, Maria Virginia; Martelli, Alma; Testai, Lara; Taglialatela, Maurizio; Calderone, Vincenzo; Ghelardini, Carla

    2017-07-15

    Hydrogen sulfide (H 2 S) is a crucial signaling molecule involved in several physiological and pathological processes. Nonetheless, the role of this gasotransmitter in the pathogenesis and treatment of neuropathic pain is controversial. The aim of the present study was to investigate the pain relieving profile of a series of slow releasing H 2 S donors (the natural allyl-isothiocyanate and the synthetics phenyl- and carboxyphenyl-isothiocyanate) in animal models of neuropathic pain induced by paclitaxel or oxaliplatin, anticancer drugs characterized by a dose-limiting neurotoxicity. The potential contribution of Kv7 potassium channels modulation was also studied. Mice were treated with paclitaxel (2.0 mg kg -1 ) i.p. on days 1, 3, 5 and 7; oxaliplatin (2.4 mg kg -1 ) was administered i.p. on days 1-2, 5-9, 12-14. Behavioral tests were performed on day 15. In both models, single subcutaneous administrations of H 2 S donors (1.33, 4.43, 13.31 μmol kg -1 ) reduced the hypersensitivity to cold non-noxious stimuli (allodynia-related measurement). The prototypical H 2 S donor NaHS was also effective. Activity was maintained after i.c.v. administrations. On the contrary, the S-lacking molecule allyl-isocyanate did not increase pain threshold; the H 2 S-binding molecule hemoglobin abolished the pain-relieving effects of isothiocyanates and NaHS. The anti-neuropathic properties of H 2 S donors were reverted by the Kv7 potassium channel blocker XE991. Currents carried by Kv7.2 homomers and Kv7.2/Kv7.3 heteromers expressed in CHO cells were potentiated by H 2 S donors. Sistemically- or centrally-administered isothiocyanates reduced chemotherapy-induced neuropathic pain by releasing H 2 S. Activation of Kv7 channels largely mediate the anti-neuropathic effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel.

    PubMed

    Mishima, Eriko; Sato, Yoko; Nanatani, Kei; Hoshi, Naomi; Lee, Jong-Kook; Schiller, Nina; von Heijne, Gunnar; Sakaguchi, Masao; Uozumi, Nobuyuki

    2016-12-01

    Voltage-dependent K + (K V ) channels control K + permeability in response to shifts in the membrane potential. Voltage sensing in K V channels is mediated by the positively charged transmembrane domain S4. The best-characterized K V channel, KvAP, lacks the distinct hydrophilic region corresponding to the S3-S4 extracellular loop that is found in other K + channels. In the present study, we evaluated the topogenic properties of the transmembrane regions within the voltage-sensing domain in KvAP. S3 had low membrane insertion activity, whereas S4 possessed a unique type-I signal anchor (SA-I) function, which enabled it to insert into the membrane by itself. S4 was also found to function as a stop-transfer signal for retention in the membrane. The length and structural nature of the extracellular S3-S4 loop affected the membrane insertion of S3 and S4, suggesting that S3 membrane insertion was dependent on S4. Replacement of charged residues within the transmembrane regions with residues of opposite charge revealed that Asp 72 in S2 and Glu 93 in S3 contributed to membrane insertion of S3 and S4, and increased the stability of S4 in the membrane. These results indicate that the SA-I function of S4, unique among K + channels studied to date, promotes the insertion of S3 into the membrane, and that the charged residues essential for voltage sensing contribute to the membrane-insertion of the voltage sensor domain in KvAP. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

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

    PubMed

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

    2018-03-01

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

  18. Recombinant Production, Reconstruction in Lipid-Protein Nanodiscs, and Electron Microscopy of Full-Length α-Subunit of Human Potassium Channel Kv7.1.

    PubMed

    Shenkarev, Z O; Karlova, M G; Kulbatskii, D S; Kirpichnikov, M P; Lyukmanova, E N; Sokolova, O S

    2018-05-01

    Voltage-gated potassium channel Kv7.1 plays an important role in the excitability of cardiac muscle. The α-subunit of Kv7.1 (KCNQ1) is the main structural element of this channel. Tetramerization of KCNQ1 in the membrane results in formation of an ion channel, which comprises a pore and four voltage-sensing domains. Mutations in the human KCNQ1 gene are one of the major causes of inherited arrhythmias, long QT syndrome in particular. The construct encoding full-length human KCNQ1 protein was synthesized in this work, and an expression system in the Pichia pastoris yeast cells was developed. The membrane fraction of the yeast cells containing the recombinant protein (rKCNQ1) was solubilized with CHAPS detergent. To better mimic the lipid environment of the channel, lipid-protein nanodiscs were formed using solubilized membrane fraction and MSP2N2 protein. The rKCNQ1/nanodisc and rKCNQ1/CHAPS samples were purified using the Rho1D4 tag introduced at the C-terminus of the protein. Protein samples were examined using transmission electron microscopy with negative staining. In both cases, homogeneous rKCNQ1 samples were observed based on image analysis. Statistical analysis of the images of individual protein particles solubilized in the detergent revealed the presence of a tetrameric structure confirming intact subunit assembly. A three-dimensional channel structure reconstructed at 2.5-nm resolution represents a compact density with diameter of the membrane part of ~9 nm and height ~11 nm. Analysis of the images of rKCNQ1 in nanodiscs revealed additional electron density corresponding to the lipid bilayer fragment and the MSP2N2 protein. These results indicate that the nanodiscs facilitate protein isolation, purification, and stabilization in solution and can be used for further structural studies of human Kv7.1.

  19. Aging-Related Hyperexcitability in CA3 Pyramidal Neurons Is Mediated by Enhanced A-Type K+ Channel Function and Expression.

    PubMed

    Simkin, Dina; Hattori, Shoai; Ybarra, Natividad; Musial, Timothy F; Buss, Eric W; Richter, Hannah; Oh, M Matthew; Nicholson, Daniel A; Disterhoft, John F

    2015-09-23

    Aging-related impairments in hippocampus-dependent cognition have been attributed to maladaptive changes in the functional properties of pyramidal neurons within the hippocampal subregions. Much evidence has come from work on CA1 pyramidal neurons, with CA3 pyramidal neurons receiving comparatively less attention despite its age-related hyperactivation being postulated to interfere with spatial processing in the hippocampal circuit. Here, we use whole-cell current-clamp to demonstrate that aged rat (29-32 months) CA3 pyramidal neurons fire significantly more action potentials (APs) during theta-burst frequency stimulation and that this is associated with faster AP repolarization (i.e., narrower AP half-widths and enlarged fast afterhyperpolarization). Using a combination of patch-clamp physiology, pharmacology, Western blot analyses, immunohistochemistry, and array tomography, we demonstrate that these faster AP kinetics are mediated by enhanced function and expression of Kv4.2/Kv4.3 A-type K(+) channels, particularly within the perisomatic compartment, of CA3 pyramidal neurons. Thus, our study indicates that inhibition of these A-type K(+) channels can restore the intrinsic excitability properties of aged CA3 pyramidal neurons to a young-like state. Significance statement: Age-related learning deficits have been attributed, in part, to altered hippocampal pyramidal neuronal function with normal aging. Much evidence has come from work on CA1 neurons, with CA3 neurons receiving comparatively less attention despite its age-related hyperactivation being postulated to interfere with spatial processing. Hence, we conducted a series of experiments to identify the cellular mechanisms that underlie the hyperexcitability reported in the CA3 region. Contrary to CA1 neurons, we demonstrate that postburst afterhyperpolarization is not altered with aging and that aged CA3 pyramidal neurons are able to fire significantly more action potentials and that this is associated with

  20. Aging-Related Hyperexcitability in CA3 Pyramidal Neurons Is Mediated by Enhanced A-Type K+ Channel Function and Expression

    PubMed Central

    Simkin, Dina; Hattori, Shoai; Ybarra, Natividad; Musial, Timothy F.; Buss, Eric W.; Richter, Hannah; Oh, M. Matthew

    2015-01-01

    Aging-related impairments in hippocampus-dependent cognition have been attributed to maladaptive changes in the functional properties of pyramidal neurons within the hippocampal subregions. Much evidence has come from work on CA1 pyramidal neurons, with CA3 pyramidal neurons receiving comparatively less attention despite its age-related hyperactivation being postulated to interfere with spatial processing in the hippocampal circuit. Here, we use whole-cell current-clamp to demonstrate that aged rat (29–32 months) CA3 pyramidal neurons fire significantly more action potentials (APs) during theta-burst frequency stimulation and that this is associated with faster AP repolarization (i.e., narrower AP half-widths and enlarged fast afterhyperpolarization). Using a combination of patch-clamp physiology, pharmacology, Western blot analyses, immunohistochemistry, and array tomography, we demonstrate that these faster AP kinetics are mediated by enhanced function and expression of Kv4.2/Kv4.3 A-type K+ channels, particularly within the perisomatic compartment, of CA3 pyramidal neurons. Thus, our study indicates that inhibition of these A-type K+ channels can restore the intrinsic excitability properties of aged CA3 pyramidal neurons to a young-like state. SIGNIFICANCE STATEMENT Age-related learning deficits have been attributed, in part, to altered hippocampal pyramidal neuronal function with normal aging. Much evidence has come from work on CA1 neurons, with CA3 neurons receiving comparatively less attention despite its age-related hyperactivation being postulated to interfere with spatial processing. Hence, we conducted a series of experiments to identify the cellular mechanisms that underlie the hyperexcitability reported in the CA3 region. Contrary to CA1 neurons, we demonstrate that postburst afterhyperpolarization is not altered with aging and that aged CA3 pyramidal neurons are able to fire significantly more action potentials and that this is associated with

  1. A Disease Mutation Causing Episodic Ataxia Type I in the S1 Links Directly to the Voltage Sensor and the Selectivity Filter in Kv Channels.

    PubMed

    Petitjean, Dimitri; Kalstrup, Tanja; Zhao, Juan; Blunck, Rikard

    2015-09-02

    The mutation F184C in Kv1.1 leads to development of episodic ataxia type I (EA1). Although the mutation has been said to alter activation kinetics and to lower expression, we show here that the underlying molecular mechanisms may be more complex. Although F184 is positioned in the "peripheral" S1 helix, it occupies a central position in the 3D fold. We show in cut-open oocyte voltage-clamp recordings of gating and ionic currents of the Shaker Kv channel expressed in Xenopus oocytes that F184 not only interacts directly with the gating charges of the S4, but also creates a functional link to the selectivity filter of the neighboring subunit. This link leads to impaired fast and slow inactivation. The effect on fast inactivation is of an allosteric nature considering that fast inactivation is caused by a linked cytosolic ball peptide. The extensive effects of F184C provide a new mechanism underlying EA. Episodic ataxia (EA) is an inherited disease that leads to occasional loss of motor control in combination with variable other symptoms such as vertigo or migraine. EA type I (EA1), studied here, is caused by mutations in a voltage-gated potassium channel that contributes to the generation of electrical signals in the brain. The mechanism by which mutations in voltage-gated potassium channels lead to EA is still unknown and there is no consistent pharmacological treatment. By studying in detail one disease-causing mutation in Kv1.1, we describe a novel molecular mechanism distinct from mechanisms described previously. This mechanism contributes to the understanding of potassium channel function in general and might lead to a better understanding of how EA develops. Copyright © 2015 the authors 0270-6474/15/3512198-09$15.00/0.

  2. APETx4, a Novel Sea Anemone Toxin and a Modulator of the Cancer-Relevant Potassium Channel KV10.1.

    PubMed

    Moreels, Lien; Peigneur, Steve; Galan, Diogo T; De Pauw, Edwin; Béress, Lászlo; Waelkens, Etienne; Pardo, Luis A; Quinton, Loïc; Tytgat, Jan

    2017-09-13

    The human ether-à-go-go channel (hEag1 or K V 10.1) is a cancer-relevant voltage-gated potassium channel that is overexpressed in a majority of human tumors. Peptides that are able to selectively inhibit this channel can be lead compounds in the search for new anticancer drugs. Here, we report the activity-guided purification and electrophysiological characterization of a novel K V 10.1 inhibitor from the sea anemone Anthopleura elegantissima . Purified sea anemone fractions were screened for inhibitory activity on K V 10.1 by measuring whole-cell currents as expressed in Xenopus laevis oocytes using the two-microelectrode voltage clamp technique. Fractions that showed activity on Kv10.1 were further purified by RP-HPLC. The amino acid sequence of the peptide was determined by a combination of MALDI- LIFT-TOF/TOF MS/MS and CID-ESI-FT-ICR MS/MS and showed a high similarity with APETx1 and APETx3 and was therefore named APETx4. Subsequently, the peptide was electrophysiologically characterized on K V 10.1. The selectivity of the toxin was investigated on an array of voltage-gated ion channels, including the cardiac human ether-à-go-go-related gene potassium channel (hERG or Kv11.1). The toxin inhibits K V 10.1 with an IC 50 value of 1.1 μM. In the presence of a similar toxin concentration, a shift of the activation curve towards more positive potentials was observed. Similar to the effect of the gating modifier toxin APETx1 on hERG, the inhibition of Kv10.1 by the isolated toxin is reduced at more positive voltages and the peptide seems to keep the channel in a closed state. Although the peptide also induces inhibitory effects on other K V and Na V channels, it exhibits no significant effect on hERG. Moreover, APETx4 induces a concentration-dependent cytotoxic and proapoptotic effect in various cancerous and noncancerous cell lines. This newly identified K V 10.1 inhibitor can be used as a tool to further characterize the oncogenic channel K V 10.1 or as a

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

    PubMed Central

    Fernández-Trillo, Jorge; Bharill, Shashank; Panyi, Gyorgy; Stühmer, Walter; Isacoff, Ehud Y.

    2017-01-01

    Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4–S5 linker). However, our recent work on channels disrupted in the S4–S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of KV10.1 revealed that the S4–S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use “split” channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in KV10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4–S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4–S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism. PMID:28360219

  4. Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels*

    PubMed Central

    Ke, Ying; Hunter, Mark J.; Ng, Chai Ann; Perry, Matthew D.; Vandenberg, Jamie I.

    2014-01-01

    The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking. PMID:24695734

  5. Loss of Functional A-Type Potassium Channels in the Dendrites of CA1 Pyramidal Neurons from a Mouse Model of Fragile X Syndrome

    PubMed Central

    Routh, Brandy N.; Johnston, Daniel

    2013-01-01

    Despite the critical importance of voltage-gated ion channels in neurons, very little is known about their functional properties in Fragile X syndrome: the most common form of inherited cognitive impairment. Using three complementary approaches, we investigated the physiological role of A-type K+ currents (IKA) in hippocampal CA1 pyramidal neurons from fmr1-/y mice. Direct measurement of IKA using cell-attached patch-clamp recordings revealed that there was significantly less IKA in the dendrites of CA1 neurons from fmr1-/y mice. Interestingly, the midpoint of activation for A-type K+ channels was hyperpolarized for fmr1-/y neurons compared with wild-type, which might partially compensate for the lower current density. Because of the rapid time course for recovery from steady-state inactivation, the dendritic A-type K+ current in CA1 neurons from both wild-type and fmr1-/y mice is likely mediated by KV4 containing channels. The net effect of the differences in IKA was that back-propagating action potentials had larger amplitudes producing greater calcium influx in the distal dendrites of fmr1-/y neurons. Furthermore, CA1 pyramidal neurons from fmr1-/y mice had a lower threshold for LTP induction. These data suggest that loss of IKA in hippocampal neurons may contribute to dendritic pathophysiology in Fragile X syndrome. PMID:24336711

  6. Distinct cellular distributions of Kv4 pore-forming and auxiliary subunits in rat dorsal root ganglion neurons.

    PubMed

    Matsuyoshi, Hiroko; Takimoto, Koichi; Yunoki, Takakazu; Erickson, Vickie L; Tyagi, Pradeep; Hirao, Yoshihiko; Wanaka, Akio; Yoshimura, Naoki

    2012-09-17

    Dorsal root ganglia contain heterogeneous populations of primary afferent neurons that transmit various sensory stimuli. This functional diversity may be correlated with differential expression of voltage-gated K(+) (Kv) channels. Here, we examine cellular distributions of Kv4 pore-forming and ancillary subunits that are responsible for fast-inactivating A-type K(+) current. Expression pattern of Kv α-subunit, β-subunit and auxiliary subunit was investigated using immunohistochemistry, in situ hybridization and RT-PCR technique. The two pore-forming subunits Kv4.1 and Kv4.3 show distinct cellular distributions: Kv4.3 is predominantly in small-sized C-fiber neurons, whereas Kv4.1 is seen in DRG neurons in various sizes. Furthermore, the two classes of Kv4 channel auxiliary subunits are also distributed in different-sized cells. KChIP3 is the only significantly expressed Ca(2+)-binding cytosolic ancillary subunit in DRGs and present in medium to large-sized neurons. The membrane-spanning auxiliary subunit DPP6 is seen in a large number of DRG neurons in various sizes, whereas DPP10 is restricted in small-sized neurons. Distinct combinations of Kv4 pore-forming and auxiliary subunits may constitute A-type channels in DRG neurons with different physiological roles. Kv4.1 subunit, in combination with KChIP3 and/or DPP6, form A-type K(+) channels in medium to large-sized A-fiber DRG neurons. In contrast, Kv4.3 and DPP10 may contribute to A-type K(+) current in non-peptidergic, C-fiber somatic afferent neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. A KCNQ1 mutation contributes to the concealed type 1 long QT phenotype by limiting the Kv7.1 channel conformational changes associated with protein kinase A phosphorylation.

    PubMed

    Bartos, Daniel C; Giudicessi, John R; Tester, David J; Ackerman, Michael J; Ohno, Seiko; Horie, Minoru; Gollob, Michael H; Burgess, Don E; Delisle, Brian P

    2014-03-01

    Type 1 long QT syndrome (LQT1) is caused by loss-of-function mutations in the KCNQ1-encoded Kv7.1 channel that conducts the slowly activating component of the delayed rectifier K(+) current (IKs). Clinically, the diagnosis of LQT1 is complicated by variable phenotypic expressivity, whereby approximately 25% of genotype-positive individuals present with concealed LQT1 (resting corrected QT [QTc] interval ≤460 ms). To determine whether a specific molecular mechanism contributes to concealed LQT1. We identified a multigenerational LQT1 family whereby 79% of the patients genotype-positive for p.Ile235Asn-KCNQ1 (I235N-Kv7.1) have concealed LQT1. We assessed the effect I235N-Kv7.1 has on IKs and the ventricular action potential (AP) by using in vitro analysis and computational simulations. Clinical data showed that all 10 patients with I235N-Kv7.1 have normal resting QTc intervals but abnormal QTc interval prolongation during the recovery phase of an electrocardiographic treadmill stress test. Voltage-clamping HEK293 cells coexpressing wild-type Kv7.1 and I235N-Kv7.1 (to mimic the patients' genotypes) showed that I235N-Kv7.1 generated relatively normal functioning Kv7.1 channels but were insensitive to protein kinase A (PKA) activation. Phosphomimetic and quinidine sensitivity studies suggest that I235N-Kv7.1 limits the conformational changes in Kv7.1 channels, which are necessary to upregulate IKs after PKA phosphorylation. Computational ventricular AP simulations predicted that the PKA insensitivity of I235N-Kv7.1 is primarily responsible for prolonging the AP with β-adrenergic stimulation, especially at slower cycle lengths. KCNQ1 mutations that generate relatively normal Kv7.1 channels, but limit the upregulation of IKs by PKA activation, likely contribute to concealed LQT1. Copyright © 2014 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  8. Vietnamese Heterometrus laoticus scorpion venom: evidence for analgesic and anti-inflammatory activity and isolation of new polypeptide toxin acting on Kv1.3 potassium channel.

    PubMed

    Hoang, Anh N; Vo, Hoang D M; Vo, Nguyen P; Kudryashova, Kseniya S; Nekrasova, Oksana V; Feofanov, Alexey V; Kirpichnikov, Mikhail P; Andreeva, Tatyana V; Serebryakova, Marina V; Tsetlin, Victor I; Utkin, Yuri N

    2014-01-01

    The scorpion Heterometrus laoticus (Scorpionidae) inhabits Indochinese peninsula and is widely distributed in South-West Vietnam. Since no human fatalities caused by H. laoticus stings were reported, no systematic characterization of the venom was earlier done. In this study we report on biological activity of the venom from H. laoticus caught in Vietnamese province An Giang. The venom manifested a very low acute toxicity with LD50 of about 190 mg/kg body weight in mice at subcutaneous (s.c.) injection and 12 mg/kg at intravenous injection. The venom analgesic effects using tail immersion and writhing tests as well as anti-inflammatory effect using carrageenan test were analyzed at doses of 9.5 and 19 mg/kg at s.c. injections. It was found that at two doses tested H. laoticus venom showed both anti-nociceptive and anti-inflammatory activity. The venom was fractionated by means of gel-filtration and reversed-phase HPLC. As a result several polypeptide toxins were isolated and new toxin hetlaxin was identified. Its amino acid sequence was determined and binding to the extracellular vestibule of the K⁺-conducting pore of Kv1.1 and Kv1.3 potassium channels was studied. Hetlaxin belongs to the scorpion alpha-toxin family and is the first toxin isolated from H. laoticus venom which possesses high affinity (K(i) 59 nM) to Kv1.3 potassium channel. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Inhibitory effects of hesperetin on Kv1.5 potassium channels stably expressed in HEK 293 cells and ultra-rapid delayed rectifier K(+) current in human atrial myocytes.

    PubMed

    Wang, Huan; Wang, Hong-Fei; Wang, Chen; Chen, Yu-Fang; Ma, Rong; Xiang, Ji-Zhou; Du, Xin-Ling; Tang, Qiang

    2016-10-15

    In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K(+) current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 μΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300μM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30μM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30μM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K(+) current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. NMR structural and dynamical investigation of the isolated voltage-sensing domain of the potassium channel KvAP: implications for voltage gating.

    PubMed

    Shenkarev, Zakhar O; Paramonov, Alexander S; Lyukmanova, Ekaterina N; Shingarova, Lyudmila N; Yakimov, Sergei A; Dubinnyi, Maxim A; Chupin, Vladimir V; Kirpichnikov, Mikhail P; Blommers, Marcel J J; Arseniev, Alexander S

    2010-04-28

    The structure and dynamics of the isolated voltage-sensing domain (VSD) of the archaeal potassium channel KvAP was studied by high-resolution NMR. The almost complete backbone resonance assignment and partial side-chain assignment of the (2)H,(13)C,(15)N-labeled VSD were obtained for the protein domain solubilized in DPC/LDAO (2:1) mixed micelles. Secondary and tertiary structures of the VSD were characterized using secondary chemical shifts and NOE contacts. These data indicate that the spatial structure of the VSD solubilized in micelles corresponds to the structure of the domain in an open state of the channel. NOE contacts and secondary chemical shifts of amide protons indicate the presence of tightly bound water molecule as well as hydrogen bond formation involving an interhelical salt bridge (Asp62-R133) that stabilizes the overall structure of the domain. The backbone dynamics of the VSD was studied using (15)N relaxation measurements. The loop regions S1-S2 and S2-S3 were found mobile, while the S3-S4 loop (voltage-sensor paddle) was found stable at the ps-ns time scale. The moieties of S1, S2, S3, and S4 helices sharing interhelical contacts (at the level of the Asp62-R133 salt bridge) were observed in conformational exchange on the micros-ms time scale. Similar exchange-induced broadening of characteristic resonances was observed for the VSD solubilized in the membrane of lipid-protein nanodiscs composed of DMPC, DMPG, and POPC/DOPG lipids. Apparently, the observed interhelical motions represent an inherent property of the VSD of the KvAP channel and can play an important role in the voltage gating.

  11. Different KChIPs compete for heteromultimeric assembly with pore-forming Kv4 subunits.

    PubMed

    Zhou, Jingheng; Tang, Yiquan; Zheng, Qin; Li, Meng; Yuan, Tianyi; Chen, Liangyi; Huang, Zhuo; Wang, KeWei

    2015-06-02

    Auxiliary Kv channel-interacting proteins 1-4 (KChIPs1-4) coassemble with pore-forming Kv4 α-subunits to form channel complexes underlying somatodendritic subthreshold A-type current that regulates neuronal excitability. It has been hypothesized that different KChIPs can competitively bind to Kv4 α-subunit to form variable channel complexes that can exhibit distinct biophysical properties for modulation of neural function. In this study, we use single-molecule subunit counting by total internal reflection fluorescence microscopy in combinations with electrophysiology and biochemistry to investigate whether different isoforms of auxiliary KChIPs, KChIP4a, and KChIP4bl, can compete for binding of Kv4.3 to coassemble heteromultimeric channel complexes for modulation of channel function. To count the number of photobleaching steps solely from cell membrane, we take advantage of a membrane tethered k-ras-CAAX peptide that anchors cytosolic KChIP4 proteins to the surface for reduction of background noise. Single-molecule subunit counting reveals that the number of KChIP4 isoforms in Kv4.3-KChIP4 complexes can vary depending on the KChIP4 expression level. Increasing the amount of KChIP4bl gradually reduces bleaching steps of KChIP4a isoform proteins, and vice versa. Further analysis of channel gating kinetics from different Kv4-KChIP4 subunit compositions confirms that both KChIP4a and KChIP4bl can modulate the channel complex function upon coassembly. Taken together, our findings show that auxiliary KChIPs can heteroassemble with Kv4 in a competitive manner to form heteromultimeric Kv4-KChIP4 channel complexes that are biophysically distinct and regulated under physiological or pathological conditions. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. New molecular targets for antiepileptic drugs: alpha(2)delta, SV2A, and K(v)7/KCNQ/M potassium channels.

    PubMed

    Rogawski, Michael A; Bazil, Carl W

    2008-07-01

    Many currently prescribed antiepileptic drugs (AEDs) act via voltage-gated sodium channels, through effects on gamma-aminobutyric acid-mediated inhibition, or via voltage-gated calcium channels. Some newer AEDs do not act via these traditional mechanisms. The molecular targets for several of these nontraditional AEDs have been defined using cellular electrophysiology and molecular approaches. Here, we describe three of these targets: alpha(2)delta, auxiliary subunits of voltage-gated calcium channels through which the gabapentinoids gabapentin and pregabalin exert their anticonvulsant and analgesic actions; SV2A, a ubiquitous synaptic vesicle glycoprotein that may prepare vesicles for fusion and serves as the target for levetiracetam and its analog brivaracetam (which is currently in late-stage clinical development); and K(v)7/KCNQ/M potassium channels that mediate the M-current, which acts a brake on repetitive firing and burst generation and serves as the target for the investigational AEDs retigabine and ICA-105665. Functionally, all of the new targets modulate neurotransmitter output at synapses, focusing attention on presynaptic terminals as critical sites of action for AEDs.

  13. Roles of somatic A-type K(+) channels in the synaptic plasticity of hippocampal neurons.

    PubMed

    Yang, Yoon-Sil; Kim, Kyeong-Deok; Eun, Su-Yong; Jung, Sung-Cherl

    2014-06-01

    In the mammalian brain, information encoding and storage have been explained by revealing the cellular and molecular mechanisms of synaptic plasticity at various levels in the central nervous system, including the hippocampus and the cerebral cortices. The modulatory mechanisms of synaptic excitability that are correlated with neuronal tasks are fundamental factors for synaptic plasticity, and they are dependent on intracellular Ca(2+)-mediated signaling. In the present review, the A-type K(+) (IA) channel, one of the voltage-dependent cation channels, is considered as a key player in the modulation of Ca(2+) influx through synaptic NMDA receptors and their correlated signaling pathways. The cellular functions of IA channels indicate that they possibly play as integral parts of synaptic and somatic complexes, completing the initiation and stabilization of memory.

  14. The roles of KCa, KATP, and KV channels in regulating cutaneous vasodilation and sweating during exercise in the heat.

    PubMed

    Louie, Jeffrey C; Fujii, Naoto; Meade, Robert D; McNeely, Brendan D; Kenny, Glen P

    2017-05-01

    We recently showed the varying roles of Ca 2+ -activated (K Ca ), ATP-sensitive (K ATP ), and voltage-gated (K V ) K + channels in regulating cholinergic cutaneous vasodilation and sweating in normothermic conditions. However, it is unclear whether the respective contributions of these K + channels remain intact during dynamic exercise in the heat. Eleven young (23 ± 4 yr) men completed a 30-min exercise bout at a fixed rate of metabolic heat production (400 W) followed by a 40-min recovery period in the heat (35°C, 20% relative humidity). Cutaneous vascular conductance (CVC) and local sweat rate were assessed at four forearm skin sites perfused via intradermal microdialysis with: 1 ) lactated Ringer solution (control); 2 ) 50 mM tetraethylammonium (nonspecific K Ca channel blocker); 3 ) 5 mM glybenclamide (selective K ATP channel blocker); or 4 ) 10 mM 4-aminopyridine (nonspecific K V channel blocker). Responses were compared at baseline and at 10-min intervals during and following exercise. K Ca channel inhibition resulted in greater CVC versus control at end exercise ( P = 0.04) and 10 and 20 min into recovery (both P < 0.01). K ATP channel blockade attenuated CVC compared with control during baseline ( P = 0.04), exercise (all P ≤ 0.04), and 10 min into recovery ( P = 0.02). No differences in CVC were observed with K V channel inhibition during baseline ( P = 0.15), exercise (all P ≥ 0.06), or recovery (all P ≥ 0.14). With the exception of K V channel inhibition augmenting sweating during baseline ( P = 0.04), responses were similar to control with all K + channel blockers during each time period (all P ≥ 0.07). We demonstrated that K Ca and K ATP channels contribute to the regulation of cutaneous vasodilation during rest and/or exercise and recovery in the heat. Copyright © 2017 the American Physiological Society.

  15. Nonreciprocal mechanisms in up- and downregulation of spinal motoneuron excitability by modulators of KCNQ/Kv7 channels.

    PubMed

    Lombardo, Joseph; Harrington, Melissa A

    2016-11-01

    KCNQ/K v 7 channels form a slow noninactivating K + current, also known as the M current. They activate in the subthreshold range of membrane potentials and regulate different aspects of excitability in neurons of the central nervous system. In spinal motoneurons (MNs), KCNQ/K v 7 channels have been identified in the somata, axonal initial segment, and nodes of Ranvier, where they generate a slow, noninactivating, K + current sensitive to both muscarinic receptor-mediated inhibition and KCNQ/K v 7 channel blockers. In this study, we thoroughly reevaluated the function of up- and downregulation of KCNQ/K v 7 channels in mouse immature spinal MNs. Using electrophysiological techniques together with specific pharmacological modulators of the activity of KCNQ/K v 7 channels, we show that enhancement of the activity of these channels decreases the excitability of spinal MNs in mouse neonates. This action on MNs results from a combination of hyperpolarization of the resting membrane potential, a decrease in the input resistance, and depolarization of the voltage threshold. On the other hand, the effect of inhibition of KCNQ/K v 7 channels suggested that these channels play a limited role in regulating basal excitability. Computer simulations confirmed that pharmacological enhancement of KCNQ/K v 7 channel activity decreases excitability and also suggested that the effects of inhibition of KCNQ/K v 7 channels on the excitability of spinal MNs do not depend on a direct effect in these neurons but likely on spinal cord synaptic partners. These results indicate that KCNQ/K v 7 channels have a fundamental role in the modulation of the excitability of spinal MNs acting both in these neurons and in their local presynaptic partners. Copyright © 2016 the American Physiological Society.

  16. RNA interference-based functional knockdown of the voltage-gated potassium channel Kv7.2 in dorsal root ganglion neurons after in vitro and in vivo gene transfer by adeno-associated virus vectors.

    PubMed

    Valdor, Markus; Wagner, Anke; Röhrs, Viola; Berg, Johanna; Fechner, Henry; Schröder, Wolfgang; Tzschentke, Thomas M; Bahrenberg, Gregor; Christoph, Thomas; Kurreck, Jens

    2018-01-01

    Activation of the neuronal potassium channel Kv7.2 encoded by the KCNQ2 gene has recently been shown to be an attractive mechanism to inhibit nociceptive transmission. However, potent, selective, and clinically proven activators of Kv7.2/Kv7.3 currents with analgesic properties are still lacking. An important prerequisite for the development of new drugs is a model to test the selectivity of novel agonists by abrogating Kv7.2/Kv7.3 function. Since constitutive knockout mice are not viable, we developed a model based on RNA interference-mediated silencing of KCNQ2. By delivery of a KCNQ2-specific short hairpin RNA with adeno-associated virus vectors, we completely abolished the activity of the specific Kv7.2/Kv7.3-opener ICA-27243 in rat sensory neurons. Results obtained in the silencing experiments were consistent between freshly prepared and cryopreserved dorsal root ganglion neurons, as well as in dorsal root ganglion neurons dissociated and cultured after in vivo administration of the silencing vector by intrathecal injections into rats. Interestingly, the tested associated virus serotypes substantially differed with respect to their transduction capability in cultured neuronal cell lines and primary dorsal root ganglion neurons and the in vivo transfer of transgenes by intrathecal injection of associated virus vectors. However, our study provides the proof-of-concept that RNA interference-mediated silencing of KCNQ2 is a suitable approach to create an ex vivo model for testing the specificity of novel Kv7.2/Kv7.3 agonists.

  17. Solution structure for Pandinus toxin K-alpha (PiTX-K alpha), a selective blocker of A-type potassium channels.

    PubMed

    Tenenholz, T C; Rogowski, R S; Collins, J H; Blaustein, M P; Weber, D J

    1997-03-11

    PiTX-K alpha, a 35-residue peptide recently isolated from the venom of Pandinus imperator, blocks the rapidly inactivating (A-type) K+ channel(s) in rat brain synaptosomes and the cloned Kv 1.2 potassium channel at very low toxin concentrations (6 nM and 32 pM, respectively) [Rogowski, R. S., Collins, J. H., O'Neil, T. J., Gustafson, T. A., Werkman, T. A., Rogawski, M. A., Tenenholz, T. C., Weber, D. J., & Blaustein, M. P. (1996) Mol. Pharmacol. 50, 1167-1177]. The three-dimensional structure of PiTX-K alpha was determined using NMR spectroscopy in order to understand its selectivity and affinity toward K+ channels. PiTX-K alpha was found to have an alpha-helix from residues 10 to 21 and two beta-strands (betaI, 26-28; betaII, 33-35) connected by a type II beta-turn to form a small antiparallel beta-sheet. Three disulfide bonds, which are conserved in all members of the charybdotoxin family (alpha-K toxins), anchor one face of the alpha-helix to the beta-sheet. The N-terminal portion of PiTX-K alpha has three fewer residues than other alpha-K toxins such as charybdotoxin. Rather than forming a third beta-strand as found for other alpha-K toxins, the N-terminal region of PiTX-K alpha adopts an extended conformation. This structural difference in PiTX-K alpha together with differences in sequence at Pro-10, Tyr-14, and Asn-25 (versus Ser-10, Trp-14, and Arg-25 in CTX) may explain why PiTX-K alpha does not block maxi-K+ channels. Differences in three-dimensional structure between PiTX-K alpha and charybdotoxin are also observed in both the tight turn and the loop that connects the first beta-strand to the alpha-helix. As a result, side chains of two residues (Tyr-23 and Arg-31) are in regions of PiTX-K alpha that probably interact with rapidly inactivating A-type K+ channels. The analogous residues in charybdotoxin are positioned differently on the toxin surface. Thus, the locations of Tyr-23 and Arg-31 side chains in PiTX-K alpha could explain why this toxin blocks A-type

  18. Computational Models for Understanding of Structure, Function and Pharmacology of the Cardiac Potassium Channel Kv11.1 (hERG).

    PubMed

    Wacker, Soren; Noskov, Sergei Yu; Perissinotti, Laura L

    2017-01-01

    The rapid delayed rectifier current IKr is one of the major K+ currents involved in repolarization of the human cardiac action potential. Various inherited or drug-induced forms of the long QT syndrome (LQTS) in humans are linked to functional and structural modifications in the IKr conducting channels. IKr is carried by the potassium channel Kv11.1 encoded by the gene KCNH2 (commonly referred to as human ether-a-go-go-related gene or hERG) [1, 2]. The first necessary step for predicting emergent drug effects on the heart is determining and modeling the binding thermodynamics and kinetics of primary and major off-target drug interactions with subcellular targets. The bulk of drugs that target hERG channels are known to have complex interactions at the atomic scale. Accordingly, one of the goals for this review is to provide comprehensive guide in the universe of computational models aiming to refine our understanding of structure-function relations in Kv11.1 and its isoforms. The special emphasis is placed on the mapping of drug binding sites and tentative mechanisms of channel inhibition and activation by drugs. An overview over recent structural models and mapping of binding sites for blockers and activators of IKr current along with the discussion on agreements and discrepancies among different models is presented. There is an apparent reciprocity or feedback loop between drug binding and action potential of the cardiac myocytes. Thus one has to connect drug binding to a particular receptor so that its functional consequences impact on the action potential duration. The natural pathway is to develop multi-scale models that connect between receptor and cellular scales. The potential for such multi-scale model development is discussed through the lens of common gating models. Accordingly, the second part of this review covers an ongoing development of the kinetic models of gating transitions and cardiac ion currents carried by hERG channels with and without drug

  19. Safety and pharmacodynamics of dalazatide, a Kv1.3 channel inhibitor, in the treatment of plaque psoriasis: A randomized phase 1b trial

    PubMed Central

    Tarcha, Eric J.; Probst, Peter; Peckham, David; Muñoz-Elías, Ernesto J.; Kruger, James G.; Iadonato, Shawn P.

    2017-01-01

    Background Dalazatide is a specific inhibitor of the Kv1.3 potassium channel. The expression and function of Kv1.3 channels are required for the function of chronically activated memory T cells, which have been shown to be key mediators of autoimmune diseases, including psoriasis. Objective The primary objective was to evaluate the safety of repeat doses of dalazatide in adult patients with mild-to-moderate plaque psoriasis. Secondary objectives were to evaluate clinical proof of concept and the effects of dalazatide on mediators of inflammation in the blood and on chronically activated memory T cell populations. Methods Patients (n = 24) were randomized 5:5:2 to receive dalazatide at 30 mcg/dose, 60 mcg/dose, or placebo twice weekly by subcutaneous injection (9 doses total). Safety was assessed on the basis of physical and neurological examination and laboratory testing. Clinical assessments included body-surface area affected, Psoriasis Area and Severity Index (PASI), and investigator and patient questionnaires. Results The most common adverse events were temporary mild (Grade 1) hypoesthesia (n = 20; 75% placebo, 85% dalazatide) and paresthesia (n = 15; 25% placebo, 70% dalazatide) involving the hands, feet, or perioral area. Nine of 10 patients in the 60 mcg/dose group had a reduction in their PASI score between baseline and Day 32, and the mean reduction in PASI score was significant in this group (P < 0.01). Dalazatide treatment reduced the plasma levels of multiple inflammation markers and reduced the expression of T cell activation markers on peripheral blood memory T cells. Limitations The study was small and drug treatment was for a short duration (4 weeks). Conclusion This study indicates that dalazatide is generally well tolerated and can improve psoriatic skin lesions by modulating T cell surface and activation marker expression and inhibiting mediators of inflammation in the blood. Larger studies of longer duration are warranted. PMID:28723914

  20. Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit.

    PubMed

    Rettig, J; Heinemann, S H; Wunder, F; Lorra, C; Parcej, D N; Dolly, J O; Pongs, O

    1994-05-26

    Structural and functional diversity of voltage-gated Kv1-type potassium channels in rat brain is enhanced by the association of two different types of subunits, the membrane-bound, poreforming alpha-subunits and a peripheral beta-subunit. We have cloned a beta-subunit (Kv beta 1) that is specifically expressed in the rat nervous system. Association of Kv beta 1 with alpha-subunits confers rapid A-type inactivation on non-inactivating Kv1 channels (delayed rectifiers) in expression systems in vitro. This effect is mediated by an inactivating ball domain in the Kv beta 1 amino terminus.

  1. A Phosphoinositide 3-Kinase (PI3K)-serum- and glucocorticoid-inducible Kinase 1 (SGK1) Pathway Promotes Kv7.1 Channel Surface Expression by Inhibiting Nedd4-2 Protein*

    PubMed Central

    Andersen, Martin Nybo; Krzystanek, Katarzyna; Petersen, Frederic; Bomholtz, Sofia Hammami; Olesen, Søren-Peter; Abriel, Hugues; Jespersen, Thomas; Rasmussen, Hanne Borger

    2013-01-01

    Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells. PMID:24214981

  2. A phosphoinositide 3-kinase (PI3K)-serum- and glucocorticoid-inducible kinase 1 (SGK1) pathway promotes Kv7.1 channel surface expression by inhibiting Nedd4-2 protein.

    PubMed

    Andersen, Martin Nybo; Krzystanek, Katarzyna; Petersen, Frederic; Bomholtz, Sofia Hammami; Olesen, Søren-Peter; Abriel, Hugues; Jespersen, Thomas; Rasmussen, Hanne Borger

    2013-12-27

    Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells.

  3. Angiotensin II reduces the surface abundance of KV 1.5 channels in arterial myocytes to stimulate vasoconstriction.

    PubMed

    Kidd, Michael W; Bulley, Simon; Jaggar, Jonathan H

    2017-03-01

    Several different voltage-dependent K + (K V ) channel isoforms are expressed in arterial smooth muscle cells (myocytes). Vasoconstrictors inhibit K V currents, but the isoform selectivity and mechanisms involved are unclear. We show that angiotensin II (Ang II), a vasoconstrictor, stimulates degradation of K V 1.5, but not K V 2.1, channels through a protein kinase C- and lysosome-dependent mechanism, reducing abundance at the surface of mesenteric artery myocytes. The Ang II-induced decrease in cell surface K V 1.5 channels reduces whole-cell K V 1.5 currents and attenuates K V 1.5 function in pressurized arteries. We describe a mechanism by which Ang II stimulates protein kinase C-dependent K V 1.5 channel degradation, reducing the abundance of functional channels at the myocyte surface. Smooth muscle cells (myocytes) of resistance-size arteries express several different voltage-dependent K + (K V ) channels, including K V 1.5 and K V 2.1, which regulate contractility. Myocyte K V currents are inhibited by vasoconstrictors, including angiotensin II (Ang II), but the mechanisms involved are unclear. Here, we tested the hypothesis that Ang II inhibits K V currents by reducing the plasma membrane abundance of K V channels in myocytes. Angiotensin II (applied for 2 h) reduced surface and total K V 1.5 protein in rat mesenteric arteries. In contrast, Ang II did not alter total or surface K V 2.1, or K V 1.5 or K V 2.1 cellular distribution, measured as the percentage of total protein at the surface. Bisindolylmaleimide (BIM; a protein kinase C blocker), a protein kinase C inhibitory peptide or bafilomycin A (a lysosomal degradation inhibitor) each blocked the Ang II-induced decrease in total and surface K V 1.5. Immunofluorescence also suggested that Ang II reduced surface K V 1.5 protein in isolated myocytes; an effect inhibited by BIM. Arteries were exposed to Ang II or Ang II plus BIM (for 2 h), after which these agents were removed and

  4. Functional expression of KCNQ (Kv7) channels in guinea pig bladder smooth muscle and their contribution to spontaneous activity

    PubMed Central

    Anderson, U A; Carson, C; Johnston, L; Joshi, S; Gurney, A M; McCloskey, K D

    2013-01-01

    Background and Purpose The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. Experimental Approach KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors. Key Results KCNQ subtypes 1–5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca2+-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20 μM) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity. Conclusions and Implications These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder. PMID:23586426

  5. KCNQ5/Kv7.5 potassium channel expression and subcellular localization in primate retinal pigment epithelium and neural retina

    PubMed Central

    Zhang, Xiaoming; Yang, Dongli

    2011-01-01

    Previous studies identified in retinal pigment epithelial (RPE) cells an M-type K+ current, which in many other cell types is mediated by channels encoded by KCNQ genes. The aim of this study was to assess the expression of KCNQ genes in the monkey RPE and neural retina. Application of the specific KCNQ channel blocker XE991 eliminated the M-type current in freshly isolated monkey RPE cells, indicating that KCNQ subunits contribute to the underlying channels. RT-PCR analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in the RPE and all five KCNQ transcripts in the neural retina. At the protein level, KCNQ5 was detected in the RPE, whereas both KCNQ4 and KCNQ5 were found in neural retina. In situ hybridization in frozen monkey retinal sections revealed KCNQ5 gene expression in the ganglion cell layer and the inner and outer nuclear layers of the neural retina, but results in the RPE were inconclusive due to the presence of melanin. Immunohistochemistry revealed KCNQ5 in the inner and outer plexiform layers, in cone and rod photoreceptor inner segments, and near the basal membrane of the RPE. The data suggest that KCNQ5 channels contribute to the RPE basal membrane K+ conductance and, thus, likely play an important role in active K+ absorption. The distribution of KCNQ5 in neural retina suggests that these channels may function in the shaping of the photoresponses of cone and rod photoreceptors and the processing of visual information by retinal neurons. PMID:21795522

  6. Voltage-gated K+ channel modulators as neuroprotective agents.

    PubMed

    Leung, Yuk-Man

    2010-05-22

    A manifestation in neurodegeneration is apoptosis of neurons. Neurons undergoing apoptosis may lose a substantial amount of cytosolic K+ through a number of pathways including K+ efflux via voltage-gated K+ (Kv) channels. The consequent drop in cytosolic [K+] relieves inhibition of an array of pro-apoptotic enzymes such as caspases and nucleases. Blocking Kv channels has been known to prevent neuronal apoptosis by preventing K+ efflux. Some neural diseases such as epilepsy are caused by neuronal hyperexcitability, which eventually may lead to neuronal apoptosis. Reduction in activities of A-type Kv channels and Kv7 subfamily members is amongst the etiological causes of neuronal hyperexcitation; enhancing the opening of these channels may offer opportunities of remedy. This review discusses the potential uses of Kv channel modulators as neuroprotective drugs.

  7. Partial blockade of Kv2.1 channel potentiates GLP-1's insulinotropic effects in islets and reduces its dose required for improving glucose tolerance in type 2 diabetic male mice.

    PubMed

    Sukma Rita, Rauza; Dezaki, Katsuya; Kurashina, Tomoyuki; Kakei, Masafumi; Yada, Toshihiko

    2015-01-01

    Glucagon-like peptide-1 (GLP-1)-based medicines have recently been widely used to treat type 2 diabetic patients, whereas adverse effects of nausea and vomiting have been documented. Inhibition of voltage-gated K(+) channel subtype Kv2.1 in pancreatic β-cells has been suggested to contribute to mild depolarization and promotion of insulin release. This study aimed to determine whether the blockade of Kv2.1 channels potentiates the insulinotropic effect of GLP-1 agonists. Kv2.1 channel blocker guangxitoxin-1E (GxTx) and GLP-1 agonist exendin-4 at subthreshold concentrations, when combined, markedly increased the insulin release and cytosolic Ca(2+) concentration ([Ca(2+)]i) in a glucose-dependent manner in mouse islets and β-cells. Exendin-4 at subthreshold concentration alone increased islet insulin release and β-cell [Ca(2+)]i in Kv2.1(+/-) mice. The [Ca(2+)]i response to subthreshold exendin-4 and GxTx in combination was attenuated by pretreatment with protein kinase A inhibitor H-89, indicating the protein kinase A dependency of the cooperative effect. Furthermore, subthreshold doses of GxTx and GLP-1 agonist liraglutide in combination markedly increased plasma insulin and improved glucose tolerance in diabetic db/db mice and NSY mice. These results demonstrate that a modest suppression of Kv2.1 channels dramatically raises insulinotropic potency of GLP-1-based drugs, which opens a new avenue to reduce their doses and associated adverse effects while achieving the same glycemic control in type 2 diabetes.

  8. Down-regulation of A-type potassium channel in gastric-specific DRG neurons in a rat model of functional dyspepsia.

    PubMed

    Li, S; Chen, J D Z

    2014-07-01

    Although without evidence of organic structural abnormalities, pain or discomfort is a prominent symptom of functional dyspepsia and considered to reflect visceral hypersensitivity whose underlying mechanism is poorly understood. Here, we studied electrophysiological properties and expression of voltage-gated potassium channels in dorsal root ganglion (DRG) neurons in a rat model of functional dyspepsia induced by neonatal gastric irritation. Male Sprague-Dawley rat pups at 10-day old received 0.1% iodoacetamide (IA) or vehicle by oral gavage for 6 days and studied at adulthood. Retrograde tracer-labeled gastric-specific T8 -T12 DRG neurons were harvested for the patch-clamp study in voltage and current-clamp modes and protein expression of K(+) channel in T8 -T12 DRGs was examined by western blotting. (1) Gastric specific but not non-gastric DRG neurons showed an enhanced excitability in neonatal IA-treated rats compared to the control: depolarized resting membrane potentials, a lower current threshold for action potential (AP) activation, and an increase in the number of APs in response to current stimulation. (2) The current density of tetraethylammonium insensitive (transiently inactivating A-type current), but not the tetraethylammonium sensitive (slow-inactivating delayed rectifier K(+) currents), was significantly smaller in IA-treated rats (65.4 ± 6.9 pA/pF), compared to that of control (93.1 ± 8.3 pA/pF). (3) Protein expression of KV 4.3 was down-regulated in IA-treated rats. A-type potassium channels are significantly down-regulated in the gastric-specific DRG neurons in adult rats with mild neonatal gastric irritation, which in part contribute to the enhanced DRG neuron excitabilities that leads to the development of gastric hypersensitivity. © 2014 John Wiley & Sons Ltd.

  9. Isoflurane depolarizes bronchopulmonary C neurons by inhibiting transient A-type and delayed rectifier potassium channels.

    PubMed

    Zhang, Zhenxiong; Zhuang, Jianguo; Zhang, Cancan; Xu, Fadi

    2013-04-01

    Inhalation of isoflurane (ISO), a widely used volatile anesthetic, can produce clinical tachypnea. In dogs, this response is reportedly mediated by bronchopulmonary C-fibers (PCFs), but the relevant mechanisms remain unclear. Activation of transient A-type potassium current (IA) channels and delayed rectifier potassium current (IK) channels hyperpolarizes neurons, and inhibition of both channels by ISO increases neural firing. Due to the presence of these channels in the cell bodies of rat PCFs, we determined whether ISO could stimulate PCFs to produce tachypnea in anesthetized rats, and, if so, whether this response resulted from ISO-induced depolarization of the pulmonary C neurons via the inhibition of IA and IK. We recorded ventilatory responses to 5% ISO exposure in anesthetized rats before and after blocking PCF conduction and the responses of pulmonary C neurons (extracellularly recorded) to ISO exposure. ISO-induced (1mM) changes in pulmonary C neuron membrane potential and IA/IK were tested using the perforated patch clamp technique. We found that: (1) ISO inhalation evoked a brief tachypnea (∼7s) and that this response disappeared after blocking PCF conduction; (2) the ISO significantly elevated (by 138%) the firing rate of most pulmonary C neurons (17 out of 21) in the nodose ganglion; and (3) ISO perfusion depolarized the pulmonary C neurons in the vitro and inhibited both IA and IK, and this evoked-depolarization was largely diminished after blocking both IA and IK. Our results suggest that ISO is able to stimulate PCFs to elicit tachypnea in rats, at least partly, via inhibiting IA and IK, thereby depolarizing the pulmonary C neurons. Copyright © 2013. Published by Elsevier B.V.

  10. Pharmacological correction of long QT-linked mutations in KCNH2 (hERG) increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum.

    PubMed

    Smith, Jennifer L; Reloj, Allison R; Nataraj, Parvathi S; Bartos, Daniel C; Schroder, Elizabeth A; Moss, Arthur J; Ohno, Seiko; Horie, Minoru; Anderson, Corey L; January, Craig T; Delisle, Brian P

    2013-11-01

    KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K(+) current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4-5 h after drug washout. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential.

  11. Pharmacological correction of long QT-linked mutations in KCNH2 (hERG) increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum

    PubMed Central

    Smith, Jennifer L.; Reloj, Allison R.; Nataraj, Parvathi S.; Bartos, Daniel C.; Schroder, Elizabeth A.; Moss, Arthur J.; Ohno, Seiko; Horie, Minoru; Anderson, Corey L.; January, Craig T.

    2013-01-01

    KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K+ current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4–5 h after drug washout. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential. PMID:23864605

  12. Role of voltage-gated K(+) channels in regulating Ca(2+) entry in rat cortical astrocytes.

    PubMed

    Wu, King-Chuen; Kuo, Chang-Shin; Chao, Chia-Chia; Huang, Chieh-Chen; Tu, Yuan-Kun; Chan, Paul; Leung, Yuk-Man

    2015-03-01

    Astrocytes have multiple functions such as provision of nourishment and mechanical support to the nervous system, helping to clear extracellular metabolites of neurons and modulating synaptic transmission by releasing gliotransmitters. In excitable cells, voltage-gated K(+) (Kv) channels serve to repolarize during action potentials. Astrocytes are considered non-excitable cells since they are not able to generate action potentials. There is an abundant expression of various Kv channels in astrocytes but the functions of these Kv channels remain unclear. We examined whether these astrocyte Kv channels regulate astrocyte "excitability" in the form of cytosolic Ca(2+) signaling. Electrophysiological examination revealed that neonatal rat cortical astrocytes possessed both delayed rectifier type and A-type Kv channels. Pharmacological blockade of both delayed rectifier Kv channels by TEA and A-type Kv channels by quinidine significantly suppressed store-operated Ca(2+) influx; however, TEA alone or quinidine alone did not suffice to cause such suppression. TEA and quinidine together dramatically enhanced current injection-triggered membrane potential overshoot (depolarization); either drug alone caused much smaller enhancements. Taken together, the results suggest both delayed rectifier and A-type Kv channels regulate astrocyte Ca(2+) signaling via controlling membrane potential.

  13. [Voltage-gated potassium channels and human neurological diseases].

    PubMed

    Jin, Hong-Wei; Wang, Xiao-Liang

    2002-01-01

    Voltage-gated potassium channels (Kv) is the largest, most complex in potassium channel superfamily. It can be divided into Kv alpha subunit and auxiliary two groups. The roles of some Kv channels types, e.g. rapidly inactivating (A-Type channel) and muscarine sensitive channels (M-type channel) are beginning to be understood. They are prominent in nervous system, acting in delicate and accurate ways to control or modify many physiological and pathological functions including membrane excitability, neurotransmitter release, cell proliferation or degeneration, signal transduction in neuronal network. Many human neurological disease pathogenesis are found to be related to mutant of Kv-channels subunit or subtype, such as, learning and memory impairing, ataxia, epilepsy, deafness, etc.

  14. Effect of Intercalated Water on Potassium Ion Transport through Kv1.2 Channels Studied via On-the-Fly Free-Energy Parametrization.

    PubMed

    Paz, S Alexis; Maragliano, Luca; Abrams, Cameron F

    2018-05-08

    We introduce a two-dimensional version of the method called on-the-fly free energy parametrization (OTFP) to reconstruct free-energy surfaces using Molecular Dynamics simulations, which we name OTFP-2D. We first test the new method by reconstructing the well-known dihedral angles free energy surface of solvated alanine dipeptide. Then, we use it to investigate the process of K + ions translocation inside the Kv1.2 channel. By comparing a series of two-dimensional free energy surfaces for ion movement calculated with different conditions on the intercalated water molecules, we first recapitulate the widely accepted knock-on mechanism for ion translocation and then confirm that permeation occurs with water molecules alternated among the ions, in accordance with the latest experimental findings. From a methodological standpoint, our new OTFP-2D algorithm demonstrates the excellent sampling acceleration of temperature-accelerated molecular dynamics and the ability to efficiently compute 2D free-energy surfaces. It will therefore be useful in large variety complex biomacromolecular simulations.

  15. Weighing the evidence for a ternary protein complex mediating A-type K+ currents in neurons.

    PubMed

    Maffie, Jonathon; Rudy, Bernardo

    2008-12-01

    The subthreshold-operating A-type K(+) current in neurons (I(SA)) has important roles in the regulation of neuronal excitability, the timing of action potential firing and synaptic integration and plasticity. The channels mediating this current (Kv4 channels) have been implicated in epilepsy, the control of dopamine release, and the regulation of pain plasticity. It has been proposed that Kv4 channels in neurons are ternary complexes of three types of protein: pore forming subunits of the Kv4 subfamily and two types of auxiliary subunits, the Ca(2+) binding proteins KChIPs and the dipeptidyl peptidase-like proteins (DPPLs) DPP6 (also known as DPPX) and DPP10 (4 molecules of each per channel for a total of 12 proteins in the complex). Here we consider the evidence supporting this hypothesis. Kv4 channels in many neurons are likely to be ternary complexes of these three types of protein. KChIPs and DPPLs are required to efficiently traffic Kv4 channels to the plasma membrane and regulate the functional properties of the channels. These proteins may also be important in determining the localization of the channels to specific neuronal compartments, their dynamics, and their response to neuromodulators. A surprisingly large number of additional proteins have been shown to modify Kv4 channels in heterologous expression systems, but their association with native Kv4 channels in neurons has not been properly validated. A critical consideration of the evidence suggests that it is unlikely that association of Kv4 channels with these additional proteins is widespread in the CNS. However, we cannot exclude that some of these proteins may associate with the channels transiently or in specific neurons or neuronal compartments, or that they may associate with the channels in other tissues.

  16. Enhancing the Predictive Power of Mutations in the C-Terminus of the KCNQ1-Encoded Kv7.1 Voltage-Gated Potassium Channel.

    PubMed

    Kapplinger, Jamie D; Tseng, Andrew S; Salisbury, Benjamin A; Tester, David J; Callis, Thomas E; Alders, Marielle; Wilde, Arthur A M; Ackerman, Michael J

    2015-04-01

    Despite the overrepresentation of Kv7.1 mutations among patients with a robust diagnosis of long QT syndrome (LQTS), a background rate of innocuous Kv7.1 missense variants observed in healthy controls creates ambiguity in the interpretation of LQTS genetic test results. A recent study showed that the probability of pathogenicity for rare missense mutations depends in part on the topological location of the variant in Kv7.1's various structure-function domains. Since the Kv7.1's C-terminus accounts for nearly 50 % of the overall protein and nearly 50 % of the overall background rate of rare variants falls within the C-terminus, further enhancement in mutation calling may provide guidance in distinguishing pathogenic long QT syndrome type 1 (LQT1)-causing mutations from rare non-disease-causing variants in the Kv7.1's C-terminus. Therefore, we have used conservation analysis and a large case-control study to generate topology-based estimative predictive values to aid in interpretation, identifying three regions of high conservation within the Kv7.1's C-terminus which have a high probability of LQT1 pathogenicity.

  17. Biphasic Somatic A-Type K+ Channel Downregulation Mediates Intrinsic Plasticity in Hippocampal CA1 Pyramidal Neurons

    PubMed Central

    Jung, Sung-Cherl; Hoffman, Dax A.

    2009-01-01

    Since its original description, the induction of synaptic long-term potentiation (LTP) has been known to be accompanied by a lasting increase in the intrinsic excitability (intrinsic plasticity) of hippocampal neurons. Recent evidence shows that dendritic excitability can be enhanced by an activity-dependent decrease in the activity of A-type K+ channels. In the present manuscript, we examined the role of A-type K+ channels in regulating intrinsic excitability of CA1 pyramidal neurons of the hippocampus after synapse-specific LTP induction. In electrophysiological recordings we found that LTP induced a potentiation of excitability which was accompanied by a two-phased change in A-type K+ channel activity recorded in nucleated patches from organotypic slices of rat hippocampus. Induction of LTP resulted in an immediate but short lasting hyperpolarization of the voltage-dependence of steady-state A-type K+ channel inactivation along with a progressive, long-lasting decrease in peak A-current density. Blocking clathrin-mediated endocytosis prevented the A-current decrease and most measures of intrinsic plasticity. These results suggest that two temporally distinct but overlapping mechanisms of A-channel downregulation together contribute to the plasticity of intrinsic excitability. Finally we show that intrinsic plasticity resulted in a global enhancement of EPSP-spike coupling. PMID:19662093

  18. SP6616 as a new Kv2.1 channel inhibitor efficiently promotes β-cell survival involving both PKC/Erk1/2 and CaM/PI3K/Akt signaling pathways.

    PubMed

    Zhou, T T; Quan, L L; Chen, L P; Du, T; Sun, K X; Zhang, J C; Yu, L; Li, Y; Wan, P; Chen, L L; Jiang, B H; Hu, L H; Chen, J; Shen, X

    2016-05-05

    Kv2.1 as a voltage-gated potassium (Kv) channel subunit has a pivotal role in the regulation of glucose-stimulated insulin secretion (GSIS) and pancreatic β-cell apoptosis, and is believed to be a promising target for anti-diabetic drug discovery, although the mechanism underlying the Kv2.1-mediated β-cell apoptosis is obscure. Here, the small molecular compound, ethyl 5-(3-ethoxy-4-methoxyphenyl)-2-(4-hydroxy-3-methoxybenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (SP6616) was discovered to be a new Kv2.1 inhibitor. It was effective in both promoting GSIS and protecting β cells from apoptosis. Evaluation of SP6616 on either high-fat diet combined with streptozocin-induced type 2 diabetic mice or db/db mice further verified its efficacy in the amelioration of β-cell dysfunction and glucose homeostasis. SP6616 treatment efficiently increased serum insulin level, restored β-cell mass, decreased fasting blood glucose and glycated hemoglobin levels, and improved oral glucose tolerance. Mechanism study indicated that the promotion of SP6616 on β-cell survival was tightly linked to its regulation against both protein kinases C (PKC)/extracellular-regulated protein kinases 1/2 (Erk1/2) and calmodulin(CaM)/phosphatidylinositol 3-kinase(PI3K)/serine/threonine-specific protein kinase (Akt) signaling pathways. To our knowledge, this may be the first report on the underlying pathway responsible for the Kv2.1-mediated β-cell protection. In addition, our study has also highlighted the potential of SP6616 in the treatment of type 2 diabetes.

  19. Immunohistochemical localization of DPP10 in rat brain supports the existence of a Kv4/KChIP/DPPL ternary complex in neurons.

    PubMed

    Wang, Wan-Chen; Cheng, Chau-Fu; Tsaur, Meei-Ling

    2015-03-01

    Subthreshold A-type K(+) currents (ISA s) have been recorded from the cell bodies of hippocampal and neocortical interneurons as well as neocortical pyramidal neurons. Kv4 channels are responsible for the somatodendritic ISA s. It has been proposed that neuronal Kv4 channels are ternary complexes including pore-forming Kv4 subunits, K(+) channel-interacting proteins (KChIPs), and dipeptidyl peptidase-like proteins (DPPLs). However, colocalization evidence was still lacking. The distribution of DPP10 mRNA in rodent brain has been reported but its protein localization remains unknown. In this study, we generated a DPP10 antibody to label DPP10 protein in adult rat brain by immunohistochemistry. Absent from glia, DPP10 proteins appear mainly in the cell bodies of DPP10(+) neurons, not only at the plasma membrane but also in the cytoplasm. At least 6.4% of inhibitory interneurons in the hippocampus coexpressed Kv4.3, KChIP1, and DPP10, with the highest density in the CA1 strata alveus/oriens/pyramidale and the dentate hilus. Colocalization of Kv4.3/KChIP1/DPP10 was also detected in at least 6.9% of inhibitory interneurons scattered throughout the neocortex. Both hippocampal and neocortical Kv4.3/KChIP1/DPP10(+) inhibitory interneurons expressed parvalbumin or somatostatin, but not calbindin or calretinin. Furthermore, we found colocalization of Kv4.2/Kv4.3/KChIP3/DPP10 in neocortical layer 5 pyramidal neurons and olfactory bulb mitral cells. Together, although DPP10 is also expressed in some brain neurons lacking Kv4 (such as parvalbumin- and somatostatin-positive Golgi cells in the cerebellum), colocalization of DPP10 with Kv4 and KChIP at the plasma membrane of ISA -expressing neuron somata supports the existence of Kv4/KChIP/DPPL ternary complex in vivo. © 2014 Wiley Periodicals, Inc.

  20. Hydrogen Sulfide Targets the Cys320/Cys529 Motif in Kv4.2 to Inhibit the Ito Potassium Channels in Cardiomyocytes and Regularizes Fatal Arrhythmia in Myocardial Infarction

    PubMed Central

    Ma, Shan-Feng; Luo, Yan; Ding, Ying-Jiong; Chen, Ying; Pu, Shi-Xin; Wu, Hang-Jing; Wang, Zhong-Feng; Tao, Bei-Bei; Wang, Wen-Wei

    2015-01-01

    Abstract Aims: The mechanisms underlying numerous biological roles of hydrogen sulfide (H2S) remain largely unknown. We have previously reported an inhibitory role of H2S in the L-type calcium channels in cardiomyocytes. This prompts us to examine the mechanisms underlying the potential regulation of H2S on the ion channels. Results: H2S showed a novel inhibitory effect on Ito potassium channels, and this effect was blocked by mutation at the Cys320 and/or Cys529 residues of the Kv4.2 subunit. H2S broke the disulfide bridge between a pair of oxidized cysteine residues; however, it did not modify single cysteine residues. H2S extended action potential duration in epicardial myocytes and regularized fatal arrhythmia in a rat model of myocardial infarction. H2S treatment significantly increased survival by ∼1.4-fold in the critical 2-h time window after myocardial infarction with a protection against ventricular premature beats and fatal arrhythmia. However, H2S did not change the function of other ion channels, including IK1 and INa. Innovation and Conclusion: H2S targets the Cys320/Cys529 motif in Kv4.2 to regulate the Ito potassium channels. H2S also shows a potent regularizing effect against fatal arrhythmia in a rat model of myocardial infarction. The study provides the first piece of evidence for the role of H2S in regulating Ito potassium channels and also the specific motif in an ion channel labile for H2S regulation. Antioxid. Redox Signal. 23, 129–147. PMID:25756524

  1. Multiple modes of a-type potassium current regulation.

    PubMed

    Cai, Shi-Qing; Li, Wenchao; Sesti, Federico

    2007-01-01

    Voltage-dependent potassium (K+) channels (Kv) regulate cell excitability by controlling the movement of K+ ions across the membrane in response to changes in the cell voltage. The Kv family, which includes A-type channels, constitute the largest group of K+ channel genes within the superfamily of Na+, Ca2+ and K+ voltage-gated channels. The name "A-type" stems from the typical profile of these currents that results form the opposing effects of fast activation and inactivation. In neuronal cells, A-type currents (I(A)), determine the interval between two consecutive action potentials during repetitive firing. In cardiac muscle, A-type currents (I(to)), control the initial repolarization of the myocardium. Structurally, A-type channels are tetramers of alpha-subunits each containing six putative transmembrane domains including a voltage-sensor. A-type channels can be modulated by means of protein-protein interactions with so-called beta-subunits that control inactivation voltage sensitivity and other properties, and by post-transcriptional modifications such as phosphorylation or oxidation. Recently a new mode of A-type regulation has been discovered in the form of a class of hybrid beta-subunits that posses their own enzymatic activity. Here, we review the biophysical and physiological properties of these multiple modes of A-type channel regulation.

  2. Inhibitory effects of pimozide on cloned and native voltage-gated potassium channels.

    PubMed

    Zhang, Zhi-Hao; Lee, Yan T; Rhodes, Kenneth; Wang, Kewei; Argentieri, Thomas M; Wang, Qiang

    2003-07-04

    The primary goal of this study was to use the cloned neuronal Kv channels to test if pimozide (PMZD), an antipsychotic drug, modulates the activity of Kv channels. In CHO cells, PMZD blocked Kv2.1, a major neuronal delayed rectifier, in a manner that depends upon time and concentration. The estimated IC50 was 4.2 microM at +50 mV. Tail current analysis shows that PMZD reduced the amplitude of the currents, with no effect on the steady-state activation curve (V(1/2) from 14.1 to 11.1 mV) or the slope (16.7 vs. 14.0 mV). From -120 to -20 mV, PMZD did not impact the deactivation kinetics of Kv2.1. PMZD also blocked Kv1.1, another neuronal delayed rectifier, with 16.1 microM of IC50. When Kv1.1 was co-expressed with Kvbeta1, approximately 50% of the Kv1.1 were converted into an inactivating A-type current and the Kv1.1/Kvbeta1 A-type currents were insensitive to PMZD. PMZD (10 microM) had minimal effect on Kv1.4, and had no effect on the M-current candidates, KCNQ2 and KCNQ3 when co-expressed in Xenopus oocytes. In hippocampal neurons, PMZD inhibited the delayed rectifiers by approximately 60%, and A-type currents were insensitive to PMZD. The results suggest that PMZD inhibits certain neuronal Kv channels in heterologous expression systems and in hippocampal neurons. PMZD was less effective on A-type currents, presumably because its ability to block requires a prolonged opening of the K channels. It is thus conceivable that the time-dependent and/or subunit-specific inhibition of Kv channels may increase the release of neurotransmitters such as serotonin and glutamate.

  3. Kv1.5 in the immune system: the good, the bad, or the ugly?

    PubMed

    Felipe, Antonio; Soler, Concepció; Comes, Núria

    2010-01-01

    For the last 20 years, knowledge of the physiological role of voltage-dependent potassium channels (Kv) in the immune system has grown exponentially. Leukocytes express a limited repertoire of Kv channels, which contribute to the membrane potential. These proteins are involved in the immune response and are therefore considered good pharmacological targets. Although there is a clear consensus about the physiological relevance of Kv1.3, the expression and the role of Kv1.5 are controversial. However, recent reports indicate that certain heteromeric Kv1.3/Kv1.5 associations may provide insight on Kv1.5. Here, we summarize what is known about this issue and highlight the role of Kv1.5 partnership interactions that could be responsible for this debate. The Kv1.3/Kv1.5 heterotetrameric composition of the channel and their possible differential associations with accessory regulatory proteins warrant further investigation.

  4. Redox modulation of A-type K+ currents in pain-sensing dorsal root ganglion neurons.

    PubMed

    Hsieh, Chi-Pan

    2008-06-06

    Redox modulation of fast inactivation has been described in certain cloned A-type voltage-gated K(+) (Kv) channels in expressing systems, but the effects remain to be demonstrated in native neurons. In this study, we examined the effects of cysteine-specific redox agents on the A-type K(+) currents in acutely dissociated small diameter dorsal root ganglion (DRG) neurons from rats. The fast inactivation of most A-type currents was markedly removed or slowed by the oxidizing agents 2,2'-dithio-bis(5-nitropyridine) (DTBNP) and chloramine-T. Dithiothreitol, a reducing agent for the disulfide bond, restored the inactivation. These results demonstrated that native A-type K(+) channels, probably Kv1.4, could switch the roles between inactivating and non-inactivating K(+) channels via redox regulation in pain-sensing DRG neurons. The A-type channels may play a role in adjusting pain sensitivity in response to peripheral redox conditions.

  5. Pharmacological modulation of the voltage-gated neuronal Kv7/KCNQ/M-channel alters the intrinsic excitability and synaptic responses of pyramidal neurons in rat prefrontal cortex slices.

    PubMed

    Peng, Hui; Bian, Xi-Ling; Ma, Fu-Cui; Wang, Ke-Wei

    2017-09-01

    The prefrontal cortex (PFC) critical for higher cognition is implicated in neuropsychiatric diseases, such as Alzheimer's disease, depression and schizophrenia. The voltage-activated Kv7/KCNQ/M-channel or M-current modulates the neuronal excitability that defines the fundamental mechanism of brain function. However, whether M-current functions to regulate the excitability of PFC neurons remains elusive. In this study, we recorded the native M-current from PFC layer V pyramidal neurons in rat brain slices and showed that it modulated the intrinsic excitability and synaptic responses of PFC pyramidal neurons. Application of a specific M-channel blocker XE991 (40 μmol/L) or opener retigabine (10 μmol/L) resulted in inhibition or activation of M-current, respectively. In the current-clamp recordings, inhibition of M-current was evidenced by the increased average spike frequency and the reduced first inter-spike interval (ISI), spike onset latency and fast afterhyperpolarization (fAHP), whereas activation of M-current caused opposite responses. Furthermore, inhibition of M-current significantly increased the amplitude of excitatory postsynaptic potentials (EPSPs) and depolarized the resting membrane potential (RMP) without affecting the miniature EPSC (mEPSC) frequency. These data demonstrate that voltage-gated neuronal Kv7/KCNQ/M-current modulates the excitability and synaptic transmission of PFC neurons, suggesting that pharmacological modulation of M-current in the PFC may exert beneficial effects on cognitive deficits implicated in the pathophysiology of neuropsychiatric disorders.

  6. Single cell analysis of voltage-gated potassium channels that determines neuronal types of rat hypothalamic paraventricular nucleus neurons.

    PubMed

    Lee, S K; Lee, S; Shin, S Y; Ryu, P D; Lee, S Y

    2012-03-15

    The hypothalamic paraventricular nucleus (PVN), a site for the integration of both the neuroendocrine and autonomic systems, has heterogeneous cell composition. These neurons are classified into type I and type II neurons based on their electrophysiological properties. In the present study, we investigated the molecular identification of voltage-gated K+ (Kv) channels, which determines a distinctive characteristic of type I PVN neurons, by means of single-cell reverse transcription-polymerase chain reaction (RT-PCR) along with slice patch clamp recordings. In order to determine the mRNA expression profiles, firstly, the PVN neurons of male rats were classified into type I and type II neurons, and then, single-cell RT-PCR and single-cell real-time RT-PCR analysis were performed using the identical cell. The single-cell RT-PCR analysis revealed that Kv1.2, Kv1.3, Kv1.4, Kv4.1, Kv4.2, and Kv4.3 were expressed both in type I and in type II neurons, and several Kv channels were co-expressed in a single PVN neuron. However, we found that the expression densities of Kv4.2 and Kv4.3 were significantly higher in type I neurons than in type II neurons. Taken together, several Kv channels encoding A-type K+ currents are present both in type I and in type II neurons, and among those, Kv4.2 and Kv4.3 are the major Kv subunits responsible for determining the distinct electrophysiological properties. Thus these 2 Kv subunits may play important roles in determining PVN cell types and regulating PVN neuronal excitability. This study further provides key molecular mechanisms for differentiating type I and type II PVN neurons. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. The Kv7.2/Kv7.3 heterotetramer assembles with a random subunit arrangement.

    PubMed

    Stewart, Andrew P; Gómez-Posada, Juan Camilo; McGeorge, Jessica; Rouhani, Maral J; Villarroel, Alvaro; Murrell-Lagnado, Ruth D; Edwardson, J Michael

    2012-04-06

    Voltage-gated K(+) channels composed of Kv7.2 and Kv7.3 are the predominant contributors to the M-current, which plays a key role in controlling neuronal activity. Various lines of evidence have indicated that Kv7.2 and Kv7.3 form a heteromeric channel. However, the subunit stoichiometry and arrangement within this putative heteromer are so far unknown. Here, we have addressed this question using atomic force microscopy imaging of complexes between isolated Kv7.2/Kv7.3 channels and antibodies to epitope tags on the two subunits, Myc on Kv7.2 and HA on Kv7.3. Initially, tsA 201 cells were transiently transfected with equal amounts of cDNA for the two subunits. The heteromer was isolated through binding of either tag to immunoaffinity beads and then decorated with antibodies to the other tag. In both cases, the distribution of angles between pairs of bound antibodies had two peaks, at around 90° and around 180°, and in both cases the 90° peak was about double the size of the 180° peak. These results indicate that the Kv7.2/Kv7.3 heteromer generated by cells expressing approximately equal amounts of the two subunits assembles as a tetramer with a predominantly 2:2 subunit stoichiometry and with a random subunit arrangement. When the DNA ratio for the two subunits was varied, copurification experiments indicated that the subunit stoichiometry was variable and not fixed at 2:2. Hence, there are no constraints on either the subunit stoichiometry or the subunit arrangement.

  8. Amyloid beta peptide as a physiological modulator of neuronal 'A'-type K+ current.

    PubMed

    Plant, Leigh D; Webster, Nicola J; Boyle, John P; Ramsden, Martin; Freir, Darragh B; Peers, Chris; Pearson, Hugh A

    2006-11-01

    Control of neuronal spiking patterns resides, in part, in the type and degree of expression of voltage-gated K(+) channel subunits. Previous studies have revealed that soluble forms of the Alzheimer's disease associated amyloid beta protein (Abeta) can increase the 'A'-type current in neurones. In this study, we define the molecular basis for this increase and show that endogenous production of Abeta is important in the modulation of Kv4.2 and Kv4.3 subunit expression in central neurones. A-type K(+) currents, and Kv4.2 and Kv4.3 subunit expression, were transiently increased in cerebellar granule neurones by the 1-40 and 1-42 forms of Abeta (100nM, 2-24h). Currents through recombinant Kv4.2 channels expressed in HEK293 cells were increased in a similar fashion to those through the native channels. Increases in 'A'-type current could be prevented by the use of cycloheximide and brefeldin A, indicating that protein expression and trafficking processes were altered by Abeta, rather than protein degredation. Endogenous Abeta production in cerebellar granule neurones was blocked using inhibitors of either gamma- or beta-secretase and resulted in decreased K(+) current. Crucially this could be prevented by co-application of exogenous Abeta (1nM), however, no change in Kv4.2 or Kv4.3 subunit expression occurred. These data show that Abeta is a modulator of Kv4 subunit expression in neurones at both the functional and the molecular level. Thus Abeta is not only involved in Alzheimer pathology, but is also an important physiological regulator of ion channel expression and hence neuronal excitability.

  9. Reduction in voltage-gated K+ channel activity in primary sensory neurons in painful diabetic neuropathy: role of brain-derived neurotrophic factor.

    PubMed

    Cao, Xue-Hong; Byun, Hee-Sun; Chen, Shao-Rui; Cai, You-Qing; Pan, Hui-Lin

    2010-09-01

    Abnormal hyperexcitability of primary sensory neurons plays an important role in neuropathic pain. Voltage-gated potassium (Kv) channels regulate neuronal excitability by affecting the resting membrane potential and influencing the repolarization and frequency of the action potential. In this study, we determined changes in Kv channels in dorsal root ganglion (DRG) neurons in a rat model of diabetic neuropathic pain. The densities of total Kv, A-type (IA) and sustained delayed (IK) currents were markedly reduced in medium- and large-, but not in small-, diameter DRG neurons in diabetic rats. Quantitative RT-PCR analysis revealed that the mRNA levels of IA subunits, including Kv1.4, Kv3.4, Kv4.2, and Kv4.3, in the DRG were reduced approximately 50% in diabetic rats compared with those in control rats. However, there were no significant differences in the mRNA levels of IK subunits (Kv1.1, Kv1.2, Kv2.1, and Kv2.2) in the DRG between the two groups. Incubation with brain-derived neurotrophic factor (BDNF) caused a large reduction in Kv currents, especially IA currents, in medium and large DRG neurons from control rats. Furthermore, the reductions in Kv currents and mRNA levels of IA subunits in diabetic rats were normalized by pre-treatment with anti-BDNF antibody or K252a, a TrkB tyrosine kinase inhibitor. In addition, the number of medium and large DRG neurons with BDNF immunoreactivity was greater in diabetic than control rats. Collectively, our findings suggest that diabetes primarily reduces Kv channel activity in medium and large DRG neurons. Increased BDNF activity in these neurons likely contributes to the reduction in Kv channel function through TrkB receptor stimulation in painful diabetic neuropathy.

  10. Contribution of kv7.4/kv7.5 heteromers to intrinsic and calcitonin gene-related peptide-induced cerebral reactivity.

    PubMed

    Chadha, Preet S; Jepps, Thomas A; Carr, Georgina; Stott, Jennifer B; Zhu, Hei-Lei; Cole, William C; Greenwood, Iain A

    2014-04-01

    Middle cerebral artery (MCA) diameter is regulated by inherent myogenic activity and the effect of potent vasodilators such as calcitonin gene-related peptide (CGRP). Previous studies showed that MCAs express KCNQ1, 4, and 5 potassium channel genes, and the expression products (Kv7 channels) participate in the myogenic control of MCA diameter. The present study investigated the contribution of Kv7.4 and Kv7.5 isoforms to myogenic and CGRP regulation of MCA diameter and determined whether they were affected in hypertensive animals. Isometric tension recordings performed on MCA from normotensive rats produced CGRP vasodilations that were inhibited by the pan-Kv7 channel blocker linopirdine (P<0.01) and after transfection of arteries with siRNA against KCNQ4 (P<0.01) but not KCNQ5. However, isobaric myography revealed that myogenic constriction in response to increases in intravascular pressure (20-80 mm Hg) was affected by both KCNQ4 and KCNQ5 siRNA. Proximity ligation assay signals were equally abundant for Kv7.4/Kv7.4 or Kv7.4/Kv7.5 antibody combinations but minimal for Kv7.5/Kv7.5 antibodies or Kv7.4/7.1 combinations. In contrast to systemic arteries, Kv7 function and Kv7.4 abundance in MCA were not altered in hypertensive rats. This study reveals, for the first time to our knowledge, that in cerebral arteries, Kv7.4 and Kv7.5 proteins exist predominantly as a functional heterotetramer, which regulates intrinsic myogenicity and vasodilation attributed to CGRP. Surprisingly, unlike systemic arteries, Kv7 activity in MCAs is not affected by the development of hypertension, and CGRP-mediated vasodilation is well maintained. As such, cerebrovascular Kv7 channels could be amenable for therapeutic targeting in conditions such as cerebral vasospasm.

  11. Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan’s syndrome and acquired neuromyotonia

    PubMed Central

    Irani, Sarosh R.; Alexander, Sian; Waters, Patrick; Kleopa, Kleopas A.; Pettingill, Philippa; Zuliani, Luigi; Peles, Elior; Buckley, Camilla; Lang, Bethan

    2010-01-01

    Antibodies that immunoprecipitate 125I-α-dendrotoxin-labelled voltage-gated potassium channels extracted from mammalian brain tissue have been identified in patients with neuromyotonia, Morvan’s syndrome, limbic encephalitis and a few cases of adult-onset epilepsy. These conditions often improve following immunomodulatory therapies. However, the proportions of the different syndromes, the numbers with associated tumours and the relationships with potassium channel subunit antibody specificities have been unclear. We documented the clinical phenotype and tumour associations in 96 potassium channel antibody positive patients (titres >400 pM). Five had thymomas and one had an endometrial adenocarcinoma. To define the antibody specificities, we looked for binding of serum antibodies and their effects on potassium channel currents using human embryonic kidney cells expressing the potassium channel subunits. Surprisingly, only three of the patients had antibodies directed against the potassium channel subunits. By contrast, we found antibodies to three proteins that are complexed with 125I-α-dendrotoxin-labelled potassium channels in brain extracts: (i) contactin-associated protein-2 that is localized at the juxtaparanodes in myelinated axons; (ii) leucine-rich, glioma inactivated 1 protein that is most strongly expressed in the hippocampus; and (iii) Tag-1/contactin-2 that associates with contactin-associated protein-2. Antibodies to Kv1 subunits were found in three sera, to contactin-associated protein-2 in 19 sera, to leucine-rich, glioma inactivated 1 protein in 55 sera and to contactin-2 in five sera, four of which were also positive for the other antibodies. The remaining 18 sera were negative for potassium channel subunits and associated proteins by the methods employed. Of the 19 patients with contactin-associated protein-antibody-2, 10 had neuromyotonia or Morvan’s syndrome, compared with only 3 of the 55 leucine-rich, glioma inactivated 1 protein

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

    PubMed

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

    2017-05-01

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

  13. Molecular cloning and functional expression of the K+ channel KV7.1 and the regulatory subunit KCNE1 from equine myocardium.

    PubMed

    Pedersen, Philip J; Thomsen, Kirsten B; Flak, Jon B; Tejada, Maria A; Hauser, Frank; Trachsel, Dagmar; Buhl, Rikke; Kalbfleisch, Theodore; DePriest, Michael Scott; MacLeod, James N; Calloe, Kirstine; Klaerke, Dan A

    2017-08-01

    The voltage-gated K + -channel K V 7.1 and the subunit KCNE1, encoded by the KCNQ1 and KCNE1 genes, respectively, are responsible for termination of the cardiac action potential. In humans, mutations in these genes can predispose patients to arrhythmias and sudden cardiac death (SCD). To characterize equine K V 7.1/KCNE1 currents and compare them to human K V 7.1/KCNE1 currents to determine whether K V 7.1/KCNE1 plays a similar role in equine and human hearts. mRNA encoding K V 7.1 and KCNE1 was isolated from equine hearts, sequenced, and cloned into expression vectors. The channel subunits were heterologously expressed in Xenopus laevis oocytes or CHO-K1 cells and characterized using voltage-clamp techniques. Equine K V 7.1/KCNE1 expressed in CHO-K1 cells exhibited electrophysiological properties that are overall similar to the human orthologs; however, a slower deactivation was found which could result in more open channels at fast rates. The results suggest that the equine K V 7.1/KCNE1 channel may be important for cardiac repolarization and this could indicate that horses are susceptible to SCD caused by mutations in KCNQ1 and KCNE1. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Effects of trimethoprim-sulfadiazine and detomidine on the function of equine Kv 11.1 channels in a two-electrode voltage-clamp (TEVC) oocyte model.

    PubMed

    Trachsel, D S; Tejada, M A; Groesfjeld Christensen, V; Pedersen, P J; Kanters, J K; Buhl, R; Calloe, K; Klaerke, D A

    2018-03-22

    The long QT syndrome (LQTS) is a channelopathy that can lead to severe arrhythmia and sudden cardiac death. Pharmacologically induced LQTS is caused by interaction between drugs and potassium channels, especially the K v 11.1 channel. Due to such interactions, numerous drugs have been withdrawn from the market or are administered with precautions in human medicine. However, some compounds, such as trimethoprim-sulfonamide combinations are still widely used in veterinarian medicine. Therefore, we investigate the effect of trimethoprim-sulfadiazine (TMS), trimethoprim, sulfadiazine, and detomidine on equine-specific K v 11.1 channels. K v 11.1 channels cloned from equine hearts were heterologously expressed in Xenopus laevis oocytes, and whole cell currents were measured by two-electrode voltage-clamp before and after drug application. TMS blocked equine K v 11.1 current with an IC 50 of 3.74 mm (95% CI: 2.95-4.73 mm) and affected the kinetics of activation and inactivation. Similar was found for trimethoprim but not for sulfadiazine, suggesting the effect is due to trimethoprim. Detomidine did not affect equine K v 11.1 current. Thus, equine K v 11.1 channels are also susceptible to pharmacological block, indicating that some drugs may have the potential to affect repolarization in horse. However, in vivo studies are needed to assess the potential risk of these drugs to induce equine LQTS. © 2018 The Authors. Journal of Veterinary Pharmacology and Therapeutics Published by John Wiley & Sons Ltd.

  15. Transient Hippocampal Down-Regulation of Kv1.1 Subunit mRNA during Associative Learning in Rats

    ERIC Educational Resources Information Center

    Kourrich, Said; Manrique, Christine; Salin, Pascal; Mourre, Christiane

    2005-01-01

    Voltage-gated potassium channels (Kv) are critically involved in learning and memory processes. It is not known, however, whether the expression of the Kv1.1 subunit, constituting Kv1 channels, can be specifically regulated in brain areas important for learning and memory processing. Radioactive in situ hybridization was used to evaluate the…

  16. A-type voltage-gated K+ currents influence firing properties of isolectin B4-positive but not isolectin B4-negative primary sensory neurons.

    PubMed

    Vydyanathan, Amaresh; Wu, Zi-Zhen; Chen, Shao-Rui; Pan, Hui-Lin

    2005-06-01

    Voltage-gated K+ channels (Kv) in primary sensory neurons are important for regulation of neuronal excitability. The dorsal root ganglion (DRG) neurons are heterogeneous, and the types of native Kv currents in different groups of nociceptive DRG neurons are not fully known. In this study, we determined the difference in the A-type Kv current and its influence on the firing properties between isolectin B4 (IB4)-positive and -negative DRG neurons. Whole cell voltage- and current-clamp recordings were performed on acutely dissociated small DRG neurons of rats. The total Kv current density was significantly higher in IB+-positive than that in IB(4)-negative neurons. Also, 4-aminopyridine (4-AP) produced a significantly greater reduction in Kv currents in IB4-positive than in IB4-negative neurons. In contrast, IB4-negative neurons exhibited a larger proportion of tetraethylammonium-sensitive Kv currents. Furthermore, IB4-positive neurons showed a longer latency of firing and required a significantly larger amount of current injection to evoke action potentials. 4-AP significantly decreased the latency of firing and increased the firing frequency in IB4-positive but not in IB4-negative neurons. Additionally, IB4-positive neurons are immunoreactive to Kv1.4 but not to Kv1.1 and Kv1.2 subunits. Collectively, this study provides new information that 4-AP-sensitive A-type Kv currents are mainly present in IB4-positive DRG neurons and preferentially dampen the initiation of action potentials of this subpopulation of nociceptors. The difference in the density of A-type Kv currents contributes to the distinct electrophysiological properties of IB4-positive and -negative DRG neurons.

  17. Analysis on the Operation Strategy of the 220kV External Transmission Channel for the Nujiang Power Grid after the Installation of Series Compensation

    NASA Astrophysics Data System (ADS)

    Liu, Xiaoxin; Feng, Peilei; Jan, Lisheng; Dai, Xiaozhong; Cai, Pengcheng

    2018-01-01

    In recent years, Nujiang Prefecture vigorously develop hydropower, the grid structure in the northwest of Yunnan Province is not perfect, which leads to the research and construction of the power grid lags behind the development of the hydropower. In 2015, the company in view of the nu river hydropower dilemma decided to change outside the nu river to send out a passage with series compensation device in order to improve the transmission capacity, the company to the main problems related to the system plan, but not too much in the region distribution network and detailed study. Nujiang power grid has unique structure and properties of the nujiang power grid after respectively, a whole rack respectively into two parts, namely power delivery channels, load power supply, the whole grid occurred fundamental changes, the original strategy of power network is not applicable, especially noteworthy is the main failure after network of independent operation problem, how to avoid the local series, emergency problem is more urgent, very tolerance test area power grid, this paper aims at the analysis of existing data, simulation, provide a reference for respectively after the operation for the stable operation of the power grid.

  18. Patients With Long-QT Syndrome Caused by Impaired hERG-Encoded Kv11.1 Potassium Channel Have Exaggerated Endocrine Pancreatic and Incretin Function Associated With Reactive Hypoglycemia

    PubMed Central

    Hyltén-Cavallius, Louise; Iepsen, Eva W.; Wewer Albrechtsen, Nicolai J.; Svendstrup, Mathilde; Lubberding, Anniek F.; Hartmann, Bolette; Jespersen, Thomas; Linneberg, Allan; Christiansen, Michael; Vestergaard, Henrik; Pedersen, Oluf; Holst, Jens J.; Kanters, Jørgen K.

    2017-01-01

    Background: Loss-of-function mutations in hERG (encoding the Kv11.1 voltage-gated potassium channel) cause long-QT syndrome type 2 (LQT2) because of prolonged cardiac repolarization. However, Kv11.1 is also present in pancreatic α and β cells and intestinal L and K cells, secreting glucagon, insulin, and the incretins glucagon-like peptide-1 (GLP-1) and GIP (glucose-dependent insulinotropic polypeptide), respectively. These hormones are crucial for glucose regulation, and long-QT syndrome may cause disturbed glucose regulation. We measured secretion of these hormones and cardiac repolarization in response to glucose ingestion in LQT2 patients with functional mutations in hERG and matched healthy participants, testing the hypothesis that LQT2 patients have increased incretin and β-cell function and decreased α-cell function, and thus lower glucose levels. Methods: Eleven patients with LQT2 and 22 sex-, age-, and body mass index–matched control participants underwent a 6-hour 75-g oral glucose tolerance test with ECG recording and blood sampling for measurements of glucose, insulin, C-peptide, glucagon, GLP-1, and GIP. Results: In comparison with matched control participants, LQT2 patients had 56% to 78% increased serum insulin, serum C-peptide, plasma GLP-1, and plasma GIP responses (P=0.03–0.001) and decreased plasma glucose levels after glucose ingestion (P=0.02) with more symptoms of hypoglycemia (P=0.04). Sixty-three percent of LQT2 patients developed hypoglycemic plasma glucose levels (<70 mg/dL) versus 36% control participants (P=0.16), and 18% patients developed serious hypoglycemia (<50 mg/dL) versus none of the controls. LQT2 patients had defective glucagon responses to low glucose, P=0.008. β-Cell function (Insulin Secretion Sensitivity Index-2) was 2-fold higher in LQT2 patients than in controls (4398 [95% confidence interval, 2259–8562] versus 2156 [1961–3201], P=0.03). Pharmacological Kv11.1 blockade (dofetilide) in rats had similar effect

  19. Exploring O2 Diffusion in A-Type Cytochrome c Oxidases: Molecular Dynamics Simulations Uncover Two Alternative Channels towards the Binuclear Site

    PubMed Central

    Oliveira, A. Sofia F.; Damas, João M.; Baptista, António M.; Soares, Cláudio M.

    2014-01-01

    Cytochrome c oxidases (Ccoxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type Ccox family, little is known about the channel(s) used by O2 to travel from the solvent/membrane to the heme a3-CuB binuclear center (BNC). Moreover, the identification of all possible O2 channels as well as the atomic details of O2 diffusion is essential for the understanding of the working mechanisms of the A-type Ccox. In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O2 molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O2 inside Ccox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O2 to reach the BNC. Both alternative O2 channels start in the membrane spanning region and terminate close to Y288I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated