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1
Al-Sabi, Ahmed, Oleg Shamotienko, Sorcha Ni Dhochartaigh, Nagesh Muniyappa, Marie Le Berre, Hamdy Shaban, Jiafu Wang, Jon T. Sack, and J. Oliver Dolly. "Arrangement of Kv1 α subunits dictates sensitivity to tetraethylammonium." Journal of General Physiology 136, no. 3 (August 30, 2010): 273–82. http://dx.doi.org/10.1085/jgp.200910398.
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Shaker-related Kv1 channels contain four channel-forming α subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 α subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels’ properties. Kv1.1 and 1.2 α genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K+ currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2–1.1-1.2-1.1). Pore-blocking petidergic toxins, α dendrotoxin, agitoxin-1, tityustoxin-Kα, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of α subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity.
2
Denisova, Kristina R., Nikita A. Orlov, Sergey A. Yakimov, Mikhail P. Kirpichnikov, Alexey V. Feofanov, and Oksana V. Nekrasova. "Atto488-Agitoxin 2—A Fluorescent Ligand with Increased Selectivity for Kv1.3 Channel Binding Site." Bioengineering 9, no. 7 (July 1, 2022): 295. http://dx.doi.org/10.3390/bioengineering9070295.
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Fluorescently labeled peptide blockers of ion channels are useful probes in studying the localization and functioning of the channels and in the performance of a search for new channel ligands with bioengineering screening systems. Here, we report on the properties of Atto488-agitoxin 2 (A-AgTx2), a derivative of the Kv1 channel blocker agitoxin 2 (AgTx2), which was N-terminally labeled with Atto 488 fluorophore. The interactions of A-AgTx2 with the outer binding sites of the potassium voltage-gated Kv1.x (x = 1, 3, 6) channels were studied using bioengineered hybrid KcsA–Kv1.x (x = 1, 3, 6) channels. In contrast to AgTx2, A-AgTx2 was shown to lose affinity for the Kv1.1 and Kv1.6 binding sites but to preserve it for the Kv1.3 site. Thus, Atto488 introduces two new functionalities to AgTx2: fluorescence and the selective targeting of the Kv1.3 channel, which is known for its pharmacological significance. In the case of A-AgTx2, fluorescent labeling served as an alternative to site-directed mutagenesis in modulating the pharmacological profile of the channel blocker. Although the affinity of A-AgTx2 for the Kv1.3 binding site was decreased as compared to the unlabeled AgTx2, its dissociation constant value was within a low nanomolar range (4.0 nM). The properties of A-AgTx2 allow one to use it for the search and study of Kv1.3 channel blockers as well as to consider it for the imaging of the Kv1.3 channel in cells and tissues.
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Large, R. J., M. A. Hollywood, G. P. Sergeant, K. D. Thornbury, S. Bourke, J. R. Levick, and N. G. McHale. "Ionic currents in intimal cultured synoviocytes from the rabbit." American Journal of Physiology-Cell Physiology 299, no. 5 (November 2010): C1180—C1194. http://dx.doi.org/10.1152/ajpcell.00028.2010.
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Hyaluronan, a joint lubricant and regulator of synovial fluid content, is secreted by fibroblast-like synoviocytes lining the joint cavity, and secretion is greatly stimulated by Ca2+-dependent protein kinase C. This study aimed to define synoviocyte membrane currents and channels that may influence synoviocyte Ca2+ dynamics. Resting membrane potential ranged from −30 mV to −66 mV (mean −45 ± 8.60 mV, n = 40). Input resistance ranged from 0.54 GΩ to 2.6 GΩ (mean 1.28 ± 0.57 GΩ; ν = 33). Cell capacitance averaged 97.97 ± 5.93 pF. Voltage clamp using Cs+ pipette solution yielded a transient inward current that disappeared in Ca2+-free solutions and was blocked by 1 μM nifedipine, indicating an L-type calcium current. The current was increased fourfold by the calcium channel activator FPL 64176 (300 nM). Using K+ pipette solution, depolarizing steps positive to −40 mV evoked an outward current that showed kinetics and voltage dependence of activation and inactivation typical of the delayed rectifier potassium current. This was blocked by the nonspecific delayed rectifier blocker 4-aminopyridine. The synoviocytes expressed mRNA for four Kv1 subtypes (Kv1.1, Kv1.4, Kv1.5, and Kv1.6). Correolide (1 μM), margatoxin (100 nM), and α-dendrotoxin block these Kv1 subtypes, and all of these drugs significantly reduced synoviocyte outward current. The current was blocked most effectively by 50 nM κ-dendrotoxin, which is specific for channels containing a Kv1.1 subunit, indicating that Kv1.1 is critical, either as a homomultimeric channel or as a component of a heteromultimeric Kv1 channel. When 50 nM κ-dendrotoxin was added to current-clamped synoviocytes, the cells depolarized by >20 mV and this was accompanied by an increase in intracellular calcium concentration. Similarly, depolarization of the cells with high external potassium solution caused an increase in intracellular calcium, and this effect was greatly reduced by 1 μM nifedipine. In conclusion, fibroblast-like synoviocytes cultured from the inner synovium of the rabbit exhibit voltage-dependent inward and outward currents, including Ca2+ currents. They thus express ion channels regulating membrane Ca2+ permeability and electrochemical gradient. Since Ca2+-dependent kinases are major regulators of synovial hyaluronan secretion, the synoviocyte ion channels are likely to be important in the regulation of hyaluronan secretion.
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D’Adamo, Maria Cristina, Antonella Liantonio, Jean-Francois Rolland, Mauro Pessia, and Paola Imbrici. "Kv1.1 Channelopathies: Pathophysiological Mechanisms and Therapeutic Approaches." International Journal of Molecular Sciences 21, no. 8 (April 22, 2020): 2935. http://dx.doi.org/10.3390/ijms21082935.
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Kv1.1 belongs to the Shaker subfamily of voltage-gated potassium channels and acts as a critical regulator of neuronal excitability in the central and peripheral nervous systems. KCNA1 is the only gene that has been associated with episodic ataxia type 1 (EA1), an autosomal dominant disorder characterized by ataxia and myokymia and for which different and variable phenotypes have now been reported. The iterative characterization of channel defects at the molecular, network, and organismal levels contributed to elucidating the functional consequences of KCNA1 mutations and to demonstrate that ataxic attacks and neuromyotonia result from cerebellum and motor nerve alterations. Dysfunctions of the Kv1.1 channel have been also associated with epilepsy and kcna1 knock-out mouse is considered a model of sudden unexpected death in epilepsy. The tissue-specific association of Kv1.1 with other Kv1 members, auxiliary and interacting subunits amplifies Kv1.1 physiological roles and expands the pathogenesis of Kv1.1-associated diseases. In line with the current knowledge, Kv1.1 has been proposed as a novel and promising target for the treatment of brain disorders characterized by hyperexcitability, in the attempt to overcome limited response and side effects of available therapies. This review recounts past and current studies clarifying the roles of Kv1.1 in and beyond the nervous system and its contribution to EA1 and seizure susceptibility as well as its wide pharmacological potential.
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Yuan, Xiao-Jian, Jian Wang, Magdalena Juhaszova, Vera A. Golovina, and Lewis J. Rubin. "Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 274, no. 4 (April 1, 1998): L621—L635. http://dx.doi.org/10.1152/ajplung.1998.274.4.l621.
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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+]cytand explored the molecular identification of voltage-gated K+(KV)- and Ca2+-activated K+(KCa)-channel genes expressed in pulmonary arterial smooth muscle cells (PASMC). Two kinetically distinct KV currents [ I K(V)], a rapidly inactivating (A-type) and a noninactivating delayed rectifier, as well as a slowly activated KCa current [ I K(Ca)] were identified. I K(V) was reversibly inhibited by 4-aminopyridine (5 mM), whereas I K(Ca) was significantly inhibited by charybdotoxin (10–20 nM). K+ channels are composed of pore-forming α-subunits and auxiliary β-subunits. Five KV-channel α-subunit genes from the Shaker subfamily (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), a KV-channel α-subunit gene from the Shab subfamily (KV2.1), a KV-channel modulatory α-subunit (KV9.3), and a KCa-channel α-subunit gene ( rSlo), as well as three KV-channel β-subunit genes (KVβ1.1, KVβ2, and KVβ3) are expressed in PASMC. The data suggest that 1) native K+ channels in PASMC are encoded by multiple genes; 2) the delayed rectifier I K(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 I K(V) may be generated by the KV1.4 channel and/or the delayed rectifier KV channels (KV1 subfamily) associated with β-subunits; and 4) the I K(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+]cytin PASMC.
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Rash, John E., Kimberly G. Vanderpool, Thomas Yasumura, Jordan Hickman, Jonathan T. Beatty, and James I. Nagy. "KV1 channels identified in rodent myelinated axons, linked to Cx29 in innermost myelin: support for electrically active myelin in mammalian saltatory conduction." Journal of Neurophysiology 115, no. 4 (April 1, 2016): 1836–59. http://dx.doi.org/10.1152/jn.01077.2015.
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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 mammalian myelinated axons.
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Gladkikh, Irina, Steve Peigneur, Oksana Sintsova, Ernesto Lopes Pinheiro-Junior, Anna Klimovich, Alexander Menshov, Anatoly Kalinovsky, et al. "Kunitz-Type Peptides from the Sea Anemone Heteractis crispa Demonstrate Potassium Channel Blocking and Anti-Inflammatory Activities." Biomedicines 8, no. 11 (November 4, 2020): 473. http://dx.doi.org/10.3390/biomedicines8110473.
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The Kunitz/BPTI peptide family includes unique representatives demonstrating various biological activities. Electrophysiological screening of peptides HCRG1 and HCRG2 from the sea anemone Heteractis crispa on six Kv1.x channel isoforms and insect Shaker IR channel expressed in Xenopus laevis oocytes revealed their potassium channels blocking activity. HCRG1 and HCRG2 appear to be the first Kunitz-type peptides from sea anemones blocking Kv1.3 with IC50 of 40.7 and 29.7 nM, respectively. In addition, peptides mainly vary in binding affinity to the Kv1.2 channels. It was established that the single substitution, Ser5Leu, in the TRPV1 channel antagonist, HCRG21, induces weak blocking activity of Kv1.1, Kv1.2, and Kv1.3. Apparently, for the affinity and selectivity of Kunitz-fold toxins to Kv1.x isoforms, the number and distribution along their molecules of charged, hydrophobic, and polar uncharged residues, as well as the nature of the channel residue at position 379 (Tyr, Val or His) are important. Testing the compounds in a model of acute local inflammation induced by the introduction of carrageenan administration into mice paws revealed that HCRG1 at doses of 0.1–1 mg/kg reduced the volume of developing edema during 24 h, similar to the effect of the nonsteroidal anti-inflammatory drug, indomethacin, at a dose of 5 mg/kg. ELISA analysis of the animals blood showed that the peptide reduced the synthesis of TNF-α, a pro-inflammatory mediator playing a leading role in the development of edema in this model.
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Imbrici, Paola, Maria Cristina D'Adamo, Antonella Cusimano, and Mauro Pessia. "Episodic ataxia type 1 mutation F184C alters Zn2+-induced modulation of the human K+ channel Kv1.4-Kv1.1/Kvβ1.1." American Journal of Physiology-Cell Physiology 292, no. 2 (February 2007): C778—C787. http://dx.doi.org/10.1152/ajpcell.00259.2006.
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Episodic ataxia type 1 (EA1) is a Shaker-like channelopathy characterized by continuous myokymia and attacks of imbalance with jerking movements of the head, arms, and legs. Although altered expression and gating properties of Kv1.1 channels underlie EA1, several disease-causing mechanisms remain poorly understood. It is likely that Kv1.1, Kv1.4, and Kvβ1.1 subunits form heteromeric channels at hippocampal mossy fiber boutons from which Zn2+ ions are released into the synaptic cleft in a Ca2+-dependent fashion. The sensitivity of this macromolecular channel complex to Zn2+ is unknown. Here, we show that this heteromeric channel possesses a high-affinity (<10 μM) and a low-affinity (<0.5 mM) site for Zn2+, which are likely to regulate channel availability at distinct presynaptic membranes. Furthermore, the EA1 mutation F184C, located within the S1 segment of the Kv1.1 subunit, markedly decreased the equilibrium dissociation constants for Zn2+ binding to the high- and low-affinity sites. The functional characterization of the Zn2+ effects on heteromeric channels harboring the F184C mutation also showed that this ion significantly 1) slowed the activation rate of the channel, 2) increased the time to reach peak current amplitude, 3) decreased the rate and amount of current undergoing N-type inactivation, and 4) slowed the repriming of the channel compared with wild-type channels. These results demonstrate that the EA1 mutation F184C will not only sensitize the homomeric Kv1.1 channel to extracellular Zn2+, but it will also endow heteromeric channels with a higher sensitivity to this metal ion. During the vesicular release of Zn2+, its effects will be in addition to the intrinsic gating defects caused by the mutation, which is likely to exacerbate the symptoms by impairing the integration and transmission of signals within specific brain areas.
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Brock, Mathew W., Chris Mathes, and William F. Gilly. "Selective Open-Channel Block of Shaker (Kv1) Potassium Channels by S-Nitrosodithiothreitol (Sndtt)." Journal of General Physiology 118, no. 1 (June 27, 2001): 113–34. http://dx.doi.org/10.1085/jgp.118.1.113.
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Large quaternary ammonium (QA) ions block voltage-gated K+ (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH2CH(OH)CH(OH)CH2SNO) produces qualitatively similar “open-channel block” in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na+ or Ca2+ channels. Inactivation-removed ShakerB (ShBΔ) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose–response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (Kd2 = 0.14 mM) is lower than that of the first molecule (Kd1 = 0.67 mM), indicating cooperativity. The half-blocking concentration (K1/2) is ∼0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about −0.3 e0) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (τ ≈ 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1–1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.
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Platoshyn, Oleksandr, Carmelle V. Remillard, Ivana Fantozzi, Mehran Mandegar, Tiffany T. Sison, Shen Zhang, Elyssa Burg, and Jason X. J. Yuan. "Diversity of voltage-dependent K+ channels in human pulmonary artery smooth muscle cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 287, no. 1 (July 2004): L226—L238. http://dx.doi.org/10.1152/ajplung.00438.2003.
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Electrical excitability, which plays an important role in excitation-contraction coupling in the pulmonary vasculature, is regulated by transmembrane ion flux in pulmonary artery smooth muscle cells (PASMC). This study examined the heterogeneous nature of native voltage-dependent K+ channels in human PASMC. Both voltage-gated K+ (KV) currents and Ca2+-activated K+ (KCa) currents were observed and characterized. In cell-attached patches of PASMC bathed in Ca2+-containing solutions, depolarization elicited a wide range of K+ unitary conductances (6–290 pS). When cells were dialyzed with Ca2+-free and K+-containing solutions, depolarization elicited four components of KV currents in PASMC based on the kinetics of current activation and inactivation. Using RT-PCR, we detected transcripts of 1) 22 KV channel α-subunits (KV1.1–1.7, KV1.10, KV2.1, KV3.1, KV3.3–3.4, KV4.1–4.2, KV5.1, KV 6.1–6.3, KV9.1, KV9.3, KV10.1, and KV11.1), 2) three KV channel β-subunits (KVβ1–3), 3) four KCa channel α-subunits ( Slo-α1 and SK2–SK4), and 4) four KCa channel β-subunits (KCaβ1–4). Our results show that human PASMC exhibit a variety of voltage-dependent K+ currents with variable kinetics and conductances, which may result from various unique combinations of α- and β-subunits forming the native channels. Functional expression of these channels plays a critical role in the regulation of membrane potential, cytoplasmic Ca2+, and pulmonary vasomotor tone.
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Zhao, Juan, Dimitri Petitjean, Georges A. Haddad, Zarah Batulan, and Rikard Blunck. "A Common Kinetic Property of Mutations Linked to Episodic Ataxia Type 1 Studied in the Shaker Kv Channel." International Journal of Molecular Sciences 21, no. 20 (October 14, 2020): 7602. http://dx.doi.org/10.3390/ijms21207602.
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(1) Background: Episodic ataxia type 1 is caused by mutations in the KCNA1 gene encoding for the voltage-gated potassium channel Kv1.1. There have been many mutations in Kv1.1 linked to episodic ataxia reported and typically investigated by themselves or in small groups. The aim of this article is to determine whether we can define a functional parameter common to all Kv1.1 mutants that have been linked to episodic ataxia. (2) Methods: We introduced the disease mutations linked to episodic ataxia in the drosophila analog of Kv1.1, the Shaker Kv channel, and expressed the channels in Xenopus oocytes. Using the cut-open oocyte technique, we characterized the gating and ionic currents. (3) Results: We found that the episodic ataxia mutations variably altered the different gating mechanisms described for Kv channels. The common characteristic was a conductance voltage relationship and inactivation shifted to less polarized potentials. (4) Conclusions: We suggest that a combination of a prolonged action potential and slowed and incomplete inactivation leads to development of ataxia when Kv channels cannot follow or adapt to high firing rates.
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Chen, Gang, Wangcai Gao, Kenneth C. Reinert, Laurentiu S. Popa, Claudia M. Hendrix, M. Elizabeth Ross, and Timothy J. Ebner. "Involvement of Kv1 Potassium Channels in Spreading Acidification and Depression in the Cerebellar Cortex." Journal of Neurophysiology 94, no. 2 (August 2005): 1287–98. http://dx.doi.org/10.1152/jn.00224.2005.
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Spreading acidification and depression (SAD) is a form of propagated activity in the cerebellar cortex characterized by acidification and a transient depression in excitability. This study investigated the role of Kv1 potassium channels in SAD using neutral red, flavoprotein autofluorescence, and voltage-sensitive dye optical imaging in the mouse cerebellar cortex, in vivo. The probability of evoking SAD was greatly increased by blocking Kv1.1 as well as Kv1.2 potassium channels by their specific blockers dendrotoxin K (DTX-K) and tityustoxin (TsTX), respectively. DTX-K not only greatly lowered the threshold for evoking SAD but also resulted in multiple cycles of spread and spontaneous SAD. The occurrence of spontaneous SAD originating from spontaneous parallel fiber-like beams of activity suggests that blocking Kv1 channels increased parallel fiber excitability. This was confirmed by the generation of parallel fiber-like beams with the microinjection of glutamate into the upper molecular layer in the presence of DTX-K. The dramatic effects of DTX-K suggest a possible connection between SAD and episodic ataxia type 1 (EA1), a Kv1.1 potassium channelopathy. The threshold for evoking SAD was significantly lowered in the Kv1.1 heterozygous knockout mouse compared with wild-type littermates. Carbamazepine and acetazolamide, both effective in the treatment of EA1, significantly decreased the likelihood of evoking SAD. Blocking GABAergic neurotransmission did not alter the effectiveness of DTX-K. The cyclin D2 null mouse, which lacks cerebellar stellate cells, also exhibited SAD. Therefore blocking Kv1 potassium channels establishes the conditions needed to generate SAD. Furthermore, the results are consistent with the hypothesis that SAD may underlie the transient attacks of ataxia characterizing EA1.
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Dinoi, Giorgia, Michael Morin, Elena Conte, Hagar Mor Shaked, Maria Antonietta Coppola, Maria Cristina D’Adamo, Orly Elpeleg, et al. "Clinical and Functional Study of a De Novo Variant in the PVP Motif of Kv1.1 Channel Associated with Epilepsy, Developmental Delay and Ataxia." International Journal of Molecular Sciences 23, no. 15 (July 22, 2022): 8079. http://dx.doi.org/10.3390/ijms23158079.
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Mutations in the KCNA1 gene, encoding the voltage-gated potassium channel Kv1.1, have been associated with a spectrum of neurological phenotypes, including episodic ataxia type 1 and developmental and epileptic encephalopathy. We have recently identified a de novo variant in KCNA1 in the highly conserved Pro-Val-Pro motif within the pore of the Kv1.1 channel in a girl affected by early onset epilepsy, ataxia and developmental delay. Other mutations causing severe epilepsy are located in Kv1.1 pore domain. The patient was initially treated with a combination of antiepileptic drugs with limited benefit. Finally, seizures and ataxia control were achieved with lacosamide and acetazolamide. The aim of this study was to functionally characterize Kv1.1 mutant channel to provide a genotype–phenotype correlation and discuss therapeutic options for KCNA1-related epilepsy. To this aim, we transfected HEK 293 cells with Kv1.1 or P403A cDNAs and recorded potassium currents through whole-cell patch-clamp. P403A channels showed smaller potassium currents, voltage-dependent activation shifted by +30 mV towards positive potentials and slower kinetics of activation compared with Kv1.1 wild-type. Heteromeric Kv1.1+P403A channels, resembling the condition of the heterozygous patient, confirmed a loss-of-function biophysical phenotype. Overall, the functional characterization of P403A channels correlates with the clinical symptoms of the patient and supports the observation that mutations associated with severe epileptic phenotype cluster in a highly conserved stretch of residues in Kv1.1 pore domain. This study also strengthens the beneficial effect of acetazolamide and sodium channel blockers in KCNA1 channelopathies.
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Hsiao, Chie-Fang, Gurvinder Kaur, Angela Vong, Harpreet Bawa, and Scott H. Chandler. "Participation of Kv1 Channels in Control of Membrane Excitability and Burst Generation in Mesencephalic V Neurons." Journal of Neurophysiology 101, no. 3 (March 2009): 1407–18. http://dx.doi.org/10.1152/jn.91053.2008.
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The function and biophysical properties of low threshold Kv1 current in control of membrane resonance, subthreshold oscillations, and bursting in mesencephalic V neurons (Mes V) were examined in rat brain stem slices (P8–P12) using whole cell current and voltage patch-clamp methods. α-dendrotoxin application, a toxin with high specificity for Kv1.1, 1.2, and 1.6 channels, showed the presence of a low-threshold K+ current that activated rapidly around −50 mV and was relatively noninactivating over a 1-s period and had a V1/2max of −36.2 mV. Other toxins, specific for individual channels containing either Kv 1.1, 1.2, or 1.3 α-subunits, were applied individually, or in combination, and showed that Kv1 channels are heteromeric, composed of combinations of subunits. In current-clamp mode, toxin application transformed the high-frequency resonant properties of the membrane into a low-pass filter and concomitantly reduced the frequency of the subthreshold membrane oscillations. During this period, rhythmical bursting was transformed into low-frequency tonic discharge. Interestingly, in a subset of neurons that did not show bursting, low doses of α-dendrotoxin (α-DTX) sufficient to block 50% of the low threshold Kv1 channels induced bursting and increased the resonant peak impedance and subthreshold oscillations, which was replicated with computer simulation. This suggests that a critical balance between inward and outward currents is necessary for bursting. This was replicated with computer simulation. Single cell RT-PCR and immunohistochemical methods confirmed the presence of Kv1.1, 1.2, and 1.6 α-subunits in Mes V neurons. These data indicate that low threshold Kv1 channels are responsible for membrane resonance, contribute to subthreshold oscillations, and are critical for burst generation.
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Nekrasova, Oksana V., Alexandra L. Primak, Anastasia A. Ignatova, Valery N. Novoseletsky, Olga V. Geras’kina, Ksenia S. Kudryashova, Sergey A. Yakimov, Mikhail P. Kirpichnikov, Alexander S. Arseniev, and Alexey V. Feofanov. "N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site." Toxins 12, no. 12 (December 16, 2020): 802. http://dx.doi.org/10.3390/toxins12120802.
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Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.
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Vivekananda, Umesh, Pavel Novak, Oscar D. Bello, Yuri E. Korchev, Shyam S. Krishnakumar, Kirill E. Volynski, and Dimitri M. Kullmann. "Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization." Proceedings of the National Academy of Sciences 114, no. 9 (February 13, 2017): 2395–400. http://dx.doi.org/10.1073/pnas.1608763114.
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Although action potentials propagate along axons in an all-or-none manner, subthreshold membrane potential fluctuations at the soma affect neurotransmitter release from synaptic boutons. An important mechanism underlying analog–digital modulation is depolarization-mediated inactivation of presynaptic Kv1-family potassium channels, leading to action potential broadening and increased calcium influx. Previous studies have relied heavily on recordings from blebs formed after axon transection, which may exaggerate the passive propagation of somatic depolarization. We recorded instead from small boutons supplied by intact axons identified with scanning ion conductance microscopy in primary hippocampal cultures and asked how distinct potassium channels interact in determining the basal spike width and its modulation by subthreshold somatic depolarization. Pharmacological or genetic deletion of Kv1.1 broadened presynaptic spikes without preventing further prolongation by brief depolarizing somatic prepulses. A heterozygous mouse model of episodic ataxia type 1 harboring a dominant Kv1.1 mutation had a similar broadening effect on basal spike shape as deletion of Kv1.1; however, spike modulation by somatic prepulses was abolished. These results argue that the Kv1.1 subunit is not necessary for subthreshold modulation of spike width. However, a disease-associated mutant subunit prevents the interplay of analog and digital transmission, possibly by disrupting the normal stoichiometry of presynaptic potassium channels.
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Wang, Yuanyuan, Xiaoyi Mo, Conghui Ping, Qian Huang, Hao Zhang, Chang Xie, Bo Zhong, Dongdong Li, and Jing Yao. "Site-specific contacts enable distinct modes of TRPV1 regulation by the potassium channel Kvβ1 subunit." Journal of Biological Chemistry 295, no. 50 (October 15, 2020): 17337–48. http://dx.doi.org/10.1074/jbc.ra120.015605.
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Transient receptor potential vanilloid 1 (TRPV1) channel is a multimodal receptor that is responsible for nociceptive, thermal, and mechanical sensations. However, which biomolecular partners specifically interact with TRPV1 remains to be elucidated. Here, we used cDNA library screening of genes from mouse dorsal root ganglia combined with patch-clamp electrophysiology to identify the voltage-gated potassium channel auxiliary subunit Kvβ1 physically interacting with TRPV1 channel and regulating its function. The interaction was validated in situ using endogenous dorsal root ganglia neurons, as well as a recombinant expression model in HEK 293T cells. The presence of Kvβ1 enhanced the expression stability of TRPV1 channels on the plasma membrane and the nociceptive current density. Surprisingly, Kvβ1 interaction also shifted the temperature threshold for TRPV1 thermal activation. Using site-specific mapping, we further revealed that Kvβ1 interacted with the membrane-distal domain and membrane-proximal domain of TRPV1 to regulate its membrane expression and temperature-activation threshold, respectively. Our data therefore suggest that Kvβ1 is a key element in the TRPV1 signaling complex and exerts dual regulatory effects in a site-specific manner.
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Pineda, Ricardo H., Christopher S. Knoeckel, Alison D. Taylor, Adriana Estrada-Bernal, and Angeles B. Ribera. "Kv1 Potassium Channel Complexes In Vivo Require Kvβ2 Subunits in Dorsal Spinal Neurons." Journal of Neurophysiology 100, no. 4 (October 2008): 2125–36. http://dx.doi.org/10.1152/jn.90667.2008.
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Whereas Kvβ2 subunits modulate potassium current properties carried by Kv1 channel complexes in heterologous systems, little is known about the contributions of Kvβ2 subunits to native potassium channel function. Using antisense approaches and in situ recordings from Xenopus embryo spinal cord neurons, we tested the in vivo roles of Kvβ2 subunits in modulation of voltage-dependent potassium current ( IKv). We focused on 1) two different populations of dorsal spinal neurons that express both Kvβ2 and Kv1 α-subunit genes and 2) the 24- and 48-h developmental period, during which IKv undergoes developmental regulation. At both 24 and 48 h, antisense methods produced efficient knock-down of both Kvβ2 protein and IKv. At both times, dominant negative suppression of Kv1 channels also eliminated IKv, indicating that Kv1 channels require Kvβ2 subunits to function in dorsal spinal neurons. Even though Kv1 channels determined the IKv values of both dorsal neuron types, comparisons of their IKv properties revealed important differences at both developmental stages. The latter results support the notion that different Kv1 α-subunits and/or posttranslational modifications underlie the IKv values of the two dorsal neuron types. Overall, the results demonstrate that Kvβ2 subunits function in vivo as obligatory subunits of Kv1 channels in at least two neuron types and two different developmental stages.
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Jerng, Henry H., Jay M. Patel, Tamor A. Khan, Benjamin R. Arenkiel, and Paul J. Pfaffinger. "Light-regulated voltage-gated potassium channels for acute interrogation of channel function in neurons and behavior." PLOS ONE 16, no. 3 (March 23, 2021): e0248688. http://dx.doi.org/10.1371/journal.pone.0248688.
Full textAbstract:
Voltage-gated potassium (Kv) channels regulate the membrane potential and conductance of excitable cells to control the firing rate and waveform of action potentials. Even though Kv channels have been intensely studied for over 70 year, surprisingly little is known about how specific channels expressed in various neurons and their functional properties impact neuronal network activity and behavior in vivo. Although many in vivo genetic manipulations of ion channels have been tried, interpretation of these results is complicated by powerful homeostatic plasticity mechanisms that act to maintain function following perturbations in excitability. To better understand how Kv channels shape network function and behavior, we have developed a novel optogenetic technology to acutely regulate Kv channel expression with light by fusing the light-sensitive LOV domain of Vaucheria frigida Aureochrome 1 to the N-terminus of the Kv1 subunit protein to make an Opto-Kv1 channel. Recording of Opto-Kv1 channels expressed in Xenopus oocytes, mammalian cells, and neurons show that blue light strongly induces the current expression of Opto-Kv1 channels in all systems tested. We also find that an Opto-Kv1 construct containing a dominant-negative pore mutation (Opto-Kv1(V400D)) can be used to down-regulate Kv1 currents in a blue light-dependent manner. Finally, to determine whether Opto-Kv1 channels can elicit light-dependent behavioral effect in vivo, we targeted Opto-Kv1 (V400D) expression to Kv1.3-expressing mitral cells of the olfactory bulb in mice. Exposure of the bulb to blue light for 2–3 hours produced a significant increase in sensitivity to novel odors after initial habituation to a similar odor, comparable to behavioral changes seen in Kv1.3 knockout animals. In summary, we have developed novel photoactivatable Kv channels that provide new ways to interrogate neural circuits in vivo and to examine the roles of normal and disease-causing mutant Kv channels in brain function and behavior.
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Imbrici, Paola, Maria Cristina D'Adamo, Alessandro Grottesi, Andrea Biscarini, and Mauro Pessia. "Episodic ataxia type 1 mutations affect fast inactivation of K+ channels by a reduction in either subunit surface expression or affinity for inactivation domain." American Journal of Physiology-Cell Physiology 300, no. 6 (June 2011): C1314—C1322. http://dx.doi.org/10.1152/ajpcell.00456.2010.
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Episodic ataxia type 1 (EA1) is an autosomal dominant disorder characterized by continuous myokymia and episodic attacks of ataxia. Mutations in the gene KCNA1 that encodes the voltage-gated potassium channel Kv1.1 are responsible for EA1. In several brain areas, Kv1.1 coassembles with Kv1.4, which confers N-type inactivating properties to heteromeric channels. It is therefore likely that the rate of inactivation will be determined by the number of Kv1.4 inactivation particles, as set by the precise subunit stoichiometry. We propose that EA1 mutations affect the rate of N-type inactivation either by reduced subunit surface expression, giving rise to a reduced number of Kv1.1 subunits in heterotetramer Kv1.1-Kv1.4 channels, or by reduced affinity for the Kv1.4 inactivation domain. To test this hypothesis, quantified amounts of mRNA for Kv1.4 or Kv1.1 containing selected EA1 mutations either in the inner vestibule of Kv1.1 on S6 or in the transmembrane regions were injected into Xenopus laevis oocytes and the relative rates of inactivation and stoichiometry were determined. The S6 mutations, V404I and V408A, which had normal surface expression, reduced the rate of inactivation by a decreased affinity for the inactivation domain while the mutations I177N in S1 and E325D in S5, which had reduced subunit surface expression, increased the rate of N-type inactivation due to a stoichiometric increase in the number of Kv1.4 subunits.
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Silverå Ejneby, Malin, Björn Wallner, and Fredrik Elinder. "Coupling stabilizers open KV1-type potassium channels." Proceedings of the National Academy of Sciences 117, no. 43 (October 13, 2020): 27016–21. http://dx.doi.org/10.1073/pnas.2007965117.
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The opening and closing of voltage-gated ion channels are regulated by voltage sensors coupled to a gate that controls the ion flux across the cellular membrane. Modulation of any part of gating constitutes an entry point for pharmacologically regulating channel function. Here, we report on the discovery of a large family of warfarin-like compounds that open the two voltage-gated type 1 potassium (KV1) channels KV1.5 and Shaker, but not the related KV2-, KV4-, or KV7-type channels. These negatively charged compounds bind in the open state to positively charged arginines and lysines between the intracellular ends of the voltage-sensor domains and the pore domain. This mechanism of action resembles that of endogenous channel-opening lipids and opens up an avenue for the development of ion-channel modulators.
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Wang, Jian-Ying, Jian Wang, Vera A. Golovina, Li Li, Oleksandr Platoshyn, and Jason Xiao-Jian Yuan. "Role of K+ channel expression in polyamine-dependent intestinal epithelial cell migration." American Journal of Physiology-Cell Physiology 278, no. 2 (February 1, 2000): C303—C314. http://dx.doi.org/10.1152/ajpcell.2000.278.2.c303.
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Polyamines are essential for cell migration during early mucosal restitution after wounding in the gastrointestinal tract. Activity of voltage-gated K+ channels (Kv) controls membrane potential ( E m) that regulates cytoplasmic free Ca2+ concentration ([Ca2+]cyt) by governing the driving force for Ca2+ influx. This study determined whether polyamines are required for the stimulation of cell migration by altering K+ channel gene expression, E m, and [Ca2+]cyt in intestinal epithelial cells (IEC-6). The specific inhibitor of polyamine synthesis, α-difluoromethylornithine (DFMO, 5 mM), depleted cellular polyamines (putrescine, spermidine, and spermine), selectively inhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression, and resulted in membrane depolarization. Because IEC-6 cells did not express voltage-gated Ca2+ channels, the depolarized E m in DFMO-treated cells decreased [Ca2+]cyt as a result of reduced driving force for Ca2+ influx through capacitative Ca2+ entry. Migration was reduced by 80% in the polyamine-deficient cells. Exogenous spermidine not only reversed the effects of DFMO on Kv1.1 channel expression, E m, and [Ca2+]cyt but also restored cell migration to normal. Removal of extracellular Ca2+ or blockade of Kv channels (by 4-aminopyridine, 1–5 mM) significantly inhibited normal cell migration and prevented the restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These results suggest that polyamine-dependent intestinal epithelial cell migration may be due partially to an increase of Kv1.1 channel expression. The subsequent membrane hyperpolarization raises [Ca2+]cyt by increasing the driving force (the electrochemical gradient) for Ca2+ influx and thus stimulates cell migration.
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Friederich, Patrick, Dietmar Benzenberg, Sokratis Trellakis, and Bernd W. Urban. "Interaction of Volatile Anesthetics with Human Kv Channels in Relation to Clinical Concentrations." Anesthesiology 95, no. 4 (October 1, 2001): 954–58. http://dx.doi.org/10.1097/00000542-200110000-00026.
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Background Recent evidence shows that inhibition of human Kv3 channels by intravenous anesthetics occurs at clinical concentrations. The effects of volatile anesthetics on these human ion channels are unknown. This study was designed to establish whether minimum alveolar concentrations (MAC) of halothane, enflurane, isoflurane, and desflurane exhibit effects on Kv3 channeLs. To obtain an indication whether these findings may be specific to Kv3 channels, the effects of enflurane and isoflurane on human Kv1.1 channels were also investigated. Methods Kv3 channels natively expressed in SH-SY5Y cells and Kv1.1 channels expressed in HEK293 cells were measured with the whole cell patch clamp technique by standard protocols. Concentrations of volatile anesthetics were determined by gas chromatography. Results Halothane, enflurane, isoflurane, and desflurane reversibly inhibited Kv3 channels in a concentration-dependent manner. Concentrations at half-maximal effect (IC50 values) ranged between 1,800 and 4,600 microM. Hill coefficients were between 1.7 and 2.5. IC50 values for inhibition of Kv1.1 channels were 2,800 and 5,200 microM, and Hill coefficients were 3.9 and 5.6 for enflurane and isoflurane, respectively. Conclusion Volatile anesthetics inhibit human Kv3 channels at clinical concentrations. At 1-3 MAC, inhibition would account on average for 2-12%. Inhibition would be highest with enflurane (between 3% and 22%) and lowest with isoflurane (between 0.2% and 3%). Kv1.1 channels would only be inhibited by enflurane at clinical concentrations (2% at 2 MAC and 8% at 3 MAC). Whether the degree of K channel inhibition by volatile anesthetics may contribute to their clinical action needs further study.
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Extrémet, Johanna, Oussama El Far, Norbert Ankri, Sarosh R. Irani, Dominique Debanne, and Michaël Russier. "An Epitope-Specific LGI1-Autoantibody Enhances Neuronal Excitability by Modulating Kv1.1 Channel." Cells 11, no. 17 (August 31, 2022): 2713. http://dx.doi.org/10.3390/cells11172713.
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Leucine-rich Glioma-Inactivated protein 1 (LGI1) is expressed in the central nervous system and its genetic loss of function is associated with epileptic disorders. Additionally, patients with LGI1-directed autoantibodies have frequent focal seizures as a key feature of their disease. LGI1 is composed of a Leucine-Rich Repeat (LRR) and an Epitempin (EPTP) domain. These domains are reported to interact with different members of the transsynaptic complex formed by LGI1 at excitatory synapses, including presynaptic Kv1 potassium channels. Patient-derived recombinant monoclonal antibodies (mAbs) are ideal reagents to study whether domain-specific LGI1-autoantibodies induce epileptiform activities in neurons and their downstream mechanisms. We measured the intrinsic excitability of CA3 pyramidal neurons in organotypic cultures from rat hippocampus treated with either an LRR- or an EPTP-reactive patient-derived mAb, or with IgG from control patients. We found an increase in intrinsic excitability correlated with a reduction of the sensitivity to a selective Kv1.1-channel blocker in neurons treated with the LRR mAb, but not in neurons treated with the EPTP mAb. Our findings suggest LRR mAbs are able to modulate neuronal excitability that could account for epileptiform activity observed in patients.
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Speake, Tracey, Jonathan D. Kibble, and Peter D. Brown. "Kv1.1 and Kv1.3 channels contribute to the delayed-rectifying K+ conductance in rat choroid plexus epithelial cells." American Journal of Physiology-Cell Physiology 286, no. 3 (March 2004): C611—C620. http://dx.doi.org/10.1152/ajpcell.00292.2003.
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The choroid plexuses secrete, and maintain the composition of, the cerebrospinal fluid. K+ channels play an important role in these processes. In this study the molecular identity and properties of the delayed-rectifying K+ (Kv) conductance in rat choroid plexus epithelial cells were investigated. Whole cell K+ currents were significantly reduced by 10 nM dendrotoxin-K and 1 nM margatoxin, which are specific inhibitors of Kv1.1 and Kv1.3 channels, respectively. A combination of dendrotoxin-K and margatoxin caused a depolarization of the membrane potential in current-clamp experiments. Western blot analysis indicated the presence of Kv1.1 and Kv1.3 proteins in the choroid plexus. Furthermore, the Kv1.3 and Kv1.1 proteins appear to be expressed in the apical membrane of the epithelial cells in immunocytochemical studies. The Kv conductance was inhibited by 1 μM serotonin (5-HT), with maximum inhibition to 48% of control occurring in 8 min ( P < 0.05 by Student's t-test for paired data). Channel inhibition by 5-HT was prevented by the 5-HT2C antagonist mesulergine (300 nM). It was also attenuated in the presence of calphostin C (a protein kinase C inhibitor). The conductance was partially inhibited by 1,2-dioctanoyl- sn-glycerol and phorbol 12-myristate 13-acetate, both of which activate protein kinase C. These data suggest that 5-HT acts at 5-HT2C receptors to activate protein kinase C, which inhibits the Kv channels. In conclusion, Kv1.1 and Kv1.3 channels make a significant contribution to K+ efflux at the apical membrane of the choroid plexus.
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Tobin, Ann A., Biny K. Joseph, Hamood N. Al-Kindi, Sulayma Albarwani, Jane A. Madden, Leah T. Nemetz, Nancy J. Rusch, and Sung W. Rhee. "Loss of cerebrovascular Shaker-type K+ channels: a shared vasodilator defect of genetic and renal hypertensive rats." American Journal of Physiology-Heart and Circulatory Physiology 297, no. 1 (July 2009): H293—H303. http://dx.doi.org/10.1152/ajpheart.00991.2008.
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The cerebral arteries of hypertensive rats are depolarized and highly myogenic, suggesting a loss of K+ channels in the vascular smooth muscle cells (VSMCs). The present study evaluated whether the dilator function of the prominent Shaker-type voltage-gated K+ (KV1) channels is attenuated in middle cerebral arteries from two rat models of hypertension. Block of KV1 channels by correolide (1 μmol/l) or psora-4 (100 nmol/l) reduced the resting diameter of pressurized (80 mmHg) cerebral arteries from normotensive rats by an average of 28 ± 3% or 26 ± 3%, respectively. In contrast, arteries from spontaneously hypertensive rats (SHR) and aortic-banded (Ao-B) rats with chronic hypertension showed enhanced Ca2+-dependent tone and failed to significantly constrict to correolide or psora-4, implying a loss of KV1 channel-mediated vasodilation. Patch-clamp studies in the VSMCs of SHR confirmed that the peak K+ current density attributed to KV1 channels averaged only 5.47 ± 1.03 pA/pF, compared with 9.58 ± 0.82 pA/pF in VSMCs of control Wistar-Kyoto rats. Subsequently, Western blots revealed a 49 ± 7% to 66 ± 7% loss of the pore-forming α1.2- and α1.5-subunits that compose KV1 channels in cerebral arteries of SHR and Ao-B rats compared with control animals. In each case, the deficiency of KV1 channels was associated with reduced mRNA levels encoding either or both α-subunits. Collectively, these findings demonstrate that a deficit of α1.2- and α1.5-subunits results in a reduced contribution of KV1 channels to the resting diameters of cerebral arteries from two rat models of hypertension that originate from different etiologies.
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Ohanyan, Vahagn, Sean M. Raph, Marc M. Dwenger, Xuemei Hu, Thomas Pucci, Gregory Mack, Joseph B. Moore, William M. Chilian, Aruni Bhatnagar, and Matthew A. Nystoriak. "Myocardial Blood Flow Control by Oxygen Sensing Vascular Kvβ Proteins." Circulation Research 128, no. 6 (March 19, 2021): 738–51. http://dx.doi.org/10.1161/circresaha.120.317715.
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Rationale: Voltage-gated potassium (Kv) channels in vascular smooth muscle are essential for coupling myocardial blood flow (MBF) with the metabolic demand of the heart. These channels consist of a transmembrane pore domain that associates with auxiliary Kvβ (voltage-gated potassium channel β)1 and Kvβ2 proteins, which differentially regulate Kv function in excitable cells. Nonetheless, the physiological role of Kvβ proteins in regulating vascular tone and metabolic hyperemia in the heart remains unknown. Objective: To test the hypothesis that Kvβ proteins confer oxygen sensitivity to vascular tone and are required for regulating blood flow in the heart. Methods and Results: Mice lacking Kvβ2 subunits exhibited suppressed MBF, impaired cardiac contractile performance, and failed to maintain elevated arterial blood pressure in response to catecholamine-induced stress. In contrast, ablation of Kvβ1.1 reduced cardiac workload, modestly elevated MBF, and preserved cardiac function during stress compared with wild-type mice. Coronary arteries isolated from Kvβ2 −/− , but not Kvβ1.1 −/− , mice had severely blunted vasodilation to hypoxia when compared with arteries from wild-type mice. Moreover, vasodilation of small diameter coronary and mesenteric arteries due to L-lactate, a biochemical marker of reduced tissue oxygenation and anaerobic metabolism, was significantly attenuated in vessels isolated from Kvβ2 −/− mice. Inducible enhancement of the Kvβ1:Kvβ2 ratio in Kv1 channels of arterial smooth muscle abolished L-lactate-induced vasodilation and suppressed the relationship between MBF and cardiac workload. Conclusions: The Kvβ proteins differentially regulate vascular tone and MBF, whereby Kvβ2 promotes, and Kvβ1.1 inhibits oxygen-dependent vasodilation and augments blood flow upon heightened metabolic demand.
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Shvetsova, Anastasia A., Dina K. Gaynullina, Olga S. Tarasova, and Rudolf Schubert. "Remodeling of Arterial Tone Regulation in Postnatal Development: Focus on Smooth Muscle Cell Potassium Channels." International Journal of Molecular Sciences 22, no. 11 (May 21, 2021): 5413. http://dx.doi.org/10.3390/ijms22115413.
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Maturation of the cardiovascular system is associated with crucial structural and functional remodeling. Thickening of the arterial wall, maturation of the sympathetic innervation, and switching of the mechanisms of arterial contraction from calcium-independent to calcium-dependent occur during postnatal development. All these processes promote an almost doubling of blood pressure from the moment of birth to reaching adulthood. This review focuses on the developmental alterations of potassium channels functioning as key smooth muscle membrane potential determinants and, consequently, vascular tone regulators. We present evidence that the pattern of potassium channel contribution to vascular control changes from Kir2, Kv1, Kv7 and TASK-1 channels to BKCa channels with maturation. The differences in the contribution of potassium channels to vasomotor tone at different stages of postnatal life should be considered in treatment strategies of cardiovascular diseases associated with potassium channel malfunction.
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Imbrici, Paola, Andrea Accogli, Rikard Blunck, Concetta Altamura, Michele Iacomino, Maria Cristina D’Adamo, Anna Allegri, et al. "Musculoskeletal Features without Ataxia Associated with a Novel de novo Mutation in KCNA1 Impairing the Voltage Sensitivity of Kv1.1 Channel." Biomedicines 9, no. 1 (January 14, 2021): 75. http://dx.doi.org/10.3390/biomedicines9010075.
Full textAbstract:
The KCNA1 gene encodes the α subunit of the voltage-gated Kv1.1 potassium channel that critically regulates neuronal excitability in the central and peripheral nervous systems. Mutations in KCNA1 have been classically associated with episodic ataxia type 1 (EA1), a movement disorder triggered by physical and emotional stress. Additional features variably reported in recent years include epilepsy, myokymia, migraine, paroxysmal dyskinesia, hyperthermia, hypomagnesemia, and cataplexy. Interestingly, a few individuals with neuromyotonia, either isolated or associated with skeletal deformities, have been reported carrying variants in the S2–S3 transmembrane segments of Kv1.1 channels in the absence of any other symptoms. Here, we have identified by whole-exome sequencing a novel de novo variant, T268K, in KCNA1 in a boy displaying recurrent episodes of neuromyotonia, muscle hypertrophy, and skeletal deformities. Through functional analysis in heterologous cells and structural modeling, we show that the mutation, located at the extracellular end of the S3 helix, causes deleterious effects, disrupting Kv1.1 function by altering the voltage dependence of activation and kinetics of deactivation, likely due to abnormal interactions with the voltage sensor in the S4 segment. Our study supports previous evidence suggesting that specific residues within the S2 and S3 segments of Kv1.1 result in a distinctive phenotype with predominant musculoskeletal presentation.
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SRAIRI-ABID, Najet, Joseba Iñaki GUIJARRO, Rym BENKHALIFA, Massimo MANTEGAZZA, Amani CHEIKH, Manel BEN AISSA, Pierre-Yves HAUMONT, Muriel DELEPIERRE, and Mohamed EL AYEB. "A new type of scorpion Na+-channel-toxin-like polypeptide active on K+ channels." Biochemical Journal 388, no. 2 (May 24, 2005): 455–64. http://dx.doi.org/10.1042/bj20041407.
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We have purified and characterized two peptides, named KAaH1 and KAaH2 (AaH polypeptides 1 and 2 active on K+ channels, where AaH stands for Androctonus australis Hector), from the venom of A. australis Hector scorpions. Their sequences contain 58 amino acids including six half-cysteines and differ only at positions 26 (Phe/Ser) and 29 (Lys/Gln). Although KAaH1 and KAaH2 show important sequence similarity with anti-mammal β toxins specific for voltage-gated Na+ channels, only weak β-like effects were observed when KAaH1 or KAaH2 (1 μM) were tested on brain Nav1.2 channels. In contrast, KAaH1 blocks Kv1.1 and Kv1.3 channels expressed in Xenopus oocytes with IC50 values of 5 and 50 nM respectively, whereas KAaH2 blocks only 20% of the current on Kv1.1 and is not active on Kv1.3 channels at a 100 nM concentration. KAaH1 is thus the first member of a new subfamily of long-chain toxins mainly active on voltage-gated K+ channels. NMR spectra of KAaH1 and KAaH2 show good dispersion of signals but broad lines and poor quality. Self-diffusion NMR experiments indicate that lines are broadened due to a conformational exchange on the millisecond time scale. NMR and CD indicate that both polypeptides adopt a similar fold with α-helical and β-sheet structures. Homology-based molecular models generated for KAaH1 and KAaH2 are in accordance with CD and NMR data. In the model of KAaH1, the functionally important residues Phe26 and Lys29 are close to each other and are located in the α-helix. These residues may constitute the so-called functional dyad observed for short α-KTx scorpion toxins in the β-sheet.
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MacDonald, Patrick E., Xiao Fang Ha, Jing Wang, Simon R. Smukler, Anthony M. Sun, Herbert Y. Gaisano, Ann Marie F. Salapatek, Peter H. Backx, and Michael B. Wheeler. "Members of the Kv1 and Kv2 Voltage-Dependent K+ Channel Families Regulate Insulin Secretion." Molecular Endocrinology 15, no. 8 (August 1, 2001): 1423–35. http://dx.doi.org/10.1210/mend.15.8.0685.
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Abstract In pancreatic β-cells, voltage-dependent K+ (Kv) channels are potential mediators of repolarization, closure of Ca2+ channels, and limitation of insulin secretion. The specific Kv channels expressed in β-cells and their contribution to the delayed rectifier current and regulation of insulin secretion in these cells are unclear. High-level protein expression and mRNA transcripts for Kv1.4, 1.6, and 2.1 were detected in rat islets and insulinoma cells. Inhibition of these channels with tetraethylammonium decreased IDR by approximately 85% and enhanced glucose-stimulated insulin secretion by 2- to 4-fold. Adenovirus-mediated expression of a C-terminal truncated Kv2.1 subunit, specifically eliminating Kv2 family currents, reduced delayed rectifier currents in these cells by 60–70% and enhanced glucose-stimulated insulin secretion from rat islets by 60%. Expression of a C-terminal truncated Kv1.4 subunit, abolishing Kv1 channel family currents, reduced delayed rectifier currents by approximately 25% and enhanced glucose-stimulated insulin secretion from rat islets by 40%. This study establishes that Kv2 and 1 channel homologs mediate the majority of repolarizing delayed rectifier current in rat β-cells and that antagonism of Kv2.1 may prove to be a novel glucose-dependent therapeutic treatment for type 2 diabetes.
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Hsu, Hsin-Te, You-Lan Yang, Wan-Chen Chen, Chi-Ming Chen, and Wun-Chang Ko. "Butylidenephthalide Blocks Potassium Channels and Enhances Basal Tension in Isolated Guinea-Pig Trachea." BioMed Research International 2014 (2014): 1–7. http://dx.doi.org/10.1155/2014/875230.
Full textAbstract:
Butylidenephthalide (Bdph, 30~300 μM), a constituent ofLigusticum chuanxiongHort., significantly enhanced tension in isolated guinea-pig trachea. In this study, we investigate the mechanism(s) of Bdph-induced contraction in the tissue. Isolated trachea was bathed in 5 mL of Krebs solution containing indomethacin (3 μM), and its tension changes were isometrically recorded. Cromakalim (3 μM), an ATP-dependent K+channel opener, significantly antagonized the Bdph-induced enhancement of baseline tension. Bdph (300 μM) also significantly antagonized cromakalim-induced relaxation. Bdph (300 μM) did not significantly influence the antagonistic effects of glibenclamide (GBC, 1 μM) and tetraethylammonium (TEA, 8 mM) against the cromakalim-induced relaxation. However, Bdph (300 μM) and 4-aminopiridine (4-AP, 5 mM), a blocker of Kv1 family of K+channels, in combination significantly rightward shifted the log concentration-relaxation curve of cromakalim. The antagonistic effect of the combination almost equals the sum of the individual effects of Bdph and 4-AP, suggesting that the antagonistic mechanism of Bdph may be similar to that of 4-AP. All calcium channel blockers influenced neither the baseline tension nor antagonistic effect of Bdph against cromakalim. In conclusion, Bdph may be similar to 4-AP, a blocker of Kv1 family of K+channels, to enhance the baseline tension of guinea-pig trachea.
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Finnegan, Thomas F., Shao-Rui Chen, and Hui-Lin Pan. "μ Opioid Receptor Activation Inhibits GABAergic Inputs to Basolateral Amygdala Neurons Through Kv1.1/1.2 Channels." Journal of Neurophysiology 95, no. 4 (April 2006): 2032–41. http://dx.doi.org/10.1152/jn.01004.2005.
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The basolateral amygdala (BLA) is the major amygdaloid nucleus distributed with μ opioid receptors. The afferent input from the BLA to the central nucleus of the amygdala (CeA) is considered important for opioid analgesia. However, little is known about the effect of μ opioids on synaptic transmission in the BLA. In this study, we examined the effect of μ opioid receptor stimulation on the inhibitory and excitatory synaptic inputs to CeA-projecting BLA neurons. BLA neurons were retrogradely labeled with a fluorescent tracer injected into the CeA of rats. Whole cell voltage-clamp recordings were performed on labeled BLA neurons in brain slices. The specific μ opioid receptor agonist, (d-Ala2, N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO, 1 μM), significantly reduced the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in 77% of cells tested. DAMGO also significantly decreased the peak amplitude of evoked IPSCs in 75% of cells examined. However, DAMGO did not significantly alter the frequency of mEPSCs or the peak amplitude of evoked EPSCs in 90% and 75% of labeled cells, respectively. Bath application of the Kv channel blockers, 4-AP (Kv1.1, 1.2, 1.3, 1.5, 1.6, 3.1, 3.2), α-dendrotoxin (Kv1.1, 1.2, 1.6), dendrotoxin-K (Kv1.1), or tityustoxin-Kα (Kv1.2) each blocked the inhibitory effect of DAMGO on mIPSCs. Double immunofluorescence labeling showed that some of the immunoreactivities of Kv1.1 and Kv1.2 were colocalized with synaptophysin in the BLA. This study provides new information that activation of presynaptic μ opioid receptors primarily attenuates GABAergic synaptic inputs to CeA-projecting neurons in the BLA through a signaling mechanism involving Kv1.1 and Kv1.2 channels.
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Yuan, Li-Lian, Xixi Chen, Kumud Kunjilwar, Paul Pfaffinger, and Daniel Johnston. "Acceleration of K+ channel inactivation by MEK inhibitor U0126." American Journal of Physiology-Cell Physiology 290, no. 1 (January 2006): C165—C171. http://dx.doi.org/10.1152/ajpcell.00206.2005.
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Voltage-dependent (Kv)4.2-encoded A-type K+ channels play an important role in controlling neuronal excitability and are subject to modulation by various protein kinases, including ERK. In studies of ERK modulation, the organic compound U0126 is often used to suppress the activity of MEK, which is a kinase immediately upstream from ERK. We have observed that the inactivation time constant of heterologously expressed Kv4.2 channels was accelerated by U0126 at 1–20 μM. This effect, however, was not Kv4 family specific, because U0126 also converted noninactivating K+ currents mediated by Kv1.1 subunits into transient ones. To determine whether U0126 exerted these effects through kinase inhibition, we tested U0125, a derivative of U0126 that is less potent in MEK inhibition. At the same concentrations, U0125 had effects similar to those of U0126 on channel inactivation. Finally, we expressed a mutant form of Kv4.2 in which three identified ERK phosphorylation sites (T602, T607, and S616) were replaced with alanines. The inactivation of K+ currents mediated by this mutant was still accelerated by U0126. Our data favor the conclusion that the increase in the rate of channel inactivation by U0126 is likely to be independent of protein kinase inhibition and instead represents a direct action on channel gating.
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Yin, Shi-Jin, Ling Jiang, Hong Yi, Song Han, Dai-Wen Yang, Mai-Li Liu, Hui Liu, Zhi-Jian Cao, Ying-Liang Wu, and Wen-Xin Li. "Different Residues in Channel Turret Determining the Selectivity of ADWX-1 Inhibitor Peptide between Kv1.1 and Kv1.3 Channels." Journal of Proteome Research 7, no. 11 (November 7, 2008): 4890–97. http://dx.doi.org/10.1021/pr800494a.
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Orlov, Nikita A., Anastasia A. Ignatova, Elena V. Kryukova, Sergey A. Yakimov, Mikhail P. Kirpichnikov, Oksana V. Nekrasova, and Alexey V. Feofanov. "Combining mKate2-Kv1.3 Channel and Atto488-Hongotoxin for the Studies of Peptide Pore Blockers on Living Eukaryotic Cells." Toxins 14, no. 12 (December 5, 2022): 858. http://dx.doi.org/10.3390/toxins14120858.
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The voltage-gated potassium Kv1.3 channel is an essential component of vital cellular processes which is also involved in the pathogenesis of some autoimmune, neuroinflammatory and oncological diseases. Pore blockers of the Kv1.3 channel are considered as potential drugs and are used to study Kv1 channels’ structure and functions. Screening and study of the blockers require the assessment of their ability to bind the channel. Expanding the variety of methods used for this, we report on the development of the fluorescent competitive binding assay for measuring affinities of pore blockers to Kv1.3 at the membrane of mammalian cells. The assay constituents are hongotoxin 1 conjugated with Atto488, fluorescent mKate2-tagged Kv1.3 channel, which was designed to improve membrane expression of the channel in mammalian cells, confocal microscopy, and a special protocol of image processing. The assay is implemented in the “mix and measure”, format and allows the screening of Kv1.3 blockers, such as peptide toxins, that bind to the extracellular vestibule of the K+-conducting pore, and analyzing their affinity.
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Sciamanna, Giuseppe, and Charles J. Wilson. "The ionic mechanism of gamma resonance in rat striatal fast-spiking neurons." Journal of Neurophysiology 106, no. 6 (December 2011): 2936–49. http://dx.doi.org/10.1152/jn.00280.2011.
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Striatal fast-spiking (FS) cells in slices fire in the gamma frequency range and in vivo are often phase-locked to gamma oscillations in the field potential. We studied the firing patterns of these cells in slices from rats ages 16–23 days to determine the mechanism of their gamma resonance. The resonance of striatal FS cells was manifested as a minimum frequency for repetitive firing. At rheobase, cells fired a doublet of action potentials or doublets separated by pauses, with an instantaneous firing rate averaging 44 spikes/s. The minimum rate for sustained firing was also responsible for the stuttering firing pattern. Firing rate adapted during each episode of firing, and bursts were terminated when firing was reduced to the minimum sustainable rate. Resonance and stuttering continued after blockade of Kv3 current using tetraethylammonium (0.1–1 mM). Both gamma resonance and stuttering were strongly dependent on Kv1 current. Blockade of Kv1 channels with dendrotoxin-I (100 nM) completely abolished the stuttering firing pattern, greatly lowered the minimum firing rate, abolished gamma-band subthreshold oscillations, and slowed spike frequency adaptation. The loss of resonance could be accounted for by a reduction in potassium current near spike threshold and the emergence of a fixed spike threshold. Inactivation of the Kv1 channel combined with the minimum firing rate could account for the stuttering firing pattern. The resonant properties conferred by this channel were shown to be adequate to account for their phase-locking to gamma-frequency inputs as seen in vivo.
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Dick, Gregory M., Ian N. Bratz, Léna Borbouse, Gregory A. Payne, Ü. Deniz Dincer, Jarrod D. Knudson, Paul A. Rogers, and Johnathan D. Tune. "Voltage-dependent K+ channels regulate the duration of reactive hyperemia in the canine coronary circulation." American Journal of Physiology-Heart and Circulatory Physiology 294, no. 5 (May 2008): H2371—H2381. http://dx.doi.org/10.1152/ajpheart.01279.2007.
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We previously demonstrated a role for voltage-dependent K+ (KV) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H2O2, as responses were attenuated by the KV channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that KV channels participate in coronary reactive hyperemia and examined the role of KV channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 ± 5% and inhibited hyperemic volume 32 ± 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or NG-nitro-l-arginine methyl ester (l-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or l-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 ± 6% block at 9 μg/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 μM correolide, a selective KV1 antagonist (76 ± 7% and 47 ± 2% block, respectively, at 1 μM). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 ± 6% block at 10 μg/min) and by correolide in vitro (29 ± 4% block at 1 μM). KV current in smooth muscle cells was inhibited by 4-AP (IC50 1.1 ± 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for KV1 family members was detected in coronary arteries. Our data indicate that KV channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine- and NO-induced vasodilation.
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Smart, Sharon L., Valeri Lopantsev, C. L. Zhang, Carol A. Robbins, Hao Wang, S. Y. Chiu, Philip A. Schwartzkroin, Albee Messing, and Bruce L. Tempel. "Deletion of the KV1.1 Potassium Channel Causes Epilepsy in Mice." Neuron 20, no. 4 (April 1998): 809–19. http://dx.doi.org/10.1016/s0896-6273(00)81018-1.
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Erisir, A., D. Lau, B. Rudy, and C. S. Leonard. "Function of Specific K+ Channels in Sustained High-Frequency Firing of Fast-Spiking Neocortical Interneurons." Journal of Neurophysiology 82, no. 5 (November 1, 1999): 2476–89. http://dx.doi.org/10.1152/jn.1999.82.5.2476.
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Fast-spiking GABAergic interneurons of the neocortex and hippocampus fire high-frequency trains of brief action potentials with little spike-frequency adaptation. How these striking properties arise is unclear, although recent evidence suggests K+ channels containing Kv3.1-Kv3.2 proteins play an important role. We investigated the role of these channels in the firing properties of fast-spiking neocortical interneurons from mouse somatosensory cortex using a pharmacological and modeling approach. Low tetraethylammonium (TEA) concentrations (≤1 mM), which block only a few known K+channels including Kv3.1-Kv3.2, profoundly impaired action potential repolarization and high-frequency firing. Analysis of the spike trains evoked by steady depolarization revealed that, although TEA had little effect on the initial firing rate, it strongly reduced firing frequency later in the trains. These effects appeared to be specific to Kv3.1 and Kv3.2 channels, because blockade of dendrotoxin-sensitive Kv1 channels and BK Ca2+-activated K+ channels, which also have high TEA sensitivity, produced opposite or no effects. Voltage-clamp experiments confirmed the presence of a Kv3.1-Kv3.2–like current in fast-spiking neurons, but not in other interneurons. Analysis of spike shape changes during the spike trains suggested that Na+ channel inactivation plays a significant role in the firing-rate slowdown produced by TEA, a conclusion that was supported by computer simulations. These findings indicate that the unique properties of Kv3.1-Kv3.2 channels enable sustained high-frequency firing by facilitating the recovery of Na+ channel inactivation and by minimizing the duration of the afterhyperpolarization in neocortical interneurons.
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Poveda, Clara, Maria Valero, Marianny Pernia, Juan Alvarado, David Ryugo, Miguel Merchan, and Jose Juiz. "Expression and Localization of Kv1.1 and Kv3.1b Potassium Channels in the Cochlear Nucleus and Inferior Colliculus after Long-Term Auditory Deafferentation." Brain Sciences 10, no. 1 (January 8, 2020): 35. http://dx.doi.org/10.3390/brainsci10010035.
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Deafness affects the expression and distribution of voltage-dependent potassium channels (Kvs) of central auditory neurons in the short-term, i.e., hours to days, but the consequences in the expression of Kvs after long-term deafness remain unknown. We tested expression and distribution of Kv1.1 and Kv3.1b, key for auditory processing, in the rat cochlear nucleus (CN), and in the inferior colliculus (IC), at 1, 15 and 90 days after mechanical lesion of the cochlea, using a combination of qRT-PCR and Western blot in the whole CN, along with semi-quantitative immunocytochemistry in the AVCN, where the role of both Kvs in the control of excitability for accurate auditory timing signal processing is well established. Neither Kv1.1/Kv3.1b mRNA or protein expression changed significantly in the CN between 1 and 15 days after deafness. At 90 days post-lesion, however, mRNA and protein expression for both Kvs increased, suggesting that regulation of Kv1.1 and Kv3.1b expression is part of cellular mechanisms for long-term adaptation to auditory deprivation in the CN. Consistent with these findings, immunocytochemistry showed increased labeling intensity for both Kvs in the AVCN at day 90 after cochlear lesion. This increase argues that up-regulation of Kv1.1 and Kv3.1b in AVCN neurons may be required to adapt intrinsic excitability to altered input over the long term after auditory deprivation. Contrary to these findings in the CN, expression levels of Kv1.1 and Kv3.1b in the IC did not undergo major changes after cochlear lesion. In particular, there was no evidence of long-term up-regulation of either Kv1.1 or Kv3.1b, supporting that such post-lesion adaptive mechanism may not be needed in the IC. These results reveal that post-lesion adaptations do not necessarily involve stereotyped plastic mechanisms along the entire auditory pathway.
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Johnson, Rosalyn P., Ahmed F. El-Yazbi, Morgan F. Hughes, David C. Schriemer, Emma J. Walsh, Michael P. Walsh, and William C. Cole. "Identification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449." Journal of Biological Chemistry 284, no. 24 (April 22, 2009): 16562–74. http://dx.doi.org/10.1074/jbc.m109.010918.
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Vascular smooth muscle Kv1 delayed rectifier K+ channels (KDR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with 449pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide 445KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro32P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing KDR channel modulation by agonists via PKA activation.
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Gittelman, Joshua X., and Bruce L. Tempel. "Kv1.1-Containing Channels Are Critical for Temporal Precision During Spike Initiation." Journal of Neurophysiology 96, no. 3 (September 2006): 1203–14. http://dx.doi.org/10.1152/jn.00092.2005.
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Low threshold, voltage-gated potassium currents ( Ikl) are widely expressed in auditory neurons that can fire temporally precise action potentials (APs). In the medial nucleus of the trapezoid body (MNTB), channels containing the Kv1.1 subunit (encoded by the Kcna1 gene) underlie Ikl. Using pharmacology, genetics and whole cell patch-clamp recordings in mouse brain slices, we tested the role of Ikl in limiting AP latency-variability (jitter) in response to trains of single inputs at moderate to high stimulation rates. With dendrotoxin-K (DTX-K, a selective blocker of Kv1.1-containing channels), we blocked Ikl maximally (≈80% with 100 nM DTX-K) or partially (≈50% with 1-h incubation in 3 nM DTX-K). Ikl was similar in 3 nM DTX-K–treated cells and cells from Kcna1−/− mice, allowing a comparison of these two different methods of Ikl reduction. In response to current injection, Ikl reduction increased the temporal window for AP initiation and increased jitter in response to the smallest currents that were able to drive APs. While 100 nM DTX-K caused the largest increases, latency and jitter in Kcna1 −/ − cells and in 3 nM DTX-K–treated cells were similar to each other but increased compared with +/+. The near-phenocopy of the Kcna1−/− cells with 3 nM DTX-K shows that acute blockade of a subset of the Kv1.1-containing channels is functionally similar to the chronic elimination of all Kv1.1 subunits. During rapid stimulation (100–500 Hz), Ikl reduction increased jitter in response to both large and small inputs. These data show that Ikl is critical for maintaining AP temporal precision at physiologically relevant firing rates.
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Skutel, Mikhail, Aleksandra Primak, Mikhail Kirpichnikov, Alexander Arseniev, Alexey Feofanov, and Oksana Nekrasova. "RFP-tagged Hongotoxin 1 and Its Interactions with KscA-Kv1.1 Hybrid Channels." Microscopy and Microanalysis 26, S2 (July 30, 2020): 1378–80. http://dx.doi.org/10.1017/s1431927620017900.
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Xiong, Weichen, Huizhen Fan, Qingye Zeng, Zhenhui Deng, Guanhui Li, Wancheng Lu, Bei Zhang, Shian Lai, Xin Chen, and Xueqing Xu. "The in vitro anticancer effects of FS48 from salivary glands of Xenopsylla cheopis on NCI-H460 cells via its blockage of voltage-gated K+ channels." Acta Pharmaceutica 73, no. 1 (January 24, 2023): 145–55. http://dx.doi.org/10.2478/acph-2023-0010.
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Abstract Voltage-gated K+ (Kv) channels play a role in the cellular processes of various cancer cells, including lung cancer cells. We previously identified and reported a salivary protein from the Xenopsylla cheopis, FS48, which exhibited inhibitory activity against Kv1.1-1.3 channels when assayed in HEK 293T cells. However, whether FS48 has an inhibitory effect on cancer cells expressing Kv channels is unclear. The present study aims to reveal the effects of FS48 on the Kv channels and the NCI-H460 human lung cancer cells through patch clamp, MTT, wound healing, transwell, gelatinase zymography, qRT-PCR and WB assays. The results demonstrated that FS48 can be effective in suppressing the Kv currents, migration, and invasion of NCI-H460 cells in a dose-dependent manner, despite the failure to inhibit the proliferation. Moreover, the expression of Kv1.1 and Kv1.3 mRNA and protein were found to be significantly reduced. Finally, FS48 decreases the mRNA level of MMP-9 while increasing TIMP-1 mRNA level. The present study highlights for the first time that blood-sucking arthropod saliva-derived protein can inhibit the physiological activities of tumour cells via the Kv channels. Furthermore, FS48 can be taken as a hit compound against the tumour cells expressing Kv channels.
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Primak, A. L., M. A. Skutel, O. V. Nekrasova, A. S. Arseniev, M. P. Kirpichnikov, and A. V. Feofanov. "Kv1 Potassium Channel Ligands Based on Hongotoxin 1 and Red Fluorescent Protein." Russian Journal of Bioorganic Chemistry 46, no. 6 (November 2020): 1011–17. http://dx.doi.org/10.1134/s1068162020060266.
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Kanemasa, T., L. Gan, T. M. Perney, L. Y. Wang, and L. K. Kaczmarek. "Electrophysiological and pharmacological characterization of a mammalian Shaw channel expressed in NIH 3T3 fibroblasts." Journal of Neurophysiology 74, no. 1 (July 1, 1995): 207–17. http://dx.doi.org/10.1152/jn.1995.74.1.207.
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1. The Shaw-like voltage-activated potassium channel Kv3.1 is expressed in neurons that generate rapid trains of action potentials. By expressing this channel in a mammalian cell line and by simulating its activation, we tested the potential role of this channel in action potential repolarization. 2. NIH 3T3 fibroblasts were stably transfected with Kv3.1 DNA. Currents recorded in these cells had a threshold of activation at approximately -10 mV, showed little inactivation, and were very sensitive to blockade by 4-aminopyridine and tetraethylammonium. 3. Kv3.1 currents activated rapidly at the onset of depolarizing voltage pulses. After an initial rapid phase of activation, which could be fit by an n4 Hodgkin-Huxley model, Kv3.1 currents expressed in fibroblasts had a second, slower phase of activation, and, in some cells, a slower phase of partial inactivation, both of which could be fit with modified n4p models. 4. Cell-attached single-channel recordings indicated that the Kv3.1 channel displays two gating behaviors, a short-open-time pattern, which occurs only at the onset of depolarization, and a long-open-time pattern, which predominates during prolonged depolarizations. 5. The amplitude of Kv3.1 currents, and the probability of channel openings, was reduced by a phorbol ester activator of protein kinase C, and the action of this agent was blocked by preincubation with the protein kinase inhibitor H7 (1-[5-isoquinolinesulfonyl]-2-methyl piperazine). In contrast, the effects of dioctanoyl glycerol, which also attenuated the currents, could not be completely blocked by H7, suggesting that diacylglycerols may act on the channel by a kinase-independent pathway. 6. Incorporation of a current with the kinetics and voltage dependence of Kv3.1 currents into a model cell with a sustained inward current showed that, in contrast to other delayed-rectifier currents such as the Shaker-like Kv1.1 and Kv1.6 channels, the level of expression of Kv3.1 currents could be varied over a wide range without attenuation of action potential height. Our results suggest that the Kv3.1 channel may provide rapidly firing neurons with a high safety factor for impulse propagation.
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Verdura, Edgard, Carme Fons, Agatha Schlüter, Montserrat Ruiz, Stéphane Fourcade, Carlos Casasnovas, Antonio Castellano, and Aurora Pujol. "Complete loss of KCNA1 activity causes neonatal epileptic encephalopathy and dyskinesia." Journal of Medical Genetics 57, no. 2 (October 5, 2019): 132–37. http://dx.doi.org/10.1136/jmedgenet-2019-106373.
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BackgroundSince 1994, over 50 families affected by the episodic ataxia type 1 disease spectrum have been described with mutations in KCNA1, encoding the voltage-gated K+ channel subunit Kv1.1. All of these mutations are either transmitted in an autosomal-dominant mode or found as de novo events.MethodsA patient presenting with a severe combination of dyskinesia and neonatal epileptic encephalopathy was sequenced by whole-exome sequencing (WES). A candidate variant was tested using cellular assays and patch-clamp recordings.ResultsWES revealed a homozygous variant (p.Val368Leu) in KCNA1, involving a conserved residue in the pore domain, close to the selectivity signature sequence for K+ ions (TVGYG). Functional analysis showed that mutant protein alone failed to produce functional channels in homozygous state, while coexpression with wild-type produced no effects on K+ currents, similar to wild-type protein alone. Treatment with oxcarbazepine, a sodium channel blocker, proved effective in controlling seizures.ConclusionThis newly identified variant is the first to be reported to act in a recessive mode of inheritance in KCNA1. These findings serve as a cautionary tale for the diagnosis of channelopathies, in which an unreported phenotypic presentation or mode of inheritance for the variant of interest can hinder the identification of causative variants and adequate treatment choice.
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Straub, Stephen V., Sylvie M. Perez, Beijing Tan, Kimberly A. Coughlan, Catherine E. Trebino, Patricia Cosgrove, Joanne M. Buxton, John M. Kreeger, and V. Margaret Jackson. "Pharmacological inhibition of Kv1.3 fails to modulate insulin sensitivity in diabetic mice or human insulin-sensitive tissues." American Journal of Physiology-Endocrinology and Metabolism 301, no. 2 (August 2011): E380—E390. http://dx.doi.org/10.1152/ajpendo.00076.2011.
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Genetic ablation of the voltage-gated potassium channel Kv1.3 improves insulin sensitivity and increases metabolic rate in mice. Inhibition of Kv1.3 in mouse adipose and skeletal muscle is reported to increase glucose uptake through increased GLUT4 translocation. Since Kv1.3 represents a novel target for the treatment of diabetes, the present study investigated whether Kv1.3 is functionally expressed in human adipose and skeletal muscle and whether specific pharmacological inhibition of the channel is capable of modulating insulin sensitivity in diabetic mouse models. Voltage-gated K+ channel currents in human skeletal muscle cells (SkMC) were insensitive to block by the specific Kv1.3 blockers 5-(4-phenoxybutoxy)psoralen (PAP-1) and margatoxin (MgTX). Glucose uptake into SkMC and mouse 3T3-L1 adipocytes was also unaffected by treatment with PAP-1 or MgTX. Kv1.3 protein expression was not observed in human adipose or skeletal muscle from normal and type 2 diabetic donors. To investigate the effect of specific Kv1.3 inhibition on insulin sensitivity in vivo, PAP-1 was administered to hyperglycemic mice either acutely or for 5 days prior to an insulin tolerance test. No effect on insulin sensitivity was observed at free plasma PAP-1 concentrations that are specific for inhibition of Kv1.3. Insulin sensitivity was increased only when plasma concentrations of PAP-1 were sufficient to inhibit other Kv1 channels. Surprisingly, acute inhibition of Kv1.3 in the brain was found to decrease insulin sensitivity in ob/ob mice. Overall, these findings are not supportive of a role for Kv1.3 in the modulation of peripheral insulin sensitivity.
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Seagar, Michael, Michael Russier, Olivier Caillard, Yves Maulet, Laure Fronzaroli-Molinieres, Marina De San Feliciano, Norah Boumedine-Guignon, et al. "LGI1 tunes intrinsic excitability by regulating the density of axonal Kv1 channels." Proceedings of the National Academy of Sciences 114, no. 29 (July 3, 2017): 7719–24. http://dx.doi.org/10.1073/pnas.1618656114.
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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.