Differentiation of potassium currents in cultured inhibitory interneurons of the rat hippocampus (identification of the potassium M-type current)

Using a patch-clamp technique in the whole-cell configuration, we identified the potassium M-type current and estimated its contribution to the integral depolarization-induced potassium current evoked in cultured hippocampal inhibitory interneurons of the rat. With the help of immunocytochemical labeling, we checked the presence of the KCNQ-family channels responsible for generation of M current in these neurons. It was demonstrated that non-inactivated potassium channels and channels with slow kinetics play the main role in the processes of repolarization of the membrane of inhibitory interneurons. In all studied cells, a potassium current non-inactivated with time and possessing kinetic parameters close to those of the M current developed in response to depolarization. In all cells, positive immunocytochemical labeling with respect to KCNQ2 channels was observed; however, its intensity varied significantly from neuron to neuron. The level of suppression of non-inactivated potassium currents by a blocker of KCNQ channels, linopirdine, varied noticeably in different cells; therefore, the level of expression of these channels in the interneurons under study is probably considerably dissimilar. The reason for incomplete suppression of the M current is perhaps the involvement of other potassium channels (e.g., those of Kv1 family) in the formation of this current. Neirofiziologiya/Neurophysiology, Vol. 38, No. 3, pp. 198–204, May–June, 2006.     

Expression of Kv1.3 potassium channels regulates density of cortical interneurons

The Kv1.3 protein is a member of the large family of voltage‐dependent K⁺ subunits (Kv channels), which assemble to form tetrameric membrane‐spanning channels that provide a selective pore for the conductance of K⁺ across the cell membrane. Kv1.3 differs from most other Kv channels in that deletion of Kv1.3 gene produces very striking changes in development and structure of the olfactory bulb, where Kv1.3 is expressed at high levels, resulting in a lower threshold for detection of odors, an increased number of synaptic glomeruli and alterations in the levels of a variety of neuronal signaling molecules. Because Kv1.3 is also expressed in the cerebral cortex, we have now examined the effects of deletion of the Kv1.3 gene on the expression of interneuron populations of the cerebral cortex. Using unbiased stereology we found an increase in the number of parvalbumin (PV) cells in whole cerebral cortex of Kv1.3^?/? mice relative to that in wild‐type mice, and a decrease in the number of calbindin (CB), calretinin (CR), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and somatostatin (SOM) interneurons. These changes are accompanied by a decrease in the cortical volume such that the cell density of PV interneurons is significantly increased and that of SOM neurons is decreased in Kv1.3^?/? animals. Our studies suggest that, as in the olfactory bulb, Kv1.3 plays a unique role in neuronal differentiation and/or survival of interneuron populations and that expression of Kv1.3 is required for normal cortical function. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73:841–855, 2013     

Kv4.2通道电流与大鼠海马神经元瞬间外向钾电流特性的比较

本研究比较了转染的Kv4.2钾电流与原代培养大鼠海马神经元上瞬间外向钾电流(IA)动力学特征。实验采用瞬时转染,细胞培养和全细胞膜片钳记录等方法。结果表明:转染的Kv4.2通道电流和海马神经元上IA均具有明显的A型电流特征。海马神经元IA的半数最大激活电位和斜率因子分别为-10.0±3.3 mV和13.9±2.6 mV;半数最大失活电位和斜率因子分别为-93.0±11.4 mV和-9.0±1.5 mV;失活后再激活恢复时间常数(T)为27.9±14.1 ms。Kv4.2的半数最大激活电位和斜率因子分别为-9.7±4.1 mV和15.8±5.7 mV;半数最大失活电位和斜率因子分别为-59.4±12.2 mV和8.0±3.1 mV;Kv4.2的灭活后再激活的恢复时间常数τ为172.8±10.0 ms。结果提示:Kv4.2通道电流可能是海马神经元上的IA电流的主要成分,但不是唯一成分。     

Effects of mibefradil on synaptic transmission in the hippocampus and on voltage-dependent currents in isolated hippocampal and thalamic neurons of the rat

The effects of a novel anti-hypertensive drug, mibefradil, on voltage-dependent currents in isolated thalamic and hippocampal neurons, as well as on synaptic transmission in the hippocampus have been studied. Mibefradil exerted a potent inhibitory action on low-threshold calcium currents in thalamic neurons (IC₅₀=160 nM). In higher concentrations (1–20 μM), this drug blocked not only low-threshold calcium current but also voltage-dependent sodium and delayed potassium currents in pyramidal hippocampal neurons. The amplitude of population action potentials in hippocampal slices decreased by 55% in the presence of 20μM mibefradil. All of the effects of mibefradil were almost completely reversible. In our experiments, the sensitivity of low-threshold calcium channels in thalamic neurons to mibefradil was higher than that observed on other objects. The ability of mibefradil to block not only calcium currents but also other types of voltage-dependent ion conductances in hippocampal neurons may be considered an essential factor that determines the specificity of the pharmacological profile of this drug.     

Role of calmodulin in neuronal Kv7/KCNQ potassium channels and epilepsy

Neuronal Kv7/KCNQ channels are critical regulators of neuronal excitability since they potently suppress repetitive firing of action potentials. These voltage-dependent potassium channels are composed mostly of Kv7.2 / KCNQ2 and KvT.3 / KCNQ3 subunits that show overlapping distribution throughout the brain and in the peripheral nervous system. They are also called ＇M-channels＇ since their inhibition by muscarinic agonists leads to a profound increase in action potential firing. Consistent with their ability to suppress seizures and attenuate chronic inflammatory and neuropathic pain, mutations in the KCNQ2 and KCNQ3 genes are associated with benign familial neonatal convulsions, a dominantly-inherited epilepsy in infancy. Recently, de novo mutations in the KCNQ2 gene have been linked to early onset epileptic encephalopathy. Notably, some of these mutations are clustered in a region of the intracellular cytoplasmic tail of Kv7.2 that interacts with a ubiquitous calcium sensor, calmodulin. In this review, we highlight the recent advances in understanding the role of calmodulin in modulating physiological function of neuronal Kv7 channels including their biophysical properties, assembly, and trafficking. We also summarize recent studies that have investigated functional impact of epilepsy-associated mutations localized to the calmodulin binding domains of Kv7.2.     

Characterization of binding site of closed-state KCNQ1 potassium channel by homology modeling, molecular docking, and pharmacophore identification

This investigation was performed to assess the importance of interaction in the binding of blockers to KCNQ1 potassium using molecular modeling. This work could be considered made up by three main steps: (1) the construction of closed-state structure of KCNQ1 through homology modeling; (2) the automated docking of three blockers: IKS-142, L-735821, and BMS-IKS, using DOCK program; (3) the generation and validation of pharmacophore for KCNQ1 ligands using Catalyst/HypoGen. The obtained results highlight the hydrophobic or aromatic residues involved in S6 transmembrane domain and the base of the pore helix of KCNQ1, confirming the mutagenesis data and pharmacophore model, and giving new suggestions for the rational design of novel KCNQ1 ligands.     

Evoked Electrical Activity and Immunocytochemical Peculiarities of Cultured Excitatory and Inhibitory Neurons of the Rat Hippocampus

Experiments were carried out on cultured hippocampal neurons using a patch-clamp technique in the whole-cell configuration. We studied the characteristics of regular series of action potentials (APs), which were generated with a low frequency by inhibitory and excitatory interneurons after their direct stimulation with long-lasting (500 msec) current pulses. Nearly all parameters of the evoked impulse activity (except the frequency of generation and duration of APs) in excitatory and inhibitory neurons were significantly different. According to immunocytochemical analysis, Kv1.2- and Kv4.2-type potassium channels were expressed in the membrane of excitatory neurons (granular cells), and somatostatin was present in all these cells. As to inhibitory interneurons, only a part of such cells (large units) demonstrated immunopositivity with respect to somatostatin. In inhibitory neurons, only Kv1.2-type potassium channels were expressed. Therefore, mechanisms responsible for the ability of hippocampal interneurons to generate impulse activity under conditions of direct stimulation (in our experiments, regular low-frequency series of APs) in inhibitory and excitatory neurons are rather dissimilar. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 207–216, May–June, 2005.     

培养的大脑皮层、海马及交感神经细胞钠、钾和钙离子通道的全细胞记录技术

目的: 建立新生大鼠大脑皮层、海马细胞及交感神经元细胞的培养方法及其钠、钾和钙通道的膜片钳全细胞记录技术.方法: 取出生1～3 d的大鼠大脑皮层、海马及交感神经节,用胰蛋白酶(0.125%)消化组织并分离出神经细胞,种植在涂有多聚赖氨酸的35 mm培养皿中,用高糖的DMEM培养液培养,一周后,镜下可见神经细胞壁光滑、完整,周围有明亮的光润,在高倍倒置显微镜下可见到完整的细胞核及均匀的胞浆,细胞间形成良好的突触连接,可用于细胞膜片钳记录.结果: 用胰蛋白酶消化分离培养的神经细胞,功能状态良好,在膜片钳全细胞记录中,易形成细胞与记录电极间的高阻抗封接,可分别记录到INa、IA、IK和ICa.结论:在神经系统电生理学研究中,此方法可应用于中枢神经系统不同脑区培养以及钠、钾和钙通道电流的记录.     

Ion channels in the regulation of autophagy

Autophagy is a cellular process in which the cell degrades and recycles its own constituents. Given the crucial role of autophagy in physiology, deregulation of autophagic machinery is associated with various diseases. Hence, a thorough understanding of autophagy regulatory mechanisms is crucially important for the elaboration of efficient treatments for different diseases. Recently, ion channels, mediating ion fluxes across cellular membranes, have emerged as important regulators of both basal and induced autophagy. However, the mechanisms by which specific ion channels regulate autophagy are still poorly understood, thus underscoring the need for further research in this field. Here we discuss the involvement of major types of ion channels in autophagy regulation.     

Long-term potentiation in hippocampal oriens interneurons: postsynaptic induction,presynaptic expression and evaluation of candidate retrograde factors

Several types of hippocampal interneurons exhibit a form of long-term potentiation (LTP) that depends on Ca²⁺-permeable AMPA receptors and group I metabotropic glutamate receptors. Several sources of evidence point to a presynaptic locus of LTP maintenance. The retrograde factor that triggers the expression of LTP remains unidentified. Here, we show that trains of action potentials in putative oriens-lacunosum-moleculare interneurons of the mouse CA1 region can induce long-lasting potentiation of stimulus-evoked excitatory postsynaptic currents that mimics LTP elicited by high-frequency afferent stimulation. We further report that blockers of nitric oxide production or TRPV1 receptors failed to prevent LTP induction. The present results add to the evidence that retrograde signalling underlies N-methyl-d-aspartate (NMDA) receptor-independent LTP in oriens interneurons, mediated by an unidentified factor.     

Involvement of KCNQ2 subunits in [3H]dopamine release triggered by depolarization and pre-synaptic muscarinic receptor activation from rat striatal synaptosomes

KCNQ2 and KCNQ3 subunits encode for the muscarinic-regulated current (I(KM)), a sub-threshold voltage-dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of I(KM) in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the I(KM) activator retigabine (0.01-30 micromol/L; Emax = 54.80 +/- 3.85%; IC50 = 0.50 +/- 0.36 micromol/L). The I(KM) blockers tetraethylammonium (0.1-3 mmol/L) and XE-991 (0.1-30 micromol/L) enhanced K+-evoked [3H]DA release and prevented retigabine-induced inhibition of depolarization-evoked [3H]DA release. Retigabine-induced inhibition of K+-evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti-KCNQ2 polyclonal antibodies, an effect prevented by antibody pre-absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1-300 micromol/L) potentiated 9 mmol/L [K+]e-evoked [3H]DA release (Emax = 155 +/- 9.50%; EC50 = 25 +/- 1.80 micromol/L). OXO (100 micromol/L)-induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1-10 nmol/L) and abolished by the M3-preferring antagonist 4-diphenylacetoxy N-methylpiperidine methiodide (1 micromol/L), but was unaffected by the M1-selective antagonist MT-7 (10-100 nmol/L) or by Pertussis toxin (1.5-3 microg/mL), which uncouples M2- and M4-mediated responses. Finally, OXO-induced potentiation of depolarization-induced [3H]DA release was not additive to that produced by XE-991 (10 micromol/L), was unaffected by retigabine (10 micromol/L), and was abolished by synaptosomal entrapment of anti-KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, I(KM) channels containing KCNQ2 subunits regulate depolarization-induced DA release and that I(KM) suppression is involved in the reinforcement of depolarization-induced DA release triggered by the activation of pre-synaptic muscarinic heteroreceptors.     

ATP-dependent potassium channels of muscle cells: Their properties,regulation, and possible functions

ATP-dependent potassium channels are present at high density in the membranes of heart, skeletal, and smooth muscle and have a lowP _open at physiological [ATP]_i. The unitary conductance is 15–20 pS at physiological [K⁺] _o , and the channels are highly selective for K⁺. Certain sulfonylureas are specific blockers, and some K channel openers may also act through these channels. K_ATP channels are probably regulated through the binding of ATP, which may in turn be regulated through changes in the ADP/ATP ratio or in pH_i. There is some evidence for control through G-proteins. The channels have complex kinetics, with multiple open and closed states. The main effect of ATP is to increase occupancy of long-lived closed states. The channels may have a role in the control of excitability and probably act as a route for K⁺ loss from muscle during activity. In arterial smooth muscle they may act as targets for vasodilators.     

Characterization of the inhibitory effects of erythromycin and clarithromycin on the HERG potassium channel

Both erythromycin and clarithromycin have been reported to cause QT prolongation and the cardiac arrhythmia torsade de pointes in humans, however direct evidence documenting that these drugs produce this effect by blocking human cardiac ion channels is lacking. The goal of this study was to test the hypothesis that these macrolide antibiotics significantly block the delayed rectifier current (I_Kr) encoded by HERG (the human ether-a-go-go-related gene) at drug concentrations, temperature and ionic conditions mimicking those occurring in human subjects. Potassium currents in HEK 293 cells stably transfected with HERG were recorded using a whole cell voltage clamp method. Exposure of cells to erythromycin reduced the HERG encoded potassium current in a concentration dependent manner with an IC₅₀ of 38.9 ± 1.2 M and Hill Slope factor of 0.4 ± 0.1. Clarithromycin produced a similar concentration-dependent block with an IC₅₀ of 45.7 ± 1.1 M and Hill Slope factor of 1.0 ± 0.1. Erythromycin (25–250 M) and clarithromycin (5 or 25 M) also produced a significant decrease in the integral of the current evoked by an action potential shaped voltage clamp protocol. The results of this study document that both erythromycin and clarithromycin significantly inhibit the HERG potassium current at clinically relevant concentrations.     

利诺吡啶对豚鼠耳蜗外毛细胞和Deiters细胞钾电流的抑制

为探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的功能性表达,我们观察并记录了KCNQ家族钾通道阻滞剂利诺吡啶对豚鼠耳蜗单离外毛细胞(outer hair cells,OHCs)和Deiters细胞总钾电流的影响。采用酶孵育加机械分离法分离豚鼠耳蜗单个OHCs和Deiters细胞：运用膜片钳技术,在全细胞模式下记录正常细胞外液中8个外毛细胞和5个Deiters细胞的总钾电流,并观察100μmol／L和200μmol／L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果观察到,在正常细胞外液中的单离外毛细胞,可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流(the K^ current activated at negative potential,IKa)两种钾电流,而在单离Deiters细胞中只记录到外向整流性钾电流。在细胞外液中,加入100μmol／L利诺吡啶后,OHCs中的四乙基二乙胺敏感的钾电流峰电流成分被抑制,稳态电流幅值减小,且电流的失活时问常数明显延长;在细胞外液中加入100μmol／L和200μmol／L利诺吡啶后,OHCs的内向性钾电流IKa被完全抑制;而细胞外液中利诺吡啶终浓度为200μmol／L时,Deiters细胞的外向整流性钾电流幅值无明显变化。由此我们推测,KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞,其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分,并构成全部的IKn,其功能是介导细胞内K^ 外流和防止细胞过度去极化;KCNQ家族钾通道不存在于豚鼠耳蜗Dciters细胞。     

Suppressive effect of an activator of ATP-dependent potassium channels, flocalin, on electrical and contractile activities of smooth muscles of the guinea-pig ureter

In experiments on isolated segments or strips obtained from the guinea-pig ureter, we showed, using a sucrose-gap technique, that application of an activator of ATP-dependent potassium channels (K_ATP), (flocalin (PF-5), suppresses generation of action potentials (APs) by ureter smooth muscle cells (SMCs). Pre-incubation of the ureter preparations under study in Krebs solution containing 1 to 10 μM PF-5 results initially in a decrease in the frequency of oscillations preceding an AP plateau, shortening of this plateau, and, later on, complete inhibition of AP generation. In the presence of PF-5, spikes induced by hyperpotassium depolarization were also inhibited, while a tonic component of such depolarization underwent a mild decrease. The data obtained indicate that PF-5 modulates the entry of Ca²⁺ ions through L-type voltage-dependent channels in the SMC membrane. Shortening of the plateau and suppression of the spikes initiated by application of an activator of voltage-dependent L-type potassium channels, Bay K 8644, can be considered a confirmation of the modulatory influence of PF-5 on voltage-dependent L-type potassium channels. It seems possible that Bay K 8644 can be used under experimental conditions for initiation and long-lasting modulation of APs generated by the ureter SMC instead of corresponding neurotransmitters. We hypothesize that voltage-dependent entry of Ca²⁺ ions into SMCs depends significantly on the PF-5-induced activation of K_ATP channels of the ureter SMCs. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 403–409, September–December, 2005.     

Activation of K+ and Cl− channels in MDCK cells during volume regulation in hypotonic media

Single-channel patch-clamp experiments were performed on MDCK cells in order to characterize the ionic channels participating in regulatory volume decrease (RVD). Subconfluent layers of cultured cells were exposed to a hypotonic medium (150 mOsm), and the membrane currents at the single-channel level were measured in cell-attached experiments. The results indicate that MDCK cells respond to a hypotonic swelling by activating several different ionic conductances. In particular, a potassium and a chloride channel appeared in the recordings more frequently than other channels, and this allowed a more detailed study of their properties in the inside-out configuration of the patch-clamp technique. The potassium channel had a linear I/V curve with a unitary conductance of 24 +/- 4 pS in symmetrical K+ concentrations (145 mM). It was highly selective for K+ ions vs. Na+ ions: PNa/PK less than 0.04. The time course of its open probability (P0) showed that the cells responded to the hypotonic shock with a rapid activation of this channel. This state of high activity was maintained during the first minute of hypotonicity. The chloride channel participating in RVD was an outward-rectifying channel: outward slope conductance of 63.3 +/- 4.7 pS and inward slope conductance of 26.1 +/- 4.9 pS. It was permeable to both Cl- and NO3- and its maximal activation after the hypotonic shock was reached after several seconds (between 30 and 100 sec). The activity of this anionic channel did not depend on cytoplasmic calcium concentration. Quinine acted as a rapid blocker of both channels when applied to the cytoplasmic side of the membrane. In both cases, 1 mM quinine reversibly reduced single-channel current amplitudes by 20 to 30%. These results indicate that MDCK cells responded to a hypotonic swelling by an early activation of highly selective potassium conductances and a delayed activation of anionic conductances. These data are in good agreement with the changes of membrane potential measured during RVD.     

External barium affects the gating of KCNQ1 potassium channels and produces a pore block via two discrete sites

The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba(2+) binding kinetics and the concentration and voltage dependence of Ba(2+) steady-state block. Our results indicate that extracellular Ba(2+) exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba(2+) site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba(2+) site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba(2+) on channel gating in low external K(+) solutions. Ba(2+) binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K(+) attenuates Ba(2+) inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K(+) channels, KCNQ1 channels display significant structural and functional uniqueness.     

Rundown and reactivation of ATP-sensitive potassium channels (KATP) in mouse skeletal muscle

Dissociated single fibers from the mouse flexor digitorum brevis (FDB) muscle were used in patch clamp experiments to investigate the mechanisms of activation and inactivation of K_ATP in mammalian skeletal muscle. Spontaneous rundown of channel activity, in many excised patches, occurred gradually over a period of 10–20 min. Application of 1.0 mm free-Ca²⁺ to the cytoplasmic side of the patch caused irreversible inactivation of K_ATP within 15 sec. Ca²⁺-induced rundown was not prevented by the presence of 1.0 m okadaic acid or 2.0 mg ml^– of an inhibitor of calcium-activated neutral proteases, a result consistent with the conclusion that phosphatases or calcium-activated neutral proteases were not involved in the rundown process. Application of 1.0 mm Mg.ATP to Ca²⁺inactivated K_ATP caused inhibition of residual activity but little or no reactivation of the channels upon washout of ATP, even in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase (10 U ml^–1). Mg.ATP also failed to reactivate K_ATP, even after only partial spontaneous rundown, despite the presence of channels that could be activated by the potassium channel opener BRL 38227. Nucleotide diphosphates (500 m; CDP, UDP, GDP and IDP) caused immediate and reversible opening of Ca²⁺-inactivated K_ATP. Reactivation of K_ATP by ADP (100 m) increased further upon removal of the nucleotide. In contrast to K_ATP from cardiac and pancreatic cells, there was no evidence for phosphorylation of K_ATP from the surface sarcolemma of dissociated single fibers from mouse skeletal muscle. The small degree of activation occasionally observed following application of 10 m or 1.0 mm Mg.ATP could have been due to the generation of ADP from ATP hydrolysis and not through phosphorylation. Data are consistent with the suggestion that Ca²⁺ inactivation of K_ATP involves a gating mechanism that can be reopened by nucleotide diphosphates.M.H. is supported by the Medical Research Council.