Myricetin Facilitates Potassium Currents and Inhibits Neuronal Activity of PVN Neurons

The effects of myricetin on hypothalamic paraventricular nucleus (PVN) neurons in rats were investigated. By whole-cell patch clamp detection in hypothalamic brain slices, we showed that the action potential frequency in type-I PVN neurons dose-dependently decreased after myricetin treatment. Further studies demonstrated that myricetin may enhance potassium currents and shifts the voltage-dependence of activation of potassium currents to more negative potentials by 6.07 mV. Using calcium free/cadmium perfusion solution could reverse myricetin-induced enhancement of potassium currents in PVN neurons. These results suggested that inhibition of hypothalamic PVN neurons by myricetin might be attributed to the enhancement of potassium currents.     

一氧化氮通过巯基亚硝化途径抑制海马神经元的延迟整流型钾电流

应用膜片钳全细胞记录模式研究了内源性一氧化氮(NO)对培养海马神经元延迟整流型钾电流的调控作用及其机制.给予NO合成酶的底物L-精氨酸(L-Arg,2mmol/L)可显著抑制海马神经元上的延迟整流型钾电流,但其同分异构体D-精氨酸(2mmol/L)对钾电流则无明显影响.并且,经一氧化氮合成酶抑制剂L-NAME(nomega-nitro-L-argininemethylester,0.5mmol/L)预处理后,L-Arg对钾电流的抑制作用消失,表明L-Arg抑制钾电流是通过产生NO而不是精氨酸本身.特异性鸟苷酸环化酶抑制剂ODQ(1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one,10!mol/L)预处理不影响L-Arg对钾电流的抑制作用,但巯基烷化剂NEM(N-ethylmaleimide,1mmol/L)预处理可完全阻断L-Arg的抑制效应.以上结果表明,内源性NO主要通过巯基亚硝化途径抑制海马神经元的延迟整流型钾电流.     

Human Adipose Cells Have Voltage-dependent Potassium Currents

The whole-cell patch-clamp method was used to study the membrane electrical properties of human adipocyte cells obtained by differentiating from precursors of human abdominal and mammary tissues. All differentiated cells exhibited outward currents with sigmoidal activation kinetics. The outward currents showed activation thresholds between –20 to –30 mV and slow inactivation. The ionic channels underlying the macroscopic current were highly selective for K⁺. Their selectivity was for typical K⁺ channels with relative permeabilities of K⁺>NH ₄ ⁺ >Cs⁺>Na⁺. No evidence of any other type of voltage-gated channel was found. The potassium currents (I _KV) were blocked reversibly by tetraethylammonium and barium. The IC ₅₀ value and Hill coefficient of tetraethylammonium inhibition of I _KV were 0.56 mM and 1.17 respectively. These results demonstrate that human adipose cells have voltage-dependent potassium currents.     

Vitamins C and E Modulate Neuronal Potassium Currents

We investigated the effects of vitamins C and E on the delayed-rectifier potassium current (IK_DR), which is important in repolarizing the membrane potential, and on the transient A-type potassium current (IK_A), which regulates neuronal firing frequency. The whole-cell patch-clamp technique was used to measure the currents from cultured Drosophila neurons derived from embryonic neuroblasts. The membrane potential was stepped to different voltages between −40 and +60 mV from a holding potential of −80 mV. IK_DR and IK_A measured in the vitamin C-containing solution (IK_DR 305 ± 16 pA, IK_A 11 ± 2 pA) were smaller than those measured in the control solution (488 ± 21 pA, IK_A 28 ± 3 pA). By contrast, IK_DR and IK_A measured in the vitamin E-containing solution (IK_DR 561 ± 21 pA, IK_A 31 ± 3 pA) were greater than those measured in the control solution (422 ± 15 pA, 17 ± 2 pA). These results indicate that vitamins C and E can modulate potassium current amplitudes and possibly lead to altered neuronal excitability.     

Modulation of Potassium Channels Inhibits Bunyavirus Infection

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K⁺) channels to infect cells. Time of addition assays using K⁺ channel modulating agents demonstrated that K⁺ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K⁺ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K⁺ channels (K_2P) were identified as the K⁺ channel family mediating BUNV K⁺ channel dependence. As several K_2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.     

大鼠背根神经节神经元上失活 BK 通道特性的研究

大电导的电压和 Ca²⁺ 激活的 K⁺ 通道 (BK 通道 ) 在哺乳动物的组织中广泛表达,起着多种多样的作用 . 目前只有少数组织中 BK 通道的性质被深入地研究,而且鲜见有失活的 BK 通道 (BK_i) 的报道,尤其是在神经元中 . 发现在大鼠小直径的背根神经节 (DRG) 神经元中,普遍存在失活的 BK 通道 . 失活的 BK 电流成分是 Ca²⁺敏感的,可以被大电导的 BK 通道特异阻断剂 ChTX 所阻断,而且木瓜蛋白酶可以从胞外改变通道失活的特性 .     

Moderate Hypoxia Influences Potassium Outward Currents in Adipose-Derived Stem Cells

Moderate hypoxic preconditioning of adipose-derived stem cells (ASCs) enhances properties such as proliferation and secretion of growth factors, representing a valuable strategy to increase the efficiency of cell-based therapies. In a wide variety of cells potassium (K⁺) channels are key elements involved in the cellular responses to hypoxia, suggesting that ASCs cultured under low oxygen conditions may display altered electrophysiological properties. Here, the effects of moderate hypoxic culture on proliferation, whole-cell currents, and ion channel expression were investigated using human ASCs cultured at 5% and 20% oxygen. Although cell proliferation was greatly enhanced, the dose-dependent growth inhibition by the K⁺ channel blocker tetraethylammonium (TEA) was not significantly affected by hypoxia. Under both normoxic and hypoxic conditions, ASCs displayed outward K⁺ currents composed by Ca²⁺-activated, delayed rectifier, and transient components. Hypoxic culture reduced the slope of the current-voltage curves and caused a negative shift in the voltage activation threshold of the whole-cell currents. However, the TEA-mediated shift of voltage activation threshold was not affected by hypoxia. Semiquantitative real-time RT-PCR revealed that expression of genes encoding for various ion channels subunits related to oxygen sensing and proliferation remained unchanged after hypoxic culture. In conclusion, outward currents are influenced by moderate hypoxia in ASCs through a mechanism that is not likely the result of modulation of TEA-sensitive K⁺ channels.     

Quinine Inhibits Mitochondrial ATP-regulated Potassium Channel from Bovine Heart

The mitochondrial ATP-regulated potassium (mitoK_ATP) channel has been suggested as trigger and effector in myocardial ischemic preconditioning. However, molecular and pharmacological properties of the mitoK_ATP channel remain unclear. In the present study, single-channel activity was measured after reconstitution of the inner mitochondrial membrane from bovine ventricular myocardium into bilayer lipid membrane. After incorporation, a potassium-selective current was recorded with mean conductance of 103 ± 9 pS in symmetrical 150 mM KCl. Single-channel activity of this reconstituted protein showed properties of the mitoK_ATP channel: it was blocked by 500 μM ATP/Mg, activated by the potassium-channel opener diazoxide at 30 μM, inhibited by 50 μM glibenclamide or 150 μM 5-hydroxydecanoic acid, and was not affected by the plasma membrane ATP-regulated potassium-channel blocker HMR1098 at 100 μM. We observed that the mitoK_ATP channel was blocked by quinine in the micromolar concentration range. The inhibition by quinine was additionally verified with the use of ⁸⁶Rb⁺ flux experiments and submitochondrial particles. Quinine inhibited binding of the sulfonylurea derivative [³H]glibenclamide to the inner mitochondrial membrane. We conclude that quinine inhibits the cardiac mitoK_ATP channel by acting on the mitochondrial sulfonylurea receptor.(P. Bednarczyk and A. Kicińska) These authors contributed equally to this work.This revised version was published online in August 2005 with a corrected cover date.     

Opposing Effects of Aluminum on Inward-Rectifier Potassium Currents in BeanRoot-Tip Protoplasts

Inward currents in root cap protoplasts of the aluminum-tolerant cultivar, Dade, of Phaseolus vulgaris L. were investigated using the whole-cell patch-clamp technique. The properties of these currents were similar to those seen in inward rectifying K⁺ channels in other plant tissues. Replacing bath K⁺ with Na⁺ nearly abolished the observed currents. Higher bath K⁺ concentrations increased inward currents. AlCl₃ in pH 4.7 bath solutions caused inward K⁺ currents to activate more rapidly and at more positive voltages when compared with AlCl₃ free solutions. In 10 μM AlCl₃ the activated inward K⁺ currents were significantly larger than in the AlCl₃-free solution at all voltages except at the most negative voltage of −174 mV and the least negative of −74 mV. In contrast, in 80 μM Al³⁺, when hyperpolarizing voltages were most negative, the inward K⁺ currents were inhibited relative to the currents in 10 μM AlCl₃. Enhancement of inward K⁺ currents by AlCl₃ is consistent with Al³⁺ binding to the external surface of the root cap protoplast, decreasing the surface charge, thus causing the channels to sense a more negative membrane potential. Inhibition of inward K⁺ currents with higher AlCl₃ concentrations and more negative voltages is consistent with Al³⁺ block of K⁺ channels.This revised version was published online in August 2005 with a corrected cover date.     

Effects of KCNQ2 Gene Truncation on M-Type Kv7 Potassium Currents

The KCNQ2 gene product, Kv7.2, is a subunit of the M-channel, a low-threshold voltage-gated K⁺ channel that regulates mammalian and human neuronal excitability. Spontaneous mutations one of the KCNQ2 genes cause disorders of neural excitability such as Benign Familial Neonatal Seizures. However there appear to be no reports in which both human KCNQ2 genes are mutated. We therefore asked what happens to M-channel function when both KCNQ2 genes are disrupted. We addressed this using sympathetic neurons isolated from mice in which the KCNQ2 gene was truncated at a position corresponding to the second transmembrane domain of the Kv7.2 protein. Since homozygote KCNQ2−/− mice die postnatally, experiments were largely restricted to neurons from late embryos. Quantitative PCR revealed an absence of KCNQ2 mRNA in ganglia from KCNQ2−/− embryos but 100–120% increase of KCNQ3 and KCNQ5 mRNAs; KCNQ2+/− ganglia showed ∼30% less KCNQ2 mRNA than wild-type (+/+) ganglia but 40–50% more KCNQ3 and KCNQ5 mRNA. Neurons from KCNQ2−/− embryos showed a complete absence of M-current, even after applying the Kv7 channel enhancer, retigabine. Neurons from heterozygote KCNQ2+/− embryos had ∼60% reduced M-current. In contrast, M-currents in neurons from adult KCNQ2+/− mice were no smaller than those in neurons from wild-type mice. Measurements of tetraethylammonium block did not indicate an increased expression of Kv7.5-containing subunits, implying a compensatory increase in Kv7.2 expression from the remaining KCNQ2 gene. We conclude that mouse embryonic M-channels have an absolute requirement for Kv7.2 subunits for functionality, that the reduced M-channel activity in heterozygote KCNQ2+/− mouse embryos results primarily from a gene-dosage effect, and that there is a compensatory increase in Kv7.2 expression in adult mice.     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

Photoperiod Modulates Fast Delayed Rectifier Potassium Currents in the Mammalian Circadian Clock

One feature of the mammalian circadian clock, situated in the suprachiasmatic nucleus (SCN), is its ability to measure day length and thereby contribute to the seasonal adaptation of physiology and behavior. The timing signal from the SCN, namely the 24 hr pattern of electrical activity, is adjusted according to the photoperiod being broader in long days and narrower in short days. Vasoactive intestinal peptide and gamma-aminobutyric acid play a crucial role in intercellular communication within the SCN and contribute to the seasonal changes in phase distribution. However, little is known about the underlying ionic mechanisms of synchronization. The present study was aimed to identify cellular mechanisms involved in seasonal encoding by the SCN. Mice were adapted to long-day (light–dark 16:8) and short-day (light–dark 8:16) photoperiods and membrane properties as well as K⁺ currents activity of SCN neurons were measured using patch-clamp recordings in acute slices. Remarkably, we found evidence for a photoperiodic effect on the fast delayed rectifier K⁺ current, that is, the circadian modulation of this ion channel’s activation reversed in long days resulting in 50% higher peak values during the night compared with the unaltered day values. Consistent with fast delayed rectifier enhancement, duration of action potentials during the night was shortened and afterhyperpolarization potentials increased in amplitude and duration. The slow delayed rectifier, transient K⁺ currents, and membrane excitability were not affected by photoperiod. We conclude that photoperiod can change intrinsic ion channel properties of the SCN neurons, which may influence cellular communication and contribute to photoperiodic phase adjustment.     

The Selective Inhibition of Delayed Potassium Currents in Nerve by Tetraethylammonium Ion

The effect of tetraethylammonium ion (TEA) on the voltage clamp currents of nodes of Ranvier of frog myelinated nerve fibers is studied. The delayed K currents can be totally abolished by TEA without affecting the transient Na currents or the leakage current in any way. Both inward and outward currents disappear. In low TEA concentrations small K currents remain with normal time constants. The dose-response relationship suggests the formation of a complex between TEA and a receptor with a dissociation constant of 0.4 mM. Other symmetrical quaternary ammonium ions have very little effect. There is no competition between TEA and agents that affect the Na currents such as Xylocaine, tetrodotoxin, or Ca ions. The pharmacological data demonstrate that the Na, K, and leakage permeabilities are chemically independent, probably because their mechanisms occupy different sites on the nodal membrane. The data are gathered and analyzed by digital computer.     

Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

Allitridi (diallyl trisulfide) is an active compound (volatile oil) from garlic. The previous studies reported that allitridi had anti-arrhythmic effect. The potential ionic mechanisms are, however, not understood. The present study was designed to determine the effects of allitridi on cardiac potassium channels expressed in HEK 293 cells using a whole-cell patch voltage-clamp technique and mutagenesis. It was found that allitridi inhibited hKv4.3 channels (IC₅₀ = 11.4 µM) by binding to the open channel, shifting availability potential to hyperpolarization, and accelerating closed-state inactivation of the channel. The hKv4.3 mutants T366A, T367A, V392A, and I395A showed a reduced response to allitridi with IC₅₀s of 35.5 µM, 44.7 µM, 23.7 µM, and 42.4 µM. In addition, allitridi decreased hKv1.5, hERG, hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells with IC₅₀s of 40.2 µM, 19.6 µM and 17.7 µM. However, it slightly inhibited hKir2.1 current (100 µM, inhibited by 9.8% at −120 mV). Our results demonstrate for the first time that allitridi preferably blocks hKv4.3 current by binding to the open channel at T366 and T367 of P-loop helix, and at V392 and I395 of S6 domain. It has a weak inhibition of hKv1.5, hERG, and hKCNQ1/hKCNE1 currents. These effects may account for its anti-arrhythmic effect observed in experimental animal models.     

A Single-File Model for Potassium Transport in Squid Giant Axon: Simulation of Potassium Currents at Normal Ionic Concentrations

A physical model for potassium transport in squid giant axon is proposed. The model is designed to explain the empirical data given by the Hodgkin-Huxley model and related experiments. It is assumed that K⁺ moves across the axon membrane by single-file diffusion through narrow pores. In the model a pore has three negatively charged sites that can be occupied alternatively by K⁺ or by a gating particle, GP⁺⁺, coming from the external surface. GP⁺⁺ is considered to be part of the membrane rather than a diffusible component of the surrounding solutions. A high activation barrier for GP⁺⁺ is supposed at the inner membrane border so that it cannot change over to the internal surface. Therefore potassium diffusion can be blocked by GP⁺⁺ penetrating into the pores. This mechanism controls the dynamic behaviour of the model. The time-dependent probabilities of the pore states are described by a system of differential equations. The rate constants in these equations depend on the ionic concentrations, the membrane voltage, and the electrostatic interaction between ions in a single pore. Detailed computational tests for normal composition of external and internal solutions show that the model agrees remarkably well with the stationary and dynamic behaviour of the Hodgkin-Huxley model. However, the hyperpolarization delay is not reproduced. A structural modification, concerning this delay and the way in which GP⁺⁺ is attached to the membrane, is proposed, and the qualitative behavior of the model at varied external and internal concentrations is discussed.     

Hydrogen Sulfide Suppresses Outward Rectifier Potassium Currents in Human Pluripotent Stem Cell-Derived Cardiomyocytes

Aim

Hydrogen sulfide (H₂S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H₂S.

Methods and Results

Cardiomyocytes of ventricular, atrial and nodal subtypes differentiated from H9 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were electrophysiologically characterized. The effect of NaHS, a donor of H₂S, on action potential (AP), outward rectifier potassium currents (I _Ks and I _Kr), L-type Ca²⁺ currents (I _CaL) and hyperpolarization-activated inward current (I _f) were determined by patch-clamp electrophysiology and confocal calcium imaging. In a concentration-dependent manner, NaHS (100 to 300 µM) consistently altered the action potential properties including prolonging action potential duration (APD) and slowing down contracting rates of ventricular-and atrial-like cardiomyocytes derived from both hESCs and hiPSCs. Moreover, inhibitions of slow and rapid I _K (I _Ks and I _Kr), I _CaL and I _f were found in NaHS treated cardiomyocytes and it could collectively contribute to the remodeling of AP properties.

Conclusions

This is the first demonstration of effects of H₂S on cardiac electrophysiology of human ventricular-like, atrial-like and nodal-like cardiomyocytes. It reaffirmed the inhibitory effect of H₂S on I _CaL and revealed additional novel inhibitory effects on I _f, I _Ks and I _Kr currents in human cardiomyocytes.     

大鼠发育过程中下丘脑神经元钾离子通道温度效应变化的研究

为了探讨出生后钾离子通道在下丘脑神经元热敏感分化过程中的作用,采用膜片钳技术研究出生一个月内SD大鼠急性分离神经元的温度效应,结果表明IK电流密度在出生后一个月内变化不大(P>0.05),而IA电流密度则呈现为升高趋势(P<0.05).同时升高温度,不同出生日期的钾通道NPo都有不同程度的升高,但相较P1d的神经元来说,温度对P18d的电压依赖性影响更大一些.同时温度对IK和IA的影响是不一样的,IA的Q10>2,所有这些显示IA通道在神经元温度敏感性的发育分化过程中起着重要的作用.