D-Tubocurarine-Sensitive Component of Calcium-Dependent Potassium Current in Guinea Pig <Emphasis Type="Italic">Taenia Coli</Emphasis> Myocytes

Using a patch-clamp technique in the whole-cell configuration, we studied transmembrane ion currents in isolated single smooth muscle cells of the guinea pig taenia coli. A depolarizing step shift of the membrane potential from −50 mV was accompanied by the appearance of an outward current. Application of d-tubocurarine (d-TK) or a nonselective blocker of voltage-dependent potassium channels, tetraethylammonium (TEA), led to a decrease in the outward current. Application of d-TK against the background of the action of TEA additionally decreased the outward current. Analysis of the current-voltage (I–V) relationships of the d-TK-sensitive current showed that this current is practically voltage-independent. At the same time, an inflection of the I–V curve of the potassium current within the segment of maximum activation of the voltage-dependent potassium current is indicative of the sensitivity of this current to the intracellular Ca²⁺ concentration. Therefore, the calcium-activated potassium current through small-conductance calcium-dependent potassium channels includes a d-TK-sensitive voltage-independent component. Using depolarizing shifts of the membrane potential, we observed high- and low-amplitude spontaneous outward currents (SOCs) in many studied cells, i.e., the effect of an increase in the conductance of calcium-dependent potassium channels as a result of periodic release of Ca²⁺ from the intracellular stores. Application of d-TK led to a decrease in the frequency of low-amplitude SOCs and exerted nearly no influence on the high-amplitude SOCs under study. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 271–277, May–June, 2005.     

Role of calcium mobilization in the regulation of spontaneous transient outward currents in porcine coronary artery myocytes

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents (STOCs) in freshly isolated porcine coronary artery smooth muscle cells (ASMCs). STOCs were recorded using the perforated whole-cell patch-clamp configuration. STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca2 -activated-K (BKCa) currents. Charybdotoxin (ChTX, 200 nmol/L), a selective blocker of BKCa channels, completely inhibited STOCs within 10 min. STOCs activity was greatly suppressed when extracellular Ca2 concentration decreased from 1.8 mmol/L to 200 nmol/L, further removal of Ca2 abolished STOCs activity. Ca2 ionophore A23187 (10 μmol/L) increased STOCs activity significantly. Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two kinds of organic L-type voltage-dependent Ca2 channels (L-VDCCs) antagonists, had little effect on STOCs. In addition, the ryanodine receptors (RyRs) agonist caffeine (5 mmol/L) significantly activated STOCs. Application of ryanodine (50 μmol/L) to block RyRs abolished STOCs, subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity. Inhibition of inositol 1,4,5-trisphosphate receptors (IP3Rs) by 2APB (40 μmol/L) greatly suppressed the activity of STOCs, application of caffeine (5 mmol/L) in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs. These results suggest that STOCs in porcine coronary ASMCs are mediated by BKCa channels. Extracellular Ca2 is essential for STOCs activity, while Ca2 entry through L-VDCCs has little effect on STOCs. Intracellular Ca2 release induced by RyRs is responsible for the regulation of STOCs, whereas IP3Rs might also be involved.     

Role of calcium mobilization in the regulation of spontaneous transient outward currents in porcine coronary artery myocytes

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents (STOCs) in freshly isolated porcine coronary artery smooth muscle cells (ASMCs). STOCs were recorded using the perforated whole-cell patch-clamp configuration. STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca²⁺-activated-K⁺ (BK_Ca) currents. Charybdotoxin (ChTX, 200 nmol/L), a selective blocker of BK_Ca channels, completely inhibited STOCs within 10 min. STOCs activity was greatly suppressed when extracellular Ca²⁺ concentration decreased from 1.8 mmol/L to 200 nmol/L, further removal of Ca²⁺ abolished STOCs activity. Ca²⁺ ionophore A23187 (10 μmol/L) increased STOCs activity significantly. Verapamil (20 μmol/L) and CdCl₂ (200 μmol/L), two kinds of organic L-type voltage-dependent Ca²⁺ channels (L-VDCCs) antagonists, had little effect on STOCs. In addition, the ryanodine receptors (RyRs) agonist caffeine (5 mmol/L) significantly activated STOCs. Application of ryanodine (50 μmol/L) to block RyRs abolished STOCs, subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity. Inhibition of inositol 1,4,5-trisphosphate receptors (IP₃Rs) by 2APB (40 μmol/L) greatly suppressed the activity of STOCs, application of caffeine (5 mmol/L) in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs. These results suggest that STOCs in porcine coronary ASMCs are mediated by BK_Ca channels. Extracellular Ca²⁺ is essential for STOCs activity, while Ca²⁺ entry through L-VDCCs has little effect on STOCs. Intracellular Ca²⁺ release induced by RyRs is responsible for the regulation of STOCs, whereas IP3Rs might also be involved.     

西洛他唑对人心房肌细胞瞬间外向钾电流的影响

目的:观察西洛他唑对人心房肌细胞瞬间外向钾电流(Ito1)的影响,探讨该药抗心律失常作用的机制.方法:二步酶解法分离人单个右心房肌细胞,应用全细胞膜片钳技术记录人心房肌细胞Ito1.结果:在保持电位-50 mV和去极化脉冲为 50 mV条件下,30 μmol/L西洛他唑显著降低Ito1,使Ito1幅值由加药前(8.16±0.70)pA/pF降至(4.84±0.60)pA/pF(P<0.01).西洛他唑在1～50 μmol/L范围内呈浓度依赖性的抑制Ito1,1 μmol/L时即产生作用,50 μmol/L时达最大效应(降低51.09%±3.00%),IC50为(13.18±2.60)μmol/L.此外,该药对Ito1的电压依赖性激活和失活曲线以及恢复曲线均无显著影响.结论:本实验结果表明西洛他唑浓度依赖性地阻滞人心房肌细胞的Ito1.     

去甲肾上腺素对大鼠肝细胞延迟外向钾电流的影响

目前为止国内外尚未见到有关大鼠肝细胞外向钾电流方面的报道。本文用全细胞膜片宿制技术观察了大鼠肝细胞延迟外向钾电流（Ik）及去甲肾上腺素等对人的影响。实验结果表明,在保持电位-50mV、指令电位＋140mV时大鼠肝细胞Ik为2．85±1.21nA。去甲肾上腺素明显降低IK,异丙肾上腺素和乙酰胆碱对IK无影响。     

Properties of the charibdotoxin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigTaenia Coli

Using the voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In our study, we identified and studied a charibdotoxin-sensitive component of Ca²⁺-dependent K⁺ current carried through the channels of high conductance (in most publications called “big conductance,”I _BK(Ca)). This component was completely blocked by 100 nM charibdotoxin and by tetraethylammonium in concentrations as low as 1 mM.I _BK(Ca) demonstrated fast kinetics of inactivation, which nearly coincided with that of Ca²⁺ current. In addition to the dependence on Ca²⁺ concentration, this current also showed voltage-dependent properties: with a rise in the level of depolarization its amplitude increased. In many cells, depolarizing shifts in the membrane potential evoke spontaneous outward currents. Such currents probably represent the secondary effect of cyclic Ca²⁺ release from the caffeine-sensitive intracellular stores that result in short-term activation of charibdotoxin-sensitive Ca²⁺-dependent K⁺ channels.     

Characteristics of the inward-rectifying potassium current in mouse ventricular myocytes and its relation to early after-depolarization

The properties of the inward-rectifying potassium current (IK1) were studied in the single myocytes isolated from adult mouse ventricles by the whole-cell patch-damp technique for the first time. Most of the properties of IK1 including channel conductances, activation, inactivation, rectification and external K sensitivity in mouse ventricular myocyte were similar to those in other species, but the current-voltage (1-V) curve of mouse ventricular myocyte showed no negative slope, i.e the slope in the range of membrane potential 50 mV positive to the reversal potential (VRev) was virtually flat and remained at a low current level ((59±39) pA). Under the superfusion of Tyrode's solution with 3mmol/L K and 3mmol/L Cs , IK1 in the above region nearly decreased to zero, and then the early after-depolarization (EAD) occurred. The results suggest that this distinctive characteristic of IK1 in mouse ventricular myocyte may relate to the high susceptibility to EA0 in mouse myocardium. The inhibition of IK1 se     

慢性吸烟大鼠气道平滑肌大电导的钙激活钾通道和Kv1.5表达的变化

复制大鼠的慢性吸烟模型,采用气道反应性的测定、HE染色、免疫组织化学染色、原位杂交和免疫印迹实验等方法,观察吸烟对大鼠支气管平滑肌大电导的钙激活的钾通道(BKca)和电压依赖性延迟整流钾通道Kv1．5蛋白和mRNA表达的影响,以阐明吸烟引起的气道高反应性发病机制中钾通道表达变化的作用。结果显示：(1)慢性吸烟可降低大鼠大气道和小气道BKca和Kv1．5蛋白和mRNA表达;(2)大气道BKca的降低程度大于Kv1．5,小气道BKca和Kv1．5的降低程度无明显差异：(3)吸烟对全肺组织BKca和Kv1．5的蛋白表达无明显影响。上述结果提示,慢性吸烟可下调大鼠气道平滑肌钾通道BKca和Kv1．5的表达水平,是导致气道高反应的机制之一。     

Adrenergic modulation of potassium currents in isolated human atrial myocytes

The adrenergic modulation of inwardly rectifying and depolarization-activated outward potassium currents was studied in single cardiac myocytes obtained from the human atrium. Membrane currents were recorded in enzymatically dissociated cells using the whole-cell voltage-clamp technique. It was observed that, in the presence or absence of atenolol (or 1 µM propranolol), 30 µM phenylephrine attenuated inwardly rectifying and depolarization-activated outward potassium currents including both transient and late-activated current. This suppressant effect of phenylephrine could be prevented by pretreatment with an -adrenoceptor antagonist. Isoproterenol (30 µM) increased the late outward potassium current and net transient outward current. It is concluded that, in human atrial myocytes, -adrenergic activation reduces depolarization-activated transient and late outward potassium current and inwardly rectifying background potassium current. -Adrenergic activation resulted in an increase in the depolarization-activated transient and late outward potassium current.     

花生四烯酸对大鼠顶叶皮层神经元延迟整流钾电流的抑制作用

目的和方法:采用全细胞式膜片钳技术,观察花生四烯酸(AA)对大鼠顶叶皮层神经元延迟整流钾电流(Ik)的影响。结果:①AA(10μmol/L)对大鼠顶叶皮层神经元Ik有抑制作用,抑制率为33.9%±8.74%(P<0.01)。②AA可使IK激活曲线的斜率因子变大且曲线向右移动,IK激活曲线的V1/2和k分别由给药前的(-55.3±0.9)mV和(10.3±0.4)mV,变为给药后的(-50.8±2.4)mV和(21.0±3.5)mV。③AA可使IK失活曲线斜率因子变大且曲线向左移动,IK失活曲线的V1/2和k分别由给药前的(-45.3±0.3)mV和(15.6±0.8)mV,变为给药后的(-70.9±1.9)mV和(36.5±2.1)mV。结论:花生四烯酸可抑制大鼠顶叶皮层神经元的延迟整流钾电流,并影响其动力学特征。     

四周模拟失重大鼠后身动脉平滑肌细胞钾电流的改变

本文采用全细胞膜片钳方法观察4周尾部悬吊大鼠（tail-suspended rats,SUS)隐动脉及肠系膜的动脉第2-6级动脉分支血管平滑肌细胞（vascular smooth muscle cells,VSMCs)钾电流密度的变化,结果表明：SUS大鼠后身动脉VSMCs的静息电位（RP）较对照大鼠（CON）后身动脉VSMCs的RP更负,SUS组隐动脉和肠系膜小鼠后身动脉VSMCs的静息电位（RP）较对照大鼠（CON）后身动脉VSMCs的RP更负,SUS组隐动脉和肠系膜小动脉VSMCs的全细胞钾电流密度较CON组显著增加,其中,SUS组的隐动脉和肠系膜小动脉VSMCs的大电导钙激活钙离子通道（BKca)和电压激活钾离子通道（Kv)电流密度较CON组的BKca和Kv电流密度均显著增加,以上结果提示,VSMCs的超极化及进一步引起的通过电压依赖性钙离子通道的钙内流减少可能是模拟失重引起后身动脉反应性降低的电生理机制之一。     

ACh对大鼠皮层体感区神经元延迟整流钾电流的抑制作用

利用全细胞膜片钳技术研究乙酰胆碱(acetylcholine,ACh)对大鼠皮层体感区神经元延迟整流钾电流(IK)的调制作用。结果表明:(1)ACh(0．1、1、10、100 μmol/L)对大鼠皮层体感区神经元IK有抑制作用,并具有剂量依赖性关系(P<0．01)。 (2)ACh可使IK激活曲线的斜率变大,并使激活曲线向超极化方向移动。IK激活曲线的半数激活电压(V1/12)和斜率因子(k)分别由给药前的(-41．8±9．7)mV和(30．7±7．2)mV变为给药后的(-122．4±38．6)mV和(42．4±7．0)mV。(3)100 μmol/L的N受体拮抗剂筒箭毒碱(tubocurarine)可减弱ACh对IK的抑制作用,在指令电压+60 mV时tubocurarine+ACh组的IK幅度下降了(16．9± 13．8)％(n=8),与10 μmol/L ACh组引起的(36．5±7．8)％的IK下降幅度相比,有极显著差异(P<0．01)。10 μmol/L的M1受体拮抗剂哌仑西平(pirenzepin)拮抗ACh对IK的抑制作用不明显(n=7,P>0．05);而10 μmol/L的M3受体拮抗剂4-DAMP可部分拮抗ACh对IK的抑制作用,并且4-DAMP+ACh组使IK的电流值下降了(26．8±4．7)％(n=6),与ACh组引起的IK电流下降相比,有显著差异(P<0．05)。(4)蛋白激酶C(protein kinase C,PKC)阻断剂chelerythrine拮抗ACh对IK的抑制作用,PKC激动剂PDBu可增强ACh对IK的抑制作用(P<0．05)。综上所述,ACh对人鼠皮层体感区神经元IK的抑制作用主要是通过烟碱受体(nAChRs)和M3受体介导,并经过PKC信号途径。     

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.     

Differential contributions of somatic and dendritic calcium-dependent potassium currents to the control of motoneuron excitability following spinal cord injury

The hyperexcitability of alpha-motoneurons and accompanying spasticity following spinal cord injury (SCI) have been attributed to enhanced persistent inward currents (PICs), including L-type calcium and persistent sodium currents. Factors controlling PICs may offer new therapies for managing spasticity. Such factors include calcium-activated potassium (KCa) currents, comprising in motoneurons an after-hyperpolarization-producing current (I _KCaN) activated by N/P-type calcium currents, and a second current (I _KCaL) activated by L-type calcium currents (Li and Bennett in J neurophysiol 97:767–783, 2007). We hypothesize that these two currents offer differential control of PICs and motoneuron excitability based on their probable somatic and dendritic locations, respectively. We reproduced SCI-induced PIC enhancement in a two-compartment motoneuron model that resulted in persistent dendritic plateau potentials. Removing dendritic I _KCaL eliminated primary frequency range discharge and produced an abrupt transition into tertiary range firing without significant changes in the overall frequency gain. However, I _KCaN removal mainly increased the gain. Steady-state analyses of dendritic membrane potential showed that I _KCaL limits plateau potential magnitude and strongly modulates the somatic injected current thresholds for plateau onset and offset. In contrast, I _KCaN had no effect on the plateau magnitude and thresholds. These results suggest that impaired function of I _KCaL may be an important intrinsic mechanism underlying PIC-induced motoneuron hyperexcitability following SCI.     

Developmental temperature selectively regulates a voltage-activated potassium current in Drosophila

Ionic currents are regulated by many conditions including disease states, ageing, learning and memory, and chronic drug treatment. Here we describe a novel phenomenon of regulation of ionic currents by developmental temperature. Raising Drosophila larvae at 28°C instead of 18°C increased one of the two voltage-activated K⁺- currents, the delayed sustained I_K, in their muscles by up to 3.5-fold, with little effect on the early transient current, I_A. Consistent with this increase in I_K, the amplitude and the duration of the action potentials were reduced. The major increase in I_K occurred between a rather abrupt interval from 25° to 28°C. The activation curve of the increased current was shifted towards hyperpolarizing potentials. There was no change in activation kinetics. This phenomenon has mechanistic implications for activity-dependent neuronal plasticity, expression of ion channels in cultured cells and heterologus systems, phototransduction, and behavior. The specificity of the regulation suggests a discrete mechanism geared to affect excitability such that it can respond to altered external stimuli such as temperature. 1994 John Wiley & Sons, Inc.     

The creatine kinase system in smooth muscle

Despite the energetic flux being much lower in smooth muscle compared to striated muscles (such as the heart and skeletal muscle) creatine kinase (CK) has been found present and active in all smooth muscles studied to date. A complete CK circuit has been identified, with CK found in the mitochondria, contractile elements, membrane pumps and the cytoplasm. CK isoenzymes are coupled to many cellular energetic processes and appears to be involved in energy production and consumption by acting as an energy transducer. The CK system responds to pathological insults and development (e.g. hypertrophy and gestation respectively) by changes in sub-cellular distribution localization, isoenzymes, and specific activity. The conclusion from these observations is that creatine kinase is intimately involved in the energetic system of smooth muscle.Abbreviations CK creatine kinase - Mi-CK mitochondrial creatine kinase - Cr creatine - PCr phosphocreatiner - NMR nuclear magnetic resonance - SHR spontaneously hypertensive rat - -GPA -guanidinopropionic acid     

高血压病患者肠系膜动脉平滑肌细胞钙激活钾通道研究

目的:研究高血压病患者肠系膜动脉平滑肌细胞钙激活钾通道(KCa)的功能活动。方法:应用膜片钳制技术内面向外式单通道记录方法。结果:①人肠系膜动脉平滑肌细胞KCa开放具有电压依赖性。KCa通道电导在高血压组、正常组分别为191.4pS、197.7pS。胞内侧应用TEA可阻断通道。②增加浴液中Ca2 浓度(从0增至10-8、10-7、5×10-7、10-6mol/L),各组KCa开放概率(Po)均呈浓度依赖性增加,高血压组Po从0.016增至0.023、0.031、0.053、0.094,正常组Po从0.004增至0.023、0.041、0.072、0.184。通道平均开放时间延长,平均关闭时间缩短。③Ca2 浓度为0时,高血压组KCa开放概率明显高于正常组,在其它Ca2 浓度下高血压组KCa开放概率等于或低于正常组。结论:高血压病患者肠系膜动脉平滑肌细胞KCa的Ca2 敏感性较低,可能促进高血压的发生。     

Genistein inhibits the inward rectifying potassium current in guinea pig ventricular myocytes

Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. Previous studies have shown that genistein has additional effects, among which the direct blocking effects on various ionic channels have recently been disclosed. Using whole-cell voltage clamp and current clamp techniques, we demonstrate that micromolar concentrations of genistein dose-dependently and reversibly inhibit the inward rectifying K(+) current, and depolarize the resting membrane potential, resulting in abnormal automaticity in guinea pig ventricular myocytes. Interestingly, another potent tyrosine kinase inhibitor, tyrphostin 51, did not produce the same inhibitory effect, while the inactive analogue of genistein, daidzein, had a similar blocking effect. We suggest that genistein directly blocks the inward rectifying K(+) current in ventricular myocytes, and one should be cautious of its pro-arrhythmic effect in clinical use.     

焦亚硫酸钠对大鼠海马CA1区神经元钾电流的影响

目的:探讨焦亚硫酸钠(SMB)、二氧化硫(SO2)及其体内衍生物(亚硫酸盐和亚硫酸氢盐)对中枢神经元钾通道的影响及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及谷胱甘肽过氧化物酶(GPx)相应的保护作用.方法:采用全细胞膜片钳技术研究了SMB对大鼠海马CA1区神经元瞬间外向钾电流(IA)和延迟整流钾电流(IK)的影响.结果:①焦亚硫酸钠可增大全细胞IA和IK,且具剂量依赖性和电压依赖性,使IA和IK增大50%的剂量分别为15.8 μmol/L和11.5μmol/L;②10 μmol/L的SMB均可显著影响IA和IK的激活过程,给药前后IA的半数激活电压分别为(-12.6±1.6)mV和(-7.0±1.3)mV(n=8,P＜0.01),IK的半数激活电压分别为(10.8±0.9)mV和(21.6±0.7)mV(n=8,P＜0.01),但不改变其斜率因子;③10μmol/L的SMB还非常显著地影响IA的失活过程,给药前后其半数失活电压分别为(-97.0±1.1)mV和(-84.4±3.3)mV(n=8,P＜0.01),但也不改变其斜率因子;④抗氧化酶SOD(1×106U/L)、CAT(2×106U/L)及GPx(105U/L)均可使SMB(10μmol/L)增大的IA和IK部分恢复.结论:SMB可显著增大IA和IK,抑制IA和IK的激活过程及IA的失活过程,从而导致胞内K 的外流增加,使胞内K 浓度降低,从而对中枢神经元功能产生不利影响.