Bertaso F, Hendry B M, Donohoe P, James A F
Cell Signalling Group, King's College London, London, United Kingdom.
Biochem Biophys Res Commun. 2000 Jun 24;273(1):10-6. doi: 10.1006/bbrc.2000.2886.
External divalent cations are known to play an important role in the function of voltage-gated ion channels. The purpose of this study was to examine the sensitivity of the voltage-gated K(+) currents of human atrial myocytes to external Ca(2+) ions. Myocytes were isolated by collagenase digestion of atrial appendages taken from patients undergoing coronary artery-bypass surgery. Currents were recorded from single isolated myocytes at 37 degrees C using the whole-cell patch-clamp technique. With 0.5 mM external Ca(2+), voltage pulses positive to -20 mV (holding potential = -60 mV) activated outward currents which very rapidly reached a peak (I(peak)) and subsequently inactivated (tau = 7.5 +/- 0.7 msec at +60 mV) to a sustained level, demonstrating the contribution of both rapidly inactivating transient (I(to1)) and non-inactivating sustained (I(so)) outward currents. The I(to1) component of I(peak), but not I(so), showed voltage-dependent inactivation using 100 msec prepulses (V(1/2) = -35.2 +/- 0.5 mV). The K(+) channel blocker, 4-aminopyridine (4-AP, 2 mM), inhibited I(to1) by approximately 76% and reduced I(so) by approximately 33%. Removal of external Ca(2+) had several effects: (i) I(peak) was reduced in a manner consistent with an approximately 13 mV shift to negative voltages in the voltage-dependent inactivation of I(to1). (ii) I(so) was increased over the entire voltage range and this was associated with an increase in a non-inactivating 4-AP-sensitive current. (iii) In 79% cells (11/14), a slowly inactivating component was revealed such that the time-dependent inactivation was described by a double exponential time course (tau(1) = 7.0 +/- 0.7, tau(2) = 90 +/- 21 msec at +60 mV) with no effect on the fast time constant. Removal of external Ca(2+) was associated with an additional component to the voltage-dependent inactivation of I(peak) and I(so) (V(1/2) = -20.5 +/- 1.5 mV). The slowly inactivating component was seen only in the absence of external Ca(2+) ions and was insensitive to 4-AP (2 mM). Experiments with Cs(+)-rich pipette solutions suggested that the Ca(2+)-sensitive currents were carried predominantly by K(+) ions. External Ca(2+) ions are important to voltage-gated K(+) channel function in human atrial myocytes and removal of external Ca(2+) ions affects I(to1) and 4-AP-sensitive I(so) in distinct ways.
已知细胞外二价阳离子在电压门控离子通道的功能中发挥重要作用。本研究的目的是检测人心房肌细胞电压门控钾电流对细胞外钙离子的敏感性。通过胶原酶消化取自接受冠状动脉搭桥手术患者的心房附件来分离肌细胞。使用全细胞膜片钳技术在37℃下从单个分离的肌细胞记录电流。在细胞外钙离子浓度为0.5 mM时，正向-20 mV的电压脉冲（钳制电位=-60 mV）激活外向电流，该电流迅速达到峰值（I(peak)），随后失活（在+60 mV时，tau = 7.5±0.7毫秒）至一个稳定水平，这表明快速失活的瞬时外向电流（I(to1)）和非失活的持续外向电流（I(so)）均有贡献。I(peak)的I(to1)成分而非I(so)成分，在使用100毫秒预脉冲时表现出电压依赖性失活（V(1/2)=-35.2±0.5 mV）。钾通道阻滞剂4-氨基吡啶（4-AP，2 mM）可抑制I(to1)约76%，并使I(so)降低约33%。去除细胞外钙离子有多种效应：(i) I(peak)降低，其方式与I(to1)电压依赖性失活向负电压方向约13 mV的偏移一致。(ii) 在整个电压范围内I(so)增加，这与一种非失活的4-AP敏感电流增加相关。(iii) 在79%的细胞（11/14）中，揭示出一个缓慢失活成分，使得时间依赖性失活由双指数时间进程描述（在+60 mV时，tau(1)=7.0±0.7，tau(2)=90±21毫秒），对快速时间常数无影响。去除细胞外钙离子与I(peak)和I(so)电压依赖性失活的一个额外成分相关（V(1/2)=-20.5±1.5 mV）。缓慢失活成分仅在无细胞外钙离子时出现，且对4-AP（2 mM）不敏感。使用富含铯离子的微电极溶液进行的实验表明，对钙离子敏感的电流主要由钾离子携带。细胞外钙离子对人心房肌细胞电压门控钾通道功能很重要，去除细胞外钙离子以不同方式影响I(to1)和4-AP敏感的I(so)。
