Josephson I R, Sperelakis N
Department of Physiology and Biophysics, University of Cincinnati, College of Medicine, Ohio 45267-0576.
J Gen Physiol. 1992 Aug;100(2):195-216. doi: 10.1085/jgp.100.2.195.
Nonlinear or asymmetric charge movement was recorded from single ventricular myocytes cultured from 17-d-old embryonic chick hearts using the whole-cell patch clamp method. The myocytes were exposed to the appropriate intracellular and extracellular solutions designed to block Na+, Ca2+, and K+ ionic currents. The linear components of the capacity and leakage currents during test voltage steps were eliminated by adding summed, hyperpolarizing control step currents. Upon depolarization from negative holding potentials the nonlinear charge movement was composed of two distinct and separable kinetic components. An early rapidly decaying component (decay time constant range: 0.12-0.50 ms) was significant at test potentials positive to -70 mV and displayed saturation above 0 mV (midpoint -35 mV; apparent valence 1.6 e-). The early ON charge was partially immobilized during brief (5 ms) depolarizing test steps and was more completely immobilized by the application of less negative holding potentials. A second slower-decaying component (decay time constant range: 0.88-3.7 ms) was activated at test potentials positive to -60 mV and showed saturation above +20 mV (midpoint -13 mV, apparent valence 1.9 e-). The second component of charge movement was immobilized by long duration (5 s) holding potentials, applied over a more positive voltage range than those that reduced the early component. The voltage dependencies for activation and inactivation of the Na+ and Ca2+ ionic currents were determined for myocytes in which these currents were not blocked. There was a positive correlation between the voltage dependence of activation and inactivation of the Na+ and Ca2+ ionic currents and the activation and immobilization of the fast and slow components of charge movement. These complementary kinetic and steady-state properties lead to the conclusion that the two components of charge movement are associated with the voltage-sensitive conformational changes that precede Na+ and Ca2+ channel openings.
采用全细胞膜片钳方法，从17日龄鸡胚心脏培养的单个心室肌细胞中记录到非线性或不对称电荷移动。将这些肌细胞置于旨在阻断钠、钙和钾离子电流的合适细胞内和细胞外溶液中。通过加入叠加的超极化控制阶跃电流，消除了测试电压阶跃期间电容电流和漏电流的线性成分。从负的钳制电位去极化时，非线性电荷移动由两个不同且可分离的动力学成分组成。一个早期快速衰减成分（衰减时间常数范围：0.12 - 0.50毫秒）在测试电位高于 -70 mV时显著，且在0 mV以上表现出饱和（中点 -35 mV；表观价态1.6 e-）。早期开启电荷在短暂（5毫秒）去极化测试步骤中部分固定，通过施加不太负的钳制电位能更完全地固定。第二个较慢衰减成分（衰减时间常数范围：0.88 - 3.7毫秒）在测试电位高于 -60 mV时被激活，且在 +20 mV以上表现出饱和（中点 -13 mV，表观价态1.9 e-）。电荷移动的第二个成分通过长时间（5秒）钳制电位固定，施加的电压范围比降低早期成分的电压范围更正。对于未阻断钠和钙电流的肌细胞，测定了钠和钙离子电流激活和失活的电压依赖性。钠和钙离子电流激活和失活的电压依赖性与电荷移动的快速和慢速成分的激活和固定之间存在正相关。这些互补的动力学和稳态特性得出结论，电荷移动的两个成分与钠和钙通道开放之前的电压敏感构象变化有关。
