Trequattrini C, Catacuzzeno L, Petris A, Franciolini F
Dipartimento Biologia Cellulare e Molecolare, Università di Perugia Via Pascoli 1, I-06100 Perugia, Italy.
Pflugers Arch. 1998 Mar;435(4):503-10. doi: 10.1007/s004240050545.
We have used the patch-clamp method in the whole-cell configuration to investigate the mechanism of block of the delayed rectifier K current (IDRK) by verapamil in embryonic chick dorsal root ganglion (DRG) neurons. Verapamil induced a dose-dependent decay of the current, without altering its activation kinetics. This observation, together with the good description of IDRK time course at various blocker concentrations with the computer simulation of a three-state chain model (closed left and right arrow open left and right arrow open-blocked), indicates that verapamil acts as a state-dependent, open-channel blocker. To account for the double-exponential time course of recovery from block, this minimal kinetics scheme was expanded to include a closed-blocked state resulting from channel closure (at hyperpolarized voltages) with verapamil still bound to it. The apparent block and unblock rate constants assessed from verapamil-induced current decay in the presence of external Na were 0.95 +/- 0.05 ms-1mM-1 and 0.0037 +/- 0.0016 ms-1, respectively. When external Na was replaced by K, only the unblock rate constant changed, to 0.02 +/- 0.009 ms-1. Under these ionic conditions it was also observed that the recovery from block was modified from the double-exponential time course in the presence of external Na (tau1 = 160 ms; tau2 = 1600 ms), to a faster single-exponential recovery (tau = 100 ms). We tested the voltage dependence of block by applying stimulation protocols aimed at eliminating bias easily introduced by the shift of the gating equilibrium and by the coupling of channel activation and block. Under these experimental conditions the resulting block rate constant was not measurably voltage dependent.
我们采用全细胞膜片钳方法，研究了维拉帕米对胚胎鸡背根神经节（DRG）神经元延迟整流钾电流（IDRK）的阻断机制。维拉帕米引起电流呈剂量依赖性衰减，而不改变其激活动力学。这一观察结果，以及通过三态链模型（关闭⇄开放⇄开放-阻断）的计算机模拟对不同阻断剂浓度下IDRK时间进程的良好描述，表明维拉帕米作为一种状态依赖性的开放通道阻断剂起作用。为了解释从阻断中恢复的双指数时间进程，这个最小动力学方案被扩展，以包括通道关闭（在超极化电压下）导致的关闭-阻断状态，此时维拉帕米仍与之结合。在存在外部Na的情况下，从维拉帕米诱导的电流衰减评估得到的表观阻断和解阻断速率常数分别为0.95±0.05 ms-1mM-1和0.0037±0.0016 ms-1。当外部Na被K取代时，只有解阻断速率常数发生变化，变为0.02±0.009 ms-1。在这些离子条件下还观察到，从阻断中恢复的过程从存在外部Na时的双指数时间进程（τ1 = 160 ms；τ2 = 1600 ms），变为更快的单指数恢复（τ = 100 ms）。我们通过应用刺激方案来测试阻断的电压依赖性，该方案旨在消除由门控平衡的移动以及通道激活和阻断的耦合容易引入的偏差。在这些实验条件下，得到的阻断速率常数没有明显的电压依赖性。
