Milnes James T, Witchel Harry J, Leaney Joanne L, Leishman Derek J, Hancox Jules C
Department of Physiology and Pharmacology, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK.
J Pharmacol Toxicol Methods. 2010 Mar-Apr;61(2):178-91. doi: 10.1016/j.vascn.2010.02.007. Epub 2010 Feb 19.
Pharmacological inhibition of cardiac potassium channels encoded by hERG (human ether-à-go-go-related gene) is associated with QT interval prolongation and torsades de pointes arrhythmia. Electrophysiological assays of hERG channel inhibition are integral to the safety testing of novel drug candidates. This study was conducted to compare, for the high affinity hERG inhibitors dofetilide and cisapride, hERG blockade between action potential (AP) and conventional (step and step-ramp) screening waveforms. Furthermore, it evaluated dynamic (pulse-by-pulse) protocol-dependence of hERG channel inhibition by these drugs.
Whole-cell patch-clamp recordings were made at 37 degrees C from hERG-expressing HEK 293 cells. Half-maximal inhibitory concentrations (IC(50) values) for I(hERG) blockade were obtained using conventional voltage clamp and action potential clamp, using previously digitised ventricular and Purkinje fibre (PF) AP waveforms.
A more marked variation in IC(50) values with different command waveforms was observed for cisapride (ranging from 7 to 72 nM) than for dofetilide (ranging from 4 to 15 nM), with higher IC(50)s obtained with AP than step or step-ramp commands. The two drugs differed little from one another in effects on voltage-dependent activation; however, I(hERG) blockade by each drug was initially voltage-dependent, but at steady-state was only voltage-dependent for cisapride. There was comparatively little difference between the two drugs in effects on I(hERG) availability or time constants of development of inactivation. Features of time-dependence of blockade and the use of protocols employing varying rest periods in drug or commands of alternating duration highlighted a pronounced ability of cisapride, but not dofetilide, to dissociate and reassociate from hERG on a pulse-by-pulse basis.
Protocols described here that demonstrated dynamic variation (drug dissociation/reassociation) in hERG channel current blockade at 37 degrees C for cisapride may have future value for investigating drug interactions with the hERG channel. Downloadable digitised ventricular and PF AP waveforms that can be used in AP clamp experiments also accompany this article.
对由hERG（人ether-à-go-go相关基因）编码的心脏钾通道进行药理学抑制与QT间期延长和尖端扭转型室性心动过速有关。hERG通道抑制的电生理测定是新型候选药物安全性测试不可或缺的一部分。本研究旨在比较高亲和力hERG抑制剂多非利特和西沙必利在动作电位（AP）和传统（阶跃和阶跃-斜坡）筛选波形下对hERG的阻断作用。此外，还评估了这些药物对hERG通道抑制的动态（逐脉冲）方案依赖性。
在37℃下，采用全细胞膜片钳记录法，从表达hERG的HEK 293细胞中进行记录。使用传统电压钳和动作电位钳，利用先前数字化的心室和浦肯野纤维（PF）AP波形，获得I(hERG)阻断的半数最大抑制浓度（IC(50)值）。
观察到西沙必利（范围为7至72 nM）的IC(50)值随不同指令波形的变化比多非利特（范围为4至15 nM）更显著，与阶跃或阶跃-斜坡指令相比，AP指令获得的IC(50)值更高。两种药物对电压依赖性激活的影响彼此差异不大；然而，每种药物对I(hERG)的阻断最初是电压依赖性的，但在稳态时仅西沙必利是电压依赖性的。两种药物对I(hERG)可用性或失活发展时间常数的影响差异相对较小。阻断的时间依赖性特征以及使用在药物或指令中采用不同静息期的方案，突出了西沙必利（而非多非利特）在逐脉冲基础上与hERG解离和重新结合的显著能力。
本文描述的方案在37℃下证明了西沙必利对hERG通道电流阻断的动态变化（药物解离/重新结合），可能对研究药物与hERG通道的相互作用具有未来价值。本文还附带了可用于AP钳实验的可下载数字化心室和PF AP波形。
