Weiss Norbert, De Waard Michel
Inserm U607, Laboratoire Canaux Calciques, Fonctions et Pathologies, 17 Rue des Martyrs, Bâtiment C3, 38054 Grenoble Cedex 09, France; Commissariat à l'Energie Atomique, Grenoble, France.
J Neurosci Methods. 2007 Feb 15;160(1):26-36. doi: 10.1016/j.jneumeth.2006.08.010. Epub 2006 Sep 20.
Direct G protein inhibition of voltage-dependent calcium channels is currently indirectly assessed by the gain of current produced by depolarizing prepulse potentials (PP). Indeed, PPs produce a channel opening- and time-dependent dissociation of G proteins from the channel that is responsible for the increase in Ca(2+) permeation. Parameters of G protein dissociation are essential to describe the characteristic landmark modifications in channel activities that underlie G protein regulation. From the kinetics and opening-dependence of this dissociation, crucial biophysical parameters are extracted such as the extent and the rate of G protein unbinding from the channel. Unfortunately, the method used so far assumes that G protein regulated channels undergo the same inactivation kinetics than control channels. Herein, we demonstrate for the first time that G protein-bound channels undergo a much slower inactivation than control channels. We thus introduce a novel simple-to-use method that avoids the use of PPs and that is not affected by potential changes in channel inactivation kinetics conferred by G protein binding. This method extracts G protein unbinding parameters from ionic currents induced by regular depolarizing pulses by separating the ionic currents due to non-regulated channels from the ionic currents that result from a progressive departure of G proteins from regulated channels.
目前，通过去极化预脉冲电位（PP）产生的电流增加来间接评估G蛋白对电压依赖性钙通道的直接抑制作用。实际上，预脉冲电位会使通道开放，并使G蛋白与通道发生时间依赖性解离，从而导致Ca(2+)通透性增加。G蛋白解离的参数对于描述通道活动中作为G蛋白调节基础的特征性标志性变化至关重要。从这种解离的动力学和开放依赖性中，提取出关键的生物物理参数，如G蛋白从通道上解离的程度和速率。不幸的是，迄今为止所使用的方法假定G蛋白调节的通道与对照通道具有相同的失活动力学。在此，我们首次证明与G蛋白结合的通道的失活比对照通道慢得多。因此，我们引入了一种新颖且易于使用的方法，该方法避免使用预脉冲电位，并且不受G蛋白结合所赋予的通道失活动力学潜在变化的影响。该方法通过将非调节通道产生的离子电流与G蛋白从调节通道逐渐解离产生的离子电流分离，从常规去极化脉冲诱导的离子电流中提取G蛋白解离参数。
