Introducing an alternative biophysical method to analyze direct G protein regulation of voltage-dependent calcium channels

Abstract

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.

The S218L familial hemiplegic migraine mutation promotes deinhibition of Cav2.1 calcium channels during direct G-protein regulation

“…2a and b, both before (I Control ) and during DAMGO application (I DAMGO ). According to our recently developed method of analysis of G-protein regulation [22,23], the Lost current traces were extracted (I Lost ) by subtracting I DAMGO from I Control . I Lost provides the time course of the lost current following G-protein activation, affected by both the recovery from G-protein inhibition following channel activation and by the inactivation process.…”

“…The two parameters that characterize the OFF components of G-protein regulation of Ca v 2.1 voltage-gated calcium channels, i.e., the time constant of current recovery (τ) and the maximal extent of current recovery from inhibition (RI), were extracted from the data using our recently published method [22,23]. Representative current traces before and under DAMGO application are shown for wild-type and S218L mutant Ca v 2.1 channels at 0-and 20-mV step depolarization from a holding potential of −90 mV (Fig.…”

“…Hence, if current inhibition finally only represents an index of the total amount of channels potentially affected by direct G-protein inhibition, current deinhibition really reflects the importance of this regulation under channel activity. In this study, ON and OFF Gprotein regulation parameters were quantified using our recently developed method [22,23]. Hence, G-protein inhibition, measured at the start of the depolarization, before the initiation of the process of recovery, is not affected by the FHM-1 S218L mutation.…”

“…The method used to extract all biophysical parameters of Gprotein regulation (GI t 0 , the initial extent of G-protein inhibition before the start of depolarization, τ, the time constant of G-protein unbinding from the channel, and RI, the extent of recovery from inhibition at the end of a 200-ms test pulse) was described in [22]. Briefly, subtracting I DAMGO from I Control results in I Lost , the evolution of the lost current under G-protein activation.…”

Rim1 modulates direct G-protein regulation of Cav2.2 channels

“…Biophysical parameters of G-protein regulation (GI t0 , the initial extent of G-protein inhibition before the start of the depolarization, τ, the time constant of G-protein unbinding from the channel, and RI, the maximal extent of recovery from inhibition) were measured and analyzed according to previously described procedures [34,36]. In brief, subtracting I DAMGO (the current recorded after DAMGO application) from I Control (the current recorded before DAMGO application) results in I Lost , the evolution of the lost current during membrane depolarization under G-protein regulation.…”