Introducing an alternative biophysical method to analyze direct G protein regulation of voltage-dependent calcium channels.
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.
Weiss
Norbert
N
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.
De Waard
Michel
M
D000818:Animals; D001703:Biophysics / Q000379:methods*; D015220:Calcium Channels / Q000502:physiology*; D004307:Dose-Response Relationship, Radiation; D004558:Electric Stimulation / Q000379:methods; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D018377:Neurotransmitter Agents / Q000494:pharmacology; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D013997:Time Factors; D014162:Transfection; D014982:Xenopus laevis
D015220:Calcium Channels; D018377:Neurotransmitter Agents; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D019204:GTP-Binding Proteins