Origin of the voltage dependence of G-protein regulation of P/Q-type Ca2+ channels.

G-protein (G beta gamma)-mediated voltage-dependent inhibition of N- and P/Q-type Ca2+ channels contributes to presynaptic inhibition and short-term synaptic plasticity. The voltage dependence derives from the dissociation of G beta gamma from the inhibited channels, but the underlying molecular and biophysical mechanisms remain largely unclear. In this study we investigated the role in this process of Ca2+ channel beta subunit (Ca-v beta) and a rigid alpha-helical structure between the alpha-interacting domain (AID), the primary Ca-v beta docking site on the channel alpha(1) subunit, and the pore-lining IS6 segment. G beta gamma inhibition of P/Q-type channels was reconstituted in giant inside-out membrane patches from Xenopus oocytes. Large populations of channels devoid of Ca-v beta were produced by washing out a mutant Ca-v beta with a reduced affinity for the AID. These beta-less channels were still inhibited by G beta gamma, but without any voltage dependence, indicating that Ca-v beta is indispensable for voltage-dependent G beta gamma inhibition. Atruncated Ca-v beta containing only the AID-binding guanylate kinase (GK) domain could fully confer voltage dependence to G beta gamma inhibition. G beta gamma did not alter inactivation properties, and channels recovered from G beta gamma inhibition exhibited the same activation property as un-inhibited channels, indicating that G beta gamma does not dislodge Ca-v beta from the inhibited channel. Furthermore, voltage-dependent G beta gamma inhibition was abolished when the rigid alpha-helix between the AID and IS6 was disrupted by insertion of multiple glycines, which also eliminated Ca-v beta regulation of channel gating, revealing a pivotal role of this rigid alpha-helix in both processes. These results suggest that depolarization-triggered movement of IS6, coupled to the subsequent conformational change of the G beta gamma-binding pocket through a rigid alpha-helix induced partly by the Ca-v beta GK domain, causes the dissociation of G beta gamma and is fundamental to voltage-dependent G beta gamma inhibition.