Distinct RGK GTPases differentially use α1- and auxiliary β-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels

Abstract

CaV1/CaV2 channels, comprised of pore-forming α1 and auxiliary (β,α2δ) subunits, control diverse biological responses in excitable cells. Molecules blocking CaV1/CaV2 channel currents (ICa) profoundly regulate physiology and have many therapeutic applications. Rad/Rem/Rem2/Gem GTPases (RGKs) strongly inhibit CaV1/CaV2 channels. Understanding how RGKs block ICa is critical for insights into their physiological function, and may provide design principles for developing novel CaV1/CaV2 channel inhibitors. The RGK binding sites within CaV1/CaV2 channel complexes responsible for ICa inhibition are ambiguous, and it is unclear whether there are mechanistic differences among distinct RGKs. All RGKs bind β subunits, but it is unknown if and how this interaction contributes to ICa inhibition. We investigated the role of RGK/β interaction in Rem inhibition of recombinant CaV1.2 channels, using a mutated β (β2aTM) selectively lacking RGK binding. Rem blocked β2aTM-reconstituted channels (74% inhibition) less potently than channels containing wild-type β2a (96% inhibition), suggesting the prevalence of both β-binding-dependent and independent modes of inhibition. Two mechanistic signatures of Rem inhibition of CaV1.2 channels (decreased channel surface density and open probability), but not a third (reduced maximal gating charge), depended on Rem binding to β. We identified a novel Rem binding site in CaV1.2 α1C N-terminus that mediated β-binding-independent inhibition. The CaV2.2 α1B subunit lacks the Rem binding site in the N-terminus and displays a solely β-binding-dependent form of channel inhibition. Finally, we discovered an unexpected functional dichotomy amongst distinct RGKs- while Rem and Rad use both β-binding-dependent and independent mechanisms, Gem and Rem2 use only a β-binding-dependent method to inhibit CaV1.2 channels. The results provide new mechanistic perspectives, and reveal unexpected variations in determinants, underlying inhibition of CaV1.2/CaV2.2 channels by distinct RGK GTPases. © 2012 Yang et al.