Distinct RGK GTPases Differentially Use α1-and Auxiliary β-Binding-Dependent Mechanisms to Inhibit CaV1.2/CaV2.2 Channels

Ca(V)1/Ca(V)2 channels, comprised of pore-forming alpha(1) and auxiliary (beta,alpha(2)delta) subunits, control diverse biological responses in excitable cells. Molecules blocking Ca(V)1/Ca(V)2 channel currents (I-Ca) profoundly regulate physiology and have many therapeutic applications. Rad/Rem/Rem2/Gem GTPases (RGKs) strongly inhibit Ca(V)1/Ca(V)2 channels. Understanding how RGKs block I-Ca is critical for insights into their physiological function, and may provide design principles for developing novel Ca(V)1/Ca(V)2 channel inhibitors. The RGK binding sites within Ca(V)1/Ca(V)2 channel complexes responsible for I-Ca inhibition are ambiguous, and it is unclear whether there are mechanistic differences among distinct RGKs. All RGKs bind beta subunits, but it is unknown if and how this interaction contributes to I-Ca inhibition. We investigated the role of RGK/beta interaction in Rem inhibition of recombinant Ca(V)1.2 channels, using a mutated beta (beta(2aTM)) selectively lacking RGK binding. Rem blocked beta(2aTM)-reconstituted channels (74% inhibition) less potently than channels containing wild-type beta(2a) (96% inhibition), suggesting the prevalence of both beta-binding-dependent and independent modes of inhibition. Two mechanistic signatures of Rem inhibition of Ca(V)1.2 channels (decreased channel surface density and open probability), but not a third (reduced maximal gating charge), depended on Rem binding to beta. We identified a novel Rem binding site in Ca(V)1.2 alpha(1C) N-terminus that mediated beta-binding-independent inhibition. The Ca(V)2.2 alpha(1B) subunit lacks the Rem binding site in the N-terminus and displays a solely beta-binding-dependent form of channel inhibition. Finally, we discovered an unexpected functional dichotomy amongst distinct RGKs- while Rem and Rad use both beta-binding-dependent and independent mechanisms, Gem and Rem2 use only a beta-binding-dependent method to inhibit Ca(V)1.2 channels. The results provide new mechanistic perspectives, and reveal unexpected variations in determinants, underlying inhibition of Ca(V)1.2/Ca(V)2.2 channels by distinct RGK GTPases.