Distinct RGK GTPases differentially use ?1- and auxiliary ?-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels.

ABSTRACT: Ca(V)1/Ca(V)2 channels, comprised of pore-forming ?(1) and auxiliary (?,?(2)?) 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 ? subunits, but it is unknown if and how this interaction contributes to I(Ca) inhibition. We investigated the role of RGK/? interaction in Rem inhibition of recombinant Ca(V)1.2 channels, using a mutated ? (?(2aTM)) selectively lacking RGK binding. Rem blocked ?(2aTM)-reconstituted channels (74% inhibition) less potently than channels containing wild-type ?(2a) (96% inhibition), suggesting the prevalence of both ?-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 ?. We identified a novel Rem binding site in Ca(V)1.2 ?(1C) N-terminus that mediated ?-binding-independent inhibition. The Ca(V)2.2 ?(1B) subunit lacks the Rem binding site in the N-terminus and displays a solely ?-binding-dependent form of channel inhibition. Finally, we discovered an unexpected functional dichotomy amongst distinct RGKs- while Rem and Rad use both ?-binding-dependent and independent mechanisms, Gem and Rem2 use only a ?-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.

SUBMITTER: Yang T 

PROVIDER: S-EPMC3349659 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature