title: Distinct RGK GTPases differentially use α1- and auxiliary β-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels.
abstract: Ca(V)1/Ca(V)2 channels, comprised of pore-forming α(1) and auxiliary (β,α(2)δ) 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 β subunits, but it is unknown if and how this interaction contributes to I(Ca) inhibition. We investigated the role of RGK/β interaction in Rem inhibition of recombinant Ca(V)1.2 channels, using a mutated β (β(2aTM)) selectively lacking RGK binding. Rem blocked β(2aTM)-reconstituted channels (74% inhibition) less potently than channels containing wild-type β(2a) (96% inhibition), suggesting the prevalence of both β-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 β. We identified a novel Rem binding site in Ca(V)1.2 α(1C) N-terminus that mediated β-binding-independent inhibition. The Ca(V)2.2 α(1B) 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 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.
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, New York, New York, United States of America. ty2190@columbia.edu
authors.forename: Tingting
authors.initials: T
authors.lastname: Yang
authors.forename: Akil
authors.initials: A
authors.lastname: Puckerin
authors.forename: Henry M
authors.initials: HM
authors.lastname: Colecraft
mesh_terms: D001665:Binding Sites; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
chemical_list: C500545:CACNA1B protein, human; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; C419385:L-type calcium channel alpha(1C); D020559:Monomeric GTP-Binding Proteins