Rad and Rem are non-canonical G-proteins with respect to the regulatory role of guanine nucleotide binding in CaV1.2 channel regulation

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Rad and Rem are Ras-like G-proteins linked to diverse cardiovascular functions and pathophysiology. Understanding how Rad and Rem are regulated is important for deepened insights into their pathophysiological roles. As in other Ras-like G-proteins, Rad and Rem contain a conserved guanine-nucleotide binding domain (G-domain). Canonically, G-domains are key control modules, functioning as nucleotide-regulated switches of G-protein activity. Whether Rad and Rem G-domains conform to this canonical paradigm is ambiguous. Here, we used multiple functional measurements in HEK293 cells and cardiomyocytes (CaV1.2 currents, Ca2+ transients, CaVβ binding) as biosensors to probe the role of the G-domain in regulation of Rad and Rem function. We utilized RadS105N and RemT94N, which are the cognate mutants to RasS17N, a dominant-negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over-expression of either RadS105N or RemT94N strongly inhibited reconstituted CaV1.2 currents to the same extent as their wild-type (wt) counterparts, contrasting with reports that RadS105N is functionally inert in HEK293 cells. Adenovirus-mediated expression of either wt Rad or RadS105N in cardiomyocytes dramatically blocked L-type calcium current (ICa,L) and inhibited Ca2+-induced Ca2+ release, contradicting reports that RadS105N acts as a dominant negative in heart. By contrast, RemT94N was significantly less effective than wt Rem at inhibiting ICa,L and Ca2+ transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both RadS105N and RemT94N had moderately reduced binding affinity for CaVβs relative to their wt counterparts. The results indicate Rad and Rem are non-canonical G-proteins with respect to the regulatory role of their G-domain in CaV1.2 regulation.

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