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Rad and Rem are non‐canonical G‐proteins with respect to the regulatory role of guanine nucleotide binding in Ca V 1.2 channel regulation
Author(s) -
Chang Donald D.,
Colecraft Henry M.
Publication year - 2015
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jp270889
Subject(s) - microbiology and biotechnology , biology , guanine , cav1.2 , nucleotide , hek 293 cells , chemistry , biochemistry , gene , protein subunit
Key points RGK (Rad, Rem, Rem2, Gem/Kir) proteins are small monomeric G‐proteins implicated in various cellular functions and disease states. All RGK proteins potently inhibit voltage‐gated calcium (Ca V ) channels. It is unclear whether RGK proteins are regulated by guanine nucleotide binding in a manner that conforms to a canonical G‐protein regulation paradigm. We utilized strategic Rad and Rem mutants together with a range of functional readouts (Ca V 1.2 currents, Ca 2+ transients, Ca V β binding) in heterologous cells and cardiomyocytes to determine whether the function of these RGK proteins is regulated in a canonical manner. Our results demonstrate that Rad and Rem are non‐canonical G‐proteins with respect to the regulatory role of their guanine nucleotide binding domain in Ca V 1.2 channel regulation. Our findings offer deepened insights into cellular mechanisms governing RGK regulation and function, and contribute towards our understanding of their pathophysiological roles.Abstract 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 (Ca V 1.2 currents, Ca 2+ transients, Ca V β binding) as biosensors to probe the role of the G‐domain in regulation of Rad and Rem function. We utilized Rad S105N and Rem T94N , which are the cognate mutants to Ras S17N , a dominant‐negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over‐expression of either Rad S105N or Rem T94N strongly inhibited reconstituted Ca V 1.2 currents to the same extent as their wild‐type (wt) counterparts, contrasting with reports that Rad S105N is functionally inert in HEK293 cells. Adenovirus‐mediated expression of either wt Rad or Rad S105N in cardiomyocytes dramatically blocked L‐type calcium current ( I Ca,L ) and inhibited Ca 2+ ‐induced Ca 2+ release, contradicting reports that Rad S105N acts as a dominant negative in heart. By contrast, Rem T94N was significantly less effective than wt Rem at inhibiting I Ca,L and Ca 2+ transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both Rad S105N and Rem T94N had moderately reduced binding affinity for Ca V βs 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 Ca V 1.2 regulation.