title: 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.
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.
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, 10032, USA.
authors.forename: Donald D
authors.initials: DD
authors.lastname: Chang
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, 10032, USA.
authors.forename: Henry M
authors.initials: HM
authors.lastname: Colecraft
mesh_terms: D000200:Action Potentials; D000595:Amino Acid Sequence; D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002478:Cells, Cultured; D006153:Guanosine Diphosphate / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); C108135:Rem protein, mouse; C490030:Rrad protein, mouse; D006153:Guanosine Diphosphate; D020559:Monomeric GTP-Binding Proteins; D018631:ras Proteins