Differential Requirement of GTP-Binding for Rem and Rad Inhibition of CaV1.2 Channels in Heart

Abstract

Ca2+ influx through L-type (CaV1.2) calcium channels in heart controls: action potential duration; muscle contraction; and gene expression. Rad/Rem/Rem2/ Gem/Kir (RGK) GTPases are Ras-like monomeric G-proteins that bind auxiliary CaV channel beta subunits and potently inhibit CaV1/CaV2 channels. RGK proteins are expressed in heart, where their expression levels changes in disease. Whether GTP-binding is important for RGK-mediated inhibition of Ca2+ current (ICa,L) in heart is controversial. Here, we investigated whether GTP binding to Rem and Rad is important for their ability to inhibit CaV1.2 channels. Both wild-type (wt) Rem and Rad dramatically inhibited recombinant CaV1.2 channels reconstituted in HEK 293 cells. Putative GTP-binding-deficient mutants (RemT94N and RadS105N) similarly inhibited recombinant ICa,L. In heart cells (Figure), over-expressing wt Rem or Rad (black lines) potently inhibited endogenous ICa,L (gray lines). Surprisingly, RemT94N (solid triangles) was functionally inert in heart, whereas RadS105N (solid squares) inhibited ICa,L to the same extent as wt Rad. The results contradict reports that RadS105N acts as a dominant negative in heart, and reveals a novel intrinsic difference between the two RGK proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide