Molecular Mechanisms, and Selective Pharmacological Rescue, of Rem-Inhibited Ca V 1.2 Channels in Heart

Abstract

Rationale: In heart, Ca 2+ entering myocytes via Ca V 1.2 channels controls essential functions, including excitation–contraction coupling, action potential duration, and gene expression. RGK GTPases (Rad/Rem/Rem2/Gem/Kir sub-family of Ras-like GTPases) potently inhibit Ca V 1.2 channels, an effect that may figure prominently in cardiac Ca 2+ homeostasis under physiological and disease conditions. Objective: To define the mechanisms and molecular determinants underlying Rem GTPase inhibition of Ca V 1.2 channels in heart and to determine whether such inhibited channels can be pharmacologically rescued. Methods and Results: Overexpressing Rem in adult guinea pig heart cells dramatically depresses L-type calcium current ( I Ca,L ) (≈90% inhibition) and moderately reduces maximum gating charge ( Q max ) (33%), without appreciably diminishing the physical number of channels in the membrane. Rem-inhibited Ca V 1.2 channels were supramodulated by BAY K 8644 (10-fold increase) compared to control channels (3-fold increase). However, Rem prevented protein kinase A–mediated upregulation of I Ca,L , an effect achieved without disrupting the sympathetic signaling cascade because protein kinase A modulation of I KS (slow component of the delayed rectifier potassium current) remained intact. In accord with its functional impact on I Ca,L , Rem selectively prevented protein kinase A– but not BAY K 8644–induced prolongation of the cardiac action potential duration. A GTP-binding-deficient Rem[T94N] mutant was functionally inert with respect to I Ca,L inhibition. A chimeric construct, Rem 265 -H, featuring a swap of the Rem C-terminal tail for the analogous domain from H-Ras, inhibited I Ca,L and Q max to the same extent as wild-type Rem, despite lacking the capacity to autonomously localize to the sarcolemma. Conclusions: Rem predominantly inhibits I Ca,L in heart by arresting surface Ca V 1.2 channels in a low open probability gating mode, rather than by interfering with channel trafficking. Moreover, Rem-inhibited Ca V 1.2 channels can be selectively rescued by BAY K 8644 but not protein kinase A–dependent phosphorylation. Contrary to findings in reconstituted systems, Rem-induced ablation of cardiac I Ca,L requires GTP-binding, but not membrane-targeting of the nucleotide binding domain. These findings provide a different perspective on the molecular mechanisms and structural determinants underlying RGK GTPase inhibition of Ca V 1.2 channels in heart, and suggest new (patho)physiological dimensions of this crosstalk.