Molecular mechanisms, and selective pharmacological rescue, of rem-inhibited CaV1.2 channels in heart

Abstract

Rationale: In heart, Ca2+ entering myocytes via Ca V1.2 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 CaV1.2 channels, an effect that may figure prominently in cardiac Ca2+ homeostasis under physiological and disease conditions. Objective: To define the mechanisms and molecular determinants underlying Rem GTPase inhibition of CaV1.2 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 (ICa,L) (≈90% inhibition) and moderately reduces maximum gating charge (Qmax) (33%), without appreciably diminishing the physical number of channels in the membrane. Rem-inhibited CaV1.2 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 ICa,L, an effect achieved without disrupting the sympathetic signaling cascade because protein kinase A modulation of IKS (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 ICa,L inhibition. A chimeric construct, Rem265-H, featuring a swap of the Rem C-terminal tail for the analogous domain from H-Ras, inhibited ICa,L and Qmax to the same extent as wild-type Rem, despite lacking the capacity to autonomously localize to the sarcolemma. Conclusions: Rem predominantly inhibits ICa,L in heart by arresting surface CaV1.2 channels in a low open probability gating mode, rather than by interfering with channel trafficking. Moreover, Rem-inhibited CaV1.2 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 ICa,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 CaV1.2 channels in heart, and suggest new (patho)physiologICal dimensions of this crosstalk. © 2010 American Heart Association, Inc.