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


RESULTS
Overexpressing Rem in adult guinea pig heart cells dramatically depresses L-type calcium current (I(Ca,L)) ( approximately 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.
Xu
Xianghua
X
Columbia University, College of Physicians and Surgeons, Department of Physiology and Cellular Biophysics, 1150 St Nicholas Avenue, New York, NY 10032, USA.
Marx
Steven O
SO
Colecraft
Henry M
HM
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000200:Action Potentials; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002120:Calcium Channel Agonists / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006168:Guinea Pigs; D015640:Ion Channel Gating / Q000187:drug effects; D008297:Male; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D002120:Calcium Channel Agonists; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D011993:Recombinant Fusion Proteins; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D006160:Guanosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D020559:Monomeric GTP-Binding Proteins