Adrenergic Ca(V)1.2 Activation via Rad Phosphorylation Converges at α(1C) I-II Loop.

RATIONALE: Changing activity of cardiac Ca(V)1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac Ca(V)1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic. OBJECTIVE: The primary goal of this study was to determine how various factors converging at the Ca(V)1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure. METHODS AND RESULTS: We generated transgenic mice with expression of Ca(V)1.2 α(1C) subunits with (1) mutations ablating interaction between α(1C) and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α(1C)), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α(1C) upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of Ca(V)β to α(1C) I-II loop and eliminated β-adrenergic agonist stimulation of Ca(V)1.2 current. In contrast, introduction of the exon 9* splice variant in the α(1C) I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β(2B) and Rad or transgenically expressed in cardiomyocytes. CONCLUSIONS: Ca(2+) channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α(1C) I-II loop. Ca(V)β binding to α(1C) stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca(2+) channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α(1C).