Transgenic expression of mutant CaV1.2 α1C subunits revealed that an intact rigid linker in the I-II loop is required for basal channel open probability and β-adrenergic stimulation.
The study reveals that CaVβ binding to α1C and Rad-mediated inhibition release by β-adrenergic agonists require an intact rigid linker in the α1C 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 .
Papa et al. (2020) studied Cardiac physiology and pathophysiology. Transgenic expression of mutant CaV1.2 α1C subunits was evaluated on CaV1.2 channel activity and response to β-adrenergic agonists. Transgenic expression of mutant CaV1.2 α1C subunits revealed that an intact rigid linker in the I-II loop is required for basal channel open probability and β-adrenergic stimulation.