Adrenergic Ca1.2 Activation via Rad Phosphorylation Converges at α I-II Loop.

RATIONALE

Changing activity of cardiac Ca1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac Ca1.2 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 Ca1.2 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 Ca1.2 α subunits with (1) mutations ablating interaction between α and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α 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 Caβ to α I-II loop and eliminated β-adrenergic agonist stimulation of Ca1.2 current. In contrast, introduction of the exon 9* splice variant in the α 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 and Rad or transgenically expressed in cardiomyocytes.

CONCLUSIONS

Ca channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α I-II loop. Caβ binding to α 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 channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α.