Proteolytic cleavage and PKA phosphorylation of α1C subunit are not required for adrenergic regulation of CaV1.2 in the heart

Calcium influx through the voltage-dependent L-type calcium channel (Ca(V)1.2) rapidly increases in the heart during fight or flight through activation of the beta-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of beta-adrenergic activation of cardiac Ca(V)1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in alpha(1C) and C-terminal proteolytic cleavage of the alpha(1C) subunit. We generated transgenic mice expressing an alpha(1C) with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for beta-adrenergic regulation of CaV1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to querywhether regulation requires their contribution in combination. We determined that acute beta-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse alpha(1C) subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant alpha(1C). Prevention of C-terminal cleavage did not alter beta-adrenergic stimulation of Ca(V)1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in alpha(1C) in beta-adrenergic stimulation of Ca(V)1.2, and show that phosphoregulatory sites on alpha(1C) are not redundant and do not each fractionally contribute to the net stimulatory effect of beta-adrenergic stimulation. Further, proteolytic cleavage of alpha(1C) is not required for beta-adrenergic stimulation of Ca(V)1.2.