Abstract
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 β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac Ca(V)1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α(1C) and C-terminal proteolytic cleavage of the α(1C) subunit. We generated transgenic mice expressing an α(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 β-adrenergic regulation of Ca(V)1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α(1C) subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α(1C) Prevention of C-terminal cleavage did not alter β-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 α(1C) in β-adrenergic stimulation of Ca(V)1.2, and show that phosphoregulatory sites on α(1C) are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α(1C) is not required for β-adrenergic stimulation of Ca(V)1.2.