PKA phosphorylation of Cav1.2 channel modulates the interaction of calmodulin with the C terminal tail of the channel.

Activity of cardiac Cav1.2 channels is enhanced by cyclic AMP-PKA signaling. In this study, we studied the effects of PKA phosphorylation on the binding of calmodulin to the fragment peptide of the proximal C-terminal tail of α1C subunit (CT1, a.a. 1509-1789 of guinea-pig). In the pull-down assay, in vitro PKA phosphorylation significantly decreased calmodulin binding to CT1 (61%) at high [Ca2+]. The phosphoresistant (CT1SA) and phosphomimetic (CT1SD) CT1 mutants, in which three PKA sites (Ser1574, 1626, 1699) were mutated to Ala and Asp, respectively, bound with calmodulin with 99% and 65% amount, respectively, compared to that of wild-type CT1. In contrast, at low [Ca2+], calmodulin-binding to CT1SD was higher (33-35%) than that to CT1SA. The distal C-terminal region of α1C (CT3, a.a. 1942-2169) is known to interact with CT1 and inhibit channel activity. CT3 bound to CT1SD was also significantly less than that to CT1SA. In inside-out patch, PKA catalytic subunit (PKAc) facilitated Ca2+ channel activity at both high and low Ca2+ condition. Altogether, these results support the hypothesis that PKA phosphorylation may enhance channel activity and attenuate the Ca2+-dependent inactivation, at least partially, by modulating calmodulin-CT1 interaction both directly and indirectly via CT3-CT1 interaction.