Convergent regulation of Ca(V)1.2 channels by direct phosphorylation and by the small GTPase RAD in the cardiac fight-or-flight response

The L-type calcium currents conducted by the cardiac Ca(V)1.2 calcium channel initiate excitation–contraction coupling and serve as a key regulator of heart rate, rhythm, and force of contraction. Ca(V)1.2 is regulated by β-adrenergic/protein kinase A (PKA)-mediated protein phosphorylation, proteolytic processing, and autoinhibition by its carboxyl-terminal domain (CT). The small guanosine triphosphatase (GTPase) RAD (Ras associated with diabetes) has emerged as a potent inhibitor of Ca(V)1.2, and accumulating evidence suggests a key role for RAD in mediating β-adrenergic/PKA upregulation of channel activity. However, the relative roles of direct phosphorylation of Ca(V)1.2 channels and phosphorylation of RAD in channel regulation remain uncertain. Here, we investigated the hypothesis that these two mechanisms converge to regulate Ca(V)1.2 channels. Both RAD and the proteolytically processed distal CT (dCT) strongly reduced Ca(V)1.2 activity. PKA phosphorylation of RAD and phosphorylation of Ser-1700 in the proximal CT (pCT) synergistically reversed this inhibition and increased Ca(V)1.2 currents. Our findings reveal that the proteolytically processed form of Ca(V)1.2 undergoes convergent regulation by direct phosphorylation of the CT and by phosphorylation of RAD. These parallel regulatory pathways provide a flexible mechanism for upregulation of the activity of Ca(V)1.2 channels in the fight-or-flight response.