Convergent regulation of Ca_V1.2 channels by direct phosphorylation and by the small GTPase RAD in the cardiac fight-or-flight response.

Abstract

The L-type calcium currents conducted by the cardiac Ca_V1.2 calcium channel initiate excitation-contraction coupling and serve as a key regulator of heart rate, rhythm, and force of contraction. Ca_V1.2 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_V1.2, 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_V1.2 channels and phosphorylation of RAD in channel regulation remain uncertain. Here, we investigated the hypothesis that these two mechanisms converge to regulate Ca_V1.2 channels. Both RAD and the proteolytically processed distal CT (dCT) strongly reduced Ca_V1.2 activity. PKA phosphorylation of RAD and phosphorylation of Ser-1700 in the proximal CT (pCT) synergistically reversed this inhibition and increased Ca_V1.2 currents. Our findings reveal that the proteolytically processed form of Ca_V1.2 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_V1.2 channels in the fight-or-flight response.