Convergent regulation of Ca1.2 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 Ca1.2 calcium channel initiate excitation-contraction coupling and serve as a key regulator of heart rate, rhythm, and force of contraction. Ca1.2 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 Ca1.2, 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 Ca1.2 channels and phosphorylation of RAD in channel regulation remain uncertain. Here, we investigated the hypothesis that these two mechanisms converge to regulate Ca1.2 channels. Both RAD and the proteolytically processed distal CT (dCT) strongly reduced Ca1.2 activity. PKA phosphorylation of RAD and phosphorylation of Ser-1700 in the proximal CT (pCT) synergistically reversed this inhibition and increased Ca1.2 currents. Our findings reveal that the proteolytically processed form of Ca1.2 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 Ca1.2 channels in the fight-or-flight response.