Convergent regulation of Cav1.2 channels by direct phosphorylation and by the small GTPase rad in the cardiac fight-or-flight response.

Abstract

The cardiac CaV1.2 calcium channel serves as a key regulator of heart rate, rhythm, and force of contraction. CaV1.2 is regulated by direct β-adrenergic/PKA-mediated phosphorylation of its auto-inhibitory C-terminal domain (CT). The small GTPase RAD (Ras associated with diabetes) has also emerged as a potent inhibitor of CaV1.2 with a key role in mediating β-adrenergic/PKA up-regulation of channel activity. However, the relative roles of direct phosphorylation of CaV1.2 channels and phosphorylation of RAD in channel regulation remain uncertain. In a tsA-201 human embryonic kidney cell heterologous expression system, both RAD and the proteolytically processed distal CT (dCT) significantly reduced peak current conducted by the C-terminal truncated CaV1.2α1800 isoform. Activation of PKA by forskolin reversed this inhibition and increased CaV1.2 currents synergistically through PKA phosphorylation of RAD and PKA phosphorylation of Ser 1700 in the proximal CT (pCT). In contrast, alanine substitution for serine at CaV1.2 PKA site Ser1700 and at RAD PKA sites (Ser25, Ser38, Ser272, Ser300) both eliminated PKA-mediated up-regulation in the presence of dCT and RAD. Our findings reveal that the proteolytically processed form of CaV1.2 undergoes convergent regulation by direct PKA phosphorylation of the CT and by PKA phosphorylation of RAD. These parallel regulatory pathways provide a flexible mechanism for up-regulation of the activity of CaV1.2 channels in the fight-or-flight response. Supported by NIH Research Grant R01HL112808.