Anchored PKA synchronizes adrenergic phosphoregulation of cardiac Cav1.2 channels.

ABSTRACT

Adrenergic modulation of voltage gated Ca2+ currents is a context specific process. In the heart Cav1.2 channels initiate excitation-contraction coupling. This requires protein kinase A (PKA) phosphorylation of the small GTPase Rad (Ras associated with diabetes) and involves direct phosphorylation of a1 subunit of the Cav1.2 at Ser1700. A contributing factor is the proximity of PKA to the channel through association with A-kinase anchoring proteins (AKAPs). Disruption of PKA anchoring by the disruptor peptide AKAP-IS prevents up-regulation of Cav1.2 currents in tsA-201 cells. Biochemical analyses demonstrate that Rad does not function as an A-kinase anchoring protein. Electrophysiological recording shows that channel mutants lacking phosphorylation sites (Cav1.2 STAA) lose responsivity to the second messenger cAMP. Measurements in cardiomyocytes isolated from Rad-/- mice show that adrenergic activation of Cav1.2 is attenuated but not completely abolished. Whole animal electrocardiography studies reveal that cardiac selective Rad knockout mice exhibited higher baseline left-ventricular ejection fraction (EF), greater fractional shortening (FS), and increased heart rate as compared to control animals. Yet, each parameter of cardiac function was slightly elevated when Rad-/- mice were treated with the adrenergic agonist isoproterenol. Thus, phosphorylation of Cav1.2 and dissociation of phospho-Rad from the channel are local cAMP responsive events that act in concert to enhance L-type calcium currents. This convergence of local PKA regulatory events at the cardiac L-type calcium channel may permit maximal ß-adrenergic influence on the fight-or-flight response.