Smooth muscle cell α2δ-1 subunits are essential for vasoregulation by CaV1.2 channels

Abstract

Rationale: Voltage-dependent L-type (CaV1.2) Ca2+ channels are a heteromeric complex formed from pore-forming α1 and auxiliary α2δ and β subunits. CaV1.2 channels are the principal Ca2+ influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte CaV1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of α2δ subunits. Objective: We investigated the functional significance of α2δ subunits in myocytes of resistance-size (100 to 200 μm diameter) cerebral arteries. Methods and Results: α2δ-1 was the only α2δ isoform expressed in cerebral artery myocytes. Pregabalin, an α2δ-1/-2 ligand, and an α2δ-1 antibody, inhibited CaV1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95% of CaV1.2 α1 and α2δ-1 subunits were present in the arterial myocyte plasma membrane. α2δ-1 knockdown using short hairpin RNA reduced plasma membrane-localized Ca V1.2 α1 subunits, caused a corresponding elevation in cytosolic CaV1.2 α1 subunits, decreased intracellular Ca2+ concentration, inhibited pressure-induced vasoconstriction (myogenic tone), and attenuated pregabalin-induced vasodilation. Prolonged (24-hour) pregabalin exposure did not alter total α2δ-1 or CaV1.2 α1 proteins but decreased plasma membrane expression of each subunit, which reduced myogenic tone. Conclusions: α2δ-1 is essential for plasma membrane expression of arterial myocyte Ca V1.2 α1 subunits. α2δ-1 targeting can block CaV1.2 channels directly and inhibit surface expression of CaV1.2 α1 subunits, leading to vasodilation. These data identify α2δ-1 as a novel molecular target in arterial myocytes, the manipulation of which regulates contractility. © 2009 American Heart Association, Inc.