Smooth Muscle Cell α ₂ δ-1 Subunits Are Essential for Vasoregulation by Ca _V 1.2 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 Investigate the functional significance of α2δ subunits in myocytes of resistance-size (100–200 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 are present in the arterial myocyte plasma membrane. α2δ-1 knockdown using shRNA reduced plasma membrane-localized CaV1.2 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 CaV1.2 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, manipulation of which regulates contractility.

TMEM16A channels generate Ca2+-activated Cl− currents in cerebral artery smooth muscle cells

“…Surface proteins were biotinylated in intact cerebral arteries as previously described (1,3,4,7). Briefly, arteries were incubated in a 1 mg/ml mixture each of EZ-Link sulfo-NHS-LC-LC-biotin and Maleimide-PEG 2-biotin reagents (Pierce) in PBS (Invitrogen) at room temperature for 1 h. Unbound biotin was removed by washing with 100 mM glycine-PBS.…”

Protein kinase a regulation of T-type Ca2+ channels in rat cerebral arterial smooth muscle

“…In vascular smooth muscle, it is typically asserted that L-type Ca 2+ channels are singularly expressed and thus the only voltagegated Ca 2+ channel of functional importance Bannister et al, 2009). Recent investigations have begun to reassess this assertion by documenting the additional expression of T-type Ca 2+ channel (Kuo et al, 2010;El-Rahman et al, 2013).…”

“…In comparison, low-voltage activated family activates at more hyperpolarized potentials and T-type channels are the sole members (Catterall, 2011). Past studies have established that L-type Ca V 1.2 channels function as the primary Ca 2+ influx route in cerebral arterial smooth muscle Bannister et al, 2009). While important to tone development, recent reports have noted that two T-type Ca 2+ channels are additionally expressed at the mRNA and protein For steady-state inactivation, currents were recorded at +10 mV after prepulses lasting 1.5 s to potentials from 270 to +20 mV.…”

Nitric oxide suppresses vascular voltage-gated T-type Ca2+ channels through cGMP/PKG signaling

“…Cytosolic [Ca 2ϩ ] is primarily set by resting membrane potential and steady-state Ca 2ϩ influx through voltage-gated Ca 2ϩ channels (16,27). While L-type Ca 2ϩ channel expression predominates (3,15), recent studies have noted that T-type Ca 2ϩ channels are additionally present. Of particular note, the T-type conductance is thought to influence arterial diameter when vessels sit at more hyperpolarized potentials (1,17).…”