Published online before print October 1, 2009, doi: 10.1161/CIRCRESAHA.109.203620
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Submitted on June 24, 2009
Revised on September 1, 2009
Accepted on September 22, 2009
Smooth Muscle Cell 
2
-1 Subunits Are Essential for Vasoregulation by CaV1.2 Channels
John P. Bannister ;
From the Department of Physiology, University of Tennessee Health Science Center, Memphis.
* To whom correspondence should be addressed. E-mail: jjaggar{at}physio1.utmem.edu.
Rationale: Voltage-dependent L-type (CaV1.2) Ca2+ channels are a heteromeric complex formed from pore-forming 
1 and auxiliary 2
and 
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 2 subunits.
Objective: We investigated the functional significance of 2 subunits in myocytes of resistance-size (100 to 200 μm diameter) cerebral arteries.
Methods and Results: 2-1 was the only 2 isoform expressed in cerebral artery myocytes. Pregabalin, an 2-1/-2 ligand, and an 2-1 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 1 and 2-1 subunits were present in the arterial myocyte plasma membrane. 2-1 knockdown using short hairpin RNA reduced plasma membrane-localized CaV1.2 1 subunits, caused a corresponding elevation in cytosolic CaV1.2 1 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 2-1 or CaV1.2 1 proteins but decreased plasma membrane expression of each subunit, which reduced myogenic tone.
Conclusions: 2-1 is essential for plasma membrane expression of arterial myocyte CaV1.2 1 subunits. 2-1 targeting can block CaV1.2 channels directly and inhibit surface expression of CaV1.2 1 subunits, leading to vasodilation. These data identify 2-1 as a novel molecular target in arterial myocytes, the manipulation of which regulates contractility.
Key words: L-type Ca2+ channels • arterial contractility