Smooth muscle cell alpha2delta-1 subunits are essential for vasoregulation by CaV1.2 channels.
BACKGROUND
Voltage-dependent L-type (Ca(V)1.2) Ca(2+) channels are a heteromeric complex formed from pore-forming alpha(1) and auxiliary alpha(2)delta and beta subunits. Ca(V)1.2 channels are the principal Ca(2+) influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte Ca(V)1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of alpha(2)delta subunits.


OBJECTIVE
We investigated the functional significance of alpha(2)delta subunits in myocytes of resistance-size (100 to 200 mum diameter) cerebral arteries.


RESULTS
alpha(2)delta-1 was the only alpha(2)delta isoform expressed in cerebral artery myocytes. Pregabalin, an alpha(2)delta-1/-2 ligand, and an alpha(2)delta-1 antibody, inhibited Ca(V)1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95% of Ca(V)1.2 alpha(1) and alpha(2)delta-1 subunits were present in the arterial myocyte plasma membrane. Alpha(2)delta-1 knockdown using short hairpin RNA reduced plasma membrane-localized Ca(V)1.2 alpha(1) subunits, caused a corresponding elevation in cytosolic Ca(V)1.2 alpha(1) subunits, decreased intracellular Ca(2+) concentration, inhibited pressure-induced vasoconstriction ("myogenic tone"), and attenuated pregabalin-induced vasodilation. Prolonged (24-hour) pregabalin exposure did not alter total alpha(2)delta-1 or Ca(V)1.2 alpha(1) proteins but decreased plasma membrane expression of each subunit, which reduced myogenic tone.


CONCLUSIONS
alpha(2)delta-1 is essential for plasma membrane expression of arterial myocyte Ca(V)1.2 alpha(1) subunits. alpha(2)delta-1 targeting can block Ca(V)1.2 channels directly and inhibit surface expression of Ca(V)1.2 alpha(1) subunits, leading to vasodilation. These data identify alpha(2)delta-1 as a novel molecular target in arterial myocytes, the manipulation of which regulates contractility.
Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
John P
JP
Bannister
Adebowale
A
Adebiyi
Guiling
G
Zhao
Damodaran
D
Narayanan
Candice M
CM
Thomas
Jessie Y
JY
Feng
Jonathan H
JH
Jaggar
D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002478:Cells, Cultured; D002536:Cerebral Arteries / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D055785:Gene Knockdown Techniques; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D000069583:Pregabalin; D020033:Protein Isoforms / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology*; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology*; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives / Q000494:pharmacology
D000927:Anticonvulsants; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D020033:Protein Isoforms; D021122:Protein Subunits; C493217:calcium channel, l type beta2c subunit, rat; D000069583:Pregabalin; D005680:gamma-Aminobutyric Acid; D002118:Calcium