Reconstitution of the voltage-sensitive calcium channel purified from skeletal muscle transverse tubules

Publisher Website
Google Scholar
Abstract
The purified calcium antagonist receptor of the voltage-sensitive calcium channel from skeletal muscle transverse tubule membrane consists of three subunits: .alpha. with Mr 135,000, .beta. with Mr 50,000, and .gamma. with Mr 33,000. Purified receptor preparations were incorporated into phosphatidylcholine (PC) vesicles by addition of PC in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and removal of detergent by molecular seive chromatography. Forty-five percent of the .alpha., .beta., and .gamma. polypeptides and the [3H]dihydropyridine/receptor complex were recovered in association with PC vesicles. The rate of dissociation of the purified and reconstituted dihydropyridine/receptor complex was identical with that in T-tubule membranes, and allosteric modulation by verapamil and diltiazem was retained. The reconstituted calcium antagonist receptor, when occupied by the calcium channel activator BAY K 8644, mediated specific 45Ca2+ and 133Ba2+ transport into the reconstituted vesicles. 45Ca2+ influx was blocked by the organic calcium antagonists PN200-110 (K0.5 = 0.2 .mu.M), D600 (K0.5 = 1.0 .mu.M), and verapamil (K0.5 = 1.5 .mu.M) and by inorganic calcium channel antagonists (La3+ > Cd2+ > Ni2+ > Mg2+) as in intact T-tubules. A close quantitative correlation was observed between the presence of the .alpha., .beta., and .gamma. subunits of the calcium antagonist receptor and the ability to mediate 45Ca2+ or 133Ba2+ flux into reconstituted vesicles. Comparison of the number of reconstituted calcium antagonist receptors and functional channels supports the conclusion that only a few percent of the purified calcium antagonist receptor polypeptides are capable of mediating calcium transport as previously demonstrated for calcium antagonist receptors in intact T-tubules.

This publication has 0 references indexed in Scilit: