Reconstitution of the voltage-sensitive calcium channel purified from skeletal muscle transverse tubules.
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 sieve 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 microM), D600 (K0.5 = 1.0 microM), and verapamil (K0.5 = 1.5 microM) and by inorganic calcium channel antagonists (La3+ greater than Cd2+ greater than Ni2+ greater than 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.
D000818:Animals; D001464:Barium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000378:metabolism / Q000494:pharmacology; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D017275:Isradipine; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D010069:Oxadiazoles / Q000378:metabolism; D010713:Phosphatidylcholines
D002121:Calcium Channel Blockers; D007473:Ion Channels; D008081:Liposomes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010069:Oxadiazoles; D010713:Phosphatidylcholines; D001464:Barium; D002118:Calcium; D017275:Isradipine