The calcium channel antagonists receptor from rabbit skeletal muscle

The Ca 2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[ 3 H]PN 200–110, (−)[ 3 H]D 888 and d‐cis ‐[ 3 H]diltiazem demonstrated that receptor sites for the three most common Ca 2+ channel markers copurified with binding stoichiometries close to 1:1:1. Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of M r 170000 under non‐reducing conditions and of two polypeptides of M r 140000 and 32000 under disulfide‐reducing conditions. Iodination of the protein of M r 170000 and immunoblots experiments with antisera directed against the differents components demonstrated that the Ca 2+ channel antagonists receptor is a complex of M r 170000 composed of a polypeptide chain of M r 140000 associated to one polypeptide chain of M r 32000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca 2+ channel was the presence of smaller polypeptide chains of M r 29000 and 25000. Peptide mapping of these polypeptide chains and analysis of their cross‐reactivity with sera directed against the proteins of M r 170000 and 32000 demonstrated that they were degradative products of the M r 32000 component. Both the large (140 kDa) and the small (32 kDa) component of the putative Ca 2+ channel are heavily glycosylated. At least 20–22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140‐kDa and 32‐kDa components originate from a single polypeptide chain of M r 170000 which is cleaved by a proteolysis upon purification is discussed.