Purification of a functional receptor for calcium‐channel blockers from rabbit skeletal‐muscle microsomes

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Abstract
The dihydropyridine receptor was purified from rabbit skeletal muscle microsomes in the presence of [3H]nitrendipine plus diltiazem or [3H](+)PN 200–110 to an apparent density of 1.5–2 nmol binding sites/mg protein. Sodium dodecyl sulfate gel electrophoresis in the absence of reducing agents yielded three peptide bands of 142, 56 and 30 kDa in a relative ratio of 11:1:1.3, whereas in the presence of 40 mM dithiothreitol bands of 142, 122, 56, 31, 26 and 22 kDa were obtained in a relative ratio of 5.5:2.2:1:0.9:14:0.09. This gel pattern was observed regardless of whether the receptor was purified as a complex with nitrendipine plus diltiazem or with (+)PN 200–110. cAMP‐dependent protein kinase phosphorylated preferentially the 142‐kDa band up to a stoichiometry of 0.82 ± 0.07 (15) mol phosphate/mol peptide. The 56‐kDa band was phosphorylated only in substoichiometric amounts.[3H]PN 200–110 bound at 4°C to one site with apparent Kd and Bmax values of 9.3 ± 1.7 nM and 2.2 ± 0.3 (3) nmol/mg protein, respectively. The binding was stereospecific and was not observed in the presence of 1 mM EGTA. Desmethoxyverapamil interfered with the binding of [3H]PN 200–110 in an apparent allosteric manner. (–)Desmethoxyverapamil inhibited the binding of [3H]PN 200–110 at 37°C and stimulated it at 18°C. In agreement with these results, (–)desmethoxyverapamil increased the dissociation rate of [3H]PN 200–110 from 0.29 min−1 to 0.38 min−1 at 37°C and decreased it threefold from 0.046 min−1 to 0.017 min−1 at 18°C. The (+)isomer of desmethoxyverapamil inhibited PN 200–110 binding at all temperatures tested. d‐cis‐Diltiazem stimulated the binding of [3H]PN 200–110 at 37°C with an apparent EC50 of 1.4 μM and decreased the dissociation rate from 0.29 min−1 to 0.11 min−1. The stimulatory effect of d‐cis‐diltiazem was temperature‐dependent and was seen only at temperatures above 18°C.These results suggest that the purified dihydropyridine receptor retains the basic properties of the membrane‐bound receptor and contains separate sites for at least dihydropyridines and phenylalkylamines.