PURIFICATION OF THE DIHYDROPYRIDINE RECEPTOR OF THE VOLTAGE-DEPENDENT CA-2+ CHANNEL FROM SKELETAL-MUSCLE TRANSVERSE TUBULES USING (+) [H-3]PN200-110

The rabbit skeletal muscle T-tubule [transverse tubule] membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[3H]PN 200-110 binding experiments. The dissociation constant of the (+)[3H]PN 200-110 solubilized-receptor complex was 0.4 .+-. 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (k1 = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 10-2 min-1). The (+)[3H]PN 200-110 receptor was substantially purified by a combination of filtration of Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [3H]PN 200-110. The purified material contained polypeptides of 142,000, 32,000 and 33,000 MW. These 3 components copurified with (+)[3H]PN 200-110 binding activity. [Implications with respect to the use of dihydropyridine derivatives to treat cardiovascular disorders are presented.].