CHARACTERIZATION AND PHOTOAFFINITY-LABELING OF RECEPTOR-SITES FOR THE CA-2+- CHANNEL INHIBITORS D-CIS-DILTIAZEM, (+/-)-BEPRIDIL, DESMETHOXYVERAPAMIL, AND (+)-PN-200-110 IN SKELETAL-MUSCLE TRANSVERSE TUBULE MEMBRANES

Abstract

In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmothoxyverapamil, d-cis-[3H]diltiazem, and (.+-.)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 .+-. 0.5, 50 .+-. 5, and 20 .+-. 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 .+-. 10 for (-)-[3H]desmethoxyverapamil, 50 .+-. 15 for for d-cis-[3H]diltiazem, and 75 .+-. 15 for (.+-.)-[3H]bepridil. The kinetics of association at 10.degree. C for the three types of tritiated compounds were relatively slow (3 .times. 105 M-1 s-1 for (-)-[3H]desmethoxyverapamil, 8 .times. 103 M-1 s-1 for d-cis-[3H]diltiazem, and 4.2 .times. 105 M-1 s-1 or (.+-.)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (.+-.)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 .+-. 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-110.