Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes.
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]desmethoxyverapamil, 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 d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[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 200-100.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001552:Benzazepines / Q000378:metabolism*; D015764:Bepridil; D001667:Binding, Competitive; D015220:Calcium Channels; D004110:Diltiazem / Q000378:metabolism*; D017275:Isradipine; D007700:Kinetics; D009132:Muscles / Q000378:metabolism*; D010069:Oxadiazoles / Q000378:metabolism*; D010777:Photochemistry; D011759:Pyrrolidines / Q000378:metabolism*; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014700:Verapamil / Q000031:analogs & derivatives* / Q000378:metabolism
D000345:Affinity Labels; D001552:Benzazepines; D015220:Calcium Channels; D010069:Oxadiazoles; D011759:Pyrrolidines; D011978:Receptors, Nicotinic; D015764:Bepridil; C043184:4-desmethoxyverapamil; D014700:Verapamil; D004110:Diltiazem; D017275:Isradipine