Identification of voltage operated calcium channels by binding studies: differentiation of subclasses of calcium antagonist drugs with 3H-nimodipine radioligand binding.
3H-Nimodipine (3H-NIM) is a high affinity radioligand suitable to study Ca2+ -channels in a variety of tissues. The binding is saturable, reversible, and stereospecific in purified bovine heart and partially purified guinea-pig brain membranes. In the latter a Bmax of 600fmol/mg protein, dissociation constants (KD) of 0.4-0.8nM and a Hill slope of 1.0 are found. At 37 degrees C the optimal pH in 50mM TRIS-HCl buffer is 7.1-7.4. The calcium channel is a metalloprotein, and the divalent cation which is essential for the binding of 3H-NIM can be removed by EDTA (EC50 20 microM); the nimodipine binding site of the channel may then be reconstituted by divalent cations with Mn2+ greater than Ca2+ greater than Mg2+ greater than Sr2+. Ca2+ -antagonist drugs can be divided into three main classes based on their interaction with the 3H-NIM binding site: Class I has one site law of mass action-displacement isotherms with 3H-NIM, Class II exhibits complex biphasic inhibition profiles and Class III drugs increase the affinity of 1,4 dihydropyridines for the Ca2+ -channel. Diltiazem is a Class III Ca2+ -antagonist. Our in vitro studies lead us to conclude that the Ca2+ -channel contains multiple regulatory sites at which drugs can act.
D000818:Animals; D001921:Brain / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000145:classification / Q000378:metabolism*; D002412:Cations / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004110:Diltiazem / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000378:metabolism; D009539:Nicotinic Acids / Q000378:metabolism*; D009553:Nimodipine; D013237:Stereoisomerism
D002121:Calcium Channel Blockers; D002412:Cations; D007473:Ion Channels; D009539:Nicotinic Acids; D009553:Nimodipine; D004110:Diltiazem