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Research Article

Identification of Voltage Operated Calcium Channels by Binding Studies: Differentiation of Subclasses of Calcium Antagonist Drugs with 3H-Nimodipine Radioligand Binding

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Pages 177-190 | Published online: 26 Sep 2008
 

Abstract

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 37C 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 3 H -NIM can be removed by EDTA (EC50 20μM); the nimodipine binding site of the channel may then be reconstituted by divalent cations with Mn2+ > Ca2+>Mg2+>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.

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