Target size analysis and molecular properties of Ca2+ channels labelled with [3H]verapamil

Abstract

[3H]Verapamil was employed to label the drug receptor sites within the Ca2+-channel in skeletal muscle microsomes which are coupled in a negative heterotropic allosteric manner to the previously characterized 1,4-dihydropyridine receptors. At 2 degrees C the Kd of a high-affinity receptor site is 45 nM and the maximum density of binding sites is 37 pmol/mg of protein. Established subcellular fractionation procedures were used to isolate transverse tubule membranes from rabbit and guinea-pig skeletal muscle. [3H]Verapamil, d-cis-[3H]diltiazem as well as 1,4-[3H]dihydropyridine receptors copurify with t-tubule membranes. The ratio of high-affinity verapamil: 1,4-dihydropyridine d-cis-diltiazem Ca2+ channel receptor sites is 4:2:1. The verapamil drug receptors are heat-labile and have essential sulfhydryl groups since they are inactivated by p-chloromercuriphenylsulfonic acid and N-ethylmaleimide. The receptors recognize the main classes of Ca2+ antagonists and agonists in a stereoselective manner. Divalent cations (Mn2+ greater than Ca2+ greater than Mg2+) are inhibitory. Target size analysis with high-energy electrons was performed and the Mr of the verapamil drug receptor site is 110000.