Open AccessTarget size analysis and molecular properties of Ca 2+ channels labelled with [ 3 H]verapamil
Author(s) -
GOLL Alexandra,
FERRY David R.,
GLOSSMANN Hartmut
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb08172.x
Subject(s) - verapamil , dihydropyridine , diltiazem , chemistry , receptor , allosteric regulation , calcium channel , ryanodine receptor , biophysics , voltage dependent calcium channel , agonist , divalent , calcium , microsome , biochemistry , biology , enzyme , organic chemistry
[ 3 H]Verapamil was employed to label the drug receptor sites within the Ca 2+ ‐channel in skeletal muscle microsomes which are coupled in a negative heterotropic allosteric manner to the previously characterized 1,4‐dihydropyridine receptors. At 2°C the K d 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. [ 3 H]Verapamil, d‐cis ‐[ 3 H]diltiazem as well as 1,4‐[ 3 H]dihydropyridine receptors copurify with t‐tubule membranes. The ratio of high‐affinity verapamil: 1,4‐dihydropyridine d‐cis ‐diltiazem Ca 2+ 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 Ca 2+ antagonists and agonists in a stereoselective manner. Divalent cations (Mn 2+ > Ca 2+ > Mg 2+ ) are inhibitory. Target size analysis with high‐energy electrons was performed and the M r of the verapamil drug receptor site is 110000.