Solubilization of the nitrendipine receptor from skeletal muscle transverse tubule membranes Interactions with specific inhibitors of the voltage‐dependent Ca2+ channel

Abstract

The nitrendipine receptor associated with the voltage-dependent Ca channel from rabbit skeletal muscle transverse tubule membranes has been solubilized by detergent extraction. A highly stable solubilized receptor preparation was obtained using 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate as detergent with phospholipids or glycerol present as stabilizing agents. Binding of [3H]nitrendipine to the solubilized receptor was reversible and saturable. At 4.degree. C the equilibrium dissociation constant of the [3H]nitrendipine receptor complex was 7 .+-. 3 nM and was close to that determined from the rate constants of association (k1 = 1.3 105 M-1 s-1) and dissociation (k-1 = 1.10 .times. 10-3 s-1) of 8.4 nM. The nitrendipine concentration that gave a half-maximal inhibition of [3H]nitrendipine binding to the solubilized receptor was 10 nM, which was similar to the values for the dissociation constant determined for the radiolabeled ligand. [3H]Nitrendipine binding to its solubilized receptor was also inhibited by other antiarrythmic drugs, such as bepridil and verapamil, and enhanced by d-cis-diltiazem. Since these drugs are apparent non-competitive inhibitors of [3H]nitrendipine binding it was concluded that these different binding sites are tightly coupled. Sucrose density sedimentation of solubilized nitrendipine receptor resulted in the separation of 3 [3H]nitrendipine binding activities with apparent sedimentation coefficients of 11.4S, 14.4S and 21S.