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

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Abstract
The nitrendipine receptor associated with the voltage‐dependent calcium 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°C the equilibrium dissociation constant of the [3H]nitrendipine receptor complex was 7±3nM 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 × 10−3 s−1) of 8.4nM. 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 radiolabelled 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 three [3H]nitrendipine binding activities with apparent sedimentation coefficients of 11.4 S. 14.4 S and 21S.