Cardiac sarcoplasmic reticulum contains a low‐affinity site for phenylakylamines

The distribution of the bovine cardiac binding sites for the organic calcium‐channel blockers was studied. Crude microsomal membranes were separated into three fractions, which contained mainly membranes derived from sarcolemma, ‘junctional’ sarcoplasmic reticulum containing transversal tubuli, and free sarcoplasmic reticulum. The high‐affinity binding site for the dihydropyridines, determined in the presence of nitrobenzylthioinosine, was enriched 12‐fold and 17‐fold in sarcolemma and junctional sarcoplasmic reticulum. The binding sites for the phenylalkylamines, determined with [ 3 H]verapamil or [ 3 H](–)desmethoxyverapamil, were enriched 1.5–3.4‐fold in sarcolemma and junctional sarcoplasmic reticulum but 6–10‐fold in free sarcoplasmic reticulum. The phenylalkylamine‐binding site, present in free sarcoplasmic reticulum, was partially destroyed by chymotrypsin or phospholipase A 2 and C treatment. Specific binding was proportional to the concentration of the added membrane protein. The binding of (–)desmethoxyverapamil was half‐maximally inhibited by 6.5 mM calcium chloride and was optimal in the presence of 5 mM EGTA. In three out of five preparations (–)desmethoxyverapamil bound to a single site with an apparent K d value of 191 ± 42.8 nM and a density of 34.5 ± 7.7 pmol/mg protein. In two out of five preparations an additional high‐affinity site ( K d ∼ 0.67 nM) was detected. The low‐affinity site bound other phenylalkylamines, but stereospecific binding of phenylalkylamines was not observed. Binding of phenylalkylamines to the low‐affinity site was inhibited by some but not all calmodulin ‘antagonists’. Furthermore dihydropyridines did not affect the binding of (–)desmethoxyverapamil suggesting that the low‐affinity site differs considerably from the high‐affinity sarcolemmal site. These results suggest that free sarcoplasmic reticulum contains a binding site for phenylalkylamines at a relative high density, which is not related to the high‐affinity site present in the voltage‐dependent calcium channel.