Effect of divalent cation chelation on dihydropyridine binding in isolated cardiac sarcolemma vesicles

The effect of divalent cation chelation on specific nitrendipine and ouabain binding has been determined in a highly enriched sarcolemma preparation isolated from canine ventricle. Maximal high-affinity nitrendipine binding measured in the absence of added calcium or magnesium was 997 +/- 103 fmol/mg protein. Nitrendipine binding in the presence of EDTA significantly decreased to 419 +/- 42 fmol/mg protein (P less than 0.001) which equates to 42.0% of control. The simultaneous presence of EDTA and A23187 in the binding buffer resulted in a decrease in nitrendipine binding to below detectable levels. These results suggest that divalent cations trapped within vesicles can support high affinity nitrendipine binding. Evaluation of dihydropyridine binding at various pH values suggested that the loss of binding below pH 7.0 and above pH 8.0 may result indirectly from a change in divalent cation binding rather than a direct effect on dihydropyridine binding per se. The maximal binding of ouabain determined in the presence of magnesium and inorganic phosphate averaged 340 +/- 7.4 pmol/mg protein. Pre-treatment of the preparation with sodium dodecyl sulfate (SDS) in order to express binding in sealed inside-out (IO) vesicles, increased ouabain binding to 471 +/- 27 pmol/mg protein. Thus, these preparations averaged 27.8% sealed IO vesicles. Addition of EDTA in the absence of magnesium in the binding buffer reduced ouabain binding to 204 +/- 7.7 and 11.7 +/- 3.5 pmol/mg protein in control and SDS-treated preparations, respectively. These findings suggest that this sarcolemma preparation consists of 43.6% sealed right-side-out (RO) vesicles which contain sufficient endogenous divalent cation trapped in the intravesicular space, to support ouabain binding. The correspondence between the percentage of ouabain binding that remains in the presence of EDTA and the percentage of nitrendipine binding observed under the same conditions is consistent with the hypothesis that divalent cations support nitrendipine binding by interaction with a site or sites accessible only from the cytoplasmic membrane surface and that nitrendipine and ouabain binding sites occur in the same vesicles (i.e., the nitrendipine binding site is of sarcolemma origin).