Abstract

We have studied allosteric effects of the Ca channel blockers d-(cis)-diltiazem, (+/-)-verapamil, and (S)- and (R)-devapamil on the specific binding of the 1,4-dihydropyridine derivative (+)-[3H]PN 200-110 to intact tissue cultured rat heart cells. In polarized cells (membrane potential, -38 +/- 4 mV) d-(cis)-diltiazem (5 microM) increased the affinity of the radiolabel 2-4-fold causing 100-187% enhancement of binding at (+)-PN 200-110 concentrations below 0.5 nM. (+/-)-Verapamil (0.1-3 microM) had a similar, although smaller, effect on (+)-PN 200-110 binding. The two enantiomers of devapamil were without effect. In depolarized cells (membrane potential, 0 mV) d-(cis)-diltiazem had a small and the phenylalkylamines a strong inhibitory effect on (+)-PN 200-110 binding, mainly due to a reduction of binding affinity. At 50% receptor occupation by the radioligand, (R)-devapamil, (+/-)-verapamil, and (S)-devapamil displaced 40, 55, and 75%, respectively, of specifically bound radiolabel. Half-maximal effects were reached with 50, 20, and 4.5 nM, respectively, of the three compounds. Compared with nominally Ca-free medium (containing 3-5 microM Ca), addition of 1.25 mM CaCl2 caused an increase in the maximal binding capacity for (+)-PN 200-110 in both polarized and depolarized cells. However, Ca had only marginal effects on the allosteric interactions between (+)-PN 200-110, d-(cis)-diltiazem, and verapamil. We conclude from our results that positive cooperative interactions between Ca channel blockers prevail under conditions in which the voltage-dependent Ca channel can fluctuate between closed, open, and inactivated states. Negative cooperativity is usually observed under conditions in which all channels are inactivated (depolarized cells, fragmented membranes). Therefore, it is impossible to predict the type and the extent of allosteric interactions in vivo from studies in cell homogenates.