Abstract

We have investigated the effects of two oppositely acting enantiomers of the 1,4-dihydropyridine derivative 202-791 on voltage-dependent Ca channels by combining electrophysiological techniques and binding studies. The (S)-enantiomer of 202-791 promoting prolonged openings of single Ca channels, and thereby increasing transmembrane Ba currents, was classified as channel activator. The (R)-enantiomer favoring a closed state of the channel, and thereby reducing Ba currents, was classified as a channel blocker. Both compounds shifted the steady state current inactivation curve toward more negative potentials. At holding potentials positive to -20 mV, the Ca channel-activating effect of the (S)-enantiomer turned over into a blocking effect. In cells with normal resting potential the combination of the two enantiomers revealed a possible positive cooperative effect resulting in an enhancement of the open state probability of the channels. At depolarized holding potentials the activator enhanced the inhibitory effect of the blocker. Binding studies in intact cells were performed by using the radiolabeled channel-blocking dihydropyridine 3H-(+)-PN 200-110. The results showed a strong increase in binding affinity but no change in binding capacity when the cells were depolarized. Analysis of the interactions of (S)- and (R)-202-791 with this radioligand indicated stimulation of 3H-(+)-PN 200-110 binding by the (S)-enantiomer in polarized cells (membrane potential -38 +/- 4 mV). This effect could be attributed to an increase in binding affinity. The (R)-enantiomer had no such positive cooperative effect, but acted as a purely competitive ligand. Depolarization to 0 mV increased the apparent affinity of both enantiomers by factors of 38 (blocker) and 12 (activator), but abolished the cooperative effect of (S)-202-791 on the binding of the radioligand. Ca ions had little effect on the binding of 3H-(+)-PN 200-110 in polarized cells. However, in the presence of the activating (S)-enantiomer, Ca transformed the usual hyperbolic binding isotherm of the radioligand into a strongly sigmoid curve. Sigmoidicity was minimal with 3-5 microM Ca and maximal with 0.5 mM Ca. Together these data demonstrate homotropic and heterotropic cooperative interactions between channel activator and channel blocker. They indicate that at least two high affinity binding sites for dihydropyridines are associated with voltage-dependent Ca channels. Voltage dependence of both--binding affinity and cooperativity--suggests that these binding sites are located close to a structural component of the channel which is involved in the potential-sensitive gating process.