The mechanism of binding of dihydropyridine calcium channel blockers to rat brain membranes.

Detailed kinetic and equilibrium studies of the binding of two radiolabeled 1,4-dihydropyridine calcium antagonists to putative calcium channels in rat brain membranes were performed. (+/-)-[3H]Nitrendipine, a racemic ligand, and (+)-[3H]isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1, 4-dihydro-2,6-dimethyl-5-methoxycarbonylpyridine-3-carboxylate (PN200-110), a pure isomer, were used and their binding properties were quantitated and compared. Analysis of equilibrium binding revealed a single high affinity component for each radioligand with the same density of binding sites for both ligands. Association rates were determined over a 60-fold range of concentration of each radioligand. For both radioligands, the pseudo-first order association time courses were biphasic with the rate of the faster component dependent on radioligand concentration and the rate of the slower component independent of both the structure of the radioligand and the concentration of the radioligand. Dissociation rates were determined after various times of association. The dissociation of the optically pure radioligand, (+)-[3H]PN200-110, was monophasic at all association times, consistent with a single bound species being present throughout association. However, (+/-)-[3H]nitrendipine dissociation was biphasic after short association times (1-10 min). The biphasic dissociation observed with (+/-)-[3H]nitrendipine is consistent with the two optical isomers binding with approximately the same association rate but having different dissociation rates. These results appear to reflect the existence of two interconvertible binding states of the putative calcium channel in the membrane, one which binds the radioligands with high affinity in a simple bimolecular reaction and one which has no detectable affinity for the ligands. This mechanism of isomerization before ligand binding has been modeled by numerical solution of the differential equations of the scheme providing estimates of the rate constants for each reaction in the scheme.