Dihydropyridine binding and calcium channel function in clonal rat adrenal medullary tumor cells

We correlated the binding of the dihydropyridines, nitrendipine and PN200-110, with their pharmacological actions on voltage-dependent membrane calcium channels. Binding was studied in clonal rat adrenal medullary cells (PC12) and in plasma membranes prepared from them. Calcium currents were studied using whole cell and single channel patch clamp methods. For both [3H]-(+/-)-nitrendipine and [3H]-(+)-PN200-110, high affinity binding sites with dissociation constants of 0.6 and 0.04 nM, respectively, were identified both in membrane fragments and in intact cells. In crude membrane preparations a low affinity nitrendipine-binding site was also found. The dissociation constant for binding at this site was affected by ionic strength and the presence of divalent cations. In 500 mM KCI, 0.1 mM CaCl2, 50 mM 3-(N-morpholino)propanesulfonic acid (pH 7.4), the KD is about 70 nM. The number of high affinity binding sites for dihydropyridines was between 30 and 100 fmol/mg of protein while the number of low affinity sites was between 30 and 70 pmol/mg of protein. In whole cells the measured number of high affinity sites was between 2000 and 4000/cell and, by extrapolation from the membrane preparation, the low affinity sites correspond to several million sites per cell. The electrophysiological effects of both of the dihydropyridines on Ca2+ currents were voltage dependent. When nitrendipine was applied, a small increase in calcium current occurred and this was followed by a decrease. The inhibitory effect was more pronounced at depolarized membrane holding potentials and was relieved by hyperpolarizing the membrane, whereas the stimulatory effect was pronounced at negative membrane holding potentials. In 10 nM nitrendipine these effects were also observed in single channels; they were not due to changes in channel conductance or dwell times in the open state but, rather, were due to changes in the probability of opening. The half-maximal inhibitory concentration (IC50) for nitrendipine was 67 nM and the IC50 for the effect of (+)-PN200-110 was 9 nM using protocols which favored the depolarized state of the channel. No excitatory effect was seen. The IC50 from electrophysiological estimates is higher than the KD for the high affinity binding site for both compounds. Using a simple model of voltage-dependent binding, we could not account for the difference. The number of functional channels calculated from the relation between whole cell and single channel calcium currents and the probability of opening was in good agreement with the number of high affinity sites calculated from the binding studies.(ABSTRACT TRUNCATED AT 400 WORDS)