Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

Abstract: The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)‐verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 ± 0.1 nM, a Bmax of 161 ± 27 fmol.mg‐1 protein, and a Hill slope of 1.07, at 25°C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: (1) nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; (2) verapamil and (‐)‐D 600 are partial competitors, with biphasic competition behavior. Thus, (+)verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (‐)‐verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (‐)‐D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50= 430 nM). However, (+)‐verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization‐induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)‐verapamil inhibits Ca2+ influx by 50% at about 500 μM, whereas it inhibits 50% of the binding at concentrations 200‐fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP‐dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d‐cis‐diltiazem, with similar IC50 values and in the same concentration range (10‐5‐10‐3M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high‐affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+‐ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.