Distinction between Ca2+ pump and Ca2+/H+ antiport activities in synaptic vesicles of sheep brain cortex

Abstract

Synaptic vesicles, isolated from a sheep brain cortex, accumulate Ca2+ in a manner that depends on the pH and pCa values. In the presence of 100 [mu]M CaCl2, most of the Ca2+ taken up by the vesicles was vanadate-inhibited (86%) at pH 7.4, whereas at pH 8.5, part of the Ca2+ accumulated (36%) was [Delta]pH-dependent (bafilomycin and CCCP inhibited) and part was insensitive to those drugs (31%). We also observed that both vanadate-sensitive and bafilomycin-sensitive Ca2+ accumulations were completely released by the Ca2+ ionophore, ionomycin, and that these processes work with high (K0.5=0.6 [mu]M) and low (K0.5=217 [mu]M) affinity for Ca2+, respectively. The [Delta]pH-dependent Ca2+ transport appears to be largely operative at Ca2+ concentrations (>100 [mu]M) which completely inhibited the vanadate-sensitive Ca2+ uptake. These Ca2+ effects on the Ca2+ accumulation were well correlated with those observed on the vanadate-inhibited Ca2+-ATPase and bafilomycin-inhibited H+-ATPase, respectively. The Ca2+-ATPase activity reached a maximum at about 25 [mu]M (pH 7.4) and sharply declined at higher Ca2+ concentrations. In contrast, Ca2+ had a significant stimulatory effect on the H+-ATPase between 250 and 500 [mu]M Ca2+ concentration. Furthermore, we found that [Delta]pH-sensitive Ca2+ transport was associated with proton release from the vesicles. About 21% of the maximal proton gradient was dissipated by addition of 607.7 [mu]M CaCl2 to the reaction medium and, if CaCl2 was present before the proton accumulation, lower pH gradients were reached. Both vanadate-inhibited and bafilomycin-inhibited systems transported Ca2+ into the same vesicle pool of our preparation, suggesting that they belong to the same cellular compartment. These results indicate that synaptic vesicles of the sheep brain cortex contain two distinct mechanisms of Ca2+ transport: a high Ca2+ affinity, proton gradient-independent Ca2+ pump that has an optimal activity at pH 7.4, and a low Ca2+ affinity, proton gradient-dependent Ca2+/H+ antiport that works maximally at pH 8.5.