Synaptic vesicle Ca2+/H+ antiport:: dependence on the proton electrochemical gradient

Synaptic vesicles isolated from sheep brain cortex accumulate Ca2+ by a mechanism of secondary active transport associated to the H+-pump activity. The process can be visualized either by measuring Ca2+-induced H+ release or Delta pH-dependent Ca2+ accumulation. We observed that the amount of Ca2+ taken up by the vesicles increases with the magnitude of the Delta pH across the membrane, particularly at Ca2+ concentrations (similar to 500 mu M) found optimal for the antiporter activity. Similarly, H+ release induced by Ca2+ increased with the magnitude of Delta pH. However, above 60% Delta pH (high H+-pump activity), the net H+ release from the vesicles decreased as the pump-mediated H+ influx exceeded the Ca2+-induced H+ efflux. We also observed that the Ca2+ /H+ antiport activity depends, essentially, on the Delta pH component of the electrochemical gradient (similar to 3 nmol Ca2+ taken up/mg protein), although the hcp component may also support some Ca2+ accumulation by the vesicles (similar to 1 nmol/mg protein) in the absence of Delta pH. Both Ca2+-induced H+ release and Delta pH-dependent Ca2+ uptake could be driven by an artificially imposed proton motive force. Under normal conditions (H+ pump-induced Delta pH), the electrochemical gradient dependence of Ca2+ uptake by the vesicles was checked by inhibition of the process with specific inhibitors (bafilomycin A(1), ergocryptin, folymicin, DCCD) of the H+-Dump activity. These results indicate that synaptic vesicles Ca2+/H+ antiport is indirectly linked to ATP hydrolysis and it is essentially dependent on the chemical component (Delta pH) of the electrochemical gradient generated by the H+-pump activity. (C) 1999 Elsevier Science B.V. All rights reserved.