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

Abstract

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 (~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 (~3 nmol Ca2+ taken up/mg protein), although the [Delta][phi] component may also support some Ca2+ accumulation by the vesicles (~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 A1, ergocryptin, folymicin, DCCD) of the H+-pump 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.