Expression of plasma membrane calcium ATPases confers Ca/H exchange in rodent synaptic vesicles.

Ca transport into synaptic vesicles (SVs) at the presynaptic terminals has been proposed to be an important process for regulating presynaptic [Ca] during stimulation as well as at rest. However, the molecular identity of the transport system remains elusive. Previous studies have demonstrated that isolated SVs exhibit two distinct Ca transport systems depending on extra-vesicular (cytosolic) pH; one is mediated by a high affinity Ca transporter which is active at neutral pH and the other is mediated by a low affinity Ca/H antiporter which is maximally active at alkaline pH of 8.5. In addition, synaptic vesicle glycoprotein 2 s (SV2s), a major SV component, have been proposed to contribute to Ca clearance from the presynaptic cytoplasm. Here, we show that at physiological pH, the plasma membrane Ca ATPases (PMCAs) are responsible for both the Ca/H exchange activity and Ca uptake into SVs. The Ca/H exchange activity monitored by acidification assay exhibited high affinity for Ca (K ~ 400 nM) and characteristic divalent cation selectivity for the PMCAs. Both activities were remarkably reduced by PMCA blockers, but not by a blocker of the ATPase that transfers Ca from the cytosol to the lumen of sarcoplasmic endoplasmic reticulum (SERCA) at physiological pH. Furthermore, we rule out the contribution of SV2s, putative Ca transporters on SVs, since both Ca/H exchange activity and Ca transport were unaffected in isolated vesicles derived from SV2-deficient brains. Finally, using a PMCA1-pHluorin construct that enabled us to monitor cellular distribution and recycling properties in living neurons, we demonstrated that PMCA1-pHluorin localized to intracellular acidic compartments and recycled at presynaptic terminals in an activity-dependent manner. Collectively, our results imply that vesicular PMCAs may play pivotal roles in both presynaptic Ca homeostasis and the modulation of H gradient in SVs.