Expression of plasma membrane calcium ATPases confers Ca 2+ /H + exchange in rodent synaptic vesicles

Ca 2+ transport into synaptic vesicles (SVs) at the presynaptic terminals has been proposed to be an important process for regulating presynaptic [Ca 2+ ] 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 2+ transport systems depending on extra-vesicular (cytosolic) pH; one is mediated by a high affinity Ca 2+ transporter which is active at neutral pH and the other is mediated by a low affinity Ca 2+ /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 2+ clearance from the presynaptic cytoplasm. Here, we show that at physiological pH, the plasma membrane Ca 2+ ATPases (PMCAs) are responsible for both the Ca 2+ /H + exchange activity and Ca 2+ uptake into SVs. The Ca 2+ /H + exchange activity monitored by acidification assay exhibited high affinity for Ca 2+ ( K m ~ 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 2+ from the cytosol to the lumen of sarcoplasmic endoplasmic reticulum (SERCA) at physiological pH. Furthermore, we rule out the contribution of SV2s, putative Ca 2+ transporters on SVs, since both Ca 2+ /H + exchange activity and Ca 2+ 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 2+ homeostasis and the modulation of H + gradient in SVs.