Expression of plasma membrane calcium ATPases confers Ca2+/H+ exchange in rodent synaptic vesicles

Yoshiyasu Ono,Kenta Sumiyama,Yoshihiro Egashira,Shigeo Takamori,Yasunori Mori

doi:10.1038/s41598-019-40557-y

Yoshiyasu Ono, Kenta Sumiyama + Show 3 more

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https://doi.org/10.1038/s41598-019-40557-y

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Abstract

Ca2+ transport into synaptic vesicles (SVs) at the presynaptic terminals has been proposed to be an important process for regulating presynaptic [Ca2+] 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 Ca2+ transport systems depending on extra-vesicular (cytosolic) pH; one is mediated by a high affinity Ca2+ transporter which is active at neutral pH and the other is mediated by a low affinity Ca2+/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 Ca2+ clearance from the presynaptic cytoplasm. Here, we show that at physiological pH, the plasma membrane Ca2+ ATPases (PMCAs) are responsible for both the Ca2+/H+ exchange activity and Ca2+ uptake into SVs. The Ca2+/H+ exchange activity monitored by acidification assay exhibited high affinity for Ca2+ (Km ~ 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 Ca2+ from the cytosol to the lumen of sarcoplasmic endoplasmic reticulum (SERCA) at physiological pH. Furthermore, we rule out the contribution of SV2s, putative Ca2+ transporters on SVs, since both Ca2+/H+ exchange activity and Ca2+ 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 Ca2+ homeostasis and the modulation of H+ gradient in SVs.

Highlights

Synaptic vesicles (SV) are storage organelles for neurotransmitters and play a key role in synaptic transmission
Evidence that SVs isolated from sheep brain cortex contain low affinity Ca2+/H+ antiport activity relied essentially on an acidification assay in which fluorescence quenching of acridine orange due to V-ATPase-dependent acidification was reversed by addition of Ca2+ 21–23
We found that PMCAs, the plasma membrane Ca2+ ATPases, confer the majority of Ca2+ transport into SVs

Summary

Introduction

Synaptic vesicles (SV) are storage organelles for neurotransmitters and play a key role in synaptic transmission. Efforts to decipher the Ca2+ transport protein(s) in mammalian SVs have revealed that at least two distinct mechanisms may exist: vanadate-sensitive P-type Calcium ATPases, which are fully active at neutral pH, and a molecularly unidentified Ca2+/H+ antiporter, which functions maximally at alkaline pH of 8.521–23. These two transport systems exhibit substantial differences in their affinities for Ca2+. The relative contributions of these proteins to Ca2+ transport into SVs has not been elucidated, and whether/how the activities of these proteins may affect ΔμH+ to influence neurotransmitter uptake remains unclear

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