Expression of plasma membrane calcium ATPases confers Ca2+/H+ exchange in rodent synaptic vesicles
Expression of plasma membrane calcium ATPases confers Ca2+/H+ exchange in rodent synaptic vesicles
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DOI：
10.1038/s41598-019-40557-y
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发表时间：
2019-03-12
影响因子：
4.6
通讯作者：
Takamori，Shigeo
中科院分区：
文献类型：
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作者：
Ono，Yoshiyasu;Mori，Yasunori;Takamori，Shigeo
在突触前末梢，Ca ~（2+）向突触囊泡（SV）的转运被认为是调节突触前[Ca ~（2+）]的重要过程。然而，运输系统的分子身份仍然难以捉摸。以前的研究表明，孤立的SV表现出两种不同的钙离子转运系统依赖于囊泡外（胞质）pH值;一个是由高亲和力的钙离子转运蛋白，这是在中性pH值的活性和其他介导的低亲和力Ca 2 +/H+反向转运蛋白，这是最大限度地在碱性pH值为8.5。此外，突触囊泡糖蛋白2s（SV 2s），一个主要的SV组件，已被提出有助于从突触前细胞质中清除Ca 2+。在这里，我们表明，在生理pH值下，质膜Ca 2 + ATP酶（PMCAs）负责Ca 2 +/H+交换活性和Ca 2+摄取到SV。通过酸化测定监测的Ca 2 +/H+交换活性表现出对Ca 2+的高亲和力（Km~ 400 nM）和对PMCAs的特征性二价阳离子选择性。这两种活动都显着减少PMCA阻滞剂，但不是由ATP酶的阻滞剂，将钙离子从细胞质转移到肌浆内质网（SERCA）在生理pH值的内腔。此外，我们排除了贡献的SV 2s，推定的钙离子转运SV，因为钙离子/H+交换活性和钙离子转运不受影响的孤立囊泡来自SV 2缺陷的大脑。最后，使用PMCA 1-pHluorin构建，使我们能够监测活神经元中的细胞分布和再循环特性，我们证明了PMCA 1-pHluorin定位于细胞内酸性隔室，并以活性依赖的方式在突触前末端再循环。总的来说，我们的研究结果表明，囊泡PMCAs可能在突触前Ca 2+稳态和SV中H+梯度的调制中发挥关键作用。
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.