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

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⁺ (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 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.

SUBMITTER: Ono Y

PROVIDER: S-EPMC6414521 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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

Ono Yoshiyasu Y Mori Yasunori Y Egashira Yoshihiro Y Sumiyama Kenta K Takamori Shigeo S

Scientific reports 20190312 1

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+ transporte ...[more]

PMID: 30862855

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Project description:Plasma membrane calcium-ATPases (PMCAs) play a critical role in regulating intracellular calcium concentration. Four genes encode PMCA proteins with alternative splicing of transcripts at three sites (A, B and C) serving to increase isoform diversity. Our previous work shows that all four PMCAs are expressed and have specific locations in human corneal epithelium (hCE). The present work examined which splice variants of PMCAs are expressed in hCE. Total RNA was extracted from hCE scraped from cadaver corneas of five different donors (two females and three males, age range 55-76 years). RT-PCR was performed using PMCA isoform-specific primers designed to amplify transcripts that included either splice site A or splice sites B and C. PMCA cDNAs were sequenced or cloned, and then sequenced. There was uniformity in the PMCA1 and PMCA4 expression profile among the five donors. Specifically, every donor expressed PMCA4 transcripts (4x at site A and 4b at site B/C). Every donor also expressed PMCA1 transcripts at sites B/C, specifically PMCA1b and PMCA1kb. In contrast, PMCA2 and PMCA3 expression varied; PCR DNAs were detected in two of five donors. One donor expressed PMCA2a and a novel PMCA2 variant we termed PMCA2((i)). PMCA3a transcript was demonstrated in a different donor. Finally, for all the donors, bands encoding site A transcripts for PMCA4 were obtained but no PCR transcripts were detected at site A for PMCA1, PMCA2 and PMCA3. This investigation showed that hCE expressed multiple splice variants of PMCA isoforms. Furthermore, this study documented the expression of the PMCA1k variant (PMCA1kb) previously only described in intestine and pancreatic beta cells and describes a novel PMCA2((i)) variant. Finally, this study suggests that the molecular configuration of PMCA1, PMCA2 and PMCA3 in the region of splice site A in hCE must be different than in other tissues since the same primers that produced site A transcripts in several other tissues were ineffective in priming PCR in hCE.

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

Publications

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

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