期刊
JOURNAL OF NEUROSCIENCE
卷 35, 期 8, 页码 3701-3710出版社
SOC NEUROSCIENCE
DOI: 10.1523/JNEUROSCI.4160-14.2015
关键词
acidification; synaptic vesicle; V-ATPase
资金
- Japan Society for the Promotion of Science (JSPS) through the Funding Program for NEXT Generation World-Leading Researchers (NEXT Program) [LS118]
- Council for Science and Technology Policy and Ministry of Education, Culture, Sports, Science, and Technology [26115716]
- Core Research for Evolutional Science and Technology of Japan Science and Technology Agency
- JSPS Core-to-Core Program, A. Advanced Research Networks
- Grants-in-Aid for Scientific Research [26115716] Funding Source: KAKEN
During synaptic vesicle (SV) recycling, the vacuolar-type H+ ATPase creates a proton electrochemical gradient (Delta mu H+) that drives neurotransmitter loading into SVs. Given the low estimates of free luminal protons, it has been envisioned that the influx of a limited number of protons suffices to establish Delta mu H+. Consistent with this, the time constant of SV re-acidification was reported to be <5s, much faster than glutamate loading (tau of similar to 15 s) and thus unlikely to be rate limiting for neurotransmitter loading. However, such estimates have relied on pHluorin-based probes that lack sensitivity in the lower luminal pH range. Here, we reexamined re-acidification kinetics using the mOrange2-based probe that should report the SV pH more accurately. In recordings from cultured mouse hippocampal neurons, we found that re-acidification took substantially longer (tau of similar to 15 s) than estimated previously. In addition, we found that the SV lumen exhibited a large buffering capacity (similar to 57 mM/pH), corresponding to an accumulation of similar to 1200 protons during re-acidification. Together, our results uncover hitherto unrecognized robust proton influx and storage in SVs that can restrict the rate of neurotransmitter refilling.
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