TY - JOUR T1 - Monitoring of Vacuolar-Type H<sup>+</sup> ATPase-Mediated Proton Influx into Synaptic Vesicles JF - The Journal of Neuroscience JO - J. Neurosci. SP - 3701 LP - 3710 DO - 10.1523/JNEUROSCI.4160-14.2015 VL - 35 IS - 8 AU - Yoshihiro Egashira AU - Miki Takase AU - Shigeo Takamori Y1 - 2015/02/25 UR - http://www.jneurosci.org/content/35/8/3701.abstract N2 - During synaptic vesicle (SV) recycling, the vacuolar-type H+ ATPase creates a proton electrochemical gradient (ΔμH+) 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 ΔμH+. Consistent with this, the time constant of SV re-acidification was reported to be &lt;5 s, much faster than glutamate loading (τ of ∼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 (τ of ∼15 s) than estimated previously. In addition, we found that the SV lumen exhibited a large buffering capacity (∼57 mm/pH), corresponding to an accumulation of ∼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. ER -