Monitoring of Vacuolar-Type H+ ATPase-Mediated Proton Influx into Synaptic Vesicles.

During synaptic vesicle (SV) recycling, the vacuolar-type H⁺ ATPase creates a proton electrochemical gradient (∆μH⁺) 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 ∆μH⁺. Consistent with this, the time constant of SV re-acidification was reported to be <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. [ABSTRACT FROM AUTHOR]