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 (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.