title: Monitoring of vacuolar-type H+ ATPase-mediated proton influx into synaptic vesicles.
abstract: 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 <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.
authors.affiliation: Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, Kyotanabe, Kyoto 610-0394, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Egashira
authors.affiliation: Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, Kyotanabe, Kyoto 610-0394, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Takase
authors.affiliation: Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, Kyotanabe, Kyoto 610-0394, Japan stakamor@mail.doshisha.ac.jp.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Takamori
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D011522:Protons; D013572:Synaptic Vesicles / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D011522:Protons; D018698:Glutamic Acid; D025262:Vacuolar Proton-Translocating ATPases
keywords: V-ATPase; acidification; synaptic vesicle