After exocytotic release of neurotransmitter from synaptic vesicles (SVs), SVs are regenerated by endocytosis, re-acidified by the V-ATPase, and refilled with neurotransmitters by the vesicular transporters. Refilling of neurotransmitters critically depends on a proton electrochemical gradient (ΔμH⁺) generated by the V-ATPase, which consists of two components, the membrane potential (ΔΨ) and the pH gradient (ΔpH). Biochemical investigations using isolated SVs and reconstitution systems have revealed that relative contributions of ΔΨ and ΔpH to drive neurotransmitter transport vary depending on neurotransmitters. For instance, transport of glutamate is predominantly driven by ΔΨ, whereas that of GABA depends on both components without clear preference. However, little is known as to how the ΔμH⁺ is built up in vivo, and if it were different depending on neurotransmitter types. Furthermore, it has been controversial how Cl^- affects glutamate transport and ΔμH⁺ in isolated SVs.
To investigate how the proton motive force is built up under physiological circumstance in a neurotransmitter-specific manner, we utilized a fluorescent protein-based pH measurement in the vesicle lumen of cultured hippocampal neurons derived from VGAT-Venus transgenic mice. Our analyses revealed that (1) re-acidification of SVs takes longer than previously reported, (2) SVs exhibit large buffering capacity which enables glutamatergic SVs to accommodate ~ 1,000 H⁺ during re-acidification, (3) GABAergic SVs has higher luminal pH and remarkably smaller buffering capacity compared to glutamatergic SVs, resulting in only ~5-fold less H⁺ influx, and (4) SVs lacking the vesicular glutamate transporter 1 show the same luminal pH with wild-type SVs, but have reduced buffering capacity. These parameters will be helpful for the mechanistic understandings of the proton-driven transmitter uptake into SVs.