Unique pH dynamics in GABAergic synaptic vesicles illuminates the mechanism and kinetics of GABA loading.
GABA acts as the major inhibitory neurotransmitter in the mammalian brain, shaping neuronal and circuit activity. For sustained synaptic transmission, synaptic vesicles (SVs) are required to be recycled and refilled with neurotransmitters using an H(+) electrochemical gradient. However, neither the mechanism underlying vesicular GABA uptake nor the kinetics of GABA loading in living neurons have been fully elucidated. To characterize the process of GABA uptake into SVs in functional synapses, we monitored luminal pH of GABAergic SVs separately from that of excitatory glutamatergic SVs in cultured hippocampal neurons. By using a pH sensor optimal for the SV lumen, we found that GABAergic SVs exhibited an unexpectedly higher resting pH (∼6.4) than glutamatergic SVs (pH ∼5.8). Moreover, unlike glutamatergic SVs, GABAergic SVs displayed unique pH dynamics after endocytosis that involved initial overacidification and subsequent alkalization that restored their resting pH. GABAergic SVs that lacked the vesicular GABA transporter (VGAT) did not show the pH overshoot and acidified further to ∼6.0. Comparison of luminal pH dynamics in the presence or absence of VGAT showed that VGAT operates as a GABA/H(+) exchanger, which is continuously required to offset GABA leakage. Furthermore, the kinetics of GABA transport was slower (τ > 20 s at physiological temperature) than that of glutamate uptake and may exceed the time required for reuse of exocytosed SVs, allowing reuse of incompletely filled vesicles in the presence of high demand for inhibitory transmission.
Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, Kyoto 610-0394, Japan;
Yoshihiro
Y
Egashira
Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, Kyoto 610-0394, Japan;
Miki
M
Takase
Laboratory of Ion Channel Pathophysiology, Graduate School of Brain Science, Doshisha University, Kyoto 610-0394, Japan;
Shoji
S
Watanabe
Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki 305-8577, Japan;
Junji
J
Ishida
Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki 305-8577, Japan;
Akiyoshi
A
Fukamizu
Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Gunma 371-8514, Japan.
Ryosuke
R
Kaneko
Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Gunma 371-8514, Japan.
Yuchio
Y
Yanagawa
Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, Kyoto 610-0394, Japan; stakamor@mail.doshisha.ac.jp.
Shigeo
S
Takamori
D000818:Animals; D005089:Exocytosis / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D009474:Neurons / Q000378:metabolism* / Q000502:physiology; D018377:Neurotransmitter Agents / Q000235:genetics / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism / Q000502:physiology; D013569:Synapses / Q000235:genetics / Q000378:metabolism; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism; D050495:Vesicular Inhibitory Amino Acid Transport Proteins / Q000235:genetics / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D018377:Neurotransmitter Agents; D050495:Vesicular Inhibitory Amino Acid Transport Proteins; C492244:Viaat protein, mouse; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid
VGAT; inhibitory neuron; synaptic vesicle