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 me...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 113; no. 38; pp. 10702 - 10707
Main Authors Egashira, Yoshihiro, Takase, Miki, Watanabe, Shoji, Ishida, Junji, Fukamizu, Akiyoshi, Kaneko, Ryosuke, Yanagawa, Yuchio, Takamori, Shigeo
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 20.09.2016
Subjects
Acidity
Amino acids
Biological Sciences
Electrochemistry
Kinetics
Neurotransmitters
pH sensors
Physiology
Reaction kinetics
inhibitory neuron
VGAT
synaptic vesicle
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Summary: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.
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Edited by Robert H. Edwards, University of California, San Francisco, CA, and accepted by Editorial Board Member Pietro De Camilli July 20, 2016 (received for review March 18, 2016)
1Present address: Laboratory for Organ Regeneration, RIKEN Center for Developmental Biology, Hyogo 650-0047, Japan.
Author contributions: Y.E. and S.T. designed research; Y.E. and M.T. performed research; S.W., J.I., A.F., R.K., and Y.Y. contributed new reagents/analytic tools; Y.E. analyzed data; and Y.E. and S.T. wrote the paper.
ISSN:0027-8424
1091-6490
1091-6490
DOI:10.1073/pnas.1604527113