Evidence for a Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters

Evidence for a Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters Plasma membrane γ-aminobutyric acid (GABA) transporters (GATs) are electrogenic transport proteins that couple the cotranslocation of Na+, Cl−, and GABA across the plasma membrane of neurons and glia. A fundamental property of the transporter that determines its ability to concentrate GABA in cells and, hence, regulate synaptic and extra-synaptic GABA concentrations, is the ion/substrate coupling stoichiometry. Here, we scrutinized the currently accepted 2 Na+:1 Cl−:1 GABA stoichiometry because it is inconsistent with the measured net charge translocated per co-substrate (Na+, Cl−, and GABA). We expressed GAT1 and GAT3 in Xenopus laevis oocytes and utilized thermodynamic and uptake under voltage-clamp measurements to determine the stoichiometry of the GABA transporters. Voltage-clamped GAT1-expressing oocytes were internally loaded with GABA, and the reversal potential (V rev) of the transporter-mediated current was recorded at different external concentrations of Na+, Cl−, or GABA. The shifts in V rev for a tenfold change in the external Na+, Cl−, and GABA concentration were 84 ± 4, 30 ± 1, and 29 ± 1 mV, respectively. To determine the net charge translocated per Na+, Cl−, and GABA, we measured substrate fluxes under voltage clamp in cells expressing GAT1 or GAT3. Charge flux to substrate flux ratios were 0.7 ± 0.1 charge/Na+, 2.0 ± 0.2 charges/Cl−, and 2.1 ± 0.1 charges/GABA. Altogether, our results strongly suggest a 3 Na+:1 Cl−:1 GABA coupling stoichiometry for the GABA transporters. The revised stoichiometry has important implications for understanding the contribution of GATs to GABAergic signaling in health and disease. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Evidence for a Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters

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Publisher
Springer US
Copyright
Copyright © 2015 by Springer Science+Business Media New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-015-9797-6
Publisher site
See Article on Publisher Site

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