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

Plasma membrane gamma-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 +/- A 4, 30 +/- A 1, and 29 +/- A 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 +/- A 0.1 charge/Na+, 2.0 +/- A 0.2 charges/Cl-, and 2.1 +/- A 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.