BPS2026 – SSME resolves three GABA-induced electrogenic events and GABA-coupling stoichimetry for Na+ and Cl- in hGAT-1

The γ-aminobutyric acid transporter 1 (hGAT-1) is the principal neuronal GABA reuptake system and a relevant therapeutic target. However, its transport stoichiometry and the role of Cl - remain debated. Using solid supported membrane electrophysiology (SSME), we resolved three distinct electrogenic events during GABA transport by hGAT-1. The fast component (E1) reflects hGAT-1 occlusion in the GABA-Na + -Cl - -bound carrier, the intermediate component (E2) corresponds to a GABA-induced Na + leak conductance, and the slow component (E3) represents Na + -coupled GABA transport. Stoichiometry assays confirmed a 2:1 Na + : GABA coupling ratio under moderate Na + gradients, while high Na + gradients disrupted this ratio due to activation of the E2 leak. Cl - was found to be essential for transporter function but not stoichiometrically coupled to GABA flux, likely serving only to stabilize Na + binding. Together, these results provide a mechanistic framework for the hGAT-1 transport cycle, resolving long-standing uncertainties on ion coupling and highlighting an uncoupled Na + conductive state as a source of variability in functional studies.