Resolving three GABA induced electrogenic events and GABA ‐coupling stoichiometry for Na ⁺ and Cl ⁻ in hGAT ‐1

GAT‐1 is a neurotransmitter:sodium symporter responsible for the reuptake of GABA from the synaptic cleft. Because of its involvement in various pathological conditions, it is an important target for drug development; however, its mechanism of action remains under debate. Using high‐resolution solid‐supported membrane‐based electrophysiology, we identified three sequential electrogenic events (E1, E2, and E3) in human GAT‐1‐mediated GABA transport. E1 reflects the transition to an occluded intermediate, upon GABA binding to the Na + bound carrier; E2 was attributed to an uncoupled Na + conductance triggered by GABA binding, while E3 represents Na + ‐coupled GABA transport. This triphasic model provides a robust electrophysiological framework for dissecting individual molecular transitions in the GAT‐1 transport cycle. We also established a 2 : 1 stoichiometry for Na + : GABA cotransport under standard conditions. However, this stoichiometric relationship breaks down under conditions of steep transmembrane Na + gradients, which promote an uncoupled Na + leak current. Our data further suggests that Cl − translocation is not coupled to GABA transport. These findings refine the current understanding of the GAT‐1 transport mechanism, providing a foundation to support the development of novel therapeutics.