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.