Structural and Mechanistic Studies of Gaba Transporter Isoform 1

Abstract

The plasma membrane neurotransmitter transporter, GAT1, is responsible for the clearance of inhibitory neurotransmitter γ-aminobutyric acid (GABA) from the synaptic cleft. Targeting GABA reuptake by inhibiting GAT1, hence prolonging GABAergic signaling is one of the strategies to treat disorders arising due to imbalance in inhibitory neurotransmission like epilepsy and anxiety. A previous study from our lab used a dDAT-based chimera (dDATGAT) to recreate GAT1’s binding site, enabling structural analysis of GAT1 inhibitors. In this work, I have structurally and functionally characterized a functional GAT1 construct. We determined the cryo-EM structure of GAT1 from Rattus norvegicus at 3.1 Å resolution in a cytosol-facing conformation utilizing a strategy of transferring the epitope for a fragment-antigen binding (Fab) interaction site, from Drosophila dopamine transporter (dDAT) to GAT1, to assist structure determination through Fab binding to the GAT1 epitope construct. The structure captured a transport cycle intermediate step of GAT1 with substrate GABA, tightly bound chloride, and a partly displaced sodium from site 1. GABA interacts in the binding pocket in a pose similar to GAT1-specific inhibitors in the dDATGAT structure. The structure reveals GABA's specific interactions, the role of a π-helix in TM10, and a bean-shaped subsite architecture that differentiates GAT1 from other NSS members. Integration with an AlphaFold2 model of the outward-open state allowed us to propose a GABA transport mechanism involving alternating access and ion coupling. This study highlights important details of GABA recognition, transport activity, and inhibition.