c-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and its levels in the synaptic space are controlled by the GABA transporter isoforms (GATs). GATs are structurally related to biogenic amine transporters but display interactions with distinct inhibitors used as anti-epileptics. In this study, we engineer the binding pocket of Drosophila melanogaster dopamine transporter to resemble GAT1 and determine high-resolution X-ray structures of the modified transporter in the substrate-free state and in complex with GAT1 inhibitors NO711 and SKF89976a that are analogs of tiagabine, a medication prescribed for the treatment of partial seizures. We observe that the primary binding site undergoes substantial shifts in subsite architecture in the modified transporter to accommodate the two GAT1 inhibitors. We also observe that SKF89976a additionally interacts at an allosteric site in the extracellular vestibule, yielding an occluded conformation. Interchanging SKF89976a interacting residue in the extracellular loop 4 between GAT1 and dDAT suggests a role for this motif in the selective control of neurotransmitter uptake. Our findings, therefore, provide vital insights into the organizational principles dictating GAT1 activity and inhibition.

The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.