Substrate and inhibitor binding of human GABA transporter 3.

GABA (g-aminobutyric acid) transporter 3 (GAT3) is primarily found in glial cells and is essential for regulating GABA homeostasis in the central nervous system by mediating GABA uptake. Consequently, GAT3 has emerged as a significant therapeutic target for the treatment of epilepsy. In this study, we present the cryoelectron microscopy (cryo-EM) structures of GAT3 bound to its substrate GABA, the selective inhibitor SNAP-5114, and in the substrate-free state. GAT3 binds to GABA in an inward-facing conformation, while SNAP-5114 occupies the GABA-binding pocket and is stabilized by extensive interactions with surrounding residues. Functional studies reveal that E66 plays a pivotal role in determining the substrate-binding mode and specificity of SNAP-5114 binding. Taken together, our study clarifies the GABA binding mechanism of GAT3 and reveals the molecular basis for the specific inhibition of SNAP-5114, offering valuable insights for developing GAT3 subtypes selective inhibitors, which hold potential as a treatment for epilepsy.