Abstract
γ-Aminobutyric acid (GABA) and glycine mediate fast synaptic inhibition by restraining neuronal excitability and stabilizing neural circuits. The vesicular inhibitory amino acid transporter (VIAAT/VGAT) loads both GABA and glycine into synaptic vesicles, but its substrate-recognition and transport mechanisms remain unclear. Here we report cryo-EM structures of human VIAAT in apo luminal-open and GABA-bound occluded states. VIAAT adopts an 11 transmembrane helix LeuT-like fold, distinct from other vesicular neurotransmitter transporters. In the central pocket, an aromatic cage comprising Phe133, Tyr221, Phe315 and Phe322 mediates recognition of GABA. Structural comparison reveals that Phe315 acts as a luminal gate, rotating away from the central pocket to open the luminal pathway for substrate release. Complemented by functional neuronal rescue experiments and a cytosolic-facing model, our findings elucidate the mechanism for VIAAT-mediated GABA transport and provide a framework for understanding how epilepsy-associated VIAAT variants impair inhibitory neurotransmission.