Structural basis for the trafficking and function mechanism of the vesicular inhibitory amino acid transporter VIAAT

The vesicular inhibitory amino acid transporter VIAAT packages γ-aminobutyric acid (GABA) and glycine into synaptic vesicles for fast inhibitory neurotransmission, yet its architecture, substrate recognition and association with trafficking-associated proteins remain poorly understood. Here we report cryo-EM structures of human VIAAT and its Caenorhabditis elegans orthologue UNC-47 at resolutions of 2.4-3.4 Å, each alone and in complex with their respective partners, LAMP5 and UNC-46, respectively. Both transporters adopt an eleven-transmembrane-helix architecture with a LeuT-like fold, but their partners engage them differently. LAMP5 binds hVIAAT primarily through its transmembrane helix, whereas the luminal domain of UNC-46 forms extensive contacts with UNC-47. A GABA-bound hVIAAT–LAMP5 structure delineates the substrate-binding pocket and local rearrangements accompanying occlusion, supported by mutational thermal-shift measurements. Comparison with a predicted cytosol-open model suggests an alternating access mechanism, while structure-based pKa calculations implicate Glu213 as the putative proton-coupling residue. Together, these findings establish a structural framework for inhibitory neurotransmitter transport and reveal evolutionary divergence in VIAAT-associated trafficking assemblies.