Luminal autoinhibitory loop and binding-site protonation regulate VAChT substrate release

The vesicular acetylcholine transporter (VAChT) packages acetylcholine (ACh) into synaptic vesicles to drive cholinergic neurotransmission, which underpins cognition, movement, and neuronal survival. Its dysfunction is implicated in disorders such as Alzheimer’s disease and myasthenia gravis. Here, we present cryo-EM structures of human VAChT in apo forms at both neutral and acidic pH, as well as in complexes with its substrate and inhibitor vesamicol (VSM). Our structures are captured in a lumen-facing, occluded conformation, wherein a unique luminal loop seals the transport pathway, a feature distinct from other SLC18 family members. Combined with molecular dynamics (MD) simulations and mutagenesis analyses, we identified three key protonation sites critical for transport and demonstrated the luminal loop acts as an intrinsic gate, controlling substrate release through autoinhibitory mechanism. Our findings elucidate the transport and inhibition mechanisms of VAChT and provide a structural framework for developing therapeutics against cholinergic dysfunction.