SLC18A3 encodes the vesicular acetylcholine transporter (VAChT), an electrogenic antiporter that exchanges acetylcholine or choline with two intravesicular protons across synaptic vesicle membranes 1. VAChT uses the proton gradient established by V-type ATPase to concentrate neurotransmitters for release via exocytosis 1. The gene shares a common locus with CHAT, encoding choline acetyltransferase, representing a unique neuronal transcriptional unit controlling cholinergic neurotransmission 1. SLC18A3 regulates quantal size at neuromuscular junctions, affecting motor neuron differentiation and hippocampal synaptic transmission underlying spatial memory 1. Pathogenic variants cause congenital myasthenic syndrome type 21 (CMS-21), a presynaptic disorder impairing acetylcholine synthesis and recycling 2. CMS-21 presents with variable severity depending on variant type; nonsense mutations produce more severe phenotypes with impaired motor and cognitive development than missense variants 3. Therapy with cholinesterase inhibitors shows partial efficacy, while 3,4-diaminopyridine demonstrates limited benefit 3. Large genomic deletions encompassing SLC18A3 and CHAT contribute to developmental delay, hypotonia, and dysphagia, suggesting pleiotropic effects beyond neuromuscular dysfunction 4. Reduced SLC18A3 expression impairs cognitive function by disrupting cholinergic signaling pathways 5.