Splice Isoform Induced Selective Inhibition of Vesicular Monoamine Transporter Assembly Revealed by Multi-level Combinatorial Analysis

Synaptic vesicle loading of monoamines represents a rate-limiting step in neurotransmission, and its regulation directly shapes dopaminergic, serotonergic, and noradrenergic signaling. Vesicular Monoamine Transporters (VMATs), key determinants of quantal content and synaptic tone, have traditionally been thought to function primarily through their full-length canonical isoforms. However, alternative splicing generates isoforms that remain largely uncharacterized in membrane transporter biology, despite growing proteogenomic evidence supporting their translation. Here, we reveal a potential mechanism by which a truncated splice isoforms of VMAT1 interferes with canonical oligomer formation. Using a combination of structural docking, atomistic lipid bilayer molecular dynamics, and Poisson-Boltzmann Surface-Area (MMPBSA) binding energy decomposition, we characterize the interactions and co-evolution between the truncated isoform and the canonical protein. While the isoform retains partial interface compatibility, it exhibits higher binding affinity for the canonical VMAT than the canonical homodimer itself. Mechanistically, this molecular latch shifts the rules of engagement from a contest of size (surface area) to one of intensity (charge), effectively resolving the David-versus-Goliath conflict: the system energetically favors the heterodimer, ensuring that the smaller isoform consistently dominates over the full-length protein. Functionally, this positions the truncated isoform as a negative regulator of VMAT oligomerization. These findings redefine VMAT assembly as a splicing-sensitive checkpoint and provide a novel mechanistic framework for understanding synaptic dysfunction in neuropsychiatric and neurodegenerative disorders.