Alternative Splicing Guided Stoichiometric Competition Model of Vesicular Polyamine Transporter Reveals Modulatory Drug Targets

Abstract Alternative splicing represents a potent mechanism for the post-transcriptional regulation of solute carrier function, yet the structural biophysics governing the Vesicular Polyamine Transporter (VPAT) interactome remain elusive. Here, we present a comprehensive thermodynamic and topological analysis of canonical VPAT and its splice variants (A0A3B3ITL9, A0A3B3IU67, A0A3B3IT67, and A0A3B3ISL3) utilizing explicit membrane Molecular Dynamics (MD) simulations coupled with MM/PBSA free energy decomposition. We identify isoform A0A3B3ITL9 (10-TM) as a specific, equipotent competitive inhibitor of canonical VPAT homodimerization. While the canonical homodimer (ΔG bind=-29.29 kcal/mol) is stabilized by a conserved C-terminal lock involving Trp333 and Tyr272, the truncated ITL9 isoform bypasses this interface. Instead, ITL9 exploits a conformational switch to recruit the VPAT N-terminus via a high-affinity electrostatic anchor at Arg20, resulting in a heterodimeric complex of identical stability (ΔG bind=-30.10 kcal/mol). Conversely, we demonstrate that other isoforms are functionally segregated from this competitive pool: A0A3B3IU67 (11-TM) is destabilized by topological mismatch within the lipid bilayer, while A0A3B3IT67 (8-TM) functions as an inert allocrite driven by obligate homodimerization. These findings define the VPAT regulatory landscape as a stoichiometric competition model, highlighting the N-terminal Arg pocket as a isoform-specific equipotent therapeutic target for modulating polyamine transport.