Serotonin, dopamine, and norepinephrine transporter assembly is selectively disrupted by a NET truncation isoform as revealed through near-million-atom simulations

Abstract Background Monoamine transporters (MATs), including the dopamine, norepinephrine, and serotonin transporters (DAT, NET, SERT), are essential regulators of synaptic neurotransmission that rely on complex oligomeric bio-assemblies for proper function. The regulatory influence of naturally occurring, alternatively spliced truncated isoforms on bio-assembly dynamics remains profoundly underexplored. Methods To decode these interactions at the atomic level, we deployed a multiscale computational framework. We integrated genomics-guided multimer predictions with massive-scale, near-million-atom molecular dynamics (MD) simulations within explicit lipid bilayers. The thermodynamic stability of these heteromeric complexes was quantified using membrane-adapted MM/PBSA calculations, which were subsequently correlated with dynamics-aware evolutionary profiling to map co-evolutionary interaction hotspots. Results Our analyses reveal that the NET-derived truncated isoform A0A804HLI4 acts as a pan-family potential inhibitor. It forms stable, exergonic heterodimers with canonical NET and DAT, thermodynamically outcompeting native homodimerization. In full tetrameric simulations, the integration of a single isoform precipitates a macro-structural disruptions of the SERT complex. The variant anchors at non-native interfaces, locking the assembly into an asymmetric, non-native state. Residue-level thermodynamic decomposition and evolutionary mapping isolated conserved structural elements (most notably Gln236) that dictate this high-affinity cross-reactivity across the SLC6 family. Conclusions Truncated MAT isoforms execute a dynamic mechanism of inhibitory effects and may systematically downregulate synaptic reuptake capacity by sequestering functional monomers. These findings establish a thermodynamically grounded, high-resolution model of isoform-induced bio-assembly disruptions. Crucially, they expose these non-canonical, isoform-driven interfaces as conserved and highly druggable targets, offering a distinct pharmacological paradigm for precision interventions in neuropsychiatric and neurodegenerative pathologies.