Recent work demonstrated the propensity of dopamine transporters (DATs) to form trimers¹, enhanced upon binding a furopyrimidine, AIM-100. AIM-100 binding promotes DAT endocytosis and thereby moderates dopaminergic transmission. Despite the neurobiological significance of these events, the molecular mechanisms that underlie the stabilization of DAT trimer and the key interactions that modulate the trimerization of DAT, and not serotonin transporter SERT, remain unclear. In the present study², we determined three structural models, termed trimer-W238, -C306 and -Y303, for possible trimerization of DATs using structural data resolved for DAT and its paralogs and structural homologs that share the LeuT fold in advanced computational modeling and simulations. The models are in accord with the versatility of LeuT fold to stabilize dimeric or higher order constructs with selected residues showing a high propensity to occupy interfacial regions. D231-W238 in the extracellular loop EL2, including the intersubunit salt-bridge forming pair D231/D232-R237 (not present in SERT) (in trimer-W238), the loop EL3 (trimers-C306 and -Y303), and W497 on the intracellularly exposed IL5 loop (trimer-C306) and its spatial neighbors (e.g. K525) near the C-terminus are computationally predicted. Site-directed mutagenesis was performed to examine the effect of these elements/sites on DAT oligomerization and endocytosis (enhanced by AIM-100), and the experimental data were further interpreted using a novel machine learning classifier Rhapsody for assessing the impact of single amino acid variants (SAVs) (see Figure 1). The study suggests the possibility of controlling the effective dopamine transport by altering the oligomerization state of DAT upon small molecule binding, as a possible intervention strategy to modulate dopaminergic signaling.

Figure 1: MD simulations reveal the probable binding poses of AIM-100 onto hDAT trimer in the IF (A) and OF (B) state. In silico saturation mutagenesis results and comparison with experimental data. (C) The membrane expression of 13 single amino acid variants (SAVs) has been measured in vitro. On the abscissa, these variants are sorted based on the change in their expression levels (left, red and light-red shade: no or partial expression; right, blue shade: normal/wild-type expression). On the y-axis, the functional impact as predicted by Rhapsody (red circles), EVmutation (blue inverted triangles) and PolyPhen-2 (green squares) is shown. The EVmutation epistatic score has been normalized so that the optimal cutoff between predicted neutral and deleterious effects matches that for Rhapsody and PolyPhen-2 (y = 0.5). Correct predictions lie within the two shaded areas, red (true positives) and blue (true negatives). (D) DAT monomer color-coded by the average pathogenicity shown in C (red/blue: high/low pathogenicity probability). The 13 mutations sites are labelled and shown in licorice.