Trimerization of dopamine transporter triggered by AIM-100 binding: Molecular mechanism and effect of mutations.

Recent work demonstrated the propensity of dopamine transporters (DATs) to form trimers or higher oligomers, 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 study, we determined three structural models, termed trimer-W238, -C306 and -Y303, for possible trimerization of DATs . To this aim, we used structural data resolved for DAT and its structural homologs that share the LeuT fold, advanced computational modeling and simulations, site-directed mutagenesis experiments and live-cell imaging assays. The models are in accord with the versatility of LeuT fold to stabilize dimeric or higher order constructs. Selected residues show a high propensity to occupy interfacial regions. Among them, 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 and experimentally confirmed to play important roles in enabling the correct folding and/or oligomerization of DATs in the presence of AIM-100. The study suggests the possibility of controlling the effective transport of dopamine by altering the oligomerization state of DAT upon small molecule binding, as a possible intervention strategy to modulate dopaminergic signaling.