Dopamine transporters (DATs) control neurotransmitter dopamine (DA) homeostasis by reuptake of excess DA, assisted by sodium and chloride ions. The recent resolution of DAT structure (dDAT) from Drosophila permits us for the first time to directly view the sequence of events involved in DA reuptake in human DAT (hDAT) using homology modeling and full-atomic microseconds accelerated simulations. Major observations are spontaneous closure of extracellular gates prompted by DA binding; stabilization of a holo-occluded intermediate; disruption of N82-N353 hydrogen bond and exposure to intracellular (IC) water triggered by Na2 dislocation; redistribution of a network of salt bridges at the IC surface in the inward-facing state; concerted tilting of IC-exposed helices to enable the release of Na(+) and Cl(-) ions; and DA release after protonation of D79. The observed time-resolved interactions confirm the conserved dynamics of LeuT-fold family, while providing insights into the mechanistic role of specific residues in hDAT.
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