Dopamine transporter (DAT) controls neurotransmitter dopamine homeostasis by pumping excess extracellular (EC) dopamine into presynaptic neurons to prevent neurotoxicity. The process is assisted by co-transport of two sodium and one chloride ions. Malfunction of DAT has been implicated in many diseases, such as Parkinson's disease and attention deficit hyperactivity disorder. Significant advances have been made in the characterization of the structure and dynamics of a prokaryotic orthologue, LeuT, although the molecular mechanism of dopamine translocation through human DAT (hDAT) remains elusive. Recently, the first eukaryotic DAT structure was resolved from Drosophila melanogaster, in the outward-facing open (OFo) state. Using homology modeling and unbiased molecular dynamics simulations, we elucidated the sequence of molecular events involved in dopamine translocation by hDAT, including the uptake from the EC medium, closure of the EC gates upon dopamine binding, passage over a holo-occluded intermediate temporarily stabilized via concerted changes in the relative orientations of the TM helices, subsequent opening of the intracellular vestibule, release of co-transported ions, and succeeding discharge of dopamine upon weakening the electrostatic attractions by aspartates near the binding-site. The transport events revealed by these hDAT simulations show striking consistency with those disclosed in recent investigation of LeuT transport cycle (1, 2). The study sheds light for the first time at the mechanism of DAT functioning at the atomic level and provides insights into methods for modulating its interactions with drugs and other substrate proteins.1. Cheng, M.H. and I. Bahar. 2014. Complete mapping of substrate translocation highlights the role of LeuT N-terminal segment in regulating transport cycle, PLoS Comput Biol. in press.2. Cheng, M.H. and I. Bahar. 2013. Coupled global and local changes direct substrate translocation by neurotransmitter-sodium symporter ortholog LeuT, Biophys J. 105:630-9.