Characterizing Outward- to Inward-Facing Transition Pathway of Dopamine Transporter

Abstract

The dopamine transporter (DAT) belongs to the family of neurotransmitter sodium symporters (NSSs), which harness transmembrane electrochemical ionic gradients to transport neurotransmitters against their chemical gradients. Dysregulation of DAT is associated with serious neurological disorders, such as Parkinson's disease, depression, anxiety and epilepsy. As with all transporters, substrate translocation through DAT follows the alternating-access mechanism in which the protein swiches between outward-facing (OF) and inward-facing (IF) states. The details of these structural changes and their coupling to chemical events such as substrate and ion binding events remain elusive. In the present study, we charactrized large-scale transition from the OF state to IF state using all-atom molecular dynamics simulations of membrane-bound models of DAT. As all crystal structures of DAT are in the OF state, the initial phase of the study included modeling of a stable IF structure of DAT in the context of a membrane using the bacterial sodium-coupled leucine symporter (LeuT) as a template. Furthermore, equilibrium simulations performed in this phase revealed a novel sodium binding site located between TM3 and TM8 helices, which are elements involved in coupling of protein structural changes to substrate binding and translocation. Using the orientations of helics TM1e/TM8e and TM1i/TM8i as collective variables, and employing two dimensional bias-exchange umbrella sampling and string method with swarms of trajectories, we characerize a structural transition pathway between the OF and IF states of DAT. The results of this study provide a deeper understanding of the functional mechanism of DAT, wiht implications to all members of the NSS family.