Transition Mechanism of the Human Dopamine Transporter via Molecular Dynamics Simulations

Dopamine reuptake by the human dopamine transporter (hDAT) is driven by Na⁺/Cl^--coupled alternating access, yet the detailed transition mechanism between outward-facing, occluded, and inward-facing states has remained elusive. Leveraging recently resolved hDAT structures and extensive molecular dynamics simulations, we construct a Markov State Model describing the dopamine transport pathway and its associated conformational transitions. This analysis resolves three novel key intermediate states: Macrostate 5, an extracellular (EC) gate-closure intermediate; Macrostate 4, the kinetic bottleneck corresponding to the ion-release and dopamine-reorientation state; and Macrostate 1, the earliest outward-facing reset state that reopens the EC gate. Together, these intermediates define a sequential transport mechanism: Na2 release → Transmembrane 1a helix (TM1a) tilt → intracellular gate opening → Na1 release → dopamine rotation → water influx → dopamine release → Cl release → outward-facing reset. The structural logic of these intermediates rationalizes longstanding mutational phenotypes and reveals new targets for probing gating kinetics and therapeutic modulation. Together, these findings provide an atomistic framework that clarifies how ion coupling, gating rearrangements, and substrate dynamics coordinate dopamine transport.