Cooperative Ionic Coordination Governs Dopamine Transporter Dynamics and Substrate Translocation: Mechanistic...

The dopamine transporter (DAT) plays a vital role in maintaining dopamine (DA) homeostasis by mediating the reuptake of DA from the synaptic cleft into presynaptic neurons, a process tightly coupled to the cotransport of two sodium ions (Na⁺) and one chloride ion (Cl^-). Although structural studies have revealed key conformational states of DAT, the precise mechanistic contributions of these ions to transporter dynamics and substrate translocation remain incompletely understood. Here, we employed extensive molecular dynamics simulations and free energy calculations to systematically investigate the cooperative roles of Na⁺ and Cl^- ions in human DAT function. Our results demonstrate that Cl^- stabilizes extracellular gate closure through coordination with residues in TM2, TM6a, and TM7, while the two Na⁺ ions reinforce intracellular gate closure via interactions with TM1a and adjacent helices. Sequential binding analysis revealed an energetically favorable and functionally coupled order of binding: Na⁺ binds first, followed by DA and then Cl^-. Mapping of the free energy landscape for DA translocation uncovered four key intermediate states, each stabilized by distinct salt bridges, hydrogen bonds, and π-π stacking interactions that shape the energy barriers along the transport pathway. These findings provide a comprehensive molecular framework for understanding ion-dependent conformational transitions in DAT and offer mechanistic insights for the rational design of therapeutics targeting DAT in neuropsychiatric disorders.