A Conserved Salt Bridge between Transmembrane Segments 1 and 10 Constitutes an Extracellular Gate in the Dopamine Transporter

Neurotransmitter transporters play an important role in termination of synaptic transmission by mediating reuptake of neurotransmitter, but the molecular processes behind translocation are still unclear. The crystal structures of the bacterial homologue, LeuT, provided valuable insight into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the “thin gate” formed by an interaction between Arg-30 and Asp-404. In the human dopamine transporter (DAT), the corresponding residues are Arg-85 and Asp-476. Here, we present results supporting the existence of a similar interaction in DAT. The DAT R85D mutant has a complete loss of function, but the additional insertion of an arginine in opposite position (R85D/D476R), causing a charge reversal, results in a rescue of binding sites for the cocaine analogue [³H]CFT. Also, the coordination of Zn²⁺ between introduced histidines (R85H/D476H) caused a ∼2.5-fold increase in [³H]CFT binding (B_max). Importantly, Zn²⁺ also inhibited [³H]dopamine transport in R85H/D476H, suggesting that a dynamic interaction is required for the transport process. Furthermore, cysteine-reactive chemistry shows that mutation of the gating residues causes a higher proportion of transporters to reside in the outward facing conformation. Finally, we show that charge reversal of the corresponding residues (R104E/E493R) in the serotonin transporter also rescues [³H](S)-citalopram binding, suggesting a conserved feature. Taken together, these data suggest that the extracellular thin gate is present in monoamine transporters and that a dynamic interaction is required for substrate transport.