Defining Proximity Relationships in the Tertiary Structure of the Dopamine Transporter

Recently, we have described a distance constraint in the unknown tertiary structure of the human dopamine transporter (hDAT) by identification of two histidines, His193 in the second extracellular loop and His375 at the top of transmembrane (TM) 7, that form two coordinates in an endogenous, high affinity Zn2+-binding site. To achieve further insight into the tertiary organization of hDAT, we set out to identify additional residues involved in Zn2+ binding and subsequently to engineer artificial Zn2+-binding sites. Ten aspartic acids and glutamic acids, predicted to be on the extracellular side, were mutated to asparagine and glutamine, respectively. Mutation of Glu396 (E396Q) at the top of TM 8 increased the IC50 value for Zn2+ inhibition of [3H]dopamine uptake from 1.1 to 530 μm and eliminated Zn2+-induced potentiation of [3H]WIN 35,428 binding. These data suggest that Glu396 is involved in Zn2+ binding to hDAT. Importantly, Zn2+ sensitivity was preserved following substitution of Glu396 with histidine, indicating that the effect of mutating Glu396 is not an indirect effect because of the removal of a negatively charged residue. The common participation of Glu396, His193, and His375 in binding the small Zn2+ ion implies their proximity in the unknown tertiary structure of hDAT. The close association between TM 7 and 8 was further established by engineering of a Zn2+-binding site between His375 and a cysteine inserted in position 400 in TM 8. Summarized, our data define an important set of proximity relationships in hDAT that should prove an important template for further exploring the molecular architecture of Na+/Cl−-dependent neurotransmitter transporters.

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