Defining proximity relationships in the tertiary structure of the dopaminetransporter - Identification of a conserved glutamic acid as a third coordinate in the endogenous Zn2+-binding site

Recently, we have described a distance constraint in the unknown tertiary s tructure of the human dopamine transporter (hDAT) by identification of two histidines, His(193) in the second extracellular loop and His(375) at the t op of transmembrane (TM) 7, that form two coordinates in an endogenous, hig h affinity Zn2+-binding site. To achieve further insight into the tertiary organization of hDAT, we set out to identify additional residues involved i n 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 Glu(396) (E396Q) at the top of TM 8 increased the IC50 value for Zn2+ inhib ition of [H-3]dopamine uptake from 1.1 to 530 mu M and eliminated Zn2+ indu ced potentiation of [H-3]WIN 35,428 binding. These data suggest that Glu(39 6) is involved in Zn2+ binding to hDAT, Importantly, Zn2+ sensitivity was p reserved following substitution of Glu(396) with histidine, indicating that the effect of mutating Glu(396) is not an indirect effect because of the r emoval of a negatively charged residue. The common participation of Glu(396 ), His(193), and His(375) in binding the small Zn2+ ion implies their proxi mity in the unknown tertiary structure of hDAT, The close association betwe en TM. 7 and 8 was further established by engineering of a Zn2+-binding sit e between His(375) and a cysteine inserted in position 400 in TM 8, Summari zed, our data define an important set of proximity relationships in hDAT th at should prove an important template for further exploring the molecular a rchitecture of Na+/Cl--dependent neurotransmitter transporters.