Abstract
Recently, we have described a distance constraint in the unknown tertiary structure of the human dopamine transporter (hDAT) by identification of two histidines, His(193) in the second extracellular loop and His(375) at the top of transmembrane (TM) 7, that form two coordinates in an endogenous, high affinity Zn(2+)-binding site. To achieve further insight into the tertiary organization of hDAT, we set out to identify additional residues involved in Zn(2+) binding and subsequently to engineer artificial Zn(2+)-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 IC(50) value for Zn(2+) inhibition of [(3)H]dopamine uptake from 1.1 to 530 microM and eliminated Zn(2+)-induced potentiation of [(3)H]WIN 35,428 binding. These data suggest that Glu(396) is involved in Zn(2+) binding to hDAT. Importantly, Zn(2+) sensitivity was preserved following substitution of Glu(396) with histidine, indicating that the effect of mutating Glu(396) is not an indirect effect because of the removal of a negatively charged residue. The common participation of Glu(396), His(193), and His(375) in binding the small Zn(2+) 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 Zn(2+)-binding site between His(375) 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.
Highlights
We have described a distance constraint in the unknown tertiary structure of the human dopamine transporter 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
His193 is nonconserved between human dopamine transporter (hDAT) and human NET (hNET), whereas His375 is conserved between the two transporters
Full Zn2ϩ susceptibility can be conveyed to the hNET by mutational transfer of only His193 [22]; Zn2ϩ-coordinating residues in addition to His193 and His375 can be expected to be conserved between hDAT and hNET
Summary
Site-directed Mutagenesis—The cDNA encoding the human dopamine transporter (hDAT) and norepinephrine transporter (hNET) were kindly provided by Dr Marc G. Transfected COS-7 cells were plated in either 24-well dishes (105 cells/well) or 12-well dishes (2–3 ϫ 105 cells/well) to achieve an uptake level of 5–10% of total added [3H]dopamine. Cells were seeded in either 24-well dishes (105 cells/well) or 12-well dishes (2–3 ϫ 105 cells/well) to achieve a binding level of 5–10% of total added [3H]WIN 35,428. Competition binding assays were performed in a final volume of 500 l of uptake buffer containing 2– 4 nM [3H]WIN 35,428 and indicated concentrations of compound to be tested. Binding was terminated after 2 h at 4 °C by washing the cells twice in 500 l of uptake buffer prior to lysis in 500 l 1% SDS for 1 h at 37 °C. Calculations —Uptake and binding data were analyzed by nonlinear regression analysis using Prism 2.0 from GraphPad Software (San Diego, CA)
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