Structural probing of a microdomain in the dopamine transporter by engineering of artificial Zn2+ binding sites

Previously, we have identified three Zn2+ binding residues in an endogenous Zn2+ binding site in the human dopamine transporter (hDAT): (193)His in ex tracellular loop 2 (ECL 2), (375)His at the external end of transmembrane s egment (TM) 7, and (396)Glu at the external end of TM 8. Here we have gener ated a series of artificial Zn2+ binding sites in a domain situated around the external ends of TMs 7 and 8 by taking advantage of the well-defined st ructural constraints for binding of the zinc(II) ion. Initially, we found t hat the Zn2+-coordinating (193)His in ECL 2 could be substituted with a his tidine inserted at the i - 4 position relative to (375)His in TM 7. In this mutant (H193K/M371H), Zn2+ potently inhibited [H-3]dopamine uptake with an IC50 value of 7 muM as compared to a value of 300 muM for the control (H19 3K). These data are consistent with the presence of an alpha -helical confi guration of TM 7. This inference was further corroborated by the observatio n that no increase in the apparent Zn2+ affinity was observed following int roduction of histidines at the i - 2, i - 3, and i - 5 positions. In contra st, introduction of histidines at positions i + 2, i + 3, and i + 4 all res ulted in potent inhibition of [H-3]dopamine uptake by Zn2+ (IC50 = 3-32 muM ). These observations are inconsistent with continuation of the helix beyon d position 375 and indicate an approximate boundary between the end of the helix and the succeeding loop. In summary, the data presented here provide new insight into the structure of a functionally important domain in the hD AT and illustrate how engineering of Zn2+ binding sites can be a useful app roach for probing both secondary and tertiary structure relationships in me mbrane proteins of unknown structure.