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

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Previously, we have identified three Zn2+ binding residues in an endogenous Zn2+ binding site in the human dopamine transporter (hDAT): 193His in extracellular loop 2 (ECL 2), 375His at the external end of transmembrane segment (TM) 7, and 396Glu at the external end of TM 8. Here we have generated 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 structural constraints for binding of the zinc(II) ion. Initially, we found that the Zn2+-coordinating 193His in ECL 2 could be substituted with a histidine inserted at the i - 4 position relative to 375His in TM 7. In this mutant (H193K/M371H), Zn2+ potently inhibited [3H]dopamine uptake with an IC50 value of 7 μM as compared to a value of 300 μM for the control (H193K). These data are consistent with the presence of an α-helical configuration of TM 7. This inference was further corroborated by the observation that no increase in the apparent Zn2+ affinity was observed following introduction of histidines at the i - 2, i - 3, and i - 5 positions. In contrast, introduction of histidines at positions i + 2, i + 3, and i + 4 all resulted in potent inhibition of [3H]dopamine uptake by Zn2+ (IC50 = 3-32 μM). These observations are inconsistent with continuation of the helix beyond 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 hDAT and illustrate how engineering of Zn2+ binding sites can be a useful approach for probing both secondary and tertiary structure relationships in membrane proteins of unknown structure.

Original languageEnglish
Issue number51
Pages (from-to)15836-15846
Number of pages11
Publication statusPublished - 26 Dec 2000

ID: 237414139