Delineating structure-function relationships in the dopamine transporter from natural and engineered Zn2+ binding sites

The dopamine transporter is member of a large family of Na+/Cl- dependent n eurotransmitter and amino acid transporters, Little is known about the mole cular basis for substrate translocation in this class of transporters as we ll as their tertiary structure remains elusive. In this report, we provide the first crude insight into the structural organization of the human dopam ine transporter (hDAT) based on the identification of an endogenous high af finity Zn2+ binding site followed by engineering of an artificial Zn2+ bind ing site. By binding to the endogenous site, Zn2+ acts as a potent non-comp etitive inhibitor of dopamine uptake mediated by the hDAT transiently expre ssed in COS-7 cells. Systematic mutagenesis of potential Zn2+ coordinating residues lead to the identification of three residues on the predicted extr acellular face of the transporter, (193)His in the second extracellular loo p, (375)His at the external end of the putative transmembrane segment (TM) 7, and (396)Glu at the external end of TM 8, forming three coordinates in t he endogenous Zn2+ binding site. The three residues are separate in the pri mary structure but their common participation in binding the small Zn(II) i on define their spatial proximity in the tertiary structure of the transpor ter. Finally, an artificial inhibitory Zn2+ binding site was engineered bet ween TM 7 and TM 8. This binding site both verify the proximity between the two domains as wells as it supports an a-helical configuration at the top of TM 8 in the hDAT. (C) 2001 Elsevier Science Inc. All rights reserved.