title: Delineating structure-function relationships in the dopamine transporter from natural and engineered Zn2+ binding sites.
abstract: The dopamine transporter is member of a large family of Na+/Cl- dependent neurotransmitter and amino acid transporters. Little is known about the molecular basis for substrate translocation in this class of transporters as well as their tertiary structure remains elusive. In this report, we provide the first crude insight into the structural organization of the human dopamine transporter (hDAT) based on the identification of an endogenous high affinity Zn2+ binding site followed by engineering of an artificial Zn2+ binding site. By binding to the endogenous site, Zn2+ acts as a potent non-competitive inhibitor of dopamine uptake mediated by the hDAT transiently expressed in COS-7 cells. Systematic mutagenesis of potential Zn2+ coordinating residues lead to the identification of three residues on the predicted extracellular face of the transporter, 193His in the second extracellular loop, 375His at the external end of the putative transmembrane segment (TM) 7, and 396Glu at the external end of TM 8, forming three coordinates in the endogenous Zn2+ binding site. The three residues are separate in the primary structure but their common participation in binding the small Zn(II) ion define their spatial proximity in the tertiary structure of the transporter. Finally, an artificial inhibitory Zn2+ binding site was engineered between TM 7 and TM 8. This binding site both verify the proximity between the two domains as wells as it supports an alpha-helical configuration at the top of TM 8 in the hDAT.
authors.affiliation: Department of Medical Physiology, The Panum Institute, University of Copenhagen, Denmark. gether@mfi.ku.dk
authors.forename: U
authors.initials: U
authors.lastname: Gether
authors.forename: L
authors.initials: L
authors.lastname: Norregaard
authors.forename: C J
authors.initials: CJ
authors.lastname: Loland
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003042:Cocaine / Q000031:analogs & derivatives* / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000032:analysis; D006639:Histidine / Q000032:analysis; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D018698:Glutamic Acid; D006639:Histidine; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D003042:Cocaine; D015032:Zinc; D004298:Dopamine