title: Dopamine Transporter Amino and Carboxyl Termini Synergistically Contribute to Substrate and Inhibitor Affinities.
abstract: Extracellular dopamine and serotonin concentrations are determined by the presynaptic dopamine (DAT) and serotonin (SERT) transporters, respectively. Numerous studies have investigated the DAT and SERT structural elements contributing to inhibitor and substrate binding. To date, crystallographic studies have focused on conserved transmembrane domains, where multiple substrate binding and translocation features are conserved. However, it is unknown what, if any, role the highly divergent intracellular N and C termini contribute to these processes. Here, we used chimeric proteins to test whether DAT and SERT N and C termini contribute to transporter substrate and inhibitor affinities. Replacing the DAT N terminus with that of SERT had no effect on DA transport Vmaxbut significantly decreased DAT substrate affinities for DA and amphetamine. Similar losses in uptake inhibition were observed for small DAT inhibitors, whereas substituting the DAT C terminus with that of SERT affected neither substrate nor inhibitor affinities. In contrast, the N-terminal substitution was completely tolerated by the larger DAT inhibitors, which exhibited no loss in apparent affinity. Remarkably, all affinity losses were rescued in DAT chimeras encoding both SERT N and C termini. The sensitivity to amino-terminal substitution was specific for DAT, because replacing the SERT N and/or C termini affected neither substrate nor inhibitor affinities. Taken together, these findings provide compelling experimental evidence that DAT N and C termini synergistically contribute to substrate and inhibitor affinities.
authors.affiliation: From the Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School, Worcester, Massachusetts 01604 and.
authors.forename: Carolyn G
authors.initials: CG
authors.lastname: Sweeney
authors.affiliation: From the Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School, Worcester, Massachusetts 01604 and.
authors.forename: Bradford P
authors.initials: BP
authors.lastname: Tremblay
authors.affiliation: the Institute for Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Stockner
authors.affiliation: the Institute for Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria.
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.affiliation: From the Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School, Worcester, Massachusetts 01604 and haley.melikian@umassmed.edu.
authors.forename: Haley E
authors.initials: HE
authors.lastname: Melikian
mesh_terms: D019943:Amino Acid Substitution; D001693:Biological Transport, Active; D002460:Cell Line; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020125:Mutation, Missense; D000072417:Protein Domains; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins
keywords: amphetamine; cocaine; dopamine transporter; kinetics; monoamine transporter; psychostimulants; reuptake; selective serotonin reuptake inhibitor (SSRI); serotonin transporter