title: An N-terminal threonine mutation produces an efflux-favorable, sodium-primed conformation of the human dopamine transporter.
abstract: The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr62 revealed T62D-human (h)DAT partitions in a predominately efflux-preferring conformation. Compared with wild-type (WT), T62D-hDAT exhibits reduced [(3)H]DA uptake and enhanced baseline DA efflux, whereas T62A-hDAT and WT-hDAT function in an influx-preferring conformation. We now interrogate the basis of the mutants' altered function with respect to membrane conductance and Na(+) sensitivity. The hDAT constructs were expressed in Xenopus oocytes to investigate if heightened membrane potential would explain the efflux characteristics of T62D-hDAT. In the absence of substrate, all constructs displayed identical resting membrane potentials. Substrate-induced inward currents were present in oocytes expressing WT- and T62A-hDAT but not T62D-hDAT, suggesting equal bidirectional ion flow through T62D-hDAT. Utilization of the fluorescent DAT substrate ASP(+) [4-(4-(dimethylamino)styryl)-N-methylpyridinium] revealed that T62D-hDAT accumulates substrate in human embryonic kidney (HEK)-293 cells when the substrate is not subject to efflux. Extracellular sodium (Na(+) e) replacement was used to evaluate sodium gradient requirements for DAT transport functions. The EC50 for Na(+) e stimulation of [(3)H]DA uptake was identical in all constructs expressed in HEK-293 cells. As expected, decreasing [Na(+)]e stimulated [(3)H]DA efflux in WT- and T62A-hDAT cells. Conversely, the elevated [(3)H]DA efflux in T62D-hDAT cells was independent of Na(+) e and commensurate with [(3)H]DA efflux attained in WT-hDAT cells, either by removal of Na(+) e or by application of amphetamine. We conclude that T62D-hDAT represents an efflux-willing, Na(+)-primed orientation-possibly representing an experimental model of the conformational impact of amphetamine exposure to hDAT.
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (R.F., B.G., K.D.L., S.L.S., A.B., M.E.G.); and Departments of Psychiatry (Y.C.) and Physiology and Biophysics (L.J.D.), Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Rheaclare
authors.initials: R
authors.lastname: Fraser
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (R.F., B.G., K.D.L., S.L.S., A.B., M.E.G.); and Departments of Psychiatry (Y.C.) and Physiology and Biophysics (L.J.D.), Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Yongyue
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (R.F., B.G., K.D.L., S.L.S., A.B., M.E.G.); and Departments of Psychiatry (Y.C.) and Physiology and Biophysics (L.J.D.), Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Bipasha
authors.initials: B
authors.lastname: Guptaroy
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (R.F., B.G., K.D.L., S.L.S., A.B., M.E.G.); and Departments of Psychiatry (Y.C.) and Physiology and Biophysics (L.J.D.), Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Kathryn D
authors.initials: KD
authors.lastname: Luderman
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (R.F., B.G., K.D.L., S.L.S., A.B., M.E.G.); and Departments of Psychiatry (Y.C.) and Physiology and Biophysics (L.J.D.), Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Stokes
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (R.F., B.G., K.D.L., S.L.S., A.B., M.E.G.); and Departments of Psychiatry (Y.C.) and Physiology and Biophysics (L.J.D.), Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Asim
authors.initials: A
authors.lastname: Beg
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (R.F., B.G., K.D.L., S.L.S., A.B., M.E.G.); and Departments of Psychiatry (Y.C.) and Physiology and Biophysics (L.J.D.), Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Louis J
authors.initials: LJ
authors.lastname: DeFelice
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (R.F., B.G., K.D.L., S.L.S., A.B., M.E.G.); and Departments of Psychiatry (Y.C.) and Physiology and Biophysics (L.J.D.), Virginia Commonwealth University, Richmond, Virginia pgnegy@umich.edu.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Gnegy
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002460:Cell Line; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D009865:Oocytes / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D013912:Threonine / Q000235:genetics* / Q000378:metabolism; D014981:Xenopus
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; C493214:SLC6A3 protein, human; D013912:Threonine; D012964:Sodium
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