title: Zinc regulates the dopamine transporter in a membrane potential and chloride dependent manner.
abstract: The dopamine transporter (DAT), a membrane protein specifically expressed by dopaminergic neurons and mediating the action of psychostimulants and dopaminergic neurotoxins, is regulated by Zn(2+) which directly interacts with the protein. Herein, we report a host-cell-specific direction of the Zn(2+) effect on wild type DAT. Whereas low mumolar Zn(2+) decreased dopamine uptake by DAT expressing HEK293 cells, it stimulated uptake by DAT expressing SK-N-MC cells. Inhibition or stimulation was lost in a DAT construct without the binding site for Zn(2+). Also reverse transport was differentially affected by Zn(2+), dependent on whether the DAT was expressed in HEK293 or SK-N-MC cells. Pre-treatment of DAT expressing cells with phorbol-12-myristate-13-acetate, an activator of protein kinase C, attenuated the inhibitory effect of Zn(2+) on uptake in HEK293 cells and increased the stimulatory effect in SK-N-MC cells. Patch-clamp experiments under non-voltage-clamped conditions revealed a significantly higher membrane potential of HEK293 than SK-N-MC cells and a reduced membrane potential after phorbol ester treatment. Lowering chloride in the uptake buffer switched the stimulatory effect of Zn(2+) in SK-N-MC cells to an inhibitory, whereas high potassium depolarization of HEK293 cells switched the inhibitory effect of Zn(2+) to a stimulatory one. This study represents the first evidence that DAT regulation by Zn(2+) is profoundly modulated by the membrane potential and chloride.
authors.affiliation: Center for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria. christian.pifl@meduniwien.ac.at
authors.forename: Christian
authors.initials: C
authors.lastname: Pifl
authors.forename: Alexandra
authors.initials: A
authors.lastname: Wolf
authors.forename: Patrick
authors.initials: P
authors.lastname: Rebernik
authors.forename: Harald
authors.initials: H
authors.lastname: Reither
authors.forename: Michael L
authors.initials: ML
authors.lastname: Berger
mesh_terms: D000661:Amphetamine / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D003042:Cocaine / Q000031:analogs & derivatives / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects* / Q000235:genetics; D009638:Norepinephrine / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000235:genetics; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D014131:Trace Elements / Q000494:pharmacology*; D014162:Transfection; D019287:Zinc Sulfate / Q000494:pharmacology*
chemical_list: D002712:Chlorides; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D050484:Norepinephrine Plasma Membrane Transport Proteins; D014131:Trace Elements; D006639:Histidine; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; C020922:4-O-methyl-12-O-tetradecanoylphorbol 13-acetate; D019287:Zinc Sulfate; D000661:Amphetamine; D003042:Cocaine; D008239:Lysine; D013755:Tetradecanoylphorbol Acetate; D009638:Norepinephrine