title: Expression and regulation of the human dopamine transporter in a neuronal cell line.
abstract: Human cocaine users exhibit increased striatal [3H]WIN35428 binding to the dopamine transporter (DAT). However, the nature of the changes induced in the DAT are complex and may not result from a simple increase in number of DAT molecules. To better understand the regulation of DAT inhibitor binding sites and their relationship to the overall process of dopamine uptake, a neuronal model system expressing the human DAT has been developed. Initial experiments were attempted with native dopaminergic neurons so as to allow examination of DAT interactions with vesicular release and storage mechanisms. Dissociated fetal rat mesencephalic neurons, of various ages and mixtures with target cells, were grown to confluence. However, [3H]WIN35428 binding was of low affinity at all levels of maturity. Following this, a simpler model was assessed, using DAT cDNA transfected into neuroblastoma-derived Neuro2A cells. Initially, no specific and little non-specific [3H]WIN35428 or [3H]paroxetine binding was found in non-transfected cells. After transfection with the human DAT inserted in the pcDNA vector, both DAT binding and dopamine uptake were significantly and stably present. Treatment with (-)cocaine, 10-6 M for 24 h, increased DAT binding and uptake, which did not occur in parallel COS-7 experiments. Other experiments with Neuro2A cells also found that dopamine uptake was down-regulated by treatment with a PKC activator. These results suggest that the transfected Neuro2A neurons should be useful for ongoing experiments examining the regulation of the DAT by assorted treatments.
authors.affiliation: Department of Psychiatry, University of Michigan, Ann Arbor, MI, USA.
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: L W
authors.initials: LW
authors.lastname: Elmer
authors.forename: K Y
authors.initials: KY
authors.lastname: Little
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000378:metabolism; D004789:Enzyme Activation; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008636:Mesencephalon / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D017374:Paroxetine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; C493216:Slc6a3 protein, rat; D017374:Paroxetine; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D011494:Protein Kinases; D003042:Cocaine; D013755:Tetradecanoylphorbol Acetate; D004298:Dopamine