title: Expression studies of naturally occurring human dopamine transporter variants identifies a novel state of transporter inactivation associated with Val382Ala.
abstract: Multiple, rare, human dopamine (DA) transporter (hDAT, SLC6A3) coding variants have been described, though to date they have been incompletely characterized. Here we present studies analyzing the function and regulation of five naturally occurring coding variants, V55A, R237Q, V382A, A559V and E602G, expressed in COS-7 and SH-SY5Y cells. All variants, except V382A, exhibited levels of surface protein expression and DA transport activity comparable to hDAT. V382A, divergent at the most highly conserved residue among reported hDAT variants, exhibited significantly diminished surface expression, likely derived from inefficient plasma membrane delivery. Moreover, a greater expression of V382A protein was required to achieve comparable levels of transport to hDAT, consistent with a loss of transport function. V382A displayed a decrease in sensitivity to phorbol ester (PMA)-induced internalization, as well as an altered substrate selectivity for DA versus norepinephrine (NE). Analysis of PMA-induced V382A internalization revealed a trafficking-independent action of PMA, consistent with the existence of a surface-localized, transport-inactive state.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Mazei-Robison
authors.forename: Randy D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D000409:Alanine / Q000235:genetics*; D000661:Amphetamine / Q000494:pharmacology; D000818:Animals; D019921:Biotinylation / Q000379:methods; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D003042:Cocaine / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D004347:Drug Interactions; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D014644:Genetic Variation / Q000502:physiology*; D006801:Humans; D051379:Mice; D008954:Models, Biological; D016296:Mutagenesis / Q000502:physiology; D009447:Neuroblastoma; D009638:Norepinephrine / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014633:Valine / Q000235:genetics*
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D010703:Phorbol Esters; C493214:SLC6A3 protein, human; D000661:Amphetamine; D014633:Valine; D003042:Cocaine; D000409:Alanine; D004298:Dopamine; D009638:Norepinephrine