title: Dopamine transporter oligomerization: impact of combining protomers with differential cocaine analog binding affinities.
abstract: Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [³H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious 'mixing' experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among five pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [³H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer. The dopamine transporter (DAT) can exist as an oligomer but it is unknown whether the protomers function independently. The present results indicate that protomers that are superpotent or deficient in cocaine analog binding can confer enhanced or reduced potency to the oligomer, respectively. In this respect, positive or negative cooperativity is revealed in the DAT oligomer.
authors.affiliation: Department of Psychiatry, New York University School of Medicine, New York, New York, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhen
authors.forename: Tamara
authors.initials: T
authors.lastname: Antonio
authors.forename: Shu-Yuan
authors.initials: SY
authors.lastname: Cheng
authors.forename: Solav
authors.initials: S
authors.lastname: Ali
authors.forename: Kymry T
authors.initials: KT
authors.lastname: Jones
authors.forename: Maarten E A
authors.initials: ME
authors.lastname: Reith
mesh_terms: D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics; D019921:Biotinylation; D003042:Cocaine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D003198:Computer Simulation; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000493:pharmacokinetics*; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012044:Regression Analysis; D013329:Structure-Activity Relationship; D014162:Transfection; D014316:Tritium / Q000493:pharmacokinetics
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D021122:Protein Subunits; D014316:Tritium; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D003042:Cocaine
keywords: 428; CFT; WIN35; cocaine analog binding; dopamine transporter oligomers; transporter point mutants