title: Relationship between conformational changes in the dopamine transporter and cocaine-like subjective effects of uptake inhibitors.
abstract: Cocaine exerts its stimulatory effect by inhibiting the dopamine transporter (DAT). However, novel benztropine- and rimcazole-based inhibitors show reduced stimulant effects compared with cocaine, despite higher affinity and selectivity for DAT. To investigate possible mechanisms, we compared the subjective effects of different inhibitors with their molecular mode of interaction at the DAT. We determined how different inhibitors affected accessibility of the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate to an inserted cysteine (I159C), which is accessible when the extracellular transporter gate is open but inaccessible when it is closed. The data indicated that cocaine analogs bind an open conformation, whereas benztropine and rimcazole analogs bind a closed conformation. Next, we investigated the changes in inhibition potency of [(3)H]dopamine uptake of the compounds at a mutant DAT (Y335A) characterized by a global change in the conformational equilibrium. We observed a close relationship between the decrease in potencies of inhibitors at this mutant and cocaine-like responding in rats trained to discriminate cocaine from saline injections. Our data suggest that chemically different DAT inhibitors stabilize distinct transporter conformations and that this in turn affects the cocaine-like subjective effects of these compounds in vivo.
authors.affiliation: Molecular Neuropharmacology Group, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. claus@neuropharm.ku.dk
authors.forename: Claus J
authors.initials: CJ
authors.lastname: Loland
authors.forename: Rajeev I
authors.initials: RI
authors.lastname: Desai
authors.forename: Mu-Fa
authors.initials: MF
authors.lastname: Zou
authors.forename: Jianjing
authors.initials: J
authors.lastname: Cao
authors.forename: Peter
authors.initials: P
authors.lastname: Grundt
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerstbrein
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.forename: Amy Hauck
authors.initials: AH
authors.lastname: Newman
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Katz
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002522:Chlorocebus aethiops; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D003627:Data Interpretation, Statistical; D004193:Discrimination Learning / Q000187:drug effects; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D020128:Inhibitory Concentration 50; D008297:Male; D008698:Mesylates; D009043:Motor Activity / Q000187:drug effects; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008698:Mesylates; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D003042:Cocaine; D000409:Alanine