title: Binding domains for blockers and substrates on the cloned human dopamine transporter studied by protection against N-ethylmaleimide-induced reduction of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) binding.
abstract: Binding sites for 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) on the human dopamine transporter expressed in C6 glioma cells were alkylated with N-ethylmaleimide (NEM), and the protective potency of the blockers cocaine, N[1-(2-benzo[b]thiophenyl) cyclohexyl]piperidine (BTCP), and benztropine, and of the substrates dopamine, d-amphetamine, and norepinephrine was measured. In general, the protective potency was lower (at least 4-5 times) than the potency in inhibiting [3H]WIN 35,428 binding with the compounds present under the same experimental conditions used for the NEM alkylation. However, the disparity was substantially greater for all substrates tested (23- to 44-fold) than for the blockers (4- to 11-fold), especially cocaine (5-fold) and BTCP (4-fold). Benztropine took an intermediate place (11-fold) between cocaine (5-fold) and BTCP (4-fold), on the one hand, and dopamine (23-fold), on the other hand. [3H]WIN 35,428 binding was best described by a one-site model under the present conditions. The results are discussed in terms of models involving blocker-induced conformational changes and overlapping nonidentical binding domains for blockers and substrates.
authors.affiliation: Department of Biomedical and Therapeutic Sciences (Formerly Basic Sciences), University of Illinois College of Medicine, Peoria 61656, USA.
authors.forename: M E
authors.initials: ME
authors.lastname: Reith
authors.forename: C
authors.initials: C
authors.lastname: Xu
authors.forename: L L
authors.initials: LL
authors.lastname: Coffey
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003042:Cocaine / Q000031:analogs & derivatives* / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D051381:Rats; 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; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D003042:Cocaine; D005033:Ethylmaleimide; D004298:Dopamine