BINDING DOMAINS FOR BLOCKERS AND SUBSTRATES ON THE CLONED HUMAN DOPAMINE TRANSPORTER STUDIED BY PROTECTION AGAINST N-ETHYLMALEIMIDE-INDUCEDREDUCTION OF A-CARBOMETHOXY-3-BETA-(4-FLUOROPHENYL)[H-3]TROPANE ([H-3]WIN-35,428) BINDING

Binding sites for 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[H-3]trop ane ([H-3]WIN 35,428) the human dopamine transporter expressed in C6 g lioma cells were alkylated with N-ethylmaleimide (NEM), and the protec tive potency of the blockers cocaine, N[1-(2-benzo[b]thiophenyl)cycloh exyl]piperidine (BTCP), and benztropine, and of the substrates dopamin e, d-amphetamine, and norepinephrine was measured. In general, the pro tective potency was lower (at least 4-5 times) than the potency in inh ibiting [H-3]WIN 35,428 binding with the compounds present under the s ame 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-fo ld) between cocaine (5-fold) and BTCP (4-fold), on the one hand, and d opamine (23-fold), on the other hand. [H-3]WIN 35,428 binding was best described by a one site model under the present conditions. The resul ts are discussed in terms of models involving blocker-induced conforma tional charges and overlapping nonidentical binding domains for blocke rs and substrates. Copyright (C) 1996 Elsevier Science Inc.