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.

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.