Binding domains for blockers and substrates on the dopamine transporter in rat striatal membranes studied by protection against N-ethylmaleimide-induced reduction of [3H]WIN 35,428 binding.

Binding sites for 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) on rat striatal membranes were alkylated with N-ethylmaleimide (NEM), and the protective potency was measured of the blockers cocaine, N[1-(2-benzo[b]thiophenyl) cyclohexyl]piperidine (BTCP), benztropine, WIN 35,428; and nomifensine, and of the substrates dopamine, norepinephrine, S(+)-amphetamine, tyramine, and metaraminol. In general, the protective potency was lower (at least 3 times) than the potency in inhibiting [3H]WIN 35,428 binding with the compounds present under the same experimental conditions used for the NEM-induced alkylation. However, the disparity was substantially greater for all substrates tested (10- to 93-fold) than for the blockers (2- to 6-fold), especially cocaine and BTCP (3-fold). [3H]WIN 35,428 binding was best described by a l-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.