Studies of the biogenic amine transporters. 1. Dopamine reuptake blockers inhibit [3H]mazindol binding to the dopamine transporter by a competitive mechanism: preliminary evidence for different binding domains.

The present study addressed the hypothesis that the DA transporter ligand, [3H]mazindol, labels multiple sites/states associated with the dopamine (DA) transporter in striatal membranes. Incubations with [3H]mazindol proceeded for 18-24 hr at 4 degrees C in 55.2 mM sodium phosphate buffer, pH 7.4, with a protease inhibitor cocktail. In order to obtain data suitable for quantitative curve fitting, it was necessary to repurify the [3H]mazindol by HPLC before a series of experiments. Under these conditions, we observed greater than 80% specific binding. The method of binding surface analysis was used to characterize the interaction of GBR12935, BTCP, mazindol, and CFT with binding site/sites labeled by [3H]mazindol. A one site model fit the data as well as the two site model: Bmax = 16911 fmol/mg protein, Kd of [3H]mazindol = 75 nM, Ki of GBR12935 = 8.1 nM, Ki of CFT = 50 nM and Ki of BTCP = 44 nM. The inhibitory mechanism (competitive or noncompetitive) of several drugs (GBR12935, CFT, BTCP, cocaine, cis-flupentixol, nomifensine, WIN35,065-2, bupropion, PCP, and benztropine) was determined. All drugs inhibited [3H]mazindol binding by a competitive mechanism. Although the ligand-selectivity of the [3H]mazindol binding site indicates that it is the uptake inhibitor recognition site of the classic DA transporter, the quantitative differences among the ligand-selectivities of different radioligands for the same site suggest that each radioligand labels different overlapping domains of the DA uptake inhibitor recognition site. It is likely that development of domain-selective drugs may further our understanding of the DA transporter.