STUDIES OF THE BIOGENIC-AMINE TRANSPORTERS .1. DOPAMINE REUPTAKE BLOCKERS INHIBIT [H-3] 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 lig and, [H-3]mazindol, labels multiple sites/states associated with the d opamine (DA) transporter in striatal membranes. Incubations with [H-3] mazindol proceeded for 18-24 hr at 4 degrees C in 55.2 mM sodium phosp hate buffer, pH 7.4, with a protease inhibitor cocktail. In order to o btain data suitable for quantitative curve fitting, it was necessary t o repurify the [H-3]mazindol by HPLC before a series of experiments. U nder these conditions, we observed greater than 80% specific binding. The method of binding surface analysis was used to characterize the in teraction of GBR12935, BTCP, mazindol, and CFT with binding site/sites labeled by [H-3]mazindol. A one site model fit the data as well as th e two site model: Bmax = 16911 fmol/mg protein, Kd of [H-3]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 severa l drugs (GBR12935, CFT, BTCP, cocaine, cis-flupentixol, nomifensine, W IN35,065-2, bupropion, PCP, and benztropine) was determined. All drugs inhibited [H-3]mazindol binding by a competitive mechanism. Although the ligand-selectivity of the [H-3]mazindol binding site indicates tha t it is the uptake inhibitor recognition site of the classic DA transp orter, 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 recog nition site. It is likely that development of domain-selective drugs m ay further our understanding of the DA transporter.