title: 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.
abstract: 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.
authors.affiliation: Clinical Psychopharmacology Section, NIDA Addiction Research Center, NIH, Baltimore, Maryland 21224.
authors.forename: C M
authors.initials: CM
authors.lastname: Dersch
authors.forename: H C
authors.initials: HC
authors.lastname: Akunne
authors.forename: J S
authors.initials: JS
authors.lastname: Partilla
authors.forename: G U
authors.initials: GU
authors.lastname: Char
authors.forename: B R
authors.initials: BR
authors.lastname: de Costa
authors.forename: K C
authors.initials: KC
authors.lastname: Rice
authors.forename: F I
authors.initials: FI
authors.lastname: Carroll
authors.forename: R B
authors.initials: RB
authors.lastname: Rothman
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D008297:Male; D008454:Mazindol / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D014316:Tritium
chemical_list: D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D014316:Tritium; D008454:Mazindol; D004298:Dopamine