title: Studies of the biogenic amine transporters. VI. Characterization of a novel cocaine binding site, identified with [125I]RTI-55, in membranes prepared from whole rat brain minus caudate.
abstract: Previous studies showed that the cocaine analog [125I]RTI-55 labels dopamine and serotonergic (5-HT) biogenic amine transporters (BATs) with high affinity. Here we characterized [125I]RTI-55 binding to membranes prepared from whole rat brain minus the caudate nuclei. Paroxetine (50 nM) was used to block [125I]RTI-55 binding to 5-HT transporter sites. Initial experiments identified drugs that displaced [125I]RTI-55 binding with moderately low slope factors. Binding surface analysis of the interaction of 3 beta-(4-chlorophenyl)tropan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-113) and 3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-122) with [125I]RTI-55 binding sites readily resolved two binding sites for [125I]RTI-55 with Kd values of 0.44 nM and 17 nM and Bmax values of 31 and 245 fmol/mg protein. Potent 5-HT and noradrenergic uptake inhibitors had low affinity for both sites. Whereas cocaine, CFT and WIN35,065-2 were 6.0-, 25- and 14-fold selective for the first site, benztropine, PCP and the novel pyrrole, (+-)-(2RS,3aSR,8bRS)-1,2,3,3a,4,8b-hexahydro- 2-benzyl-1-methylindeno-[1,2-b]pyrrole resorcylate [(+-)-HBMP, formerly called (+-)-RTI-4793-14], were moderately selective for the second site. A single binding site with the characteristics of site 1 was resolved using COS cells transiently expressing the cloned rat dopamine transporter. Lesion studies with 6-hydroxydopamine and 5,7-dihydroxytryptamine were conducted to test the hypothesis that site 1 and site 2 are physically distinct. The data showed that these neurotoxins differentially decreased [125I]RTI-55 binding to sites 1 and 2. The differential distribution of sites 1 and 2 in rat brain provides further support for this hypothesis. Viewed collectively, these data show that [125I]RTI-55 labels a novel binding site in rat brain membranes, termed DATsite2, which is not associated with the classic dopamine, serotonin or norepinephrine transporters.
authors.affiliation: Clinical Pharmacology Section, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland, USA.
authors.forename: R B
authors.initials: RB
authors.lastname: Rothman
authors.forename: M L
authors.initials: ML
authors.lastname: Silverthorn
authors.forename: M H
authors.initials: MH
authors.lastname: Baumann
authors.forename: C B
authors.initials: CB
authors.lastname: Goodman
authors.forename: J L
authors.initials: JL
authors.lastname: Cadet
authors.forename: D
authors.initials: D
authors.lastname: Matecka
authors.forename: K C
authors.initials: KC
authors.lastname: Rice
authors.forename: F I
authors.initials: FI
authors.lastname: Carroll
authors.forename: J B
authors.initials: JB
authors.lastname: Wang
authors.forename: G R
authors.initials: GR
authors.lastname: Uhl
mesh_terms: D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002421:Caudate Nucleus; D003042:Cocaine / Q000031:analogs & derivatives* / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins; D014316:Tritium
chemical_list: D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D014316:Tritium; D012701:Serotonin; C069723:2beta-carbomethoxy-3beta-(4-iodophenyl)tropane; D003042:Cocaine; D004298:Dopamine