STUDIES OF THE BIOGENIC-AMINE TRANSPORTERS .6. CHARACTERIZATION OF A NOVEL COCAINE BINDING-SITE, IDENTIFIED WITH [I-125] RTI-55, IN MEMBRANES PREPARED FROM WHOLE RAT-BRAIN MINUS CAUDATE

Previous studies showed that the cocaine analog [(1)25I]RTI-55 labels dopamine and serotonergic (5-HT) biogenic amine transporters (BATs) wi th high affinity. Here we characterized [I-125]RTI-55 binding to membr anes prepared from whole rat brain minus the caudate nuclei. Paroxetin e (50 nM) was used to block [I-125]RTI-55 binding to 5-HT transporter sites. Initial experiments identified drugs that displaced [I-125]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)tr opan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-122) with [I-125]RTI-55 binding sites readily resolved two binding sites for [I- 125]RTI-55 with K-d values of 0.44 nM and 17 nM and B-max values of 31 and 245 fmol/mg protein. Potent 5-HT and noradrenergic uptake inhibit ors had low affinity for both sites. Whereas cocaine, CFT and WIN35,06 5-2 were 6.0-, 25- and 14-fold selective for the first site, benztropi ne, PCP and the novel pyrrole, b-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 trans iently expressing the cloned rat dopamine transporter. Lesion stud ies with 6-hydroxydopamine and 5,7-dihydroxytryptamine were conducted to test the hypothesis that site 1 and site 2 are physically distinct. Th e data showed that these neurotoxins differentially decreased [I-125]R TI-55 binding to sites 1 and 2. The differential distribution of sites 1 and 2 in rat brain provides further support for this hypothesis. Vi ewed collectively, these data show that [I-125]RTI-55 labels a novel b inding site in rat brain membranes, termed DAT(site2), which is not as sociated with the classic dopamine, serotonin or norepinephrine transp orters.