Studies of the biogenic amine transporters. VI. Characterization of a novel cocaine binding site, identified with [¹²⁵1]RTI-55, in membranes prepared from whole rat brain minus caudate

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 3β-(4- chlorophenyl)tropan-2β-carboxylic acid phenyl ester hydrochloride (RTI-113) and 3β-(4-iodophenyl)tropan-2β-carboxylic acid phenyl ester hydrochloride (RTI-122) with [¹²⁵I]RTI-55 binding sites readily resolved two binding sites for [¹²⁵I]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 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 DAT(site2), which is not associated with the classic dopamine, serotonin or norepinephrine transporters.