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

R. B. Rothman, M. L. Silverthorn, M. H. Baumann, C. B. Goodman, J. L. Cadet, D. Matecka, K. C. Rice, F. I. Carroll, J. B. Wang, G. R. Uhl, J. S. Partilla, C. M. Dersch

Research output: Contribution to journalArticle

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 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.

Original languageEnglish (US)
Pages (from-to)385-395
Number of pages11
JournalJournal of Pharmacology and Experimental Therapeutics
Volume274
Issue number1
StatePublished - 1995
Externally publishedYes

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Biogenic Amines
Cocaine
Binding Sites
Membranes
Brain
Dopamine Plasma Membrane Transport Proteins
Carboxylic Acids
Serotonin
Esters
Benztropine
5,7-Dihydroxytryptamine
Norepinephrine Plasma Membrane Transport Proteins
Paroxetine
Serotonin Plasma Membrane Transport Proteins
Pyrroles
RTI 55
Caudate Nucleus
Oxidopamine
COS Cells
Neurotoxins

ASJC Scopus subject areas

  • Pharmacology

Cite this

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. / Rothman, R. B.; Silverthorn, M. L.; Baumann, M. H.; Goodman, C. B.; Cadet, J. L.; Matecka, D.; Rice, K. C.; Carroll, F. I.; Wang, J. B.; Uhl, G. R.; Partilla, J. S.; Dersch, C. M.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 274, No. 1, 1995, p. 385-395.

Research output: Contribution to journalArticle

Rothman, RB, Silverthorn, ML, Baumann, MH, Goodman, CB, Cadet, JL, Matecka, D, Rice, KC, Carroll, FI, Wang, JB, Uhl, GR, Partilla, JS & Dersch, CM 1995, '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', Journal of Pharmacology and Experimental Therapeutics, vol. 274, no. 1, pp. 385-395.
Rothman, R. B. ; Silverthorn, M. L. ; Baumann, M. H. ; Goodman, C. B. ; Cadet, J. L. ; Matecka, D. ; Rice, K. C. ; Carroll, F. I. ; Wang, J. B. ; Uhl, G. R. ; Partilla, J. S. ; Dersch, C. M. / 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. In: Journal of Pharmacology and Experimental Therapeutics. 1995 ; Vol. 274, No. 1. pp. 385-395.
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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 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.",
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AU - Baumann, M. H.

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AU - Partilla, J. S.

AU - Dersch, C. M.

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N2 - 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.

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