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Titolo:
Biophysical characterization of the cocaine binding pocket in the serotonin transporter using a fluorescent cocaine analogue as a molecular reporter
Autore:
Rasmussen, SGF; Carroll, FI; Maresch, MJ; Jensen, AD; Tate, CG; Gether, U;
Indirizzi:
Univ Copenhagen, Panum Inst, Dept Med Physiol 12 5 22, Div Cellular & Mol Physiol, DK-2200 Copenhagen N, Denmark Univ Copenhagen Copenhagen DenmarkN siol, DK-2200 Copenhagen N, Denmark Res Triangle Inst, Res Triangle Pk, NC 27709 USA Res Triangle Inst Res Triangle Pk NC USA 27709 Triangle Pk, NC 27709 USA MRC, Mol Biol Lab, Cambridge CB2 2QH, England MRC Cambridge England CB2 2QH , Mol Biol Lab, Cambridge CB2 2QH, England
Titolo Testata:
JOURNAL OF BIOLOGICAL CHEMISTRY
fascicolo: 7, volume: 276, anno: 2001,
pagine: 4717 - 4723
SICI:
0021-9258(20010216)276:7<4717:BCOTCB>2.0.ZU;2-N
Fonte:
ISI
Lingua:
ENG
Soggetto:
BIOGENIC-AMINE TRANSPORTERS; HUMAN DOPAMINE TRANSPORTER; TRANSMEMBRANE DOMAIN; HIGH-AFFINITY; ADRENERGIC-RECEPTOR; I-125 RTI-55; MICE LACKING; SITE; SUBSTRATE; RECOGNITION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
35
Recensione:
Indirizzi per estratti:
Indirizzo: Gether, U Univ Copenhagen, Panum Inst, Dept Med Physiol 12 5 22, Div Cellular & Mol Physiol, DK-2200 Copenhagen N, Denmark Univ Copenhagen Copenhagen Denmark N 200 Copenhagen N, Denmark
Citazione:
S.G.F. Rasmussen et al., "Biophysical characterization of the cocaine binding pocket in the serotonin transporter using a fluorescent cocaine analogue as a molecular reporter", J BIOL CHEM, 276(7), 2001, pp. 4717-4723

Abstract

To explore the biophysical properties of the binding site for cocaine and related compounds in the serotonin transporter SERT, a high affinity cocaine analogue (3 beta-(4-methylphenyl)tropane-2 beta -carboxylic acid N-(N-methyl-N- (4-nitrobenzo-2-oxa-1,3-diazol-7-yl) ethanol amine ester hydrochloride (RTI-233); K-I = 14 nM) that contained the environmentally sensitive fluorescent moiety 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was synthesized. Specific binding of RTI-233 to the rat serotonin transporter, purified from Sf-9insect cells, was demonstrated by the competitive inhibition of fluorescence using excess serotonin, citalopram, or RTI-55 (B beta -carbomethoxy-3 beta-(4-iodophenyl)tropane). Moreover, specific binding was evidenced by measurement of steady-state fluorescence anisotropy, showing constrained mobility of bound RTI-233 relative to RTI-233 free in solution. The fluorescence of bound RTI-233 displayed an emission maximum (lambda (max)) of 532 nm, corresponding to a 4-nm blue shift as compared with the lambda (max) of RTI-233 in aqueous solution and corresponding to the lambda (max) of RTI-233 in 80% dioxane, Collisional quenching experiments revealed that the aqueous quencher potassium iodide was able to quench the fluorescence of RTI-233 in the binding pocket (K-SV = 1.7 M-1), although not to the same extent as freeRTI-233 (K-SV = 7.2 M-1). Conversely, the hydrophobic quencher 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) quenched the fluorescence of bound RTI-233 more efficiently than free RTI-233. These data are consistent with a highly hydrophobic microenvironment in the binding pocket for cocaine-like uptake inhibitors. However, in contrast to what has been observed for small-molecule binding sites in, for example, G protein-coupled receptors, the boundcocaine analogue was still accessible for aqueous quenching and, thus, partially exposed to solvent.

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Documento generato il 05/07/20 alle ore 05:35:50