Biophysical characterization of the cocaine binding pocket in the serotonin transporter using a fluorescent cocaine analogue as a molecular reporter

Citation
Sgf. 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
Citations number
35
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
276
Issue
7
Year of publication
2001
Pages
4717 - 4723
Database
ISI
SICI code
0021-9258(20010216)276:7<4717:BCOTCB>2.0.ZU;2-N
Abstract
To explore the biophysical properties of the binding site for cocaine and r elated compounds in the serotonin transporter SERT, a high affinity cocaine analogue (3 beta-(4-methylphenyl)tropane-2 beta -carboxylic acid N-(N-meth yl-N- (4-nitrobenzo-2-oxa-1,3-diazol-7-yl) ethanol amine ester hydrochlorid e (RTI-233); K-I = 14 nM) that contained the environmentally sensitive fluo rescent moiety 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was synthesized. Specif ic binding of RTI-233 to the rat serotonin transporter, purified from Sf-9 insect cells, was demonstrated by the competitive inhibition of fluorescenc e using excess serotonin, citalopram, or RTI-55 (B beta -carbomethoxy-3 bet a-(4-iodophenyl)tropane). Moreover, specific binding was evidenced by measu rement of steady-state fluorescence anisotropy, showing constrained mobilit y of bound RTI-233 relative to RTI-233 free in solution. The fluorescence o f bound RTI-233 displayed an emission maximum (lambda (max)) of 532 nm, cor responding to a 4-nm blue shift as compared with the lambda (max) of RTI-23 3 in aqueous solution and corresponding to the lambda (max) of RTI-233 in 8 0% dioxane, Collisional quenching experiments revealed that the aqueous que ncher potassium iodide was able to quench the fluorescence of RTI-233 in th e binding pocket (K-SV = 1.7 M-1), although not to the same extent as free RTI-233 (K-SV = 7.2 M-1). Conversely, the hydrophobic quencher 2,2,6,6-tetr amethylpiperidine-N-oxyl (TEMPO) quenched the fluorescence of bound RTI-233 more efficiently than free RTI-233. These data are consistent with a highl y hydrophobic microenvironment in the binding pocket for cocaine-like uptak e inhibitors. However, in contrast to what has been observed for small-mole cule binding sites in, for example, G protein-coupled receptors, the bound cocaine analogue was still accessible for aqueous quenching and, thus, part ially exposed to solvent.