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
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.