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 (3beta-(4-methylphenyl)tropane-2beta-carboxylic acid N-(N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine 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-9 insect cells, was demonstrated by the competitive inhibition of fluorescence using excess serotonin, citalopram, or RTI-55 (2beta-carbomethoxy-3beta-(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 free RTI-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 bound cocaine analogue was still accessible for aqueous quenching and, thus, partially exposed to solvent.
Highlights
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(4-methylphenyl)tropane-2-carboxylic acid N-(N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine ester hydrochloride (RTI-233); KI ؍14 nM) that contained the environmentally sensitive fluorescent moiety 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was synthesized
Cysteine-scanning mutagenesis of transmembrane segment 3 in the SERT has suggested that two residues (Ile-172 and Tyr-176) in the middle of the transmembrane segment could be in close proximity to the cocaine binding site [20], no direct contact sites have been established between cocaine and specific transporter residues
Cocaine is one the most widely abused psychostimulants, only little is known about the molecular mechanisms underlying the inhibitory effect of cocaine at the monoamine transporters (SERT, dopamine transporter (DAT), and norepinephrine transporter (NET))
Summary
Synthesis of 3-(4-Methylphenyl)tropane-2-carboxylic Acid N-(Nmethyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine Ester Hydrochloride (RTI-233)—Oxalyl chloride (2.0 M in CH2Cl2; 0.80 ml, 1.60 mmol) was added dropwise to a stirred solution of 3-(4-methylphenyl)tropane-2-carboxylic acid (RTI-374) [23] (200 mg, 0.77 mol) in CH2Cl2 (20 ml) under an argon atmosphere at 25 °C. Ligand Binding Assays—Binding experiments on rSERT expressed in Sf-9 insect cell membranes and of the purified transporter were performed using 125I-RTI-55 (PE Biosystems) as radioligand. For the emission scan, quenching, and anisotropy experiments, 20 pmol of purified rSERT was incubated in 100 l of digitonin buffer (25 mM Hepes, pH 7.5, with 0.1% digitonin, 100 mM NaCl) in the presence of 1 M RTI-233 and, when indicated, 1 mM 5-HT, 10 M RTI 55, or 10 M citalopram for 30 min at 4 °C before separation of bound from unbound on 2 ml of Sephadex G-50 columns. The anisotropy measurements were carried out using the Constant Wavelength Analysis program with the excitation set at 480 nm and emission measured at 532 nm for RTI-233 bound to rSERT and 536 nm for free RTI-233 in digitonin buffer (integration time 10 s). The anisotropy was stable for at least 15 min at the indicated temperatures, indicating negligible dissociation of ligand under the experimental conditions used
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