Biophysical characterization of the cocaine binding pocket in the serotonin transporter using a fluorescent cocaine analogue as a molecular reporter
| Autor: | Christopher G. Tate, Søren G. F. Rasmussen, Anne Dam Jensen, F. Ivy Carroll, Ulrik Gether, Martin J. Maresch |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Serotonin
Stereochemistry Nerve Tissue Proteins Citalopram Spodoptera Transfection Biochemistry chemistry.chemical_compound Non-competitive inhibition Cocaine Animals Binding site Molecular Biology Cocaine binding Fluorescent Dyes Serotonin Plasma Membrane Transport Proteins Aqueous solution Quenching (fluorescence) Binding Sites Membrane Glycoproteins Chemistry Membrane Transport Proteins Tropane Cell Biology Fluorescence Recombinant Proteins Rats Spectrometry Fluorescence Carrier Proteins human activities Fluorescence anisotropy Protein Binding |
| Zdroj: | The Journal of biological chemistry. 276(7) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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