title: Biophysical characterization of the cocaine binding pocket in the serotonin transporter using a fluorescent cocaine analogue as a molecular reporter.
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.
authors.affiliation: Division of Cellular and Molecular Physiology, Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors.forename: S G
authors.initials: SG
authors.lastname: Rasmussen
authors.forename: F I
authors.initials: FI
authors.lastname: Carroll
authors.forename: M J
authors.initials: MJ
authors.lastname: Maresch
authors.forename: A D
authors.initials: AD
authors.lastname: Jensen
authors.forename: C G
authors.initials: CG
authors.lastname: Tate
authors.forename: U
authors.initials: U
authors.lastname: Gether
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D015283:Citalopram / Q000378:metabolism; D003042:Cocaine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000235:genetics; D014162:Transfection
chemical_list: C429017:8-methyl-3-(4-methylphenyl)tropane--2-carboxylic acid N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine ester; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D015283:Citalopram; D012701:Serotonin; C069723:2beta-carbomethoxy-3beta-(4-iodophenyl)tropane; D003042:Cocaine