Biophysical Characterization of the Cocaine Binding Pocket in the Serotonin Transporter Using a Fluorescent Cocaine Analogue as a Molecular Reporter
2001; Elsevier BV; Volume: 276; Issue: 7 Linguagem: Inglês
10.1074/jbc.m008067200
ISSN1083-351X
AutoresSøren G. F. Rasmussen, F. Ivy Carroll, Martin J. Maresch, Anne D. Jensen, Christopher G. Tate, Ulrik Gether,
Tópico(s)Receptor Mechanisms and Signaling
ResumoTo explore the biophysical properties of the binding site for cocaine and related compounds in the serotonin transporter SERT, a high affinity cocaine analogue (3β-(4-methylphenyl)tropane-2β-carboxylic acidN-(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 (2β-carbomethoxy-3β-(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 (λmax) of 532 nm, corresponding to a 4-nm blue shift as compared with the λmax of RTI-233 in aqueous solution and corresponding to the λmax 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 = 1.7m−1), although not to the same extent as free RTI-233 (K SV = 7.2m−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. To explore the biophysical properties of the binding site for cocaine and related compounds in the serotonin transporter SERT, a high affinity cocaine analogue (3β-(4-methylphenyl)tropane-2β-carboxylic acidN-(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 (2β-carbomethoxy-3β-(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 (λmax) of 532 nm, corresponding to a 4-nm blue shift as compared with the λmax of RTI-233 in aqueous solution and corresponding to the λmax 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 = 1.7m−1), although not to the same extent as free RTI-233 (K SV = 7.2m−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. serotonin transporter rat SERT norepinephrine transporter dopamine transporter 7-nitrobenzo-2-oxa-1,3-diazole 5-hydroxytryptamine 2,2,6,6-tetramethylpiperidine-N-oxyl 2β-carbomethoxy-3β(4-iodophenyl)tropane 3β-(4-methylphenyl)tropane-2β-carboxylic acidN-(N-methyl-N-(4-nitrobenzo2-oxa-1,3-diazol-7-yl)ethanolamine ester Cocaine is one of the most widely abused psychostimulants, causing major medical and socioeconomic problems (1Caine S.B. Nat. Neurosci. 1998; 1: 90-92Crossref PubMed Scopus (37) Google Scholar). Currently, there is no effective treatment against cocaine addiction available; therefore, clarifying the molecular mechanisms underlying the psychostimulatory effects and addictive properties of cocaine should prove critical for potential development of future therapeutic strategies. Cocaine and related drugs act by inhibiting clearance of released monoamine neurotransmitters from the synaptic cleft (2Horn A.S. Prog. Neurobiol. 1990; 34: 387-400Crossref PubMed Scopus (219) Google Scholar, 3Giros B. Caron M.G. Trends Pharmacol. Sci. 1993; 14: 43-49Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 4Povlock S.L. Amara S.G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 1-28Crossref Google Scholar). This clearance of monoamines occurs via three distinct but highly homologous monoamine transporters, the serotonin transporter (SERT),1 the dopamine transporter (DAT), and the norepinephrine transporter (NET) (2Horn A.S. Prog. Neurobiol. 1990; 34: 387-400Crossref PubMed Scopus (219) Google Scholar, 3Giros B. Caron M.G. Trends Pharmacol. Sci. 1993; 14: 43-49Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 4Povlock S.L. Amara S.G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 1-28Crossref Google Scholar). Cocaine binds with high affinity to all three transporters and is generally believed to act as a competitive blocker of substrate translocation (4Povlock S.L. Amara S.G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 1-28Crossref Google Scholar, 5Carroll F.I. Lewin A.H. Kuhar M.J. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 263-295Crossref Google Scholar). Several studies have provided evidence that inhibition of the DAT is the predominant mechanism behind the stimulatory effects and addictive properties of cocaine (6Ritz M.C. Lamb R.J. Goldberg S.R. Kuhar M.J. Science. 1987; 237: 1219-1223Crossref PubMed Scopus (2039) Google Scholar, 7Giros B. Jaber M. Jones S.R. Wightman R.M. Caron M.G. Nature. 1996; 379: 606-612Crossref PubMed Scopus (2076) Google Scholar, 8Wise R.A. Annu. Rev. Neurosci. 1996; 19: 319-340Crossref PubMed Scopus (674) Google Scholar). However, this hypothesis has been challenged by recent studies on mice in which the DAT gene has been deleted (1Caine S.B. Nat. Neurosci. 1998; 1: 90-92Crossref PubMed Scopus (37) Google Scholar). Despite the absence of the DAT gene, it was surprisingly observed that these mice self-administered cocaine, indicating a possible important role of also the SERT and NET (9Rocha B.A. Fumagalli F. Gainetdinov R.R. Jones S.R. Ator R. Giros B. Miller G.W. Caron M.G. Nat. Neurosci. 1998; 1: 132-137Crossref PubMed Google Scholar, 10Xu F. Gainetdinov R.R. Wetsel W.C. Jones S.R. Bohn L.M. Miller G.W. Wang Y.M. Caron M.G. Nat. Neurosci. 2000; 3: 465-471Crossref PubMed Scopus (378) Google Scholar). The SERT belongs together with DAT and NET to a family of Na+/Cl−-dependent solute carriers that are characterized functionally by their dependence on the presence of Na+ and Cl− in the extracellular fluid (3Giros B. Caron M.G. Trends Pharmacol. Sci. 1993; 14: 43-49Abstract Full Text PDF PubMed Scopus (490) Google Scholar,11Nelson N. J. Neurochem. 1998; 71: 1785-1803Crossref PubMed Scopus (324) Google Scholar). All Na+/Cl−-dependent carriers are believed to share a common topology characterized by the presence of 12 transmembrane segments connected by alternating extracellular and intracellular loops with an intracellular location of the N and C terminus (3Giros B. Caron M.G. Trends Pharmacol. Sci. 1993; 14: 43-49Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 4Povlock S.L. Amara S.G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 1-28Crossref Google Scholar, 11Nelson N. J. Neurochem. 1998; 71: 1785-1803Crossref PubMed Scopus (324) Google Scholar). Despite intense efforts, including many mutagenesis studies (12Kitayama S. Shimada S. Xu H. Markham L. Donovan D.M. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7782-7785Crossref PubMed Scopus (349) Google Scholar, 13Barker E.L. Perlman M.A. Adkins E.M. Houlihan W.J. Pristupa Z.B. Niznik H.B. Blakely R.D. J. Biol. Chem. 1998; 273: 19459-19468Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 14Barker E.L. Moore K.R. Rakhshan F. Blakely R.D. J. Neurosci. 1999; 19: 4705-4717Crossref PubMed Google Scholar, 15Norregaard L. Frederiksen D. Nielsen E.O. Gether U. EMBO J. 1998; 17: 4266-4273Crossref PubMed Scopus (136) Google Scholar, 16Loland C.J. Norregaard L. Gether U. J. Biol. Chem. 1999; 274: 36928-36934Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 17Itokawa M. Lin Z. Cai N.S. Wu C. Kitayama S. Wang J.B. Uhl G.R. Mol. Pharmacol. 2000; 57: 1093-1103PubMed Google Scholar, 18Lin Z. Itokawa M. Uhl G.R. FASEB J. 2000; 14: 715-728Crossref PubMed Scopus (58) Google Scholar) and studies using photoaffinity labeling (19Vaughan R.A. Mol. Pharmacol. 1995; 47: 956-964PubMed Google Scholar), surprisingly little is known about the binding site for cocaine-like substances in the monoamine transporters. Although 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 (20Chen J.G. Sachpatzidis A. Rudnick G. J. Biol. Chem. 1997; 272: 28321-28327Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), no direct contact sites have been established between cocaine and specific transporter residues. Hence, it is not yet clear whether the cocaine binding site is deeply buried inside the transmembrane core of the transporter molecule or if the binding site is partially or fully exposed on the transporter surface. In this study we have investigated the biophysical nature of the cocaine binding site in the rat SERT (rSERT). For this purpose, a cocaine analogue, which contained the environmentally sensitive fluorescent moiety, nitrobenzoxadiazol, were synthesized (see Fig. 1). Moreover, an epitope-tagged version of the rSERT was expressed in Sf-9 insect cells and purified to obtain a transporter preparation that provided a sufficiently high signal-to-noise ratio for characterizing the fluorescent properties of the ligand bound to the rSERT. Our subsequent spectroscopic analysis of the bound fluorescent ligand provided evidence for a highly hydrophobic binding pocket. However, collisional quenching experiments showed that, despite the hydrophobic character of the binding crevice, the bound cocaine analogues were still accessible for aqueous quenching consistent with a partially exposed binding site. These findings contrast the observations for small-molecule ligand binding sites in other membrane proteins, such as, for example, G protein-coupled receptors, where the binding sites for small-molecule ligands are known to be deeply embedded in the transmembrane core of the molecule and entirely inaccessible to aqueous quenching (21Tota R.T. Strader C.D. J. Biol. Chem. 1990; 265: 16891-16897Abstract Full Text PDF PubMed Google Scholar, 22Turcatti G. Zoffmann S. Lowe III, J.A. Drozda S.E. Chassaing G. Schwartz T.W. Chollet A. J. Biol. Chem. 1997; 272: 21167-21175Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Oxalyl chloride (2.0 m in CH2Cl2; 0.80 ml, 1.60 mmol) was added dropwise to a stirred solution of 3β-(4-methylphenyl)tropane-2β-carboxylic acid (RTI-374) (23Carroll F.I. Kotian P. Dehghani A. Gray J.L. Kuzemko M.A. Parham K.A. Abraham P. Lewin A.H. Boja J.W. Kuhar M.J. J. Med. Chem. 1995; 38: 379-388Crossref PubMed Scopus (199) Google Scholar) (200 mg, 0.77 mol) in CH2Cl2 (20 ml) under an argon atmosphere at 25 °C. After stirring for 1 h, the CH2Cl2 was removed by reduced pressure. A solution of the acid chloride andN-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)aminoethanol (458 mg, 1.93 mmol) in CH2Cl2 (10 ml) under argon was treated slowly with triethylamine (0.5 ml, 3.5 mmol) and stirred at 25 °C for 38 h. Removal of CH2Cl2 by rotary evaporation afforded an orange-red residue, which was purified by column chromatography on silica gel (4.5 × 18 cm) with an ethyl acetate/methyl alcohol gradient elution (100:0–50:50; 100 ml). Removal of the eluant by reduced pressure gave a red solid (270 mg, 73%), melting point 184–186 °C. The free base (270 mg) in CH2Cl2 was treated with ethereal HCl (1.0m, 3 ml) to yield the hydrochloride salt (282 mg), melting point >250 °C (decomposition); [α]Dd20 −35° (C = 0.1g/100 ml methanol). The analysis of C25H30ClN5O5·0.5 H2O was C, 57.23; H, 5.95; N, 12.35 (calculated) and C, 57.22; H, 6.32; N, 12.18 (experimental). The rSERT, tagged at the N terminus with the c-myc epitope and at the C terminus with 10 histidines, was inserted into baculovirus expression vector pVL1392 (Invitrogen, San Diego, CA) as described (24Tate C.G. Whiteley E. Betenbaugh M.J. J. Biol. Chem. 1999; 274: 17551-17558Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The resulting construct was expressed in Sf-9 insect cells, and baculovirus encoding the tagged rSERT was isolated by plaque purification followed by several rounds of amplification to obtain a high titer virus stock (∼109 plaque-forming units/ml) (24Tate C.G. Whiteley E. Betenbaugh M.J. J. Biol. Chem. 1999; 274: 17551-17558Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Baculovirus encoding canine calnexin was generated as described previously (24Tate C.G. Whiteley E. Betenbaugh M.J. J. Biol. Chem. 1999; 274: 17551-17558Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). For purification, insect cells were grown in 1000-ml cultures in SF 900 II medium supplemented with 5% (v/v) heat-inactivated fetal calf serum and 0.1 mg/ml gentamicin (all products purchased from Life Technologies, Inc. The cell cultures were infected at a density of 2 × 106 cells/ml by inoculating a 1:25 dilution of a rSERT high titer virus stock plus 1:15 dilution of a high titer calnexin virus stock. The cells were harvested 48 h later by centrifugation (10 min at 5000 × g) and kept at −80 °C until purification. A significant fraction of the rSERT expressed in Sf-9 cells was previously found to be misfolded (24Tate C.G. Whiteley E. Betenbaugh M.J. J. Biol. Chem. 1999; 274: 17551-17558Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 25Tate C.G. Blakely R.D. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar). However, coinfection with the molecular chaperone, calnexin, was demonstrated to increase the fraction of correctly folded protein through assisted folding in the endoplasmatic reticulum of properly glycosylated SERT, which stabilizes the transporter at the cell surface (24Tate C.G. Whiteley E. Betenbaugh M.J. J. Biol. Chem. 1999; 274: 17551-17558Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 25Tate C.G. Blakely R.D. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar). 25 ml of Sf-9 cell cultures at a density of 3 × 106 cells/ml in 125-ml disposable Erlenmeyer flasks (Costar, Acton, MA) were infected for 48 h with 1.0 ml of a high titer rSERT virus stock encoding rSERT. The cells were harvested by centrifugation (2000 ×g for 5 min), washed once in phosphate-buffered saline, and homogenized using 25 strokes with a Dounce homogenizer in 50 mmNa2HPO4/NaH2PO4, pH 7.4, containing 1 mm EDTA, 10 μg/ml benzamidine (Sigma), 10 μg/ml leupeptin (Sigma), and 0.5 mmphenylmethylsulfonyl fluoride(Sigma). The lysate was centrifuged for 5 min at 500 × g, and the resulting supernatant was centrifuged at 40,000 × g for 30 min at 4 °C. The membrane pellet was resuspended in ice-cold sodium phosphate buffer (50 mmNa2HPO4/NaH2PO4, pH 7.4, 1 mm EDTA) containing the above-mentioned protease inhibitors followed by an additional round of centrifugation and resuspension. Protein was determined using the Bio-Rad DC protein assay kit (Bio-Rad). The membranes were diluted to 1 mg of protein/ml in buffer before storage at −80 °C. The transporter was purified using a two-step purification procedure, which will be described in further detail elsewhere. 2S. G. F. Rasmussen and U. Gether, manuscript in preparation. Briefly, one pellet of Sf-9 cells from a 1000-ml infected culture was resuspended in ice-cold lysis buffer (25 mm Hepes, pH 7.5, with 10 μm desipramine (Research Biochemicals International, Natick, MA), 10 μg/ml leupeptin (Sigma), 10 μg/ml benzamidine (Sigma), and 1 mm phenylmethylsulfonyl fluoride (Sigma)) followed by centrifugation at 30,000 ×g for 30 min at 4 °C. The lysed cells were resuspended in solubilization buffer (25 mm Hepes, pH 7.5, with 1% digitonin (Calbiochem), 30% glycerol, 100 mm NaCl, 10 μm desipramine, 10 μg/ml leupeptin, 10 μg/ml benzamidine, and 1 mm phenylmethylsulfonyl fluoride), homogenized in a Dounce homogenizer, and stirred for 2 h at 4 °C. Upon centrifugation (30,000 × g for 30 min at 4 °C), the supernatant containing the solubilized transporter was purified by nickel chromatography using chelating Sepharose (Amersham Pharmacia Biotech). Binding to the resin was carried out in batch for 2 h at 4 °C under constant rotation. The transporter was eluted from the nickel resin in 200 mm imidazole. The eluted transporter was further purified by concanavalin A chromatography. Binding to the concanavalin A resin (Amersham Pharmacia Biotech) was carried out in batch, and elution was done in 250 mmα-methyl-mannopyranoside (Sigma). The purified transporter was concentrated using Centricon-30 spin concentrators (Amicon, Beverly, MA). The specific activity of the purified transporter was ∼3 nmol/mg of protein, corresponding to around 20% purity (active protein). Protein was determined using the detergent-insensitive Bio-Rad DC protein assay kit (Bio-Rad). The purified transporter was routinely analyzed by 10% SDS-polyacrylamide gel electrophoresis and visualized by standard silver staining from which functional protein (125I-RTI-55 binding activity) were assessed to be around two-thirds of total SERT on the stain. In general, ∼2 nmol of purified SERT could be obtained from a 1000-ml culture. 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. In competition binding assays on membranes, 5 μg of membrane protein was assayed in a total volume of 250 μl using a sodium phosphate buffer (50 mmNa2HPO4/NaH2PO4, pH 7.4) containing 0.25 nm125I-RTI-55 and increasing concentrations of competing ligands, 5-HT, citalopram, cocaine, RTI-233, or RTI-55. Citalopram was kindly provided by Klaus Gundertofte, Lundbeck A/S, Denmark. Cocaine and 5-HT were obtained from Research Biochemicals International. The membranes were incubated for 2 h at room temperature before separation of bound from unbound by rapid filtration over glass fiber filters (FilterMat B, Wallac, Turku, Finland) using a Tomtec 96-well cell harvester. MeltiLex Melt-on scintillator sheets (Wallac) were used for counting of the filter in a Wallac Tri-Lux β scintillation counter. Competition binding experiments on purified transporter (15 fmol of transporter) were performed in digitonin buffer (25 mm Hepes, pH 7.5, containing 0.1% digitonin, and 100 mm NaCl) in a total volume of 100 μl using 0.25 nm of 125I-RTI-55 and increasing concentrations of unlabeled ligands. The binding assays were incubated at room temperature for 30 min before separation of bound from unbound on 2 ml of Sephadex G-50 columns (Amersham Pharmacia Biotech). The eluate was collected directly in 4-ml counting vials (Wallac) using 1000 μl of ice-cold digitonin buffer. Scintillation fluid (HiSafe, Wallac) was added, and the vials were counted in a Wallac Tri-Lux β scintillation counter. All determinations in the binding assays were done in triplicate. Binding data were analyzed by nonlinear regression analysis using Prism 2.0 from GraphPad Software, San Diego, CA. Fluorescence spectroscopy was performed on a SPEX Fluoromax-2 spectrofluorometer connected to a PC equipped with the Datamax 2.2 software package (Jobin Yvon Inc., Edison, NJ). In all experiments, the excitation and emission bandpass were set at 5 nm. For the emission scan, quenching, and anisotropy experiments, 20 pmol of purified rSERT was incubated in 100 μl of digitonin buffer (25 mm Hepes, pH 7.5, with 0.1% digitonin, 100 mm NaCl) in the presence of 1 μm RTI-233 and, when indicated, 1 mm 5-HT, 10 μm RTI 55, or 10 μm citalopram for 30 min at 4 °C before separation of bound from unbound on 2 ml of Sephadex G-50 columns. The fraction of bound was obtained by eluting with 1000 μl of ice-cold digitonin buffer. A 400-μl sample of the eluate was transferred to a 5 × 5-mm quartz cuvette (Helma, Mulheim, Germany) for the subsequent spectroscopic measurements. The absorption by the nonfluorescent ligands, 5-HT, citalopram, and RTI-55 is less than 0.01 at the used concentrations excluding any “inner filter” effect. The emission scans were performed either on 400 μl of Sephadex G-50-separated samples obtained as described above or directly on buffer/dioxane samples containing RTI-233. Excitation was 480 nm, and emission was measured from 495 to 625 nm with an integration time of 0.3 s/nm. All emission spectra are averages of three consecutive scans. Photobleaching was negligible under the experimental conditions used. The emission spectra were corrected for any background fluorescence by routinely subtracting control spectra on buffer alone. Stock solutions (1.0m) of the hydrophilic quencher potassium iodide containing 10 mm Na2S2O3 were prepared freshly for each round of experiments. The hydrophobic quencher 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) was dissolved in 10% Me2SO at a concentration of 100 mm and immediately used. The experiments were performed on either 400 μl of the Sephadex G-50-separated samples and prepared as described above or directly on buffer samples containing RTI-233. To correct for dilution/ionic strength effects on fluorescence, measurements were performed in parallel using a 1.0 m stock of potassium chloride (KCl) and a 10% Me2SO stock for the potassium iodide-and TEMPO-quenching experiments, respectively. Ten μl of quencher (potassium iodide or TEMPO) or control solution (potassium chloride or 10% Me2SO) was added sequentially followed by thorough mixing after each addition and subsequent recording of fluorescence using the Constant Wavelength Analysis program in the Datamax software package. The excitation wavelength was 480 nm, and the emission wavelength was either 532 nm for the recordings on RTI-233 bound to rSERT or 536 nm for the recordings on RTI-233 alone in digitonin buffer. A complete experiment was performed in 8 min, during which dissociation of ligand was negligible. In the experiments with TEMPO, the fluorescence intensities were corrected as described (26London E. Anal. Biochem. 1986; 154: 57-63Crossref PubMed Scopus (34) Google Scholar) for inner filter effects caused by the absorption by TEMPO at the used excitation and emission wavelengths. The corrected data were plotted according to the Stern-Volmer equation,F o/F = 1 +K sv[Q], whereF o/F is the ratio of fluorescence intensity in the absence and presence of quencher (Q), andK sv is the Stern-Volmer quenching constant (27Lakovicz J.R. Principles of Fluorescence Spectroscopy. Kluwer Academic Publishers/Plenum Publishing Group, New York1999: 238-318Google Scholar). The SPEX Fluoromax-2 fluorometer was equipped with an automated L-format polarization accessory including two Glan-Thomson UV polarizers placed in the sample chamber to enable polarized excitation and emission detection. 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). Concurrent measurements of the emission intensity with the excitation-side polarizer in the vertical position (V) and the emission-side polarizer in either the V or horizontal position (H) were carried out. The measurements were collected at 4, 20, and 37 °C by perfusion of water with adequate temperature through the thermostatted cuvette holder. The data were converted to anisotropy according to the equation, A = (I VV −GI VH)/(I VV + 2GI VH), where I VV is the intensity measured with both the excitation-side and emission-side polarizer in the vertical position (V), I VH is the intensity measured with the excitation-side polarizer in the vertical position (V) and the emission-side polarizer in the horizontal position (H), and G is the ratio of the sensitivities of the detection system for vertically and horizontally polarized light (S V/S H) (27Lakovicz J.R. Principles of Fluorescence Spectroscopy. Kluwer Academic Publishers/Plenum Publishing Group, New York1999: 238-318Google Scholar). The anisotropy was stable for at least 15 min at the indicated temperatures, indicating negligible dissociation of ligand under the experimental conditions used. To obtain a fluorescent analogue of cocaine with preserved high affinity for the rSERT, the environmentally sensitive fluorescent moiety 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was connected to the 2β position of the tropane backbone in RTI-374 (3β-(4-methylphenyl)tropane-2β-carboxylic acid) usingN-methylethanolamine as the linker as described under “Experimental Procedures” (Fig. 1) (5Carroll F.I. Lewin A.H. Kuhar M.J. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 263-295Crossref Google Scholar). Importantly, very large 2β-carboalkoxy groups have been shown to be rather well tolerated in both cocaine and in the 3-phenyltropane analogue, RTI-55 (5Carroll F.I. Lewin A.H. Kuhar M.J. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 263-295Crossref Google Scholar). Notably, the fluorescent compound, RTI-233, differed, like RTI-55 and the parent 2β-carbomethoxy compound RTI-374 (3β-(4-methylphenyl)tropane-2β-carboxylic acid), from cocaine by having the aromatic ring directly connected to the 3 position of the tropane ring (Fig. 1). The 3-phenyltropane-type of structure was chosen since it is known to increase binding affinity as compared with that of cocaine itself (5Carroll F.I. Lewin A.H. Kuhar M.J. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 263-295Crossref Google Scholar). As shown in Fig. 2, RTI-233 bound with high affinity to the rSERT expressed in Sf-9 cell membranes displaying a KI value of 6 nm for inhibition of 125I-RTI-55 binding (Fig. 2 and TableI). In comparison, the structurally related compound, RTI-55, and cocaine itself displayed KI values of 0.22 and 109 nm, respectively, for inhibition of 125I-RTI-55 binding.Table IBinding properties of purified rSERT in comparison to rSERT in Sf-9 membranesrSERT in Sf-9 membranesPurified rSERTK d[S.E. interval]K i [S.E. interval]Hill slopeK d [S.E. interval]K i [S.E. interval]Hill slopenmnmnmnmRTI-550.22 [0.187–0.260]−1.050.84 [0.771–0.915]−1.055-HT1112 [931–1327]−1.08441 [406–479]−0.99Cocaine109 [106.8–112.0]−1.02200 [194.0–205.6]−0.98RTI-2336.0 [5.06–7.04]−1.1613.7 [12.80–14.60]−0.93Citalopram1.0 [0.96–1.05]−1.012.4 [2.11–2.69]−1.06Competition binding on rSERT expressed in Sf-9 insect cell membranes and on purified transporter using 0.25 nm125I-RTI-55 as radioligand. Values are from three independent experiments performed in triplicates. The K d values were calculated by the equation K d = IC50 − [radioligand]. The IC50 values used for calculation ofK i values were obtained from means of pIC50values determined by nonlinear regression analysis using Prism from GraphPad software (San Diego, CA) and the S.E. interval from pIC50 ± S.E. K i values were calculated according to the equation K i = IC50/(1 + L/K d) where L is concentration of radioligand. Open table in a new tab Competition binding on rSERT expressed in Sf-9 insect cell membranes and on purified transporter using 0.25 nm125I-RTI-55 as radioligand. Values are from three independent experiments performed in triplicates. The K d values were calculated by the equation K d = IC50 − [radioligand]. The IC50 values used for calculation ofK i values were obtained from means of pIC50values determined by nonlinear regression analysis using Prism from GraphPad software (San Diego, CA) and the S.E. interval from pIC50 ± S.E. K i values were calculated according to the equation K i = IC50/(1 + L/K d) where L is concentration of radioligand. The rSERT, expressed in Sf-9 insect cells, was purified by nickel chromatography followed by concanavalin A chromatography. The purified transporter bound RTI-233 with an affinity that was almost identical to that measured in the Sf-9 cell membranes (Fig. 2 and Table I). The KI for RTI-233 was 14 nm for the purified versus 6 nm for the transporter in membranes, as determined from competition binding assays with 125I-RTI-55 (Fig. 2 and Table I). Similarly, the KI values for serotonin and the two blockers RTI-55 and citalopram fo
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