Photoaffinity Labeling with a Neuroactive Steroid Analogue
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m213168200
ISSN1083-351X
AutoresRamin Darbandi-Tonkabon, William R. Hastings, Chun‐min Zeng, Gustav Akk, Brad D. Manion, John Bracamontes, Joseph H. Steinbach, Steven Mennerick, Douglas F. Covey, Alex S. Evers,
Tópico(s)Ion channel regulation and function
ResumoNeuroactive steroids modulate the function of γ-aminobutyric acid, type A (GABAA) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3α,5β-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABAA receptors as evidenced by its ability to inhibit binding of [35S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents inXenopus oocytes and human endothelial kidney 293 cells expressing GABAA receptor subunits (α1β2γ2). [3H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of ∼35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [3H]6-AziP concentration of 1.9 μm, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [3H]6-AziP. The β2and β3 subunits of the GABAA receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABAA receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABAA receptor may be mediated by binding to an accessory protein, VDAC-1. Neuroactive steroids modulate the function of γ-aminobutyric acid, type A (GABAA) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3α,5β-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABAA receptors as evidenced by its ability to inhibit binding of [35S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents inXenopus oocytes and human endothelial kidney 293 cells expressing GABAA receptor subunits (α1β2γ2). [3H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of ∼35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [3H]6-AziP concentration of 1.9 μm, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [3H]6-AziP. The β2and β3 subunits of the GABAA receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABAA receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABAA receptor may be mediated by binding to an accessory protein, VDAC-1. γ-aminobutyric acid GABA, type A 3α,5β-6-azi-3-hydroxypregnan-20-one 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid human endothelial kidney matrix-assisted laser desorption ionization time-of-flight t-butylbicyclophosphorothionate voltage-dependent anion channel tetrahydrofuran electrospray ionization Certain endogenous pregnane steroids and their structural analogues are potent anesthetics in vertebrates (1Selye H. Proc. Soc. Exp. Biol. Med. 1941; 46: 116-121Crossref Scopus (342) Google Scholar, 2Atkinson R.M. Davis B. Pratt M.A. Sharpe H.M. Tomich E.G. J. Med. Chem. 1965; 8: 426-432Crossref PubMed Scopus (70) Google Scholar). These neuroactive steroids produce a rapid and reversible depression of the central nervous system indicating that their actions, unlike those of other steroid hormones, are not mediated by transcriptional regulation. In the 1980s it was demonstrated that neuroactive steroids could modulate the function of γ-aminobutyric acid (GABA),1 type A (GABAA) receptors in the central nervous system (3Majewska M.D. Harrison N.L. Schwartz R.D. Barker J.L. Paul S.M. Science. 1986; 232: 1004-1007Crossref PubMed Scopus (2018) Google Scholar, 4Harrison N.L. Simmonds M.A. Brain Res. 1984; 323: 287-292Crossref PubMed Scopus (449) Google Scholar, 5Harrison N.L. Vicini S. Barker J.L. J. Neurosci. 1987; 7: 604-609Crossref PubMed Google Scholar). Low concentrations of the steroids potentiate the actions of GABA whereas higher concentrations directly open the GABAA receptor ion channel (6Cottrell G.A. Lambert J.J. Peters J.A. Br. J. 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Nathan D. Kalkbrenner M. Nilsson K.R. Hu Y. Zorumski C.F. Evers A.S. J. Pharmacol. Exp. Ther. 2000; 293: 1009-1116PubMed Google Scholar) for their abilities to produce anesthesia and to modulate GABAAreceptor function. These studies showed that both steroid anesthesia and steroid modulation of GABAA receptor function are highly enantioselective, particularly in the case of allopregnanolone. This indicates that neuroactive steroids most likely act via binding to specific recognition sites on the GABAA receptor protein complex, because the enantiomeric pairs have identical physical properties but mirror image shapes. Potentiation of GABA action by neuroactive steroids does not require any specific GABAAsubunit (11Sanna E. Murgia A. Casula A. Biggio G. Mol. Pharmacol. 1997; 51: 484-490PubMed Google Scholar) although the absence of the δ subunit does decrease the sensitivity of the receptor to neuroactive steroids (12Mihalek R.M. Banerjee P.K. Korpi E.R. Quinlan J.J. Firestone L.L. Mi Z.P. Lagenaur C. Tretter V. Sieghart W. Anagnostaras S.G. Sage J.R. Fanselow M.S. Guidotti A. Spigelman I. Li Z. DeLorey T.M. Olsen R.W. Homanics G.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12905-12910Crossref PubMed Scopus (450) Google Scholar). Radioligand binding studies and electrophysiological studies indicate that the putative neuroactive steroid binding sites are not identical to or overlapping with the identified binding sites for benzodiazepines (13Turner D.M. Ransom R.W. Yang J.S. Olsen R.W. J. Pharmacol. Exp. Ther. 1989; 248: 960-966PubMed Google Scholar), GABA (13Turner D.M. Ransom R.W. Yang J.S. Olsen R.W. J. Pharmacol. Exp. Ther. 1989; 248: 960-966PubMed Google Scholar) or picrotoxin (3Majewska M.D. Harrison N.L. Schwartz R.D. Barker J.L. Paul S.M. Science. 1986; 232: 1004-1007Crossref PubMed Scopus (2018) Google Scholar) or to the putative binding site for barbiturates (13Turner D.M. Ransom R.W. Yang J.S. Olsen R.W. J. Pharmacol. Exp. 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Sci. 1995; 16: 162-168Abstract Full Text PDF PubMed Scopus (453) Google Scholar): (i) direct or analogue photolabeling and (ii) generating chimeric subunits between anesthetic-sensitive and insensitive subunits to identify regions of the GABAAreceptor involved in binding and then using site-directed mutagenesis to identify specific amino acids required for anesthetic effect. For example photoaffinity labeling has been successfully used in locating the benzodiazepine (17Mohler H. Battersby M.K. Richards J.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1666-1670Crossref PubMed Scopus (218) Google Scholar, 18Duncalfe L.L. Carpenter M.R. Smillie L.B. Martin I.L. Dunn S.M.J. J. Biol. Chem. 1996; 271: 9209-9214Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and muscimol (19Smith G.B. Olsen R.W. J. Biol. Chem. 1994; 269: 20380-20387Abstract Full Text PDF PubMed Google Scholar) binding sites on the GABAA receptor. Generation of chimeric subunits followed by site-directed mutagenesis has led to the identification of specific amino acids that are critical for the actions of fluorinated ether (20Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.D. Finn S.E. Mascia M.D. Valenzueala C.F. Hanson K.K. Greenblatt E.D. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1102) Google Scholar) and alkane anesthetics (21Greenblatt E.P. Meng X. Anesthesiology. 2001; 94: 1026-1033Crossref PubMed Scopus (16) Google Scholar), etomidate (22Hill-Venning C. Belelli D. Paters J.A. Lambert J.J. Br. J. Pharmacol. 1997; 120: 749-756Crossref PubMed Scopus (205) Google Scholar, 23Belleli D. Lambert J.J. Peters J.A. Wafford K. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 92: 11031-11036Crossref Scopus (351) Google Scholar), and propofol (24Krasowski M.D. Koltchine V.V. Rick C.E. Ye Q. Finn S.E. Harrison N.L. Mol. Pharmacol. 1998; 53: 530-538Crossref PubMed Scopus (246) Google Scholar). Although there remains controversy as to whether these critical amino acids (located at the extracellular end of M2 and M3) are part of a binding site, evidence is accumulating that there is an anesthetic binding pocket formed by residues from M1, M2, M3, and M4 (25Yamakura T. Bertaccini E. Trudell J.R. Harris R.A. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 23-51Crossref PubMed Scopus (237) Google Scholar, 26Jenkins A. Greenblatt E.P. Faulkner J.H. Bertaccini E. Light A. Lin A. Andreasen A. Viner A. Trudell J.R. Harrison N.L. J. Neurosci. 2001; 21: RC136Crossref PubMed Google Scholar). These mutations do not affect the actions of neuroactive steroids (20Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.D. Finn S.E. Mascia M.D. Valenzueala C.F. Hanson K.K. Greenblatt E.D. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1102) Google Scholar). There has been one report that a large region (between the N terminus and the M2 segment) in a β subunit can affect neuroactive steroid action (27Rick C.E. Ye Q. Finn S.E. Harrison N.L. Neuroreport. 1998; 9: 379-383Crossref PubMed Scopus (58) Google Scholar), but no further studies have substantiated or refined this observation. Thus molecular biological studies have, to date, failed to identify candidate regions or sites on the GABAAreceptor that may contribute to a neuroactive steroid binding site. To identify binding sites for the neuroactive steroids, we have developed a steroid anesthetic analogue, 6-AziP, that functions as a photolabeling reagent. Here we describe the synthesis and the characterization of this photolabeling reagent and identification of the major photolabeled protein in rat brain membranes. Rat brains were purchased from Pel-freez (Rogers, AK) and stored until use at −20 °C. Cerebella and brain stem were trimmed from the frozen brains, and the cerebral hemispheres were used to prepare membranes with minor modification of previously described methods (28Hawkinson J. Drewe J.A. Kimbrough C.L. Chen J. Hogenkamp D.J. Lan N.J. Gee K.W. Shen K. Whittemore E.R. Woodward R.M. Mol. Pharmacol. 1996; 49: 897-906PubMed Google Scholar). Briefly, brains were immersed in ice-cold 0.32 m sucrose (10 ml/g) and homogenized using a Teflon pestle in a motor-driven homogenizer. The homogenate was centrifuged for 10 min at 1,500 × g, and the pellet was discarded. The supernatant was centrifuged for 30 min at 10,000 × g to obtain the P2 pellet, which was washed three times with 50 mm potassium phosphate/200 mm NaCl, pH 7.4. The pellet was resuspended in 50 mm potassium phosphate/200 mm NaCl, pH 7.4, and recollected by centrifugation for 20 min at 10,000 ×g. The final pellet was resuspended using a Teflon homogenizer and stored at −80 °C. QT-6 and HEK 293 cells were maintained in culture using standard methods (29Paradiso K. Sabey K. Evers A.S. Zorumski C.F. Covey D.F. Steinbach J.H. Mol. Pharmacol. 2000; 58: 341-351Crossref PubMed Scopus (64) Google Scholar). The cells were passaged at subconfluent densities and were not passaged more than 15 times. Stably transfected cells were produced by standard methods (30Sabey K. Paradiso K. Zhang J. Steinbach J.H. Mol. Pharmcol. 1999; 55: 58-66Crossref PubMed Scopus (59) Google Scholar). In brief, rat α1 subunit cDNA (provided by Dr. A. Tobin) was transferred to pcDNA3 (Invitrogen) and epitope-tagged with the Myc epitope between amino acids 4 and 5 of the predicted mature peptide. Human β1 cDNA (provided by Dr. P. Whiting) was epitope-tagged with the FLAG epitope and transferred to pcDNA3. Quail QT-6 cells were transfected using the calcium phosphate precipitation method as described (31Ueno S. Zorumski C. Bracamontes J. Steinbach J.H. Mol. Pharmacol. 1996; 50: 931-938PubMed Google Scholar), and cells resistant to G418 were selected. A population of cells expressing high levels of surface FLAG and Myc epitopes were selected by sequential rounds of immunoselection using anti-FLAG antibody (M2; Sigma) and anti-Myc antibody (9E10; Invitrogen) (30Sabey K. Paradiso K. Zhang J. Steinbach J.H. Mol. Pharmcol. 1999; 55: 58-66Crossref PubMed Scopus (59) Google Scholar). The enriched population of cells was not cloned. To maximize receptor expression, 2 mm sodium butyrate and 5 mm aminopurine were added to the transfected cells 48 h prior to harvest. Membranes were generated from harvested cells (cells were harvested, and membranes were made in the presence of 0.01 mg/ml soybean trypsin inhibitor, 0.01 mg/ml ova trypsin inhibitor, 0.1 mg/ml bacitracin, 1 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, and 5 mmEDTA) by homogenization in a Tekmar tissue homogenizer followed by centrifugation at 22,000 × g. Membranes were stored at −80 °C for subsequent use. [3H]6-Azi-pregnanolone ([3H]6-AziP) was prepared by multi-step synthesis from commercially available progesterone as outlined in Fig.1. Progesterone was first converted into its 3,20-diketal. The double bond of the 3,20-diketal was then subjected to a hydroboration reaction to introduce a 6-hydroxyl group into the steroid. The 6-hydroxyl group was then oxidized to a 6-keto group. The 6-keto group was then converted into the 6-diaziryl group, and the diketal protecting groups were then removed. Selective reduction of the 3-keto group in the 6-azi-3,20-diketone precursor yields 6-AziP. Selective reduction of the 3-keto group in the same precursor with sodium borotritiide yields [3-3H]6-AziP. Details of the synthetic chemistry are described under “Experimental Procedures.” Melting points were determined on a micro hot stage and are uncorrected. NMR spectra were recorded at ambient temperature at 300 MHz (1H) or 75 MHz (13C). For 1H NMR and 13C NMR spectra, the internal references were tetramethylsilane (δ = 0.00 ppm) and CDCl3 (δ = 77.00 ppm). Elemental analyses were carried out by M-H-W Laboratories, Phoenix, AZ. Solvents were either used as purchased or dried and purified by standard methodology. Extraction solvents were dried with anhydrous Na2SO4 and removed on a rotary evaporator under water aspirator vacuum. Column chromatography was performed using flash column grade silica gel (32–63 μm) purchased from Scientific Adsorbents, Atlanta, GA. A solution of borane-tetrahydrofuran complex (1.7 ml, 1 m borane in tetrahydrofuran) was added dropwise at room temperature to a stirred solution of 3,20-diketal1 (234.4 mg, 0.58 mmol; prepared from progesterone in the usual manner (32Antonucci R. Bernstein S. Lenhard R. Sax K.J. Williams J.H. J. Org. Chem. 1952; 17: 1369-1374Crossref Scopus (18) Google Scholar)) in tetrahydrofuran (THF; 30 ml). The resulting mixture was stirred at room temperature for 5 h. After cooling to 0 °C in an ice-water bath, 3 n aqueous sodium hydroxide (1.7 ml) and 30% aqueous hydrogen peroxide (1.7 ml) were added. Immediately following the addition of the hydrogen peroxide, the cooling bath was removed, and the mixture was stirred overnight. After removal of the THF on a rotary evaporator, the residue was mixed with diethyl ether (Et2O; 200 ml), and the Et2O was washed with water and brine and then dried and removed. The residue was purified by column chromatography (silica gel e1uted with 5–20% ethyl acetate in hexanes) to give products 2 (131.1 mg),3 (47.3 mg), and 4 (41.6 mg) in a ratio of 3.2:1.1:1 in a total yield of 90%. (5β,6β)-6-Hydroxypregnane-3,20-dione, cyclic bis(1,2-ethanediyl acetal) (2) was obtained as a white solid, and properties were as follows: m.p. 201–203 °C; IR (KBr) 3529, 2942, 2874, 1052, 1018 cm−1; 1H NMR (CDCl3) δ 3.77–3.94 (8H, m), 3.64 (1H, bs), 1.23 (3H, s), 1.07 (3H, s), 0.71 (3H, s); 13C NMR (CDCl3) δ 111.89, 109.34, 72.52, 65.04, 64.12, 64.01, 63.07, 58.20, 56.13, 47.50, 42.00, 40.00, 39.55, 35.62, 34.48, 34.19, 34.02, 29.94, 29.50, 25.13, 24.36, 23.55, 22.81, 20.44, 12.88. The elemental analysis results for C25H40O5 were as follows. Predicted: C, 71.39%; H, 9.59%; found: C, 71.52%; H, 9.53%. (5α,6α)-6-Hydroxypregnane-3,20-dione, cyclic bis(1,2-ethanediyl acetal) (3) was obtained as a white solid, and properties were as follows: m.p. 186–188 °C; IR (KBr) 3461, 2935, 2874, 1089, 1042 cm−1; 1H NMR (CDCl3) δ 3.78–3.94 (8H, m), 3.31 (1H, dt, J = 10.5 Hz, 4.2 Hz), 1.22 (3H, s), 0.76 (3H, s), 0.68 (3H, s); 13C NMR (CDCl3) δ 111.91, 109.20, 69.56, 65.10, 64.12, 64.07, 63.13, 58.14, 55.98, 53.43, 50.62, 41.93, 41.66, 39.36, 36.18 (2 × C), 33.69, 32.03, 30.91, 24.40, 23.60, 22.76, 20.74, 12.87, 12.43. Elemental analysis results for C25H40O5 were as follows. Predicted: C, 71.39%; H, 9.59%; found: C, 71.24%; H, 9.68%. (5α)-5-Hydroxypregnane-3,20-dione, cyclic bis(1,2-ethanediyl acetal) (4) was obtained as a white solid that was only partially characterized. The stereochemistry assigned to the hydroxyl group in the structure should be considered as tentative. 1H NMR (CDCl3) δ 3.78–3.95 (8H, m), 2.25 (1H, d,J = 14.1 Hz), 1.22 (3H, s), 0.87 (3H, s), 0.68 (3H, s);13C NMR (CDCl3) δ 111.91, 110.13, 74.71, 65.10, 64.31, 63.93, 63.11, 58.14, 56.42, 43.16, 41.82, 40.20, 39.50, 34.42, 34.05, 29.88, 28.83, 28.15, 24.37, 23.58, 22.81, 21.20, 16.39, 12.81. Pyridinium dichromate (5.63 g, 14.97 mmol) was added to a solution of steroid 2 (1.68 g, 4.00 mmol) in dichloromethane (30 ml). The mixture was stirred at room temperature for 10 h and diluted with Et2O. The Et2O solution was washed with water and then brine and dried. Solvent removal gave a residue that was purified by column chromatography on silica gel (10% ethyl acetate in hexanes) to give steroid 5(1.64 g, 98.3%) as a white solid. Properties were as follows: m.p. 133–135 °C; IR (KBr) 2949, 2867, 1708, 1089 cm−1;1H NMR (CDCl3) δ 3.85–4.02 (8H, m), 2.36 (1H, dd, J = 13.5 Hz, 5.2 Hz), 1.30 (3H, s), 0.87 (3H, s), 0.75 (3H, s); 13C NMR (CDCl3) δ 213.78, 111.75, 108.02, 65.13, 64.33, 64.30, 63.16, 58.36, 58.08, 56.71, 42.61, 42.43, 39.32, 39.24, 37.82, 36.33, 34.31, 33.25, 30.09, 24.43, 23.41, 22.88, 22.78, 20.82, 12.82. Elemental analysis results for C25H38O5 were as follows. Predicted: C, 71.74%; H, 9.15%; found: C, 71.67%; H, 9.29%. Anhydrous ammonia gas was bubbled into a stirred solution of steroid 5 (304.1 mg, 0.73 mmol) in absolute MeOH (25 ml) at 0 °C until the MeOH was saturated. The solution was further stirred at 0 °C for 2 h. Then, a solution of hydroxylamine-O-sulfonic acid (391.4 mg, 3.46 mmol) in MeOH (6 ml) was added at 0 °C, and the mixture was stirred at room temperature overnight. After filtration to remove precipitated ammonium sulfate the filtrate was mixed with MeOH (30 ml), ethyl acetate (5 ml), and triethylamine (1 ml). Iodine dissolved in MeOH was then added dropwise while stirring until a yellow color persisted. The mixture was diluted with Et2O (300 ml), and the Et2O was washed successively with 10% aqueous sodium thiosulfate, water, and brine and dried. The residue obtained after solvent removal was purified by column chromatography on silica gel (5% ethyl acetate in hexanes) to give a mixture of diazirines (56.3 mg, 51.1% yield), recovered starting material, and the 5α-epimer of the starting material (196.9 mg total for the combined 5β- and 5α-epimers). Treatment of the mixture of diazirines (56.3 mg) withp-toluenesulfonic acid (35.7 mg) in acetone (10 ml) gave, after column chromatography on silica gel (10% ethyl acetate in hexanes), the purified products 6 (16.1 mg) and 7(20.9 mg). Product 6 (16.1 mg) was a solid, and properties were as follows: m.p. 136–138 °C; IR (KBr) 2942, 2908, 1718, 1708, 1351, 1150 cm−1; 1H NMR (CDCl3) δ 2.70 (1H, dd, J = 15.3 Hz, 13.2 Hz), 2.56 (1H, t,J = 9.3 Hz), 2.14 (3H, s), 1.29 (3H, s), 0.69 (3H, s), 0.62 (1H, dd, J = 13.2 Hz, 4.8 Hz), 0.38 (1H, dd,J = 13.5 Hz, 3.6 Hz); 13C NMR (CDCl3) δ 210.09, 209.20, 63.42, 56.19, 50.29, 44.11, 40.41, 38.68, 37.16, 36.66, 36.36, 35.77, 33.78, 32.40, 31.38, 28.29, 23.98, 22.78, 22.23, 20.88, 13.28. Elemental analysis results for C21H30N2O2 were as follows. Predicted: C, 73.65%; H, 8.83%; N, 8.18%; found: C, 73.76%; H, 8.73%; N, 8.16%. Product 7 (20.9 mg) was a solid, and properties were as follows: decomposed on heating above 152 °C; IR (KBr) 2949, 2867, 1704, 1354, 1235 cm−1; 1H NMR (CDCl3) δ 2.53 (1H, t, J = 9.0 Hz), 2.13 (3H, s), 1.35 (3H, s), 0.68 (3H, s), 0.48 (1H, dd, J = 13.8 Hz, 4.2 Hz); 13C NMR (CDCl3) δ 209.87, 209.29, 63.39, 55.86, 53.02, 46.54, 43.99, 39.47, 38.51, 37.74, 37.66, 37.30, 36.95, 33.61, 31.35, 28.74, 23.98, 22.66, 21.21, 13.28, 12.20. Lithium tri-t-butoxyaluminium hydride (37.3 mg, 0.15 mmol) in THF (0.3 ml) was added to a stirred solution of steroid 6 (49.3 mg, 0.14 mmol) in THF (10 ml) at 0 °C. The reaction was stirred at 0 °C until monitoring by silica gel thin layer chromatography (30% ethyl acetate in hexanes) showed that the starting material had reacted completely. The reaction was quenched by adding acetone (0.5 ml). Water (5 ml) was then added, and the product was extracted into Et2O. Solvent removal gave a residue that was purified by column chromatography on silica gel (15% ethyl acetate in hexanes) to give azisteroid 8 (40.4 mg, 81.5%) as a white solid. Properties were as follows: m.p. 95–97 °C; IR (KBr) 3392, 2935, 2867, 1701, 1354, 1059 cm−1;1H NMR (CDCl3) δ 3.51 (1H, m), 2.54 (1H, t,J = 9.3 Hz), 2.13 (3H, s), 1.18 (3H, s), 0.64 (3H, s), 0.28 (1H, dd, J = 14.4 Hz, 4.2 Hz), 0.20 (1H, dd,J = 12.9 Hz, 4.2 Hz); 13C NMR (CDCl3) δ 209.50, 70.31, 63.51, 56.29, 48.52, 44.17, 39.64, 38.77, 36.28, 34.42, 33.86, 32.90, 31.38, 31.20, 29.61, 28.97, 24.01, 22.93, 22.72, 20.44, 13.23. Elemental analysis results for C21H32N2O2 were as follows. Predicted: C, 73.22%; H, 9.63%; N, 8.13%; found: C, 73.16%; H, 9.30%; N, 8.38%. [3H]NaBH4 in 1 ml of 0.1 m NaOH (Amersham Biosciences, 25 mCi, specific activity 23 Ci mmol−1) was added to a stirred solution of azisteroid 6 (2.046 mg, 5.98 μmol) in ethanol (2 ml). The mixture was stirred at room temperature for 3 h. A drop of acetic acid and then water (1 ml) were added. The mixture was extracted with Et2O (4 × 1.5 ml). The combined organic phases were evaporated under a stream of air, and the residue was dissolved in chloroform (300 μl) and subjected to preparative TLC (hexane:ethyl acetate, 1:1). Radioactive bands on the dried plate were visualized by photoimaging. The only detectable products were [3H]azisteroid 9 and a band of slightly higher mobility, presumably the epimeric 3α-hydroxysteroid, [3-3H](3α,5β)-6-Azi-3-hydroxypregnan-20-one (10), in a ratio of 2.4:1. The radioactive bands were scraped from the plate, and the silica gel was packed in a small pipette and washed with ethyl acetate (4 ml). [3H]Azisteroid 9 (8.2 mCi) and presumed [3H]azisteroid 10 (3.5 mCi) were obtained after solvent removal. The purified radiolabeled products were stored in ethanol at −20 °C. To assure the purity of 6-AziP and [3H]6-AziP, each of the steroids was analyzed by thin layer chromatography before and after ultraviolet irradiation. Ethanolic solutions of the steroids were irradiated for 5 min using a 450-watt Hanovia medium pressure mercury lamp. The steroid samples were applied to reverse-phase silica chromatography plates (Fisher Scientific, Pittsburgh, PA) and developed with a mobile phase of 95% acetonitrile/5% H2O. Detection was facilitated by charring the plates following an aerosol application of 5% sulfuric acid/95% ethanol. The Rf values for non-irradiated 6-AziP was 0.53, whereas the Rfvalue for irradiated 6-AziP was 0.42. Using a charred TLC plate as a guide, TLC plates containing [3H]6-AziP were scored into 5-mm bands and scraped. Radiolabeled steroid was extracted from silica into chloroform:methanol (2:1) and analyzed by scintillation spectrometry using Scintilene scintillation mixture (Fisher Scientific, Pittsburgh, PA). TLC was also conducted using straight phase HP-K high performance silica gel TLC plates (Whatman catalog number 4807-700) and a 1:1 hexane:ethyl acetate solvent system. [35S]TBPS binding assays were performed using previously described methods (10Covey D.F. Nathan D. Kalkbrenner M. Nilsson K.R. Hu Y. Zorumski C.F. Evers A.S. J. Pharmacol. Exp. Ther. 2000; 293: 1009-1116PubMed Google Scholar,33Hawkinson J.E. Kimbrough C.L. Belelli D. Lambert J.J. Purdy R.H. Lan N.C. Mol. Pharmacol. 1994; 46: 977-985PubMed Google Scholar) with modification. Briefly, aliquots of membrane suspension (0.5 mg/ml final protein concentration in assay) were incubated with 5 μm GABA (Sigma), 2–4 nm[35S]TBPS (60–100 Ci/mmol; PerkinElmer Life Sciences), and 5-μl aliquots of steroid in Me2SO solution (final steroid concentrations ranged from 1 nm to 10 μm), in a total volume of 1 ml of 200 mmNaCl, 50 mm potassium phosphate buffer, pH 7.4. Control binding was defined as binding observed in the presence of 0.5% Me2SO and the absence of steroid; all assays contained 0.5% Me2SO. Nonspecific binding was defined as binding observed in the presence of 200 μm picrotoxin and ranged from 6.1 to 14.3% of total binding. Assay tubes were incubated for 2 h at room temperature. A Brandel (Gaithersburg, MD) cell harvester was used for filtration of the assay tubes through Whatman/GF/C filter paper. Filter paper was rinsed with 4 ml of ice-cold buffer three times and dissolved in 4 ml of ScintiVerse II (Fisher Scientific, Pittsburgh, PA). Radioactivity bound to the filters (B) was measured by liquid scintillation spectrometry, and data were fit using Sigma Plot to the Hill equation, B= Bmax/{1 + ([C]/IC50)n}, whereBmax is control binding, [C] is steroid concentration, IC50 is the half-maximal inhibitor concentration, and n is the Hill coefficient. Each data point was determined in triplicate. [3H]muscimol binding assays were performed using a previously described method with minor modification (4Harrison N.L. Simmonds M.A. Brain Res. 1984; 323: 287-292Crossref PubMed Scopus (449) Google Scholar, 34Lopez-Colome A.M. McCarthy M. Beyer C. Eur. J. Pharmacol. 1990; 176: 297-303Crossref PubMed Scopus (48) Google Scholar). Briefly, membranes were thawed and washed four times in 20 mm potassium phosphate, 100 mm KCl, 0.1 mm EDTA, pH 7.4, to remove endogenous GABA. Binding incubations contained aliquots of membrane suspension (0.5 mg/ml final protein concentration in assay), 5 nm[3H]muscimol (28 Ci/mmol; PerkinElmer Life Sciences), 10 μm steroid, and 20 mm potassium phosphate, 100 mm KCl, 0.1 mm EDTA, pH 7.4, in a total volume of 0.5 ml. Assay tubes were incubated for 1 h at 4 °C in the dark. Control binding was defined as binding observed in the presence of 0.5% Me2SO and the absence of steroid; all assays contained 0.5% Me2SO. Nonspecific binding was defined as binding observed in the presence of 100 μmGABA. Assays were conducted in the presence or the absence of 10 μm 6-AziP or pregnanolone. Membranes were collected on Whatman/GF/C glass filter paper using a Brandel cell harvester (Gaithersburg, MD). Radioactivity bound to the filters was measured by liquid scintillation spectrometry using ScintiVerse II (Fisher Scientific, Pittsburgh, PA). Each data point was determined in triplicate. Stage V-VI oocytes were harvested from sexually mature female Xenopus laevis (Xenopus One, Northland, MI) anesthetized with 0.1% tricaine (3-aminobenzoic acid ethyl ester), according to institutionally approved protocols. Oocytes were defolliculated by shaking for 20 min at 37 °C in collagenase (2 mg/ml) dissolved in calcium-free solution containing the following (in mm): 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES at pH 7.4. Capped mRNA encoding rat GABAA receptor α1, β2, and γ2L subunit
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