Photoaffinity Labeling with a Neuroactive Steroid Analogue

6-AZI-PREGNANOLONE LABELS VOLTAGE-DEPENDENT ANION CHANNEL-1 IN RAT BRAIN*

Ramin Darbandi-TonkabonDagger , William R. HastingsDagger , Chun-Min Zeng§, Gustav AkkDagger , Brad D. ManionDagger , John R. BracamontesDagger , Joseph H. SteinbachDagger , Steven J. Mennerick, Douglas F. Covey§, and Alex S. EversDagger §||

From the Departments of Dagger  Anesthesiology, § Molecular Biology and Pharmacology, and  Psychiatry, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, December 26, 2002, and in revised form, January 29, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

Neuroactive steroids modulate the function of gamma -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, 3alpha ,5beta -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 in Xenopus oocytes and human endothelial kidney 293 cells expressing GABAA receptor subunits (alpha 1beta 2gamma 2). [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 beta 2 and beta 3 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

Certain endogenous pregnane steroids and their structural analogues are potent anesthetics in vertebrates (1, 2). 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 gamma -aminobutyric acid (GABA),1 type A (GABAA) receptors in the central nervous system (3-5). Low concentrations of the steroids potentiate the actions of GABA whereas higher concentrations directly open the GABAA receptor ion channel (6, 7). These observations led to the hypothesis that neuroactive steroids produce anesthesia by activating GABAA receptors and thus enhancing inhibitory synaptic transmission. The strong correlation between the ability of various neuroactive steroid analogues to modulate GABAA receptors and their ability to produce anesthesia strongly supports this hypothesis (8).

The mechanism by which neuroactive steroids modulate GABAA receptor function remains unclear. In previous work we have tested the enantiomers of allopregnanolone (9) and pregnanolone (10) for their abilities to produce anesthesia and to modulate GABAA receptor 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 GABAA subunit (11) although the absence of the delta  subunit does decrease the sensitivity of the receptor to neuroactive steroids (12). 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 (13), GABA (13) or picrotoxin (3) or to the putative binding site for barbiturates (13, 14).

A variety of anesthetic agents, including propofol, etomidate, benzodiazepines, and the halogenated alkanes and ethers, have also been shown to modulate GABAA receptor function (15). Specific binding sites for some of these agents have been identified using two approaches (16): (i) direct or analogue photolabeling and (ii) generating chimeric subunits between anesthetic-sensitive and insensitive subunits to identify regions of the GABAA receptor 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 (17, 18) and muscimol (19) 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 (20) and alkane anesthetics (21), etomidate (22, 23), and propofol (24). 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 (25, 26). These mutations do not affect the actions of neuroactive steroids (20). There has been one report that a large region (between the N terminus and the M2 segment) in a beta  subunit can affect neuroactive steroid action (27), 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 GABAA receptor 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

Membrane Preparation-- 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 (28). 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.

Tissue Culture-- QT-6 and HEK 293 cells were maintained in culture using standard methods (29). The cells were passaged at subconfluent densities and were not passaged more than 15 times. Stably transfected cells were produced by standard methods (30). In brief, rat alpha 1 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 beta 1 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 (31), 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) (30). 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 mM EDTA) by homogenization in a Tekmar tissue homogenizer followed by centrifugation at 22,000 × g. Membranes were stored at -80 °C for subsequent use.

Chemical Synthesis-- [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."


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Fig. 1.   Chemical synthesis of 6-AziP. Synthetic scheme for the preparation of 6-AziP (8) and [3H]6-AziP (9) from progesterone.

General Methods-- 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 (delta  = 0.00 ppm) and CDCl3 (delta  = 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.

Hydroboration of Pregnane-3,20-dione, Cyclic Bis(1,2-ethanediyl acetal) (1)-- 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-diketal 1 (234.4 mg, 0.58 mmol; prepared from progesterone in the usual manner (32)) 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%.

(5beta ,6beta )-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) delta  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) delta  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%.

(5alpha ,6alpha )-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) delta  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) delta  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%.

(5alpha )-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) delta  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) delta  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.

Pregnane-3,6,20-Trione, Cyclic 3,20-Bis(1,2-ethanediylacetal) (5)-- 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) delta  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) delta  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%.

(5beta )-6-Azipregnane-3,20-dione (6) and (5alpha )-6-Azipregnane-3,20-dione (7)-- 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 5alpha -epimer of the starting material (196.9 mg total for the combined 5beta - and 5alpha -epimers). Treatment of the mixture of diazirines (56.3 mg) with p-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) delta  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) delta  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) delta  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) delta  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.

(3alpha ,5beta )-6-Azi-3-hydroxypregnan-20-one (8)-- 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) delta  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) delta  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%.

[3-3H](3alpha ,5beta )-6-Azi-3-hydroxypregnan-20-one (9)-- [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 3alpha -hydroxysteroid, [3-3H](3alpha ,5beta )-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.

Thin Layer Chromatography-- 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 Rf value 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-- [35S]TBPS binding assays were performed using previously described methods (10, 33) 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 mM NaCl, 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}, where Bmax 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-- [3H]muscimol binding assays were performed using a previously described method with minor modification (4, 34). 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 µM GABA. 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.

Xenopus Oocyte Expression Studies-- 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 alpha 1, beta 2, and gamma 2L subunits was transcribed in vitro using the mMESSAGE mMachine Kit (Ambion, Austin, TX) from linearized pBluescript vectors containing receptor coding regions. Subunit transcripts were injected in equal parts (20-40 ng total RNA) 8-24 h after defolliculation. Before experiments, oocytes were incubated up to 5 days at 18 °C in ND96 medium containing the following (in mM): 96 NaCl, 1 KCl, 1 MgCl2, 2 CaCl2, and 10 HEPES at pH 7.4, supplemented with pyruvate (5 mM), penicillin (100 units/ml), streptomycin (100 µg/ml), and gentamycin (50 µg/ml). The cDNAs for the rat GABAA receptor subunits were provided by A. Tobin, University of California, Los Angeles, CA (alpha 1), P. Malherbe, Hoffmann-La Roche (beta 2), and C. Fraser, National Institute on Alcohol Abuse and Alcoholism (gamma 2L).

Oocyte Electrophysiology-- Two-electrode voltage-clamp experiments were performed with a Warner OC725 amplifier 2-5 days after RNA injection. The extracellular recording solution was ND96 medium with no supplements. Intracellular recording pipettes were filled with 3 M KCl and had open tip resistances of ~1 megohm. Drugs were applied from a common tip via a gravity-driven multi-barrel drug delivery system. Cells were clamped at -70 mV for all experiments, and current at the end of 20-30-s drug applications was measured for quantification of current amplitudes. Potentiator and GABA were co-applied with no pre-equilibration of potentiator.

Single-channel Recording-- Single-channel currents were recorded and analyzed as described previously (35). In brief, the currents were recorded from HEK 293 cells transiently transfected with alpha 1beta 1gamma 2L subunits in the cell-attached configuration at pipette potentials of +60 to +85 mV. The receptors were activated by desensitizing concentrations of GABA. The analysis was carried out on single-channel clusters using the QUB suite (36).

Electrophoresis, Western Blot, and Gel Slicing-- Polyacrylamide gel electrophoresis was performed using 10% polyacrylamide gels, under reducing conditions (37). After electrophoresis, the gels were stained, sliced, or used for Western blot. Gels were silver-stained (38) using modified ammoniacal silver stain (Amersham Biosciences) or stained with Coomassie Blue using a Novex Colloidal Coomassie G-250 kit (Invitrogen).

For gel slicing, the gels were cut in vertical columns and sliced in 1-mm horizontal slices using a DE 113 manual gel slicer (Hoeffer Scientific Instruments, San Francisco, CA). Slices were digested with 4 ml of tissue solubilizer consisting of 3a20TM and TS-2 (ratio 9:1) for 24 h, and the radioactivity in each slice was determined by scintillation spectrometry.

For Western blotting, proteins from the SDS-PAGE gels were transferred onto TropifluorTM polyvinylidene difluoride membrane. For immunoblotting with the bd17 (anti-GABAA receptor beta 2, beta 3 subunit) monoclonal antibody (Roche Molecular Biochemicals), the membranes were blocked with 5% dried milk for 1 h, washed three times with Tris-buffered saline (0.05%)-Tween 20 and incubated overnight with bd17 antibody (20 µg/ml). The membranes were then incubated for 2 h with peroxidase-conjugated anti-mouse IgG (1:1000). Immunoreactive bands were visualized using the ECL-plus Western blotting detection system (Amersham Biosciences). For immunoblotting with the anti-VDAC-1 monoclonal antibody (Oncogene Research Products), the membranes were blocked with 5% dried milk for 1 h, washed three times with Tris-buffered saline (0.05%)-Tween 20, and incubated overnight with anti-VDAC-1 antibody (2 µg/ml). The membranes were then incubated for 2 h with peroxidase-conjugated anti-mouse IgG (1:1000). Immunoreactive bands were visualized using the ECL-plus Western blotting detection system (Amersham Biosciences). Immunoblotting with the anti-VDAC-1 polyclonal antibody (Oncogene Research Products) was performed as described above, with the exception that peroxidase-conjugated anti-rabbit IgG (1:1000) was used as secondary antibody.

Photolabeling-- For photolabeling, rat brain membranes were placed in a quartz cuvette in buffer (50 mM potassium phosphate buffer, pH 7.4, 150 mM NaCl, 5 µM GABA) at a concentration of 400 µg of membrane protein/ml and pre-incubated with [3H]6-AziP (±competitor compound) for 90 min at 4 °C in the dark. These are the same buffer conditions in which 6-AziP modulates [35S]TBPS binding in rat brain membranes, assuring that the steroid will have access to its site of action. The cuvette was then placed in a photoreactor at a distance of 8 cm from the source. The photoreactor uses a 450-watt Hanovia medium pressure mercury lamp as the light source. The lamp is cooled by a circulating cold water jacket, and the light is filtered through a 1.5-cm-thick saturated copper sulfate solution. This filter absorbs all light of wavelength < 315 nM (39). The samples were routinely irradiated for 3 min and continuously cooled to 4 °C. The membrane concentration and duration of irradiation were based on preliminary experiments showing that [3H]6-AziP is completely photolyzed and that label incorporation is maximal under these conditions (see "Results"). Following irradiation the membranes were harvested by centrifugation and solubilized in SDS sample buffer (312.5 mM Tris-HCl, 5% SDS, 0.5 M dithiothreitol, 50% glycerol, and 0.1% bromphenol blue) and analyzed by electrophoresis on a 10% SDS-PAGE gel. The gels were sliced, and radioactivity was measured in each slice. The data were analyzed as described by Bureau and Olsen (40), integrating the area under the graph of radioactivity versus slice number and plotting the resultant data as a concentration-effect curve for each peak observed on the gel.

Autoradiography-- For autoradiography, photolabeled membranes were lysed in SDS sample buffer, and labeled proteins were separated by SDS-PAGE. The resultant gels were fixed for 30 min in isopropanol:water:acetic acid (25:65:10) at room temperature and then dried under vacuum. The dried gels were placed in cassettes and exposed to [3H]-sensitive ultrafilm (Eastman Kodak Co. Biomax light film) at -70 °C for periods ranging from 5 days to 2 weeks.

Immunoprecipitation-- Membranes were lysed in 1 ml of 1% Triton X-100, 150 mM NaCl, 50 mM Tris, 1 mM EDTA, pH 7.4 (lysis buffer), for 60 min at 4 °C. The suspension was then centrifuged at 16,000 × g, and the supernatant (lysate) was retained for immunoprecipitation experiments. For precipitation of GABAA receptors, bd17 monoclonal antibody (Roche Molecular Biochemicals) directed against the beta 2-beta 3 subunits of the GABAA receptor was used. Lysate was incubated with bd17 antibody overnight at 4 °C, and anti-mouse IgG attached to agarose beads was used as secondary antibody (41). VDAC-1 was immunoprecipitated using monoclonal antibody directed against the N terminus of VDAC-1 (Oncogene Research Products). Lysate was incubated with VDAC-1 antibody overnight at 4 °C, and protein A and G attached to agarose beads was used as secondary antibody. Immunoprecipitated protein was eluted from the agarose beads by boiling in 5 × SDS sample buffer for 10 min.

Two-dimensional Electrophoresis-- Photolabeled membranes were washed with buffer and pooled to yield the requisite amount of protein (500 µg-2 mg) for each two-dimensional gel. The pelleted membranes were resuspended in lysis buffer (40 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS, 2% SB-310, 2 mM tributyl phosphine, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of pepstatin A, chymostatin, leupeptin, and antipain) at a concentration of 2.0 mg protein/ml lysis buffer (42). The suspension was sonicated and centrifuged at 150,000 × g for 45 min at 4 °C. Protein in the lysate was measured with a Bradford protein assay. The lysate was then mixed with rehydration buffer (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB-310, 40 mM Tris, 0.2% ampholyte, 50 mM dithiothreitol) and used to rehydrate a 13-cm immobilized pH gradient Dry Strip in a stripholder for 16 h at 20 °C. The strip was focused on an IPGphor isoelectric focusing system (Amersham Biosciences) using the following protocol: 200 V for 30 min (gradient), 8000 V for 2.5 h (gradient), and 8000 V for 50,000-80,000 V-h. Following the isoelectric focusing, the strips were incubated in equilibration buffer (50 mM Tris-Cl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 1% dithiothreitol) and then placed on top of a 10% SDS-polyacrylamide gel that was run for 3 h at 200 V. The final gels were either processed for autoradiography or stained with silver stain or Coomassie Blue. In initial experiments the photolabeled spots were identified by autoradiography. In subsequent experiments, two samples were processed in tandem, one in which membranes were photolabeled with non-tritiated 6-AziP and one in which membranes were photolabeled with [3H]6-AziP. The samples were simultaneously focused and subjected to electrophoresis in the same run on a multi-gel electrophoresis apparatus. The non-radioactive gel was stained with Coomassie Blue. The gel with tritiated sample was silver-stained, and all stained spots in the area identified in the original autoradiography were excised, digested, and analyzed by scintillation spectrometry. In this way, protein spot(s) covalently labeled by [3H]6-AziP could be precisely identified. The spots on the Coomassie Blue-stained gel corresponding to spots containing maximum radioactivity were manually excised. The excised samples were prepared for mass spectrometry by in-gel proteolytic digestion (43) and subsequently analyzed by MALDI-TOF/MS and ESI/MS. MALDI-TOF analysis was performed at Protein and Nucleic Acid Chemistry Laboratories (PNACL) at Washington University School of Medicine. The gel pieces were digested with Promega (Madison, WI) sequencing grade-modified trypsin. The recovered tryptic peptides were analyzed by MALDI-TOF mass spectrometry on an Applied Biosystems (Foster City, CA) Voyager DE-PRO mass spectrometer. Automated data acquisition and automated data base searching was performed with Applied Biosystems Proteomics Solution 1 (PS1) software. ESI/MS sequence analysis was performed at Harvard Microchemistry Facility by microcapillary reverse-phase HPLC nanoelectrospray tandem mass spectrometry (µLC/MS/MS) on a Finnigan LCQ DECA quadruple ion trap mass spectrometer.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

Characterization of 6-AziP-- Initial thin layer chromatography experiments were performed to assess the purity of [3H]6-AziP. The results show that virtually all of the radioactivity in non-irradiated samples of [3H]6-AziP migrated as a single band with the same Rf value as unlabeled 6-AziP (Rf = 0.54). None of the radioactivity in the irradiated sample (Rf = 0.45) migrated with 6-AziP (Rf = 0.42); all of the counts migrated at a higher Rf (0.54) presumably as an ethanol adduct. To determine optimal time for irradiation, the UV-induced decay of a 5.8 mM ethanolic solution of 6-AziP was monitored during an irradiation period of 300 s. The diazirine band at 352 nm decayed exponentially as a function of time with a time constant of 12 s.

Biological Activity of 6-AziP-- To determine whether 6-AziP has the biological activity of a neuroactive steroid, electrophysiological and radioligand binding studies were performed. Fig. 2A shows the effect 10 µM 6-AziP on the current elicited by 2 µM GABA in oocytes expressing GABAA (alpha 1beta 2gamma 2L) receptors. In the absence of GABA, 6-AziP (10 µM) did not elicit any detectable current. 6-AziP reversibly tripled the peak amplitude of the GABA response, without changing the baseline membrane current level as evidenced with the wash-out step. Fig. 2C summarizes the potentiation by 6-AziP relative to responses gated by 2 µM GABA alone in an oocyte expressing the alpha 1beta 2gamma 2L subunit combination, voltage-clamped at -70 mV. 6-AziP potentiated GABA-elicited currents with an EC50 of 2.8 µM. In the same assay, pregnanolone produces an 5.3-fold enhancement of the peak amplitude of the GABA response with an EC50 of 1.1 µM.


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Fig. 2.   Biological activity of 6-AziP. The effect of 6-AziP on GABA responses in Xenopus oocytes is shown. A, responses to 2 µM GABA alone and in the presence of increasing concentrations of 6-AziP are shown in an oocyte expressing the alpha 1beta 2gamma 2L subunit combination and voltage-clamped at -70 mV. 6-AziP reversibly tripled the peak amplitude of the GABA response, without changing the baseline membrane current level. B, single-channel clusters elicited by 50 µM GABA in the absence and presence of 10 µM 6-AziP. Open events are shown upwards. The two upper traces show entire clusters at lower time resolution, whereas the two lower traces show segments (indicated by bars over the upper traces) at expanded resolution. The scale bar shows 1 pA (all traces) and 500 ms (upper row) or 67 ms (lower row). For the figure, data were refiltered and resampled at 1-ms intervals. C, the response to 2 µM GABA in the presence 6-AziP normalized to the response in the same oocyte to 2 µM GABA alone is shown (------). For comparison, the mean open times from single-channel records normalized to the mean duration in the presence of 50 µM GABA alone (triangle ) are shown on the same axes. Data are shown as mean ± 1 S.E. The curve through the oocyte data shows an arbitrary fit of the Hill equation with maximal normalized response 3.0, EC50 2.8 µM, and Hill coefficient fixed to 2.0. The dotted lines show error limits for the mean open time recorded with 50 µM GABA alone.

We also looked at the effect of 6-AziP on single-channel currents elicited by 50 µM GABA in cell-attached patches recorded from the HEK 293 cells transiently transfected with alpha 1beta 2gamma 2L subunits. Sample clusters, recorded in the absence and presence of 10 µM steroid, are shown in Fig. 2B. The normalized mean intracluster open interval durations in the absence and presence of 1-10 µM 6-AziP are given in Fig. 2C. The data show that the application of steroid leads to an increase in the channel mean open duration. The mean open duration is 2.5 ± 0.3 ms (4 patches) under control conditions and 3.5 (1 patch), 5.0 ± 0.3 (3 patches), and 8.6 ± 1.7 ms (2 patches) in the presence of 1, 5, or 10 µM 6-AziP, respectively. Comparison of single-channel open durations with the potentiation of whole-cell currents (Fig. 2C) suggests that the potentiation of whole-cell currents in oocytes can be largely accounted for by an increase in the channel mean open durations.

Effect of 6-AziP on [35S]TBPS Binding-- Neuroactive steroids are known to allosterically inhibit binding of the caged convulsant of [35S]TBPS to the picrotoxin binding site of GABAA receptors (3). Fig. 3A shows that 6-AziP completely inhibits [35S]TBPS binding in rat brain membranes with an IC50 of 331 ± 43 nM and a Hill slope of 0.90 ± 0.09. For comparison, pregnanolone inhibits [35S]TBPS binding with an IC50 of 70 nM.


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Fig. 3.   Effect of 6-AziP on [35S]TBPS binding and [3H]muscimol binding. A, 6-AziP inhibits [35S]TBPS binding in rat brain membranes () with an IC50 of 331 ± 43 nM and a Hill Slope of 0.90 ± 0.09 and in QT-6 alpha 1mycbeta 1FLAG cells (open circle ) with an IC50 of 186 ± 35 nM and a Hill slope of 0.81 ± 0.10. B, the effects of 6-AziP and pregnanolone on [3H]muscimol binding in rat brain membranes. 10 µM 6-AziP produced a 242 ± 11% enhancement of specific muscimol binding to rat brain membranes. This is similar to the 227 ± 10% enhancement produced by pregnanolone.

Effects of 6-AziP on [3H]Muscimol Binding-- Neuroactive steroids are also positive allosteric modulators of [3H]muscimol binding to GABAA receptors (4, 34). To determine whether 6-AziP produces effects similar to those of other neuroactive steroids and to obtain further evidence that 6-AziP acts at the putative steroid binding site, the effects of 6-AziP and pregnanolone on [3H]muscimol binding were examined in rat brain membranes. 6-AziP (10 µM) produced an 242% ± 11 enhancement of specific muscimol binding to rat brain membranes. This is similar to the 227% ± 10 enhancement produced by pregnanolone (Fig. 3B). Collectively the electrophysiological and radioligand binding studies indicate that 6-AziP has the typical biological activity of a neuroactive steroid, although it has modest efficacy (in the electrophysiological assay) and a lesser potency in comparison to pregnanolone. We were unable to test the effects of 6-AziP as an in vivo anesthetic because of limited supply of the compound.

Photolabeling-- To determine the optimal irradiation time for photolabeling membranes, membranes were incubated with [3H]6-AziP for varying periods of time (5-300 s), and covalent incorporation of radioactivity in the membranes was monitored. These experiments showed that photoincorporation of [3H]6-AziP into rat brain membrane was maximal at 180 s of irradiation. Accordingly a 3-min irradiation period was used in all subsequent experiments. To determine the potential protein sites of [3H]6-AziP incorporation, rat brain membranes were photolabeled with varying concentrations (0-30 µM) of [3H]6-AziP and analyzed by SDS-PAGE with gel slicing. As shown in Fig. 4A, radioactivity was incorporated into two major protein bands, one at ~35 kDa and one at ~60 kDa. Both peaks were labeled in a concentration-dependent fashion. The 35-kDa peak was half-maximally labeled at a [3H]6-AziP concentration of 1.9 µM, whereas the 60-kDa peak was labeled at higher concentrations (Fig. 4B). Because the 35-kDa protein was labeled at 6-AziP concentrations at which the steroid is biologically active, our initial efforts have focused on identification and characterization of this protein.


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Fig. 4.   Photolabeling of rat brain membranes and QT-6 cells. A, rat brain membranes were pre-incubated with 1 (), 3 (), and 10 (black-triangle) µM [3H]6-AziP and irradiated for 3 min. The samples were separated by SDS-PAGE gel and evaluated by gel slicing with determination of the radioactivity in each slice. [3H]6-AziP produced concentration-dependent photolabeling of protein bands of 35 and 60 kDa in rat brain membranes. B, radioactivity in the 35-kDa (open circle ) and 60-kDa (black-triangle) bands (area under curve) was measured as a function of [3H]6-AziP concentration. The curves were fit with the Michaelis-Menten equation plus a nonspecific (linear) component. The 35-kDa peak was half-maximally labeled at a [3H]6-AziP concentration of 1.9 µM, whereas the 60-kDa peak was half-maximally labeled at a [3H]6-AziP concentration of 30 µM. C, membranes from QT-6 cells expressing alpha 1mycbeta 1FLAG GABAA subunits () (transfected) and from non-transfected QT-6 cells () (control) were photolabeled with 10 µM [3H]6-AziP. Gel slice analysis of SDS-PAGE of these samples shows that protein bands at 35 and 60 kDa were photolabeled to a similar extent in both types of membranes.

Prevention of Photolabeling by Pregnanolone-- To confirm that [3H]6-AziP labeled a specific binding site on the 35-kDa protein, we examined whether photolabeling could be prevented by co-incubation with other steroids. Photolabeling of the 35-kDa protein by [3H]6-AziP (10 µM) was not prevented by co-incubation with super maximal concentrations of progesterone (100 nM), testosterone (100 nM), estradiol (100 nM), or dexamethasone (100 nM), indicating that it was not binding a classical steroid receptor or a plasma membrane variant thereof (44, 45) (data not shown). In contrast, photolabeling was inhibited in a concentration-dependent manner by pregnanolone (3alpha -OH-5beta -pregnan-20-one) (Fig. 5A). The extent of inhibition and the concentration of pregnanolone required to inhibit photolabeling varied as a function of 6-AziP concentration, consistent with a competitive interaction (Fig. 5B). It should be noted that given the complicated nature of the interaction between [3H]6-AziP and pregnanolone (the number of binding sites and the 6-AziP concentration vary as a function of time during the experiment) it is not possible to definitively conclude that pregnanolone prevents photolabeling via a competitive mechanism.


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Fig. 5.   Prevention of photolabeling by pregnanolone in rat brain membrane. A, photolabeling of the 35-kDa protein () with 300 nM [3H]6-AziP was largely prevented when incubations were performed in the presence of 30 µM pregnanolone (triangle ). This suggests that [3H]6-AziP labels a specific binding site on the 35-kDa protein. B, rat brain membranes were photolabeled with 0.3 (black-triangle) or 1.0 (black-square) µM [3H]6-AziP in the presence of varying concentrations (1-30 µM) of pregnanolone. The figure shows labeling of the 35-kDa band (area under the curve) as a function of pregnanolone concentration. Curves were fit to the Hill equation with a fixed maximum of 100% and the minimum as an independent variable. The extent of inhibition and the concentration of pregnanolone required to inhibit photolabeling varied as a function of [3H]6-AziP concentration, consistent with a competitive interaction. The 0.3 µM [3H]6-AziP curve has an EC50 of 1.91 ± 0.17 µM with a minimum labeling of 30%, and the 1.0 µM [3H]6-AziP curve has an EC50 of 3.82 ± 0.23 µM with a minimum labeling of 55%.

Photolabeling of QT-6 Cells-- One possible explanation for the photolabeling data is that the 60-kDa protein is a GABAA receptor subunit and that the 35-kDa protein is a proteolytic fragment of a GABAA receptor subunit. To explore this possibility, we examined membranes from QT-6 cells transfected with alpha 1mycbeta 1FLAG GABAA subunits (transfected) and from non-transfected QT-6 cells (wild-type). To prove that 6-AziP modulated GABAA receptors expressed in QT-6 cells, we first examined the ability of 6-AziP to inhibit [35S]TBPS binding in the membranes. As shown in Fig. 3A, 6-AziP inhibits [35S]TBPS binding with an IC50 of 186 ± 35 nM and a Hill slope of 0.81 ± 0.10. Wild-type QT-6 cells had no detectable [35S]TBPS binding indicating the absence of GABAA receptors. Fig. 4C shows the results of photolabeling transfected and wild-type QT-6 cell membranes with 10 µM [3H]6-AziP. Gel slice analysis of SDS-PAGE of these samples shows that the 35- and 60-kDa proteins were photolabeled in both types of membranes. This indicates that the 60-kDa protein is unlikely to be a GABAA receptor subunit, and the 35-kDa protein is unlikely to be a GABAA receptor subunit fragment.

Identification of the 35-kDa Protein-- To identify the 35-kDa protein, we isolated the photolabeled protein using two-dimensional electrophoresis (Fig. 6). Two major spots of radioactivity were observed on autoradiograms of membranes photolabeled with [3H]6-AziP. These spots had approximate pI values of 8.5 and 8.6. Additional minor radiolabeled spots were observed at the same molecular weight, most likely representing spot trains (post-translationally modified versions of the same protein varying slightly in pI value). Spots on Coomassie Blue-stained gel corresponding to the spots identified on the autoradiogram were excised and identified by MALDI-TOF/MS. The only identified protein in both spots was VDAC-1 (GenBankTM accession number 10720404). The protein spots were also analyzed by ESI/MS/MS. The major identified protein in both spots was again voltage-dependent anion channel-1. In spot A (pI = 8.5) voltage-dependent anion channel-2 (VDAC-2; GenBankTM accession number 13786202) was also identified, and in spot B (pI = 8.6), voltage-dependent anion channel-3 (VDAC-3; GenBankTM accession number 13786204) was also identified.


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Fig. 6.   Identification of the 35-kDa protein. Rat brain membranes were photolabeled with either tritiated or non-tritiated 6-AziP and analyzed by two-dimensional electrophoresis. The gel labeled with [3H]6-AziP was analyzed by autoradiography. The gel photolabeled with cold 6-AziP was silver-stained. The stained spots (A and B) corresponding to radiolabeled spots were excised, digested with trypsin, and analyzed by MALDI-TOF/MS. The only identified protein in both spots was VDAC-1 (GenBankTM accession number 10720404). The protein spots were also analyzed by ESI/MS/MS. The major identified protein in both spots was again voltage-dependent anion channel-1. In spot A VDAC-2 (GenBankTM accession number 13786202) was also identified, and in spot B, VDAC-3 (GenBankTM accession number 13786204) was also identified.

Immunoprecipitation of VDAC-1-- To confirm that the proteins identified by mass spectrometry were the same proteins that were photolabeled, immunoprecipitation studies were performed (Fig. 7). A monoclonal antibody directed against the N terminus of VDAC-1 was used for immunoprecipitation. The VDAC-1 antibody precipitated radiolabeled 35-kDa protein, confirming that VDAC-1 is photolabeled by [3H]6-AziP. Control experiments in which primary antibody was omitted did not show precipitation of any radioactivity. Interestingly, the radiolabeled 60-kDa protein was also precipitated by the VDAC-1 antibody, suggesting that the two labeled proteins (35- and 60-kDa) are associated physically. In some membrane labeling experiments and in some VDAC-1 immunoprecipitation experiments a radiolabeled band of ~20 kDa was also observed. This 20-kDa band was infrequently observed and was thus attributed to proteolysis of one of the labeled proteins. It should be noted that the VDAC-1 antibody immunoprecipitated only a fraction of the radioactivity in the 35-kDa peak. This is due, at least in part, to the fact that the immunoprecipitation was not quantitative. Studies using a polyclonal antibody to immunoblot VDAC-1 showed that under the conditions employed only a fraction of VDAC-1 is immunoprecipitated by the monoclonal antibody. It is also possible that 35-kDa proteins other than VDAC-1 (including VDAC-2 and VDAC-3) are photolabeled by [3H]6-AziP.


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Fig. 7.   Immunoprecipitation of VDAC-1. Rat brain membranes (open circle ) photolabeled with [3H]6-AziP were solubilized in 1% Triton X-100. A monoclonal antibody directed at the N terminus of VDAC-1 was used for immunoprecipitation. The VDAC-1 antibody immunoprecipitates (black-square) the radiolabeled 35-kDa protein, confirming that VDAC-1 is photolabeled by [3H]6-AziP. Control experiments (black-triangle) in which primary antibody was omitted did not show precipitation of any radioactivity. The radiolabeled 60-kDa protein was also precipitated by the VDAC-1 antibody, suggesting that the two labeled proteins (35 and 60 kDa) are physically associated. The 20-kDa band was infrequently observed and was thus attributed to proteolysis of one of the labeled proteins.

To look for a possible relationship between VDAC-1 and the GABAA receptor, VDAC-1 was immunoprecipitated from a detergent lysate of rat brain membrane. The precipitated protein was separated on SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and analyzed by Western blot using bd17 antibody directed against the beta 2 and beta 3 subunits of the GABAA receptor. The VDAC-1 antibody co-immunoprecipitated GABAA receptors, as evidenced by bd17 staining of proteins corresponding to the beta 2 and beta 3 subunits of the GABAA receptor. Control experiments in which primary antibody was omitted did not result in immunoprecipitation of any identifiable GABAA receptor. These results indicate that VDAC-1 is physically associated with the GABAA receptor (Fig. 8).


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Fig. 8.   Co-immunoprecipitation of GABAA receptor subunits. Monoclonal antibody to VDAC-1 was used to immunoprecipitate VDAC-1 from a detergent lysate of rat brain membrane. The immunoprecipitated protein was analyzed by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Western blot was performed using bd17 antibody directed against the beta 2 and beta 3 subunits of the GABAA receptor. The VDAC-1 antibody co-immunoprecipitates GABAA receptors, as evidenced by bd17 staining of proteins corresponding to the beta 2 and beta 3 subunits of the GABAA receptor. Control experiments in which primary antibody was omitted did not result in immunoprecipitation of any identifiable GABAA receptor.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

Identification of the neuroactive steroid binding sites responsible for anesthesia remains an unsolved problem. Although the GABAA receptor is an important functional target, chimeric and site-directed mutagenesis approaches have failed to identify candidate regions or binding sites on the GABAA receptor protein. There are several potential explanations for the failure of molecular biological approaches. (i) A binding site may involve multiple non-contiguous portions of the protein, foiling chimeric strategies. (ii) There may be multiple binding sites each mediating discrete but functionally additive effects. The functional assays used to screen mutated receptors may thus lack sensitivity. (iii) The binding site(s) may be on a protein other than the GABAA receptor.

In this study we have used analogue photoaffinity labeling, a direct approach to identifying neuroactive steroid binding sites that avoids all of the aforementioned problems. The photoaffinity labeling reagent we have developed, 6-AziP, has several positive attributes including the fact that the diazirine group is photolyzed at a wavelength (350-360 nm) that is not damaging to proteins and generates carbenes capable of reacting with virtually any amino acid (46).

Importantly, 6-AziP has the typical neuroactive steroid effects on GABAA receptor function as evidenced by whole-cell and single-channel patch clamp electrophysiology (Fig. 2) and by radioligand binding assays (Fig. 3). A potential shortcoming of 6-AziP is that it is less efficacious and potent as a modulator of GABAA receptor function than naturally occurring neurosteroids such as pregnanolone. In this regard, one must consider the possibility that 6-AziP identifies only a subset of the neuroactive steroid binding sites that modulate GABAA receptor function.

The major finding reported in this study is that 6-AziP photolabels VDAC-1 in rat brain in a robust and specific manner. The fact that VDAC-1 is saturably labeled with [3H]6-AziP, coupled with the observation that pregnanolone prevents photolabeling, indicates that 6-AziP interacts with a discrete binding site on VDAC-1 and is not merely adsorbed to an abundant protein. VDAC-2 and VDAC-3 were also identified in protein spots extracted from two-dimensional gels. It remains to be determined whether either of these isoforms is also photolabeled by 6-AziP. VDAC-1 (also known as mitochondrial porin) is a pore-forming protein, abundant in the outer mitochondrial membrane (47). It is a member of a small family of homologous proteins (VDAC-1-3) that enables permeability to the outer mitochondrial membrane (48). It is a large conductance anion-selective channel that is thought to be important in various aspects of mitochondrial function, possibly including apoptosis (49-51).

The fact that VDAC-1 is predominantly localized to mitochondria raises a question as to the possible mechanism through which neuroactive steroid binding to VDAC-1 could modulate GABAA receptor function. Our data indicate that antisera to VDAC-1 co-immunoprecipitate GABAA receptors from a detergent lysate of rat brain, indicating a strong physical association between the two proteins (Fig. 8). This is consistent with the earlier observation that VDAC-1 and/or VDAC-2 co-purified with GABAA receptors in a multi-step purification procedure of detergent-solubilized mammalian brain. In this study, antisera to VDAC also immunoprecipitated functional detergent-solubilized GABAA receptors (52). Although it remains to be demonstrated that VDAC-1 and GABAA receptors are physically associated in an intact cell (as opposed to a detergent lysate), it is plausible that GABAA receptors could either bind to VDAC-1 in mitochondria that abut against the post-synaptic membrane or could bind to a non-mitochondrial form of VDAC-1. In this regard, there is some evidence that VDAC may be present in plasma membranes and/or endosomes. Using electrophysiological and biochemical techniques, porin was identified within isolated caveolae and caveolae-like domains (53, 54). VDAC-1 has also been identified morphologically in post-synaptic density, suggesting possible co-localization with post-synaptic neurotransmitter receptors such as the GABAA receptor (55).

Mechanistically, it is implausible that VDAC-1 functions as a steroid-modulated ion channel in the plasma membrane; its huge pore size and conductance would be incompatible with maintaining cellular integrity. VDAC-1 could be part of a multi-protein complex with the GABAA receptor in which neuroactive steroid binding to VDAC-1 allosterically modulates GABAA receptor function. This would be analogous to the mechanism through which inhalational anesthetics bind to the scaffolding protein, PSD-95/SAP90, and thus modulate N-methyl-D-aspartate receptor function (56). It is also worth noting that endogenous neurosteroid binding to mitochondrial VDAC could be an important mechanism for regulation of mitochondrial function. It will be important to understand the effects of neuroactive steroid binding to VDAC in terms of both anesthetic effects and mitochondrial regulation. The existence of VDAC-1-deficient mice should facilitate delineation of the functional significance of neuroactive steroid binding sites on VDAC-1 (57).

One important question is whether neuroactive steroids bind to the GABAA receptor protein itself. Our findings indicate that 6-AziP does not photolabel the GABAA receptor. Although 6-AziP does label a 60-kDa protein, this protein is labeled equally in HEK 293 cells expressing and not expressing GABA receptors. Additionally, quantitative immunoprecipitation of [3H]6-AziP photolabeled, epitope-tagged GABAA receptors (alpha 1,beta 1FLAG) from HEK 293 cells failed to precipitate any radiolabeled protein (data not shown). As stated earlier, it is possible that 6-AziP does not label all neuroactive steroid binding sites, and a different analogue photolabel could bind to a GABAA receptor subunit. We are currently in the process of identifying the 60-kDa protein and will assess its relationship to the GABAA receptor.

    CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

6-AziP is the first effective neuroactive steroid analogue photoaffinity labeling reagent. The identification of VDAC-1 as a neuroactive steroid-binding protein suggests the possibility that neuroactive steroids may modulate GABAA receptor function by binding to an accessory protein. Exploration of the functional interaction between VDAC-1 and the GABAA receptor, as well as identification of other proteins photolabeled by 6-AziP, should shed light on the molecular mechanisms of neuroactive steroid action.

    ACKNOWLEDGEMENTS

We thank Ann Benz and Amanda Shute for technical assistance and Dr. Charles Zorumski for helpful discussions.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants PO1-GM47969 (to J. H. S., D. F. C., and A. S. E.) and AA12952 (to S. J. M.) and by a grant from the Klingenstein Foundation (to S. J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8054, St. Louis, MO 63110. Tel.: 314-454-8702; Fax: 314-454-5572; E-mail: eversa@notes.wustl.edu.

Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M213168200

    ABBREVIATIONS

The abbreviations used are: GABA, gamma -aminobutyric acid; GABAA, GABA, type A; 6-AziP, 3alpha ,5beta -6-azi-3-hydroxypregnan-20-one; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HEK, human endothelial kidney; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TBPS, t-butylbicyclophosphorothionate; VDAC, voltage-dependent anion channel; THF, tetrahydrofuran; ESI, electrospray ionization.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

1. Selye, H. (1941) Proc. Soc. Exp. Biol. Med. 46, 116-121
2. Atkinson, R. M., Davis, B., Pratt, M. A., Sharpe, H. M., and Tomich, E. G. (1965) J. Med. Chem. 8, 426-432[Medline] [Order article via Infotrieve]
3. Majewska, M. D., Harrison, N. L., Schwartz, R. D., Barker, J. L., and Paul, S. M. (1986) Science 232, 1004-1007[Medline] [Order article via Infotrieve]
4. Harrison, N. L., and Simmonds, M. A. (1984) Brain Res. 323, 287-292[CrossRef][Medline] [Order article via Infotrieve]
5. Harrison, N. L., Vicini, S., and Barker, J. L. (1987) J. Neurosci. 7, 604-609[Abstract]
6. Cottrell, G. A., Lambert, J. J., and Peters, J. A. (1987) Br. J. Pharmacol. 90, 491-500[Abstract]
7. Barker, J. L., Harrison, N. L., Lange, G. D., and Owen, D. G. (1987) J. Physiol. 386, 485-501[Abstract]
8. Harrison, N. L., Majewska, M. D., Harrington, J. W., and Barker, J. L. (1987) J. Pharmacol. Exp. Ther. 241, 346-353[Abstract]
9. Wittmer, L. L., Hu, Y., Kalkbrenner, M., Evers, A. S., Zorumski, C., and Covey, D. F. (1996) Mol. Pharmacol. 50, 1581-1586[Abstract]
10. Covey, D. F., Nathan, D., Kalkbrenner, M., Nilsson, K. R., Hu, Y., Zorumski, C. F., and Evers, A. S. (2000) J. Pharmacol. Exp. Ther. 293, 1009-1116[Abstract/Free Full Text]
11. Sanna, E., Murgia, A., Casula, A., and Biggio, G. (1997) Mol. Pharmacol. 51, 484-490[Abstract/Free Full Text]
12. Mihalek, 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., and Homanics, G. E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12905-12910[Abstract/Free Full Text]
13. Turner, D. M., Ransom, R. W., Yang, J. S., and Olsen, R. W. (1989) J. Pharmacol. Exp. Ther. 248, 960-966[Abstract]
14. Callachan, H., Cottrell, G. A., Hather, N. Y., Lambert, J. J., Nooney, J. M., and Peters, J. A. (1987) Proc. R. Soc. Lond. B Biol. Sci. 231, 359-369[Medline] [Order article via Infotrieve]
15. Franks, N. P., and Lieb, W. R. (1994) Nature 367, 607-614[CrossRef][Medline] [Order article via Infotrieve]
16. Smith, G. B., and Olsen, R. W. (1995) Trends Pharmacol. Sci. 16, 162-168[CrossRef][Medline] [Order article via Infotrieve]
17. Mohler, H., Battersby, M. K., and Richards, J. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 1666-1670[Abstract]
18. Duncalfe, L. L., Carpenter, M. R., Smillie, L. B., Martin, I. L., and Dunn, S. M. J. (1996) J. Biol. Chem. 271, 9209-9214[Abstract/Free Full Text]
19. Smith, G. B., and Olsen, R. W. (1994) J. Biol. Chem. 269, 20380-20387[Abstract/Free Full Text]
20. Mihic, 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., and Harrison, N. L. (1997) Nature 389, 385-389[CrossRef][Medline] [Order article via Infotrieve]
21. Greenblatt, E. P., and Meng, X. (2001) Anesthesiology 94, 1026-1033[CrossRef][Medline] [Order article via Infotrieve]
22. Hill-Venning, C., Belelli, D., Paters, J. A., and Lambert, J. J. (1997) Br. J. Pharmacol. 120, 749-756[Abstract]
23. Belleli, D., Lambert, J. J., Peters, J. A., Wafford, K., and Whiting, P. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 92, 11031-11036[CrossRef]
24. Krasowski, M. D., Koltchine, V. V., Rick, C. E., Ye, Q., Finn, S. E., and Harrison, N. L. (1998) Mol. Pharmacol. 53, 530-538[Abstract/Free Full Text]
25. Yamakura, T., Bertaccini, E., Trudell, J. R., and Harris, R. A. (2001) Annu. Rev. Pharmacol. Toxicol. 41, 23-51[CrossRef][Medline] [Order article via Infotrieve]
26. Jenkins, A., Greenblatt, E. P., Faulkner, J. H., Bertaccini, E., Light, A., Lin, A., Andreasen, A., Viner, A., Trudell, J. R., and Harrison, N. L. (2001) J. Neurosci. 21, RC136[Medline] [Order article via Infotrieve]
27. Rick, C. E., Ye, Q., Finn, S. E., and Harrison, N. L. (1998) Neuroreport 9, 379-383[Medline] [Order article via Infotrieve]
28. Hawkinson, J., Drewe, J. A., Kimbrough, C. L., Chen, J., Hogenkamp, D. J., Lan, N. J., Gee, K. W., Shen, K., Whittemore, E. R., and Woodward, R. M. (1996) Mol. Pharmacol. 49, 897-906[Abstract]
29. Paradiso, K., Sabey, K., Evers, A. S., Zorumski, C. F., Covey, D. F., and Steinbach, J. H. (2000) Mol. Pharmacol. 58, 341-351[Abstract/Free Full Text]
30. Sabey, K., Paradiso, K., Zhang, J., and Steinbach, J. H. (1999) Mol. Pharmcol. 55, 58-66[Abstract/Free Full Text]
31. Ueno, S., Zorumski, C., Bracamontes, J., and Steinbach, J. H. (1996) Mol. Pharmacol. 50, 931-938[Abstract]
32. Antonucci, R., Bernstein, S., Lenhard, R., Sax, K. J., and Williams, J. H. (1952) J. Org. Chem 17, 1369-1374
33. Hawkinson, J. E., Kimbrough, C. L., Belelli, D., Lambert, J. J., Purdy, R. H., and Lan, N. C. (1994) Mol. Pharmacol. 46, 977-985[Abstract]
34. Lopez-Colome, A. M., McCarthy, M., and Beyer, C. (1990) Eur. J. Pharmacol. 176, 297-303[CrossRef][Medline] [Order article via Infotrieve]
35. Steinbach, J. H., and Akk, G. (2001) J. Physiol. 537, 715-733[Abstract/Free Full Text]
36. Qin, F., Auerbach, A., and Sachs, F. (1996) Biophys. J. 70, 264-280[Abstract]
37. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
38. Hatzimanikatis, V., Choe, L. H., and Lee, K. H. (1999) Biotech. Prog. 15, 312-318[CrossRef][Medline] [Order article via Infotrieve]
39. Katzenellenbogen, J. A., Johnson, H. J., Jr., Carlson, K. E., and Myres, H. N. (1974) Biochemistry. 13, 2986-2994[Medline] [Order article via Infotrieve]
40. Bureau, M. H., and Olsen, R. W. (1993) J. Neurochem. 61, 1479-1491[Medline] [Order article via Infotrieve]
41. Watson, R. E., Wiegand, S. J., Clough, R. W., and Hoffman, G. E. (1985) Peptides 7, 155-159[CrossRef]
42. Fountoulakis, M., Schuller, E., Hardmeier, R., Berndt, P., and Lubec, G. (1999) Electrophoresis 20, 3572-3579[CrossRef][Medline] [Order article via Infotrieve]
43. Jimenez, C. R., Huang, L., Qiu, Y., and Burlingame, A. L. (2000) Current Protocols in Protein Science , pp. 16.14.11-16.14.15, John Wiley & Sons, Inc., New York
44. Tsai, M. J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486[CrossRef][Medline] [Order article via Infotrieve]
45. Schmidt, B. M. W., Gerdes, D., Feuring, M., Falkenstein, E., Christ, M., and Wehling, M. (2000) Front. Neuroendocrinol. 21, 57-94[CrossRef][Medline] [Order article via Infotrieve]
46. Brunner, J., and Semenza, G. (1981) Biochemistry 20, 7174-7182[Medline] [Order article via Infotrieve]
47. Colombini, M., Blachly-Dyson, E., and Forte, M. (1996) Ion Channels 4, 169-202[Medline] [Order article via Infotrieve]
48. Rostovtseva, T., and Colombini, M. (1996) J. Biol. Chem. 271, 28006-28008[Abstract/Free Full Text]
49. Rostovtseva, T., and Colombini, M. (1997) Biophys. J. 75, 1954-1962
50. Shimizu, S., Konishi, A., Kodama, T., and Tsujimoto, Y. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3100-3105[Abstract/Free Full Text]
51. Shimizu, S., Matsuoka, Y., Shinohara, Y., Yoneda, Y., and Tsujimoto, Y. (2001) J. Cell Biol. 152, 237-250[Abstract/Free Full Text]
52. Bureau, M. H., Khrestchatisky, M., Heeren, M. A., Zambrowicz, E. B., Kim, H., Grisar, T. M., Colombini, M., Tobin, A. J., and Olsen, R. W. (1992) J. Biol. Chem. 267, 8679-8684[Abstract/Free Full Text]
53. Bathori, G., Parolini, I., Tombola, F., Szabo, I., Messina, A., Oliva, M., De Pinto, V., Lisanti, M., Sargiacomo, M., and Zoratti, M. (1999) J. Biol. Chem. 274, 29607-29612[Abstract/Free Full Text]
54. Bathori, G., Parolini, I., Szabo, I., Tombola, F., Messina, A., Oliva, M., Sargiacomo, M., De Pinto, V., and Zoratti, M. (2000) J. Bioenerg. Biomembr. 32, 79-89[CrossRef][Medline] [Order article via Infotrieve]
55. Moon, J., Jung, Y. W., Ko, B. H., De Pinto, V., Jin, I., and Moon, S. (1999) Neuroreport 10, 443-447[Medline] [Order article via Infotrieve]
56. Tao, Y., and Johns, R. A. (2001) Anesthesiology 94, 1010-1015[Medline] [Order article via Infotrieve]
57. Anflous, K., Armstrong, D. D., and Craigen, W. J. (2001) J. Biol. Chem. 276, 1954-1960[Abstract/Free Full Text]


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