 |
INTRODUCTION |
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
-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
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
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 |
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
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
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.
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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 (
= 0.00 ppm) and
CDCl3 (
= 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%.
(5
,6
)-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)
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)
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%.
(5
,6
)-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)
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)
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%.
(5
)-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)
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)
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)
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)
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%.
(5
)-6-Azipregnane-3,20-dione (6) and
(5
)-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 5
-epimer of
the starting material (196.9 mg total for the combined 5
- and
5
-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)
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)
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)
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)
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.
(3
,5
)-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)
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)
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](3
,5
)-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 3
-hydroxysteroid,
[3-3H](3
,5
)-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
1,
2, and
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 (
1), P. Malherbe, Hoffmann-La Roche (
2), and C. Fraser,
National Institute on Alcohol Abuse and Alcoholism
(
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
1
1
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
2,
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
2-
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 |
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 (
1
2
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
1
2
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 1 2 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 ( ) 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.
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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
1
2
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
1myc 1FLAG cells ( ) 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.
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|
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 ( ) 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 ( ) and 60-kDa ( ) 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
1myc 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.
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|
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 (3
-OH-5
-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 ( ). 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 ( ) or 1.0 ( ) 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%.
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|
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
1myc
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.
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|
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 ( ) 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 ( ) the 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. 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.
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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
2 and
3 subunits of the
GABAA receptor. The VDAC-1 antibody co-immunoprecipitated GABAA receptors, as evidenced by bd17
staining of proteins corresponding to the
2 and
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 2 and
3 subunits of the GABAA receptor. The VDAC-1
antibody co-immunoprecipitates GABAA receptors, as
evidenced by bd17 staining of proteins corresponding to the
2 and 3 subunits of the GABAA
receptor. Control experiments in which primary antibody was omitted did
not result in immunoprecipitation of any identifiable GABAA
receptor.
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|
 |
DISCUSSION |
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 (
1,
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 |
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.