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The Journal of Neuroscience, May 1, 2002, 22(9):3366-3375
3
-Hydroxypregnane Steroids Are Pregnenolone Sulfate-Like
GABAA Receptor Antagonists
Mingde
Wang1,
Yejun
He1,
Lawrence N.
Eisenman5,
Christopher
Fields1,
Chun-Min
Zeng3,
Jose
Mathews1,
Ann
Benz1,
Tao
Fu1,
Erik
Zorumski1,
Joe Henry
Steinbach2,
Douglas F.
Covey3,
Charles F.
Zorumski1, 4, and
Steven
Mennerick1, 4
Departments of 1 Psychiatry,
2 Anesthesiology, 3 Molecular Biology and
Pharmacology, 4 Anatomy and Neurobiology, and
5 Neurology, Washington University School of Medicine, St.
Louis, Missouri 63110
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ABSTRACT |
Endogenous neurosteroids have rapid actions on ion
channels, particularly GABAA receptors, which are
potentiated by nanomolar concentrations of 3
-hydroxypregnane
neurosteroids. Previous evidence suggests that 3
-hydroxypregnane
steroids may competitively antagonize potentiation induced by their
3 diastereomers. Because of the potential importance of antagonists
as experimental and clinical tools, we characterized the functional
effect of 3-hydroxysteroids. Although 3-hydroxysteroids reduced
the potentiation induced by 3-hydroxysteroids, 3-hydroxysteroids
acted noncompetitively with respect to potentiating steroids and
inhibited the largest degrees of potentiation most effectively.
Potentiation by high concentrations of barbiturates was also reduced by
3-hydroxysteroids. 3-Hydroxysteroids are also direct,
noncompetitive GABAA receptor antagonists.
3-Hydroxysteroids coapplied with GABA significantly inhibited
responses to 
15 µM GABA. The profile of block was
similar to that exhibited by sulfated steroids, known blockers of
GABAA receptors. This direct, noncompetitive effect of
3-hydroxysteroids was sufficient to account for the apparent
antagonism of potentiating steroids. Mutated receptors exhibiting
decreased sensitivity to sulfated steroid block were insensitive to
both the direct effects of 3-hydroxysteroids on GABAA
responses and the reduction of potentiating steroid effects. At
concentrations that had little effect on GABAergic synaptic currents,
3-hydroxysteroids and low concentrations of sulfated steroids
significantly reversed the potentiation of synaptic currents induced by
3-hydroxysteroids. We conclude that 3-hydroxypregnane steroids
are not direct antagonists of potentiating steroids but rather are
noncompetitive, likely state-dependent, blockers of GABAA
receptors. Nevertheless, these steroids may be useful functional
blockers of potentiating steroids when used at concentrations that do
not affect baseline neurotransmission.
Key words:
neurosteroids; inhibitory postsynaptic current; GABAA receptors; pregnenolone sulfate; anesthetic; hippocampal culture
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INTRODUCTION |
GABAA
receptors mediate most fast inhibitory neurotransmission in the
CNS. Many important neuroactive compounds, including benzodiazepines, barbiturates, neuroactive steroids, and other general
anesthetics, allosterically interact with GABAA
receptors and thereby influence the balance between neuronal excitation and inhibition (Macdonald and Olsen, 1994
). These drugs enhance the
activity produced by low concentrations of GABA and/or directly gate
GABAA receptor channels in the absence of GABA
(Majewska, 1992).
Neuroactive steroids are of particular interest, because
they are synthesized in the CNS and periphery and are present in the
CNS at concentrations that may endogenously modulate
GABAA receptor function (Robel and Baulieu,
1994). (3,5)-3-Hydroxypregnan-20-one (35P),
(3,5)-3-hydroxypregnan-20-one (35P),
(3,5)-3,21-dihydroxypregnan-20-one (35THDOC), and
(3,5)-3,21-dihydroxypregnan-20-one (35THDOC) act as potent,
efficacious positive modulators of GABAA
receptors (Gasior et al., 1999; Lambert et al., 2001).
Other endogenous neuroactive steroids, such as pregnenolone
sulfate (PS) and (3,5)-3-hydroxypregnan-20-one sulfate
(35PS), inhibit GABA responses at high nanomolar to micromolar
concentrations (for review, see Lambert et al., 2001). The role of
endogenous neuroactive steroids in modulating
GABAA receptor function remains unclear because
of the lack of specific antagonists at the steroid-modulating sites.
Precise sites of action of potentiating steroids on the
GABAA receptor have also remained elusive
(Lambert et al., 2001). Identification of antagonists may help to
clarify interactions between potentiating steroids and the
GABAA receptor.
Several previous studies explored potential steroid
antagonists against potentiation by neuroactive steroids. In studies of [3H]flunitrazepam binding, which is
a validated measure of GABAA receptor
potentiation (Turner et al., 1989), both
(3,5)-3-hydroxypregnan-20-one (35P) and
(3,5)-3-hydroxypregnan-20-one (35P) produced insignificant changes in [3H]flunitrazepam binding
when administered alone. However, both competitively
antagonized the potentiation of
[3H]flunitrazepam binding by 35P
and 35P (Prince and Simmonds, 1992, 1993). In
electrophysiological studies, 35P antagonized the
35P-induced enhancement of GABA current (Garrett and Gan, 1998;
Maitra and Reynolds, 1998). Similarly, 35P and 35P
diminished the inhibitory effects of 35P and 35P on
population spikes evoked in rat hippocampal CA1 stratum pyramidale
(Wang et al., 2000).
In the present study, we examined the effects of a series of endogenous
and synthetic 3-hydroxypregnane and 3-hydroxyandrostane steroids
on GABA-induced currents in Xenopus oocytes expressing recombinant GABAA receptors containing 1,
2, and 
2L subunits and on GABA-mediated synaptic responses in
cultured rat hippocampal neurons. We report here that
3-hydroxypregnane steroids exhibit block of
GABAA receptor function that is dependent on GABA
concentration, similar to block by sulfated steroids. The direct effect
on GABAA receptors explains the apparent ability
of 3-hydroxysteroids to antagonize the effects of positive modulators.
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MATERIALS AND METHODS |
Chemicals. (3,5)-3,21-dihydroxypregnan-20-one,
(3,5)-3-hydroxypregnan-20-one sulfate (35PS),
(3,5)-3-hydroxypregnan-20-one sulfate (35PS), and
35PS were obtained from Steraloids Inc. (Newport, RI), and
flumazenil was from Roche (Basel, Switzerland). (3,5)-3-Hydroxypregnan-20-one sulfate (35PS) was
synthesized as described previously (Park-Chung et al., 1997)
and was a generous gift of Dr. Robert H. Purdy (Scripps Research
Institute, La Jolla, CA).
(3,5,17)-3-hydroxyandrostane-17-carbonitrile (35ACN) was synthesized as described previously (Han et al., 1996). Synthesis of (3,5,7,17)-3-hydroxy-7-methylpregnan-20-one
[(7Me)35P] was also described previously (Zeng et
al., 2001). All of the remaining chemicals were from RBI/Sigma (St.
Louis, MO). The steroids were dissolved in DMSO. Pentobarbital was
dissolved in 0.1% NaOH. All chemicals were then diluted in saline for
experiments. The concentration of DMSO in experimental solutions was
0.1%.
Hippocampal microcultures. Primary microisland cultures of
hippocampal cells were prepared from 1- to 3-d-old postnatal Sprague Dawley rats using established methods (Mennerick et al., 1995). Under
halothane anesthesia, rats were decapitated, and the hippocampi were
dissected and cut into 500-µm-thick transverse slices. The slices
were dissociated with 1 mg/ml papain in oxygenated Leibovitz L-15
medium and mechanical trituration in modified Eagle's medium containing 5% horse serum, 5% fetal calf serum, 17 mM D-glucose, 400 µM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Isolated cells were plated onto 35 mm plastic
culture dishes at a density of 75 cells/mm2. Before plating, culture dishes
were coated with a layer of 0.15% agarose, dried overnight, and
sprayed with small droplets of rat tail collagen using a microatomizer
(Thomas Scientific, Swedeboro, NJ). The agarose layer serves as a
nonpermissive background for cell adhesion. Cultures were treated with
cytosine arabinoside (5-10 µM) after 3 d
in vitro to halt glial proliferation. Electrophysiological recordings were performed 8-15 d after plating.
Culture electrophysiology. Whole-cell recordings were
performed on solitary, inhibitory, hippocampal microculture neurons, using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA)
interfaced to a Pentium III-based computer via a Digidata 1200 acquisition board (Axon Instruments). Recordings were at room
temperature. Electrodes had resistances of 1.5-4 M
for whole-cell recordings. Access resistance was electronically compensated 90-100%. Autaptic release of neurotransmitter was stimulated in voltage-clamped solitary neurons with a 2 msec voltage pulse to 0 mV from a holding potential of 
70 mV. This stimulation protocol elicits an escaped action potential in the partially clamped axons that triggers transmitter release (Bekkers and Stevens, 1991; Mennerick et al., 1995). Whenever possible, at least three traces in each experimental condition were acquired for analysis. For all experiments, the interval
between data sweeps was 25 sec for synaptic responses. Control
conditions were interleaved with experimental conditions to
counterbalance any time-dependent changes. Data sampling frequency was
5-10 kHz.
At the time of experiments, culture medium was replaced with an
extracellular recording solution consisting of (in mM): 138 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, and 0.025 D-APV, pH 7.25. Solutions were exchanged via a local
multibarrel perfusion pipette, with a common delivery port placed 0.5 mm from the cell under study. The standard pipette solution for
autaptic responses contained (in mM): 140 KCl, 4 NaCl, 0.5 CaCl2, 5 EGTA, and 10 HEPES, pH 7.25. IPSCs were
easily distinguished from AMPA receptor-mediated EPSCs under these
conditions by the >10-fold slower 10-90% decay times of IPSCs. There
was no effect of either 3-hydroxy (500 nM 35P) or
3-hydroxy (10 µM 35THDOC) neurosteroids on the
amplitude or decay of AMPA receptor-mediated EPSCs (n = 4). IPSCs exhibited decays that were fitted by two or three exponential components, and potentiating steroid had most prominent effects on the
slow components of decay (Zorumski et al., 1998). For ease of comparing
effects of several drugs among cells with heterogeneous kinetics,
we used the model-independent 10-90% decay time of IPSCs as our
primary measure of IPSC duration (see Fig. 10).
Expression in Xenopus oocytes. Stage V-VI
oocytes were harvested from sexually mature female Xenopus
laevis (Xenopus One, Northland, MI) under 0.1%
tricaine (3-aminobenzoic acid ethyl ester) anesthesia. Oocytes were
defolliculated by shaking for 20 min at 37°C in collagenase (2 mg/ml)
dissolved in calcium-free solution containing (in
mM): 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES, 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 of total RNA) 8-24 hr after defolliculation. Oocytes were incubated up to 5 d at 18°C in ND96 medium
containing 96 mM NaCl, 1 mM
KCl, 1 mM MgCl2, 2 mM CaCl2 and 5 mM HEPES at pH 7.4, supplemented with 5 mM pyruvate, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 50 µg/ml gentamycin. The cDNAs for the rat
GABAA receptor subunits were provided by A. Tobin
(1) (University of California, Los Angels, CA), P. Malherbe (2)
(Hoffman-La Roche, Basal, Switzerland), and C. Fraser (2L)
(National Institute on Alcohol Abuse and Alcoholism, Bethesda,
MD). The V256S mutation, a point mutation in the 1 subunit at
the second residue N terminal to the beginning of transmembrane domain
2 (position 2'), was characterized previously and shown to dramatically
reduce pregnenolone sulfate block of GABAA
receptors (Akk et al., 2001).
Oocyte electrophysiology. Two-electrode voltage-clamp
experiments were performed with an Axoclamp 2B amplifier or Warner
Instruments (Hamden, CT) OC725 amplifier 2-5 d 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 M.
Drugs were applied from a common tip via a gravity-driven multibarrel
drug-delivery system. Drugs were always coapplied with GABA and were
not preapplied in the absence of GABA. Cells were clamped at 70 mV
for all experiments, and current at the end of 20-30 sec drug
applications was measured for quantification of current amplitudes. In
three oocytes, we pretreated cells with a low concentration of
potentiating steroid (35P, 100 nM for 40 sec) before coapplying GABA. This protocol resulted in little difference in response size compared with the standard (coapplication only) protocol used for all figures (101 ± 11%
potentiation with pretreatment, followed by coapplication; 85 ± 12% with coapplication alone). Likewise, pretreatment with a moderate
concentration of blocking steroid (35THDOC, 6 µM for 40 sec) yielded similar block at the end
of 20 µM GABA coapplication as with
coapplication alone (36 ± 4% depression with pretreatment vs
36 ± 2% depression with coapplication alone) (see Fig. 6).
Data analysis. Data acquisition and analysis were performed
with pClamp software (Axon Instruments). Data plotting and curve fitting were done with Sigma Plot software (SPSS, Chicago, IL). Data
are presented in the text and figures as mean ± SE. Statistical differences were determined using a two-tailed Student's t
test or a one-way ANOVA. The percentage of modulation of
GABA-activated current was calculated as
(IM IN)/IN,
where IN (normalizing current) and
IM (measured current) are the
amplitudes of the GABA-activated current in the absence and presence of
the test substance, respectively. Fitting of the dose-response
relationships were performed using the Hill equation as follows:
I = Imax × Cn/(EC50n + Cn), where C is the
concentration of steroid (or GABA) (see Fig. 6),
Imax is the maximum current amplitude,
EC50 is the concentration of steroid (or GABA)
that produces 50% of Imax, and
n is the Hill coefficient.
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RESULTS |
Noncompetitive antagonism of steroid and barbiturate potentiation
by 3-hydroxypregnane steroids
To characterize the action of 3-hydroxypregnane steroids at
GABAA receptors, we examined their putative
antagonist profile against GABA-potentiating neurosteroids. Figure
1 shows the pregnane-androstane steroid
ring system, and the chiral centers at carbon 3 (C3) and C5 are
highlighted. The steroid shown is a 3,5 steroid. The steroids
used in the present study varied in the side chain group at C17 (Fig.
1), but these side groups all conformed to the general rule that the
side chain contained a hydrogen bond acceptor (Covey et al., 2001).
This side chain group at C17 in the configuration is necessary (but
not sufficient) for GABAA receptor potentiation (Lambert et al., 2001). Potentiation by neuroactive steroids is also
critically dependent on a hydrogen bond donor in the configuration at C3 (Lambert et al., 2001). For naturally occurring steroids, such as
35P, 35P, and 35THDOC, this hydrogen bond donor is a
hydroxyl group. We tested the hypothesis in the current work that the
3 diastereomers of these potentiating neurosteroids are competitive
antagonists of the potentiators (Prince and Simmonds, 1992, 1993).
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Figure 1.
Steroid structure, with emphasis
(arrows) on the chiral centers at C3 and C5. The
structure shown is 3,5,17. 3-Hydroxypregnane steroids have
been suggested to antagonize the potentiating actions of steroids with
a 3-hydroxy configuration. For the steroids tested in this work, the
R group was CN, COCH3, or COCH2OH.
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Figure 2A shows raw
traces from a Xenopus oocyte expressing a combination of
122L GABAA receptor subunits. We applied
a low concentration of GABA (2 µM) alone, or we
coapplied (with GABA) varied concentrations of 35P plus or minus
10 µM 35P. Note that 35P
significantly inhibited only the response to the high concentration of
potentiating steroid. Figure 2B shows a similar
experiment using another 3-hydroxypregnane steroid,
35THDOC, which exhibited a similar profile of apparent
antagonism. Figure 2C-E shows summaries of the effect of
three different 3-hydroxypregnane steroids (35P,
35THDOC, and 35P) on potentiation by 35P. Table
1 gives values from fits to the Hill
equation of the concentration-response curves shown in Figure
2C-E. Note that the EC50 values for
potentiation were not substantially affected by the presence of the
3-hydroxysteroid, suggesting a noncompetitive mechanism of reduced
potentiation. The similarity of the effects of the three different
steroids (Fig. 2C-E) suggests that neither the
configuration of the hydrogen atom at C5 nor the structure of the
hydrogen bond acceptor group at C17 is critical to the blocking
effect.
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Figure 2.
35P and 35THDOC reversed the effect of
high concentrations of GABA potentiating steroids. A,
Sample traces showing that 10 µM 35P inhibited
potentiation by high but not low concentrations of 35P.
B, Similar traces for 10 µM 35THDOC.
C-E, Summary of effect of 35P
(C), 35THDOC (D), and
35P (E) from oocytes tested with a range of
35P concentrations. C, Summary of the effect of 10 µM 35P against increasing concentrations of
35P. Normalized responses in this and subsequent figures were
calculated as follows: (IM IN)/IN,
where IM is the amplitude of the measured
current in a given experimental condition, and
IN is the normalizing current. For these
data and data in D and E,
IN was the response to 2 µM
GABA in the absence of modulator. The solid lines
represent least-squares fits of the data to the Hill equation as
follows: I = Imax × Cn/(EC50n + Cn), where C is the
concentration of potentiator, EC50 is the concentration of
potentiator that produced half-maximum potentiation, and
n is the Hill coefficient. Parameters of the fit for
potentiators in the absence (filled circles)
and presence (open squares) of 35P are
given in Table 1. D, Summary of the effect of 10 µM 35THDOC under similar experimental
conditions to those in C. E,
Concentration-response relationship for 35P in the presence and
absence of 35P (10 µM). F,
Concentration-response relationship of the benzodiazepine agonist
lorazepam in the presence and absence of the benzodiazepine antagonist
flumazenil. The solid line through lorazepam
concentration-response values (circles) is the best fit
of the Hill equation, with an EC50 of 76.7 nM and a Hill coefficient of 1.2 (n = 7). The solid line through
lorazepam-flumazenil interaction values (squares) is
the best fit of the Hill equation, with an EC50 of 348.5 nM and a Hill coefficient of 1.1 (n = 7).
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We were surprised by the apparently noncompetitive nature of the
interaction between the 3-hydroxysteroids and 35P (Fig. 2C-E). To be sure that our protocol was capable of
detecting a competitive interaction between potentiator and antagonist,
we examined the effect of the benzodiazepine antagonist flumazenil on
the effect of a potentiating benzodiazepine lorazepam. Flumazenil (100 nM) competitively inhibited the GABA-enhancing
effect of lorazepam by shifting lorazepam concentration-response
curves to the right (n = 7) (Fig.
2F). The EC50 of lorazepam
increased from 76.7 to 348.5 nM in the presence
of 100 nM flumazenil (Fig. 2F).
The Hill coefficients (Fig. 2F, see legend) and
maximal response were quite close between two lorazepam
concentration-response curves. This control experiment suggests that
our experimental protocol should have detected a competitive
interaction between a steroid potentiator and antagonist if present.
We explored the actions of several other 3-hydroxysteroids at 10 µM, and, consistent with the results of Figure 2, we
found little structural specificity in the action against the
potentiating neurosteroid 35P (Fig.
3A). Addition of an methyl
group at C7 did not affect block of potentiation, nor did substitution of a carbonitrile group for the acetyl side group at C17 (Fig. 3A).
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Figure 3.
Effects of a series 3-hydroxysteroids on 2 µM GABA responses in the presence and absence of
35P. A, Effects of several 3-hydroxysteroids
(10 µM) against potentiation induced by 35P (3 µM) in the presence of 2 µM GABA. For these
data, the normalizing response (IN,
representing 0 on the y-axis) was the current in the
combined presence of 2 µM GABA plus 3 µM
35P (n = 6). B, Effects of
several 3-hydroxysteroids on responses to 2 µM GABA
alone. Most compounds were effectively inert at 10 µM,
except for 35P, which slightly potentiated responses to 2 µM GABA. IN represented the
response to 2 µM GABA alone (n = 5).
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We also examined this panel of 3-hydroxysteroids (using a fixed
concentration of 10 µM) against the response to 2 µM GABA alone (Fig. 3B). We found that most
3 steroids were inert, but, as reported previously (Puia et al.,
1990; Woodward et al., 1992; Kokate et al., 1994), 10 µM 35P mildly potentiated GABA responses. For many of the remaining experiments, we used 35THDOC as our standard 3-hydroxysteroid because of its relative inertness against GABA alone (Fig. 3B), the slightly better blocking activity
than some other 3-hydroxysteroids (Fig. 3A), and its
commercial availability.
Given that the interaction between 3-hydroxysteroids and 35P
was noncompetitive, we examined whether this noncompetitive interaction
held true for other potentiating neurosteroids. Indeed, Figure
4A-C shows that
35THDOC similarly inhibited potentiation by 5- and
5-reduced neuroactive steroids. A noncompetitive profile was evident
for all steroid potentiators. When we tested other classes of GABA
potentiators, we were surprised to find that barbiturate (Fig.
5B,C)
but not benzodiazepine (Fig. 5A,C)
potentiation was inhibited by 3-hydroxysteroids. The
potentiation induced by 150 µM pentobarbital
was significantly reduced by 10 µM
35THDOC (Fig. 5B,C). As with
steroid potentiation, the effect on pentobarbital potentiation was
dependent on potentiator concentration. The potentiation by 50 µM pentobarbital (121 ± 25%) was not
affected by 35THDOC (130 ± 28%; n = 13)
(data not shown). Also, we found that maximum lorazepam potentiation (1 µM; 142 ± 8% potentiation) was
unaffected by 35THDOC (138 ± 14% potentiation) (Fig.
5C).
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Figure 4.
Effect of 3-hydroxypregnane steroids on other
5-reduced and 5-reduced steroid potentiators of GABAA
responses. A, Concentration dependence of 35THDOC
potentiation in the absence (filled circles) and
presence (open squares) of 10 µM
35THDOC. GABA (2 µM) was coapplied with steroids,
and the response to 2 µM GABA was used as the normalizing
response (IN). B,
C, Similar analyses using the 5-reduced steroids
35P and 35THDOC as potentiators. In all
panels, the solid line through the
concentration-response values in the absence and presence of
35THDOC is the best fit of the Hill equation. Parameters of the
fits and n values are given in Table 1.
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Figure 5.
Effect of 3-hydroxysteroids on two other
classes of GABAA receptor potentiators. A,
Effect of 35THDOC on potentiation by the benzodiazepine lorazepam
in an oocyte. B, Effect of 35THDOC on potentiation
by the barbiturate pentobarbital in another oocyte. C,
Summary of the effects of 10 µM 35THDOC on
pentobarbital (150 µM; n = 13) and
lorazepam (1 µM; n = 5) potentiation.
Note that the more robust potentiation by the barbiturate was more
effectively antagonized. *p < 0.01 indicates
significant inhibition.
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At high concentrations, 3-hydroxysteroids can weakly gate the
GABAA receptor (Barker et al., 1987). We
investigated whether this direct gating was antagonized by 3-hydroxy
steroids. We found that 35P (30 µM) in the absence
of GABA gated a current with an amplitude 34 ± 4% of response to
2 µM GABA in the same cell. Consistent with a lack of
direct antagonism between 3-hydroxysteroids and effects of
3-hydroxysteroids, we observed no significant reduction in this
steroid-gated current when 35THDOC was coapplied with 30 µM 35P (30 ± 6% of the response to 2 µM GABA; n = 4).
Direct 3-hydroxysteroid inhibition of GABA responses mimics the
action of sulfated steroids and explains steroid antagonism
The pattern of noncompetitive inhibition of potentiation, the lack
of structural specificity to the blocking effects, and the promiscuity
of the effects of 3-hydroxysteroids against at least two classes of
potentiators suggest that the mechanism of 3-hydroxysteroids does
not result from a direct interaction with potentiating steroids sites
on the GABAA receptor. Among all three classes of
potentiators, a pattern emerged in which only strong potentiation was
inhibited by 3-hydroxysteroids (effects on only high concentrations
of steroids and barbiturates, no effect on the modest benzodiazepine
potentiation). This pattern suggested the hypothesis that block by
3-hydroxysteroids may correlate with opening of
GABAA receptors.
To test this hypothesis, we examined the effect of 3-hydroxysteroids
on responses to GABA alone over a range of GABA concentrations. We
found that responses to low concentrations (<15 µM) of
GABA were unaffected by 10 µM 35THDOC (Fig.
6A,B).
However, responses to 15 µM GABA were
significantly depressed by 10 µM 35THDOC. The profile of 35THDOC effects against GABA was similar to that observed against potentiation by 3-hydroxysteroids (Fig.
6B) (compare Figs. 2D, 4). The
effect of 35THDOC was concentration dependent, with an
IC50 against 20 µM GABA
responses of 6.8 µM (n = 6)
(Fig. 6C). We again tested the panel of 3-hydroxysteroids shown in Figure 3 and found that, at a fixed concentration of 10 µM, all similarly blocked currents in response
to 20 µM GABA (n = 6) (data not
shown). IC50 values for other
3-hydroxysteroids against 20 µM GABA were as
follows: 35P, 12.8 µM (n = 5); 35P, 14.4 µM (n = 5); and 35ACN, 9.5 µM (n = 6).
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Figure 6.
3-Hydroxypregnane steroids block responses to
high concentrations of GABA. A, Sample traces showing
lack of effect of 10 µM 35THDOC on responses to 2 µM GABA but inhibition of responses to 20 µM GABA. Drugs were coapplied for 20 sec. Note the change
in vertical calibration bars between the
left and right panels. B,
GABA concentration-response curves in the absence
(filled circles) and presence (open
squares) of 10 µM 35THDOC. Each
point is calculated relative to the normalizing response
(IN) activated by 2 µM
GABA. The solid line through GABA
concentration-response values (circles) is the best fit
of the Hill equation, with an EC50 of 12.6 µM
and a Hill coefficient of 2.6 (n = 8). The
solid line through GABA plus 35THDOC interaction
values (squares) is the best fit of the Hill equation,
with an EC50 of 9.4 µM and a Hill coefficient
of 2.1 (n = 8). C. The graph shows the
concentration-response curve of 35THDOC against 20 µM GABA. The normalizing current
(IN) was the response to 20 µM GABA. The solid lines are the fit of
the Hill equation, with an IC50 of 6.8 µM and
a Hill coefficient of 0.8, with maximum inhibition of 1.0
(n = 6).
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It is well known that another class of endogenous neurosteroids,
steroids sulfated at C3, block GABAA
receptor-mediated responses (Majewska et al., 1988). Block is not
dependent on the stereochemistry of the sulfate group at C3, with
3-sulfated steroids blocking GABA responses with similar potency as
3-sulfated steroids (Park-Chung et al., 1999). Block is not
dependent on the presence of a sulfate group, because steroids
containing other anionic groups also block GABA currents (Park-Chung et
al., 1999). This class of blockers exhibits additional structural
nonspecificity, because the charged group can be placed at C24 rather
than C3 (Mennerick et al., 2001). Previous work has also shown that
charge is not essential for blocking activity, because
dehydroepiandrosterone sulfate and dehydroepiandrosterone both block
GABAA receptors (Demirgoren et
al., 1991; Le Foll et al., 1997). We addressed the possibility that
3-hydroxysteroids are GABAA receptor blockers,
acting similarly to sulfated steroids. As a first test of this
hypothesis, we repeated the experiments shown in Figure 4, substituting
300 nM 35PS for 10 µM 35THDOC.
The 3-sulfated steroid produced inhibition of steroid potentiation
that was essentially indistinguishable from the inhibition produced by
the 3-hydroxysteroids (Fig.
7A1,A2). In
addition, 35PS clearly inhibited responses to high GABA
concentrations more effectively than it inhibited responses to low
concentrations of GABA (Fig. 7B). When examined against a
fixed GABA concentration of 20 µM, the
IC50 for 35PS block was ~190
nM (n = 6) (Fig. 7C),
much lower than the IC50 for 35THDOC under
the same conditions (Fig. 6C). Pregnenolone sulfate was also
quite potent, with an IC50 against 20 µM GABA responses of 385 nM (data not shown).
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Figure 7.
The action of 3-hydroxysteroids is similar to
that of sulfated steroid block. A, Inhibition of
potentiation by a sulfated steroid. A1, Sample traces
showing the very small effect of 300 nM 35PS against
potentiation by 1 µM 35P but stronger inhibition
against GABA responses potentiated by 10 µM 35P.
A2, Summary of experiments like that shown in
A1, in which multiple concentrations of potentiator were
examined. GABA (2 µM) was coapplied with varied
concentrations of 35P either without (filled
circles) or with (open squares) 300 nM 35PS. Parameters of fits to the Hill equation and
experimental n are given in Table 1. B,
Sulfated steroid block, like 3-hydroxysteroid block, is dependent on
GABA concentration. Shown are concentration- response curves for
GABA in the absence (filled circles) and presence
(open squares) of 300 nM 35PS. In the
absence of steroid, the EC50 for GABA was 12.5 µM, with a Hill coefficient of 2.8 (n = 7). In the presence of steroid, the GABA EC50 was 11.4 µM, with a Hill coefficient of 3.0 (n = 7). The normalized maximum response was decreased from 13.9 to 7.8 in
the presence of steroid. C, Against a fixed GABA
concentration of 20 µM, 35PS produced a
concentration-dependent inhibition of responses. The solid
line is a fit of the Hill equation, with an IC50 of
189 nM and a Hill coefficient of 0.7 (n = 6).
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To further explore whether the action of 3-hydroxypregnane steroids
is similar to that of sulfated steroids, we used a recently characterized point mutation causing resistance to sulfated steroid block (Akk et al., 2001). The mutant carries a valine to serine substitution at position 256 of the 1 subunit, located on the cytoplasmic side of transmembrane domain 2. Because we observed that
the degree of block by both 3-hydroxysteroids and 3-sulfated steroids was dependent on GABA concentration, we examined the GABA
concentration-response profile for wild-type and mutated receptors. We
found that the mutation caused a large (~20-fold) leftward shift of
the GABA concentration-response relationship. To ensure that we
examined the effect of 3-hydroxysteroids and sulfated steroids under
conditions that should promote block, we examined the effect of the
steroids against 5 µM GABA in mutated receptors, a
concentration that produced nearly maximum responses (Fig.
8A, arrow).
Comparison was made with wild-type responses at 20 µM, a concentration somewhat higher than the
EC50 (Fig. 8A,
arrow).
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Figure 8.
A point mutation in the 1 subunit (V256S)
reduces sulfate and 3-hydroxysteroid block of GABAA
receptors. A, GABA concentration-response curves for
wild-type (filled squares; n = 5) and mutated (open squares; n = 6) GABAA receptors. Responses at 1 µM were
set to 1 for both wild-type and mutated receptors, and other responses
in the same oocyte were normalized to this response. Note that the
left y-axis corresponds to the wild-type
normalized responses, and the right
y-axis corresponds to the normalized responses from
mutated receptors. Absolute amplitudes of maximum responses did not
notably differ between wild-type and mutated receptors, but the
mutation resulted in an ~20-fold leftward shift in the GABA
EC50, from 9.6 to 0.53 µM, resulting
in the apparently larger normalized maximum responses for wild-type
receptors. Arrows indicate concentrations of GABA used
in subsequent studies of steroid block of GABAA receptors
(B, C). B, Sulfated
steroid effects (1 µM) in wild-type (WT,
filled bars; n = 5) and 1V256S
mutated (open bars; n = 5)
GABAA receptors. C, 3-Hydroxysteroid
effects (10 µM) in wild-type (WT,
filled bars; n = 6) and 1V256S
(open bars; n = 9) mutated
GABAA receptors.
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Figure 8B (filled bars) shows a
summary of the effect of various sulfated steroids at a fixed
concentration of 1 µM against responses to 20 µM GABA in wild-type receptors. All sulfated
steroids blocked more than half the current under these conditions. In contrast, none of the sulfated steroids significantly inhibited responses of mutated receptors, despite the use of a functionally higher GABA concentration than used for the wild-type receptors (Fig.
8B, open bars). We found a similar pattern
when a panel of 3-hydroxysteroids were examined against wild-type
and mutated receptors (Fig. 8C). In contrast, we found no
difference in the ability of other GABAA receptor
antagonists (gabazine, bicuculline, and picrotoxin) to inhibit mutated
receptors (n = 8) (data not shown). These results are
consistent with the idea that 3-hydroxysteroids and sulfated
steroids block GABAA receptors similarly, albeit with different potencies.
To address whether the direct effects on GABAA
receptors are sufficient to explain the apparent antagonism of
potentiating steroids, in the same oocytes, we matched the size of GABA
currents produced by a moderate concentration of GABA alone to that
produced by 2 µM GABA plus potentiating steroid (Fig.
9A,C).
We found that 20 µM GABA produced responses of
similar amplitude to those produced by 3 µM
35P plus 2 µM GABA. Responses to 20 µM GABA alone or the combination of 2 µM GABA plus 35P were inhibited to a
similar degree by 10 µM 35THDOC (Fig.
9A,C). These results are consistent with the idea that the direct effect on GABAA
receptors can account for the apparent antagonism of potentiating
steroid effects.
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Figure 9.
Direct GABAA receptor block explains
3-hydroxysteroid inhibition of potentiation. A,
Comparison of responses "potentiated" by increasing GABA
concentration or by adding 3 µM 35P. The
traces show that 20 µM GABA and 3 µM 35P cause a similar increase in current relative
to 2 µM GABA. 35THDOC (10 µM)
inhibited both increased currents to a similar degree.
B, Mutated receptors are potentiated by
3-hydroxysteroids, but steroid potentiation is not inhibited by
3-hydroxysteroids. C, Summary of the blocking actions
of 35THDOC on 20 µM GABA-activated current and
potentiation of 2 µM GABA-activated current by 3 µM 35P (n = 7) in oocytes
expressing wild-type receptors. The bar graph represents current
amplitudes normalized to the 2 µM GABA response.
D, The bar graph represents a summary of the lack of
effect of 35THDOC on potentiation by 35P in mutated
receptors. Normalizing current was the response activated by 0.1 µM GABA alone (n = 8).
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As another test of whether direct block of GABAA
receptor function can account for the apparent antagonism of
potentiating steroids, we examined steroid potentiation of 1 V256S
mutated receptors and the effect of 3-hydroxypregnane steroids on
potentiation. For studies of potentiation at wild-type receptors, we
typically used 2 µM GABA, approximately fivefold below
the GABA EC50. For studies of potentiation at
mutated receptors, we used 100 nM GABA to functionally
match the GABA concentration with that used on wild-type receptors
(Fig. 8A). We found that 3 µM 35P robustly potentiated currents
generated by mutated GABAA receptors. No additional potentiation was observed at 10 µM
35P (n = 8) (data not shown). In contrast to
wild-type receptors, we observed no significant antagonism of this
potentiation by 35THDOC (Fig. 9, compare
B,D with C).
Actions of 3-hydroxysteroids and sulfated steroids at
GABA synapses
Although these experiments show that 3-hydroxypregnane steroids
are not true antagonists of potentiating steroids and act through an
action similar to sulfated steroids, it is possible that either class
of steroid could be a useful functional antagonist against postsynaptic
potentiation at synapses. It is thought that a high GABA concentration
is present at the synapse but only briefly (~1 msec) (Maconochie et
al., 1994; Jones and Westbrook, 1995). If binding and action of
3-hydroxysteroids or sulfated steroids is dependent on GABA
concentration and time, it is possible that blocking steroids are
relatively inert against normal GABA transmission but more effective
against responses potentiated by 3-hydroxysteroids or other
postsynaptic potentiators. In pilot experiments on cultured hippocampal
neurons, we found that 2 µM 35PS produced
inhibition of responses to exogenous GABA that was equivalent to 10 µM 35THDOC (data not shown). This suggests that, in
hippocampal cells, there is only approximately fivefold
difference in potency between the 3-hydroxysteroid and the sulfated
steroid compared with the 35-fold difference observed in oocytes (Figs.
6C, 7C). We therefore examined the effect of 10 µM 35THDOC and 2 µM 35PS on IPSCs generated by recurrent
(autaptic) synapses formed in culture (Mennerick et al., 1995). We
found that 35THDOC and 35PS at these concentrations had
little effect on IPSCs generated in the absence of postsynaptic modulator (13 ± 12% decrease in 10-90% decay time for
35THDOC; 20 ± 9% decrease for 35PS;
n = 6) (Fig.
10A,C).
Despite the relative inertness of the steroids on baseline IPSCs, the
same concentrations significantly inhibited the ability of a
potentiating steroid (35P, 0.5 µM) to
prolong IPSCs (Fig. 10B,C). Also,
consistent with a direct effect on GABAA
receptors rather than a competitive inhibition of the potentiating
steroid, 30 µM 35THDOC alone significantly speeded the 10-90% decay time of baseline IPSCs (38 ± 14%; n = 15). Neither 10 nor 30 µM 35THDOC had any appreciable effect on
the peak IPSC amplitude (4 ± 3 and 8 ± 4% increase, respectively). In summary, these data are consistent with the idea that
3-hydroxysteroids and sulfated steroids share a similar mechanism
and suggest that these blockers might be useful under some
circumstances as functional antagonists of postsynaptic potentiation at
GABAergic synapses.
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Figure 10.
Similar synaptic effect of 35THDOC and
35PS. A, Autaptic IPSCs were elicited from
solitary GABAergic hippocampal neurons in microcultures with 2 msec
voltage pulse to 0 mV from a holding potential of 70 mV. The
traces represent responses obtained in the absence and
presence of 10 µM 35THDOC. Note that the drug alone
only slightly affected the IPSC. B, The
traces represent responses obtained in the absence and
presence of 0.5 µM 35P and 0.5 µM
35P plus 10 µM 35THDOC. Note that 35P
significantly prolonged the decay time course of the IPSC.
35THDOC inhibited the prolongation induced by potentiating
steroid. C, Summary of the effect of 10 µM
35THDOC and 2 µM 35PS on IPSCs in the absence
and presence of 0.5 µM 35P (n = 6 neurons). For this analysis, we used 10-90% decay times, which
averaged 315 ± 71 msec in the control situation. We also fit IPSC
decays with multiple exponential components (Zorumski et al., 1998).
Weighted time constants
( ai I, where
ai is the fractional amplitude and
I is the time constant of each exponential component)
were also used to quantify the data (Jones and Westbrook, 1997). Raw
values from the weighted time constant analysis were 137 ± 30 msec (control), 119 ± 20 msec (35THDOC alone), 101 ± 18 msec (35PS alone), 486 ± 63 msec (35P alone),
176 ± 20 msec (35P plus 35THDOC), and 266 ± 48 msec (35P plus 35PS).
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DISCUSSION |
We characterized the actions of 3-hydroxypregnane steroids,
which previous reports suggested are competitive antagonists against
3-hydroxypregnane steroid potentiators. Although the 3-hydroxysteroids behave as functional inhibitors of
GABAA receptor potentiation, we find that the
antagonism is noncompetitive with respect to 3-hydroxypregnane
steroids. In fact, 3-hydroxysteroids are also noncompetitive
antagonists of GABAA receptors themselves by
actions similar to that of sulfated steroids. Both sulfated steroids
and 3-hydroxysteroids block GABAA receptors
more effectively under conditions that promote channel opening,
suggesting that the direct antagonism of GABA responsiveness may
represent ligand-dependent or state-dependent block. This direct action
at GABAA receptors accounts for the apparent
antagonism of potentiating steroid effects.
Our results contrast with those reported by others using different
methods. Using [3H]flunitrazepam
binding, it was shown previously that 35P competitively reduced
the increase in binding produced by potentiating steroids (Prince and
Simmonds, 1992, 1993). Other studies suggest that 35P acts as a
partial agonist at the potentiating steroid site (Pignataro and Fiszer
de Plazas, 1997). Our results do not support the competitive
interaction, observed in binding studies, between 3-hydroxypregnane
steroids and 3-hydroxypregnane steroids.
Antagonism of potentiation by 3-hydroxysteroids has also been
reported in previous electrophysiological studies (Garrett and Gan,
1998; Maitra and Reynolds, 1998), but the nature of the interaction
between 3- and 3-hydroxysteroids was not explored in any of these
studies. In previous studies, 35P marginally potentiated or had
no effect on responses to GABA alone at GABA concentrations less than
EC50, similar to our r
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ABSTRACT
INTRODUCTION
