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-Aminobutyric Acid A Receptor:
Evidence for a Positive Modulatory Site
Department of Anatomy and Neurobiology (K.L.W., J.B.T., J.E.K., S.M.R.), Departments of Neurology and Pediatrics (S.M.R.), Department of Molecular Biology and Pharmacology (D.F.C.), Washington University School of Medicine, Saint Louis, Missouri 63110, Department of Neuroscience, University of Alabama at Birmingham, Birmingham, Alabama 35294 (D.S.W.), Department of Neurology, University of Texas, Houston, Texas 77225, and Neurogen Corporation, Branford, Connecticut 06425 (G.W.)
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Summary |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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The 
-aminobutyric acid-A (GABAA) receptor complex is
allosterically modulated by a variety of substances, some of clinical importance. Barbiturates and neurosteroids augment GABA-currents and
also directly gate the channel. A variety of -butyrolactone analogues also modulate GABA-induced currents, with some potentiating and others inhibiting. Because several -thiobutyrolactone analogues have biphasic effects on GABA currents, experiments with wild-type and
picrotoxinin-insensitive GABAA receptors were performed to analyze whether some -thiobutyrolactones interact with two
distinguishable sites on the GABAA receptor.
-Ethyl--methyl--thiobutyrolactone inhibited GABA-induced
currents at low concentrations (0.001-1 mM), but
potentiated GABA-induced currents at higher concentrations (3-10
mM) in wild-type 
122-subunit containing
ionophores. The related -ethyl--methyl--thiobutyrolactone
potentiated submaximal GABA currents in wild-type receptors at both low
and high concentrations (0.1-10 mM). Mutations in the
second transmembrane domain of 1, 2, or 2 conferred
picrotoxinin-insensitivity onto GABAA receptor complexes.
When these mutated 1, 2, or 2 subunits were incorporated into
the receptor complex, -ethyl--methyl--thiobutyrolactone potentiated GABA currents over the entire concentration range (0.1-10
mM). Neither the potentiating activity nor the
EC50 of -ethyl--methyl--thiobutyrolactone changed
in the mutant receptors. Further studies demonstrated that the
mutations did not affect the EC50 of chlordiazepoxide or
phenobarbital. These and our earlier results identify a modulatory site
on the GABAA receptor distinct from that interacting with
barbiturates, benzodiazepines, and steroids. Additionally, they show
that the -butyrolactones probably interact at two different sites on
the ionophore to produce opposite effects on GABA-mediated current.
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Introduction |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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The GABAA receptor is responsible for most fast inhibitory neurotransmission in the central nervous system. Consequently, this receptor has been targeted for the pharmacological control of anxiety, sleep, and epilepsy. Numerous natural and synthetic compounds interact with the GABAA receptor at distinct, yet incompletely defined, sites. These compounds include barbiturates, benzodiazepines, neurosteroids, and picrotoxin (1, 2).
-Butyrolactones and the related TBLs are a group of convulsant and
anticonvulsant agents that interact with the GABAA
receptor, but not at the benzodiazepine or barbiturate sites (3, 4). Behavioral studies have demonstrated the protective effects of these
compounds against various seizure models (5). Displacement studies with
[35S]TBPS, a ligand binding at the picrotoxinin site (6),
suggested an interaction between the TBLs and the picrotoxinin site on
the GABAA receptor. The hypothesis that TBLs interacted
with the GABAA receptor at the picrotoxinin site as either
agonists or inverse agonists (i.e., blockers or potentiators of
GABA-induced currents, respectively) explained most of the findings
(7).
The agonist/inverse agonist concept was not novel, because benzodiazepine receptor ligands can negatively (e.g., DMCM) and positively (e.g., diazepam) modulate channel behavior (8). The benzodiazepine receptor ligands are thought to act at just one site (9, 10).
Several new observations led to a re-evaluation of the TBL agonist/inverse agonist model. Holland and colleagues (11, 12) showed biphasic effects of several TBLs on GABA currents. They found that the same compound could both block and potentiate GABA currents, albeit with different time and concentration dependencies. They also observed noncompetitive interactions between [35S]TBPS and some TBLs, suggesting two sites of interaction: a negative modulatory site (the picrotoxinin site) and a yet-to-be-defined, positive modulatory site (the lactone site). In the present studies picrotoxinin-insensitive GABAA receptor mutants were used to test this hypothesis. If the two-site model for lactone interaction with the receptor were correct, the biphasic nature of the TBLs on GABA currents should disappear, when one site was ablated.
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Materials and Methods |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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GABAA subunits.
The cDNAs encoding
GABAA subunits 1 were provided by A. Tobin (University
of California, Los Angeles), 2 by P. Malherbe (Hoffman-La Roche,
Switzerland), and 2L by C. Fraser (National Institute on
Alcohol Abuse and Alcoholism).
Mutagenesis. Mutations were created as previously described (13).
Compounds.
GABA, chlordiazepoxide hydrochloride, and
phenobarbital were obtained from Sigma (St. Louis, MO), and DMCM was
obtained from Research Biochemicals International (Natick, MA).
-EMTBL and -EMTBL were synthesized by previously described
methods and had the appropriate analytical and spectroscopic properties
(5).
cRNA preparation.
The cRNA transcripts were prepared in the
following manner. The 5
- and 3-untranslated region of cDNA templates
were removed to improve channel expression, and an optimal Kozak (14)
sequence (CCACC) was added upstream of the initiator methionine to
enhance mRNA binding to the 40 S ribosomal subunit. cDNA sequences used in the study were verified by automated nucleotide sequence analysis using the Applied Biosystems Incorporated (ABI; Foster City, CA) Prism
Dye Terminator cycle sequencing system and an ABI 373A DNA sequencer.
The modified cDNA templates were linearized with the appropriate
restriction enzyme, and transcripts were prepared with Ambion's
mMessage mMachine kit (Austin, TX). As a final check, each cRNA
preparation was subjected to either polyacrylamide or agarose gel
analysis to verify size and quantity.
Oocyte collection and injection.
Oocyte preparation was
modified from the methods of Gurley et al. (13).
Xenopus laevis oocytes were collected from mature, pigmented
females obtained from Xenopus One (Northland, MI). After removal, the
oocytes were placed in an OR-2 solution containing 100 units of
penicillin and 100 µg/ml streptomycin obtained from Sigma and 2 mg/ml
collagenase I from Boehringer-Mannheim (Indianapolis, IN). The oocytes
were agitated vigorously until the follicular layer was removed.
Usually, the oocytes were injected the same day with a 20-60-ng
mixture of , , and subunits, using the Drummond Nanojector
(Broomall, PA). The oocytes were incubated for 1-3 days in L-15 medium
(GIBCO, Grand Island, NY) supplemented with 100 units/ml penicillin,
100 µg/ml streptomycin, and 50 µg/ml gentamicin sulfate (Sigma).
Electrophysiology and data analysis.
The electrophysiology
was carried out using Warner Instrument's 725C (Hamden, CT)
two-electrode voltage clamp with a virtual ground bath clamp.
Electrodes filled with 3 M KCl (resistance < 1 M
)
were used to impale the oocytes. The oocytes were clamped at a
potential of 
70 mV. Peak currents were recorded, low pass filtered at
20-30 Hz by an 8-pole Bessel filter (Frequency Devices, Haverhill,
MA), digitized by a TL-1 DMA interface (Axon Instruments, Foster City,
CA), and stored using Axon Instruments' AxoTape. The oocytes were
perfused with a solution containing the following: 90 mM
NaCl, 1 mM KCl, 2 mM
MgCl2, 5 mM HEPES and 1 mM
CaCl2 (pH 7.3). Solutions were bath applied by a
gravity-driven perfusion system until the response reached plateau or
until a rapidly desensitizing response peaked, which took anywhere from
3 to 45 sec approximately. No correction in peak amplitude was made for
quickly desensitizing responses that were common at high GABA
concentrations, high drug concentrations, and also in mutated
receptors. In the determination of the EC50 values of
-EMTBL, -EMTBL, and chlordiazepoxide, the GABA EC10
was used as a control. Data were fit using a logistic equation
(SigmaPlot; Jandel Scientific, Sausalito, CA), y = [(a d)/(1 + (x/EC50)nH)] + d, where y is the response, a is the
asymptotic maximum, d is the asymptotic minimum,
x is the ligand concentration, and nH
is the Hill coefficient. In situations in which the 10 mM response was smaller than the 3 mM response,
the 10 mM value was not used in the fitting procedure. This
value was ignored, as it probably reflected some degree of both channel
block and desensitization in addition to the potentiation; fitting such
complex behavior is beyond the limits of the logistic equation. Even
after elimination of the 10 mM response, concentration
responses for -EMTBL and -EMTBL were fit well using the logistic
equation and allowing the maximum to float.
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Results |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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GABA sensitivity.
To confirm that the double point mutations
in the TM2 did not drastically alter the behavior of the channel, the
GABA responses of the mutant ionophores were examined. Wild-type
122 GABAA receptors had an EC50 value
of 42 µM; the GABA EC50 values of the
channels containing mutated subunits changed by less than 5-fold (Fig.
1A). The apparent affinity of 1M22
and 12M2, but not 122M, for
GABA was significantly different from that of wild-type channels (ANOVA
with Dunnett's test; p < 0.05). Although these shifts
could reflect changes in GABA affinity for the receptor, they could
also have resulted from altered GABA gating (15, 16).
|
Picrotoxin and picrotoxinin sensitivity of wild-type
122.
Two point mutations in the TM2 of the 1, 2, or
2 GABA subunit or a single point mutation in the 2 subunit
produce picrotoxin-insensitivity (13). The pharmacology of the
GABAA receptor was studied using the approximate GABA
EC10 as a control. Oocytes expressing the wild-type
GABAA receptor were inhibited by both picrotoxin and picrotoxinin (the active component of picrotoxin), with
IC50 values of 1.2 and 0.8 µM, respectively
(data not shown).
12M2 showed a similar
insensitivity to picrotoxin and picrotoxinin (Fig. 1B) (13). The other
two mutant complexes (1M22 and
122M) also were picrotoxinin-insensitive (Fig.
1B). Because of the small magnitude of currents observed when two or
more mutated subunits were co-injected, such receptor complexes were
not studied.
-EMTBL modulation of wild-type and mutant receptors.
-EMTBL both inhibits and potentiates GABA currents in primary
cultures of hippocampal neurons (12). At concentrations less than 1 mM, -EMTBL inhibited GABA currents in wild-type
receptors; at 1 mM, -EMTBL partially relieved its own
block; finally, at 3 and 10 mM -EMTBL, the macroscopic
current was potentiated relative to control (Fig. 2A).
This biphasic modulation was even more clearly seen in the
concentration response curve (Fig. 2C).
|
-EMTBL did not
inhibit GABA currents in the picrotoxinin-insensitive channels (Fig.
2B). Below 300 µM -EMTBL, GABA currents were no different from control or only slightly potentiated. Above this concentration range, potentiation was robust and occasionally greater
than that seen in the wild-type (Fig. 2C).
-EMTBL modulation of wild-type and mutant receptors.
-EMTBL is one of the most potent and efficacious anticonvulsant TBL
analogues. It does not produce biphasic modulation like -EMTBL,
suggesting it does not interact strongly with the picrotoxinin receptor. With oocytes expressing wild-type 122
GABAA receptors, -EMTBL potentiated submaximal GABA
currents. At 10 mM, -EMTBL exhibited less potentiation
than at 3 mM and also exhibited off-currents (Fig.
3A).
|
-EMTBL's activity was unchanged in the mutated receptor complexes
(Fig. 3B). Potentiation was observed over the entire concentration range, even though the 10 mM response once again fell off.
Quantitatively, the EC50 values were relatively unaffected:
-EMTBL's EC50 in the picrotoxinin-insensitive channels
differed by less than 20% from wild-type. Yet, the efficacy for one of
the complexes (1M22) was 50% greater than
wild-type (Fig. 3C).
Chlordiazepoxide and phenobarbital modulation.
Double point
mutations in the TM2 sufficed to eliminate picrotoxinin modulation of
the channel; moreover, GABA concentration responses from two types of
picrotoxinin-insensitive channels were significantly different from
wild-type channels. To address whether these mutations affected more
than picrotoxinin and -EMTBL modulation, we examined three other
allosteric modulators of the channel: a benzodiazepine agonist,
benzodiazepine inverse agonist, and a barbiturate. Each compounds'
activity was qualitatively unaffected by the double point mutations in
the TM2. Chlordiazepoxide's EC50 was not changed by more
than 4-fold by the mutations (Table 1);
only the EC50 for 1M22 was
statistically different from the wild-type EC50. The efficacy, however, was quite variable, with the 1 mutant giving the
most robust response (Table 2).
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2
mutant DMCM's efficacy was unchanged, confirming that the point
mutations abolished only picrotoxinin's negative modulation (Table 2).
Phenobarbital could not be modeled using the logistic equation, because
its concentration response reflects not only potentiation of GABA-gated
currents, but also direct gating by phenobarbital. Hence, only its
efficacy is reported, but there were no significant differences
(one-way ANOVA: p = 0.42) in phenobarbital efficacy by
mutations in the TM2 (Table 2).
Flumazenil inhibition.
Last, flumazenil was used to test
whether the lactone interacted with the ionophore at the benzodiazepine
receptor. Flumazenil was unable to block -EMTBL potentiation in a
wild-type 122-containing ionophore, providing additional
evidence that the lactone site is distinct from the benzodiazepine site
(Fig. 4).
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| |
Discussion |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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The results in this study alter our early view that lactones
modulate GABAA currents through action at a single site on
GABAA channels, the picrotoxinin receptor. Because
picrotoxinin contains a -butyrolactone ring as part of its
structure, it seemed reasonable that the neuroactive -butyrolactone
analogues could interact at the GABAA receptor through the
picrotoxinin binding site (18, 19). Supporting evidence for this
hypothesis came from [35S]TBPS displacement studies that
revealed a high affinity, competitive interaction between
-substituted TBLs and the picrotoxinin receptor. Moreover, the
anticonvulsant -substituted TBLs also displayed an ability to
displace TBPS, albeit with less potency (20). Furthermore, modeling of
picrotoxinin and different lactones demonstrated potential regions for
interaction with the channel interior (19). Therefore, the
agonist/inverse agonist model of lactone activity seemed firmly
grounded both experimentally and theoretically.
Recent findings prompted us to consider the hypothesis that lactones
may interact at two sites (11, 12). To demonstrate clearly that some
TBLs interact with two sites, one of the sites had to be functionally,
even if not physically, absent. With the picrotoxinin-like effect
absent, -EMTBL only potentiated GABA currents. The affinity and
potentiation activity of -EMTBL was unchanged, suggesting that the
-substituted lactones have, at best, weak interactions with the
picrotoxinin site.
Two issues were considered in interpreting the data. Inasmuch as the
mutations were introduced into the TM2, a region of the GABAA subunits critical for proper channel function, it is
possible that these mutations could have conformational consequences
that led to the loss of the biphasic activity of -EMTBL. Although the observed picrotoxinin insensitivity could have resulted from disruption of the picrotoxinin binding site, it could also be explained
by restricted access to a binding site, or altered channel conformation
after picrotoxinin binding. If conformational changes were disturbed by
the mutations, then the loss of -EMTBL's biphasic activity would be
difficult to ascribe to two binding sites. If this were the case, the
activity of other modulators might be expected to change as well. Given
that the mutants' responses to GABA, -EMTBL, chlordiazepoxide,
DMCM, and phenobarbital are qualitatively similar to responses in the
wild-type channel, these picrotoxinin-insensitive channels do not
appear to alter the conformational equilibria. Therefore, these
mutations appear to target specifically the picrotoxinin site and do
not alter macroscopic responses to other modulators of
GABAA currents.
A second concern in interpreting the data was the decrease in potentiation seen at 10 mM lactone (Figs. 2B and 3, A and B). The fall-off in potentiation can be attributed to desensitization or to block at a second site at high drug concentrations (12). The contribution of desensitization can be inferred from similar fall-offs in current seen with high concentration GABA responses (10 mM), chlordiazepoxide (100 µM; data not shown), and phenobarbital (3 mM; data not shown). The other consideration, a second block site, is reflected by the off-currents observed with the washout of high lactone concentrations (arrows in Figs. 2, A and B, and 3, A and B). Similar block has also been seen with washout of 3 mM phenobarbital, so this is not necessarily unique to the lactones.
We believe that our data can be most parsimoniously explained by a lactone site that is distinct from the barbiturate, benzodiazepine, and steroid sites (4, 7, 21-23). Our next goal is to localize the lactone site and determine the physical explanation for its influence on GABAA channel conductance. We believe that this site represents a logical target for drugs that will be effective therapy for epilepsy, spasticity, and sleep disorders.
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Footnotes |
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Received December 19, 1996; Accepted March 31, 1997
This work was supported in part by National Institutes of Health Grants NS14834, NS07027, and AA09212, the Monsanto Fund, and the Seay Fellowship.
Send reprint requests to: Steven M. Rothman, M.D., Department of Neurology, St. Louis Children's Hospital, One Children's Place, Saint Louis, MO 63110. E-mail: rothman{at}kids.wustl.edu
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Abbreviations |
|---|
GABAA, -aminobutyric
acid-A;
TBL, -thiobutyrolactone;
DMCM, methyl-6,7-dimethoxy-4-ethyl--carboline-3-carboxylate;
TBPS, tert-butyl-bicyclophosphorothionate;
-EMTBL, -ethyl--methyl--thiobutyrolactone;
-EMTBL, -ethyl--methyl--thiobutyrolactone;
TM2, second transmembrane
domain;
ANOVA, analysis of variance;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
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References |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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