Department of Pharmacology, University of Bern, Bern, Switzerland
(E.S., R.B.); Brain Research Institute, Section of Biochemistry and
Molecular Biology, University of Vienna, Wien, Austria (R.F., W.S.);
and Institut de Chimie des Substances Naturelles, Centre National de la
Recherche Scientifique, Gif-sur-Yvette Cedex, France (R.R., R.H.D.)
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Introduction |
Several
types of drug are known to exert their effect via the inhibitory
neurotransmitter GABAA receptor (Sieghart, 1995
). Among them are the benzodiazepines, whose site of action has recently been reviewed (Sigel and Buhr, 1997). Enhancement of the action of the
GABAA receptor results in sedation/hypnosis,
muscle relaxation, anxiolysis, and prevention of convulsions, whereas
inhibition of the GABAA receptor results in
opposite effects. GABAA receptors and their
molecular biology and relatedness to other ligand-activated ion
channels has been extensively reviewed (Burt and Kamatchi, 1991; Dunn
et al., 1994; Macdonald and Olsen, 1994; Rabow et al., 1995; Barnard et
al., 1998).
Recently, a novel stimulatory ligand of the GABAA
receptor, (+)-ROD188, with a novel site of action has been described
(Thomet et al., 2000). (+)-ROD188 has been synthesized as described by Razet et al. (2000a,b) along with about 60 structurally similar substances in the search for novel ligands of the GABA binding site.
None of the compounds acted at the GABA binding site, but many of them
were positive allosteric modulators of the GABAA receptor. Because we could not investigate all 60 compounds, we selected two families of similar structure (Fig.
1). The first family of compounds, here
termed type R1 (ROD164A, ROD185), seemed to interact with the
benzodiazepine binding site, as indicated by
[3H]flunitrazepam displacement in binding
studies and inhibition by a benzodiazepine antagonist. The other family
of compounds, here termed type R2 (ROD222 and ROD259), did not seem to
interact with the benzodiazepine binding site. In this study, we
investigated the relationship of their binding sites with the
benzodiazepine binding site.
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Materials and Methods |
Construction of Receptor Subunits.
The cDNAs encoding the
1, 
2, and 
2S subunits of the rat GABAA
receptor channel have been described elsewhere (Lolait et al., 1989;
Malherbe et al., 1990a,b). For cell transfection, the cDNAs were
subcloned into the polylinker of pBC/CMV. This expression vector allows
high-level expression of a foreign gene under control of the
cytomegalovirus promoter and an SP6 promotor for in vitro transcription. The subunit was cloned into the EcoRI and
the and subunits were subcloned into the SmaI site
of the polylinker by standard techniques.
Expression and Functional Characterization.
Xenopus
laevis oocytes were prepared, injected, defolliculated, and
currents recorded as described previously (Sigel, 1987; Sigel et al.,
1990). Briefly, oocytes were injected with 50 nl of cRNA dissolved in 5 mM K-HEPES, pH 6.8. This solution contained the transcripts coding for
the different subunits at a concentration of 10 nM for 1, 10 nM for
2, and 50 nM for 2 for the triple subunit combination, and of 75 nM for each 1 and 2 for the dual subunit combination. RNA
transcripts were synthesized from linearized plasmids encoding the
desired protein using the message machine kit (Ambion, Austin, TX)
according to the recommendation of the manufacturers. A poly(A) tail of
about 300 residues was added to the transcripts by using yeast poly(A)
polymerase [U.S. Biochemical Corp. (Cleveland, OH) or Amersham
Pharmacia Biotech (Piscataway, NJ)]. The cRNA combinations were
coprecipitated in ethanol and stored at 
20°C. Transcripts were
quantified on agarose gels after staining with Radiant Red RNA Stain
(Bio-Rad, Richmond, CA) by comparing staining intensities with various
amounts of molecular mass markers (RNA-Ladder; Life
Technologies, Grand Island, NY). Electrophysiological
experiments were performed by the two-electrode voltage-clamp method at
a holding potential of 80 mV. The medium contained 90 mM NaCl, 1 mM
KCl, 1 mM MgCl2, 1 mM
CaCl2, and 10 mM Na-HEPES, pH 7.4. GABA was
applied for 20 s without or in combination with other drugs and a
washout period of 4 min was allowed to ensure full recovery from
desensitization. The perfusion solution (6 ml/min) was applied through
a glass capillary with an inner diameter of 1.35 mm, the mouth of which
was placed about 0.4 mm from the surface of the oocyte. The rate of
solution change under our conditions has been estimated 70% within
less than 0.5 s (Sigel et al., 1990). A GABA concentration that
elicited 1 to 5% (50-500 nA) of the maximal current amplitude was
used. Currents were also elicited with 200 µM pentobarbital in the
absence of GABA. They had an amplitude in the range of 30 to 180 nA.
The perfusion system was cleaned between drug applications by washing
with dimethyl sulfoxide to avoid contamination. Data are given as
mean ± S.E.M. (number of experiments).
Binding Assays.
For binding studies HEK-293 cells (CRL 1573;
American Type of Culture Collection, Manassas, VA) were maintained in
Dulbecco's modified Eagle's medium (Life Technologies) supplemented
with 10% fetal calf serum (JHR Biosciences, Lenexa, KS), 2 mM
glutamine, 50 µM -mercaptoethanol, 100 units/ml penicillin G, and
100 µg/ml streptomycin in 75-cm2 Petri dishes
by using standard cell culture techniques.
HEK-293 cells (3 × 106) were transfected
with a total of 21 µg of cDNA encoding for the rat 1, 2, and
2 subunits (ratio 1:1:1) subcloned individually into pCDM8
expression vectors, using the calcium phosphate precipitation method
(Chen and Okayama, 1988). The medium was changed 20 h after
transfection and the HEK-293 cells were harvested 48 h after
transfection by scraping into phosphate-buffered saline. Cells were
centrifuged at 12,000g for 10 min and the cell pellet was
homogenized in 50 mM Tris-citrate buffer, pH 7.4, by using an
Ultraturrax, followed by three centrifugation (200,000g for
20 min) resuspension cycles, and were then used for ligand binding
studies or were stored at 20°C.
For binding assays, membranes from rat forebrain or membranes from
transiently transfected HEK-293 cells were centrifuged and resuspended
in 50 mM Tris-citrate buffer, pH 7.4, at a protein concentration of
about 1 mg/ml as measured by the bicinchoninic acid-protein assay kit
of Pierce (Rockford, IL) with bovine serum albumin as standard.
Membranes (0.5 ml) were then incubated in a total of 1 ml of a solution
containing 50 mM Tris-citrate buffer, pH 7.4, 150 mM NaCl, and various
concentrations of [3H]flunitrazepam, 5 nM
[3H]muscimol, or 2 nM
[35S]TBPS in the absence or presence of 10 µM
diazepam, 10 µM GABA, or 10 µM TBPS or various concentrations of
the ROD compounds for 90 min at 4°C (Zezula et al., 1996). Membranes
were then filtered through Whatman GF/B filters. The filters were
rinsed twice with 5 ml of ice-cold 50 mM Tris-citrate buffer. Filters
were transferred to scintillation vials and subjected to scintillation
counting after addition of 3.5 ml of Hydrofluor (National Diagnostics, Manville, NJ) scintillation fluid. Nonspecific binding determined in
the presence of 10 µM unlabeled compounds was subtracted from total
[3H]flunitrazepam,
[3H]muscimol, or
[35S]TBPS binding, respectively, to result in
specific binding.
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Results |
Two Families of ROD Compounds, R1 and R2, Allosterically Stimulate
GABAA Receptors.
In the search for novel ligands of
the GABA binding site of GABAA receptors, we
investigated numerous compounds with a structure similar to the
competitive antagonist of GABA, bicuculline. The structure of some
selected compounds is shown in Fig. 1. None of these compounds was able
to interact with the GABA binding site as indicated by the lack of
displacement of [3H]muscimol from rat brain
membranes (data not shown). However, some, but not all compounds were
able to displace [3H]flunitrazepam. Although
ROD164A, ROD185, and ROD178B were active in the low micromolar range,
(+)-ROD188 had a reduced activity and ROD222 and ROD259 none at all
(Table 1). Scatchard analysis of
[3H]flunitrazepam binding to recombinant
122 receptors in the absence or presence of ROD185 or
(+)-ROD188 indicated a competitive interaction of these compounds with
the [3H]flunitrazepam binding site (Fig.
2). Based on the ability to displace
[3H]flunitrazepam we named ROD164A and ROD185
R1 compounds and ROD222 and ROD259 R2 compounds.
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TABLE 1
Binding properties of ROD compounds
Data (means ± S.E.M. from at least three experiments performed in
triplicates) are from rat forebrain membranes. The concentration of
[3H]flunitrazepam or [35S]TBPS is 2 nM.
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Fig. 2.
Scatchard analysis of [3H]flunitrazepam
binding in the absence and presence of ROD185 (A) or (+)-ROD188 (B).
Membranes from HEK-293 cells transfected with 122
GABAA receptor were incubated with various concentrations
of [3H]flunitrazepam in the absence ( ) and presence
( ) of 100 nM ROD185 (A) or 300 µM (+)-ROD188 (B). Results are from
one experiment in triplicates. The experiment was repeated twice with
similar results. Bmax values were not
significantly different in the absence or presence of ROD185 or
(+)-ROD188.
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ROD164A and ROD185 were able to displace
[35S]TBPS binding at micromolar concentrations,
indicating an allosteric effect on GABAA
receptors at concentrations higher than that interacting with the
benzodiazepine binding site. ROD178B, ROD222, and ROD259 exhibited only
a weak displacing activity at the [35S]TBPS
binding site (Table 1).
To investigate functional effects of these compounds, recombinant
122 GABAA receptors were expressed in
X. laevis oocytes. None of the investigated compounds at a
concentration of 100 µM opened the channel by itself, but all
compounds except ROD178B allosterically enhanced
GABAA receptors in a concentration-dependent way
(Fig. 3). With ROD164A and ROD185
significant enhancement was seen at 0.1 µM, and for (+)-ROD188,
ROD222, and ROD259 at 1 µM.
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Fig. 3.
Allosteric enhancement of currents elicited by GABA
in 122 GABAA receptors by ROD164A, ROD185,
ROD178B, (+)-ROD188, ROD222, and ROD259. Recombinant rat
GABAA receptors were expressed in X. laevis
oocytes. Application of 2 µM GABA alone resulted in approximately 5%
of the maximal current amplitude. Increasing concentrations of the
compounds were coapplied with GABA. The figure shows cumulative
concentration response curves. Values are shown as mean ± S.E.M.
of three to four oocytes.
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We next investigated the effect of the benzodiazepine antagonist
Ro15-1788 on this current enhancement. Although the enhancement by
ROD164A (Fig. 4), ROD185, and (+)-ROD188
was partially sensitive to 1 µM Ro15-1788, the enhancement by ROD222
and ROD259 was largely resistant to the benzodiazepine antagonist (Fig.
5; Table
2). Quantitative comparison of the degree
of inhibition cannot be made because different concentrations of ROD
compounds were used. The standard concentration was 20 µM. Despite
the higher concentration of ROD164A, inhibition by Ro15-1788 was larger
than for (+)-ROD188, ROD222, and ROD259. ROD185 with its high affinity
to the benzodiazepine binding site was used at a lower concentration to
allow competition with Ro15-1788 to occur.
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Fig. 4.
Enhancement by ROD164A is inhibited by a
benzodiazepine antagonist. The concentration of GABA eliciting
approximately 5% of the maximal current amplitude at recombinant rat
122 GABAA receptors was determined first. ROD164A
(100 µM) markedly enhanced these currents. When 1 µM Ro 15-1788 was
coapplied with 100 µM ROD164A, the enhancement was inhibited to a
large extent.
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Fig. 5.
Inhibition by 1 µM Ro15-1788 of the enhancement by
ROD compounds. The current elicited by GABA was enhanced by either
ROD164A or ROD185, (+)-ROD188, ROD222, and ROD259 at the concentrations
indicated in Table 2. Enhancement was standardized to 100%.
Subsequently, the same concentration of the ROD compound was coapplied
with 1 µM Ro15-1788 and the residual relative enhancement
calculated.
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TABLE 2
Inhibition by Ro15-1788
Currents activated by a GABA concentration that elicited 1 to 5% of
the maximal current amplitude were standardized to 100%. The relative
current amplitude in the presence of drug after subtraction of the
current induced by GABA alone (100%) is given. All experiments were
carried out with recombinant 122 GABAA receptors
expressed in X. laevis oocytes. Peak values were taken
except for the numbers in italics, where values were taken at the end
of the 20-s drug application period, reflecting a time dependence of
drug action.
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Diazepam enhances currents elicited by GABA via the benzodiazepine
binding site. We were interested whether this enhancement was additive
to the enhancement by ROD compounds. Although the current enhancement
by ROD185 was not additive with the one by diazepam, enhancement by
ROD222 was additive (Table 3).
Interestingly, (+)-ROD188 potentiated the effect of diazepam. Whether
this effect was caused by its interaction with the ROD222 site or with
an additional site cannot be decided by the data available.
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TABLE 3
Coenhancement by diazepam and ROD compounds
Currents activated by a GABA concentration that elicited 1 to 5% of
the maximal current amplitude were standardized to 100%. The relative
current amplitude in the presence of drug after subtraction of the
current induced by GABA alone (100%) is given. All experiments were
carried out with recombinant 122 GABAA receptors
expressed in X. laevis oocytes.
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R1 Type Compounds Act at an Additional Site Beside the
Benzodiazepine Binding Site.
We next tested the properties of the
different ROD compounds on 12 GABAA
receptors. Ligands of the benzodiazepine binding site are known to
depend on the presence of a subunit (Pritchett et al., 1989; Sigel
et al., 1990; Günther et al., 1995) for their interaction with
GABAA receptors. Unexpectedly, not only R2 type compounds but also R1 type compounds strongly enhanced 12
GABAA receptor-mediated currents (Fig.
6). We therefore analyzed the inhibition
by the benzodiazepine antagonist Ro15-1788 of the stimulatory effect of
ROD164A on GABA-induced chloride current in 122 receptors in
more detail. To determine whether we are dealing here with competitive
or noncompetitive inhibition, it would be desirable to construct a
Schild plot. However, due to solubility problems, we were limited in
the drug concentration range and were unable to do such an analysis.
Therefore, we studied the concentration-dependent inhibition by
Ro15-1788 of the GABA-induced current enhanced by several different
concentrations of ROD164A. For a competitive inhibition we would expect
here Hill slopes of 1 and a parallel shift of the inhibition curves
to the right with increasing concentrations of ROD164A. The results
shown in Fig. 7 are not compatible with a
competitive interaction of ROD164A with Ro15-1788 at a common binding
site. The nature of the curves, especially at higher concentrations of
ROD164A is difficult to understand. Ro15-1788 completely inhibits the
enhancement by classical benzodiazepines at concentrations less than
0.1 µM (Sigel and Baur, 1988). Judging by the extent of inhibition at
these concentrations of Ro15-1788, stimulatory effects of ROD164A via
the benzodiazepine binding site amount to less than 20% at all
concentrations of ROD164A tested. The inhibition of the current
enhancement by ROD164A at higher concentrations of Ro15-1788 thus could
have been caused by an allosteric effect of Ro15-1788 on an additional
site for ROD164A.
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Fig. 6.
Enhancement by ROD164A, ROD185, (+)-ROD188, ROD222,
and ROD259 in 122 ( ) and in 12 ( ) recombinant
GABAA receptors. The concentration of GABA eliciting
approximately 5% of the maximal current amplitude at the corresponding
GABAA receptor was determined first. This concentration was
then coapplied with 100 µM the substance to be tested. For all the
tested substances enhancement was comparable in 122 and
12 receptors.
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Fig. 7.
Concentration-dependent inhibition in 122
receptors by Ro15-1788 of the enhancement by different concentrations
of ROD164A. Currents elicited by GABA were enhanced by either 2, 10, or
100 µM ROD164A in separate experiments and inhibited by increasing
concentrations of Ro15-1788. Each of the three cumulative inhibition
experiments was carried out with three oocytes, thus each point
represents mean ± S.E.M. (n = 3).
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Interaction between Different ROD Compounds.
ROD178B only
marginally enhanced GABA-induced currents, displaced
[3H]flunitrazepam at micromolar concentrations,
and exhibited only a weak effect on [35S]TBPS
binding. We hypothesized that ROD178B might be an antagonist of
stimulatory ROD compounds. Figure 8 shows
that the enhancement by ROD164A is indeed strongly inhibited by
ROD178B. Table 4 summarizes results on
the inhibition by ROD178B of the enhancement by different ROD
compounds. R2 type compounds are affected to a smaller extent compared
with ROD164A, whereas ROD185 is almost unaffected during the early
phase of the exposure to GABA. For ROD185 relative inhibition increased
during time of drug application and was much larger 20 s after
beginning of the application (Table 4). The reason for this property
that was not observed for type R2 compounds is not clear. Presumably,
the open conformation of the channel accelerates the action of ROD178B.
ROD178B surprisingly also inhibited the enhancement by diazepam very
strongly. From their respective concentrations and from their
respective affinity to the benzodiazepine binding site, an inhibition
of less than 50% would have been expected. This inhibition must
therefore be partially due to an allosteric effect of ROD178B on the
benzodiazepine binding site or on channel gating.
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Fig. 8.
Enhancement by ROD164A is inhibited by ROD178B. The
concentration of GABA eliciting approximately 5% of the maximal
current amplitude at recombinant rat 122 GABAA
receptors was determined first. ROD164A (10 µM) markedly enhanced
these currents. When 100 µM ROD178B was coapplied with 10 µM
ROD164A, the enhancement was inhibited to a large extent.
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TABLE 4
Inhibition by ROD178B
Currents activated by a GABA concentration that elicited 1 to 5% of
the maximal current amplitude were standardized to 100%. The relative
current amplitude in the presence of drug after subtraction of the
current induced by GABA alone (100%) is given. All experiments were
carried out with recombinant 122 GABAA receptors
expressed in X. laevis oocytes. Peak values were taken
except for the numbers in italic, where values were taken at the end of
the 20-s drug application period, reflecting a time dependence of drug
action.
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We further investigated the mode of inhibition by ROD178B of the
current enhancement by ROD164A and ROD259. This investigation is
complicated by the fact that neither the affinity of ROD164A and ROD259
nor the affinity of ROD178B for the receptor is known. Figure
9A shows the concentration-dependent
enhancement by ROD164A in the absence of ROD178B and in its presence at
different concentrations. The data are compatible with a competitive
action of ROD164A and ROD178B. Figure 9B shows the
concentration-dependent enhancement by ROD259 in the absence of ROD178B
and in its presence at different concentrations. These data are not
compatible with a competitive interaction of ROD178B and ROD259. When a
fixed concentration of ROD259 was applied repetitively to the same
oocyte in the presence of increasing concentrations of ROD178B, the
observed inhibition was very small compared with the values given in
Table 4 (data not shown). The same phenomenon was observed with ROD164A
although to a smaller degree. The reason for this discrepancy is not
clear, but obviously repetitive application to an oocyte is not
possible in this case.
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Fig. 9.
Mode of inhibition by ROD178B of the current
enhancement by ROD164A and ROD259. A, concentration-dependent
stimulation by ROD164A in the absence of ROD178B () and in its
presence at 10 µM () and 100 µM (). B,
concentration-dependent enhancement by ROD259 in the absence of ROD178B
() and in its presence at 10 µM () and 100 µM ().
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We also investigated the relationship between type R1 compounds and
type R2 compounds. Currents induced by GABA were enhanced with ROD185,
(+)-ROD188, or ROD222 alone and subsequently in combination with ROD185
in the latter two cases. Concentrations of the compounds were chosen
for a submaximal enhancement. Current enhancement by ROD185 was clearly
not additive with that by (+)-ROD188 or ROD222, indicating a common
site of action or a common way of influencing channel gating (Table
5). The data on the inhibition by ROD178B
of current enhancement by ROD164A and ROD259 argue for the second
possibility.
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TABLE 5
Coenhancement by ROD compounds
Currents activated by a GABA concentration that elicited 1 to 5% of
the maximal current amplitude were standardized to 100%. The relative
current amplitude in the presence of drug after subtraction of the
current induced by GABA alone (100%) is given. All experiments were
carried out with recombinant 122 GABAA receptors
expressed in X. laevis oocytes.
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Relation to the Pentobarbital Binding Site.
Previously, it has
been shown that (+)-ROD188 is able to enhance pentobarbital-induced
currents (Thomet et al., 2000). Here, we demonstrate that currents
elicited by pentobarbital in the absence of GABA were also enhanced by
both ROD185 and ROD259 (Table 6). The
enhancement by ROD185, but not that by ROD259, could be significantly
inhibited by 1 µM Ro15-1788, a benzodiazepine antagonist. Similarly,
diazepam enhanced the currents elicited by pentobarbital in a
benzodiazepine antagonist-sensitive way (Table 6). The concentration
used for the benzodiazepine antagonist is more than 1000-fold in excess
of the concentration used for the half-maximal occupancy of the
benzodiazepine binding site. Interestingly, 1 µM Ro15-1788 by itself
inhibited pentobarbital-induced currents. This indicates that Ro15-1788
is not simply a benzodiazepine antagonist, but at higher concentrations
can allosterically modulate currents elicited by pentobarbital. This is
an important observation that is reminiscent to the work by Ueno et al.
(1997), who observed an inhibition of these currents by the competitive
GABA antagonist bicuculline. It may be concluded from our observation
that barbiturate action is not only allosterically coupled to the GABA
site but also to the benzodiazepine binding site.
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TABLE 6
Modulation of pentobarbital-induced currents
Currents activated by 200 µM pentobarbital was standardized to 100%.
The relative current amplitude in the presence of drug after
subtraction of the current induced by pentobarbitol alone (100%) is
given. All experiments were carried out with recombinant 122
GABAA receptors expressed in X. laevis oocytes.
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Discussion |
All ROD compounds described here except ROD178B allosterically
enhance currents activated by GABA in recombinant
GABAA receptors. It was our aim to analyze the
sites of action of these compounds relative to the benzodiazepine
binding site. The action of (+)-ROD188 has previously been investigated
in more detail (Thomet et al., 2000). It seems to interact with the
GABAA receptor at a site independent of the site
for benzodiazepines or loreclezole and of the agonistic site for
pentobarbital or neurosteroids.
Novel Enhancers May Be Grouped in R1 and R2 Compounds.
Based
on their ability to competitively displace
[3H]flunitrazepam in binding studies, ROD164A
and ROD185 were grouped in the R1 class compounds and compounds ROD222
and ROD259 that are not able to displace
[3H]flunitrazepam in binding studies were
combined in the R2 class. (+)-ROD188 represents an intermediate.
Enhancement of currents elicited by GABA by R1, but not R2 class
compounds, was antagonized by the benzodiazepine antagonist Ro15-1788.
The enhancement by R2, but not that by R1 compounds was additive to
that by the benzodiazepine diazepam. Thus, based on the observations,
it was tempting to postulate that R1, but not R2 compounds are novel
ligands of the benzodiazepine binding site.
R1 Compounds Act Both at the Benzodiazepine and at the R1
Site.
The following observations prompted us to postulate an
additional site of action for R1 compounds. First, R1 compounds are able to modulate [35S]TBPS binding and to
strongly activate GABA-induced currents at much higher concentrations
than those necessary for the interaction with the benzodiazepine
binding site as predicted from binding studies. Second, R1 (as well as
R2) compounds, in contrast to benzodiazepines (Pritchett et al., 1989;
Sigel et al., 1990; Günther et al., 1995), enhance currents
elicited by GABA in recombinant 12 receptors. Third, results from
an analysis of the inhibition of various concentrations of the R1 type
compound ROD164A by the benzodiazepine antagonist were not compatible
with a competitive interaction. We concluded that R1 type compounds not
only act at the benzodiazepine binding site but also at another site
that we call R1. ROD178B must be classified as an R1 compound because it is structurally closely related to ROD185 and also has
[3H]flunitrazepam displacing activity.
R2 Compounds Use a Separate Site from R1 Compounds.
Indicated
by the lack of [3H]flunitrazepam displacing
activity in binding studies, the lack of antagonism by the
benzodiazepine antagonist Ro15-1788 in functional enhancement
experiments, by the additivity of enhancement with diazepam, as well as
by the finding that R2 compounds are able to modulate 12
receptors, we conclude that R2 type compounds act at a site different
from benzodiazepines and have a different effector mechanism.
What is the relationship between the binding sites for R1 and R2 type
compounds? ROD178B inhibited the current enhancement by both compounds.
Current enhancement by the two types of compound was not additive.
Analysis of the inhibition by ROD178B of current enhancement by ROD164A
and ROD259 argues for independent sites, because inhibition at the R1
site seemed to be of a competitive nature. In contrast, inhibition at
the R2 site seemed to be due to a negative allosteric modulation.
Allosteric Effects of a Benzodiazepine Antagonist on
Pentobarbital-Induced Currents.
An important observation was made
during the study of interaction of the R1 and R2 type compounds with
pentobarbital. Namely, it was discovered that currents elicited by
pentobarbital in the absence of GABA were inhibited by the
benzodiazepine antagonist Ro15-1788. Furthermore, diazepam was able to
enhance these currents. This indicates that the actions of barbiturates
and those by benzodiazepines are in allosteric interaction. The
possibility that Ro15-1788 may induce a conformation different from the
resting state of the receptor has been discussed by Williams and Akabas
(2000) on the basis of an altered accessibility of a cysteine reagent to the transmembrane region M3 in the presence of this agent. Previously, it has also been shown that competitive GABA antagonists are able to inhibit pentobarbital-induced currents (Ueno et al., 1997).
Taken together, the actions of R1 compounds, benzodiazepines, and
barbiturates are in allosteric interaction with each other, whereas R2
compounds are not.
Summary.
The scheme shown in Fig.
10 summarizes our findings. The
benzodiazepine, R1, and R2 binding sites are distinct. Similarly, the
R1 and R2 sites must be distinct from the pentobarbital binding site
that directly opens chloride ion channels. R1 and R2 type use a common
pathway to affect channel gating. From the detailed investigation of
the inhibition by Ro15-1788 of the current enhancement by ROD164A, we
conclude that R1 and the benzodiazepine binding site interact
allosterically. In summary, we provide strong evidence for the
presence of additional allosteric sites on the
GABAA receptor. The location of the novel sites
remains to be shown.
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Fig. 10.
Scheme summarizing the relationship of the
benzodiazepine binding site with the barbiturate site and the R1 and R2
binding sites.
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This study was supported by the European Union Grant
BIO4-CT96-0585 (BBW 96.0010) and Grant 3100-053599.98/1 from the Swiss National Science Foundation.
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Abstract
Introduction
