Two neighboring residues of loop A of the alpha1 subunit point towards the benzodiazepine binding site of GABAA receptors.
ABSTRACT Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GABA(A) receptors. It is important to understand how these ligands are positioned in this binding site. We are especially interested here in the conformation of loop A of the alpha(1)beta(2)gamma(2) GABA(A) receptor containing a key residue for the interaction of benzodiazepines: alpha(1)H101. We describe a direct interaction of alpha(1)N102 with a diazepam- and an imidazobenzodiazepine-derivative. Our observations help to better understand the conformation of this region of the benzodiazepine pocket in GABA(A) receptor.
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ABSTRACT: GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site at the α/γ subunit interface on some of these receptors. Diazepam has sedative, hypnotic, anxiolytic, muscle relaxant and anticonvulsant effects. It acts by potentiating the current evoked by the agonist GABA. Understanding specific interaction of benzodiazepines in the binding pocket of different GABAA receptor isoforms might help to separate these divergent effects. As a first step we characterized the interaction between diazepam and the major GABAA receptor isoform α1β2γ2. We mutated several amino acid residues on the γ2-subunit assumed to be located near or in the benzodiazepine binding pocket individually to cysteine and studied the interaction with three ligands that are modified with a cysteine-reactive isothiocyanate group (-NCS). When the reactive NCS group is in apposition to the cysteine residue this leads to a covalent reaction. In this way, three amino acid residues, γ2Tyr58, γ2Asn60 and γ2Val190 were located relative to classical benzodiazepines in their binding pocket on GABAA receptors.ACS Chemical Biology 06/2014; · 5.36 Impact Factor
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ABSTRACT: We present a full-length α(1)β(2)γ(2) GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and the backbone of β(2)S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy calculations. Muscimol key interactions are predicted to be α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and β(2)F200. Furthermore, we argue that a water molecule could mediate further interactions between muscimol and the backbone of β(2)S156 and β(2)Y157. DZP is predicted to bind with interactions comparable to those of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines α(1)T206 and γ(2)T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important α(1)H101 and the N-methyl group near α(1)Y159, α(1)T206, and α(1)Y209. We present a binding mode of DZP in which the pending phenyl moiety of DZP is buried in the binding pocket and thus shielded from solvent exposure. Our full length GABA(A) receptor is made available as Model S1.PLoS ONE 01/2013; 8(1):e52323. · 3.53 Impact Factor
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ABSTRACT: High throughput discovery of ligand scaffolds for target proteins can accelerate development of leads and drug candidates enormously. Here we describe an innovative workflow for the discovery of high affinity ligands for the benzodiazepine-binding site on the so far not crystallized mammalian GABAA receptors. The procedure includes chemical biology techniques that may be generally applied to other proteins. Prerequisites are a ligand that can be chemically modified with cysteine-reactive groups, knowledge on amino acid residues contributing to the drug binding pocket and either crystal structures of proteins homologous to the target protein, or better of the target itself. Part of the protocol is virtual screening that without additional rounds of optimization in many cases only results in low affinity ligands, even when a target protein has been crystallized. Here we show how the integration of functional data into structure-based screening dramatically improves the performance of the virtual screening. Thus, lead compounds with 14 different scaffolds were identified on the basis of an updated structural model of the diazepam bound state of the GABAA receptor. Some of these compounds show considerable preference for the α3β2γ2 GABAA receptor subtype.ACS Chemical Biology 06/2014; · 5.36 Impact Factor
Two neighboring residues of loop A of the a1subunit point towards
the benzodiazepine binding site of GABAAreceptors
Kelly R. Tana, Roland Baura, Anne Gonthierb, Maurice Goeldnerb, Erwin Sigela,*
aInstitute of Biochemistry and Molecular Medicine, University of Bern, Bu ¨hlstrasse 28, CH-3012 Bern, Switzerland
bLaboratoire de Chimie Bioorganique, Unite ´ Mixte de Recherche, 7175 LC1 CNRS, Faculte ´ de Pharmacie,
Universite ´ Louis Pasteur Strasbourg, 74 Route du Rhin, 67401 Illkirch Cedex, France
Received 17 July 2007; revised 13 August 2007; accepted 29 August 2007
Available online 6 September 2007
Edited by Jesus Avila
tive, anxiolytic, muscle relaxant, and anticonvulsant effects by
acting through specific high affinity binding sites on some GA-
BAAreceptors. It is important to understand how these ligands
are positioned in this binding site. We are especially interested
here in the conformation of loop A of the a1b2c2GABAArecep-
tor containing a key residue for the interaction of benzodiaze-
pines: a1H101. We describe a direct interaction of a1N102
with a diazepam- and an imidazobenzodiazepine-derivative.
Our observations help to better understand the conformation of
this region of the benzodiazepine pocket in GABAAreceptor.
? ? 2007 Federation of European Biochemical Societies. Pub-
lished by Elsevier B.V. All rights reserved.
Benzodiazepines are widely used drugs exerting seda-
Keywords: Diazepam; c-Aminobutyric acid (GABA);
c-Aminobutyric acid type A receptor (GABAAreceptor);
Imidazobenzodiazepine; Proximity-accelerated chemical
c-Aminobutyric acid type A (GABAA) receptors are the ma-
jor inhibitory neurotransmitter receptors in the brain. They are
chloride ion channels composed of five subunits, which are
classified into six subunit families [1–3]. a1b2c2is the major
adult receptor isoform [1,2] and its subunit stoichiometry is
2a:2b:1c [4–10]. We concentrated on this major receptor iso-
Benzodiazepines are widely used drugs, which bind to GA-
BAAreceptors with high affinity . These molecules are di-
vided in positive and negative allosteric modulators and
antagonists. Mutagenesis studies identified the cleft between
a and c subunits as the binding pocket for benzodiazepines
[12–14]. a1H101 was identified as the target of photoaffinity
labeling by [3H] flunitrazepam  and a1Y209 as the target
of [3H] Ro15-4513 . Pharmacophore modeling attempted
to describe the shape of the binding pocket [17,18].
The benzodiazepine binding site is constituted of six loops
from A to F. The important residue a1H101 forming part of
loop A has previously been shown to molecularly interact with
diazepam  and imidazobenzodiazepine derivatives . We
investigated here the direct surrounding of this residue. For
this purpose, we used the proximity-accelerated chemical cou-
pling reaction. We mutated the four neighboring residues of
a1H101 to cysteine and combined the four mutant receptors
individually with a cysteine reactive diazepam- (NCS com-
pound) or a cysteine reactive imidazobenzodiazepine-deriva-
tive (Imid-NCS compound). We show that both compounds
react covalently with a1N102C and not with the other residues.
Our work suggests that this residue is pointing to the benzodi-
azepine binding pocket.
2. Materials and methods
We used two substances, an imidazobenzodiazepine, Imid-NCS
compound that is similar to Ro15-4513 except that the –N3group is
replaced by a –NCS group and the NCS compound which derives from
diazepam with the –Cl atom replaced by a –NCS group (Fig. 1). Syn-
thesis and sample preparation for both compounds are detailed else-
2.2. Transfection of recombinant GABAAreceptors in HEK 293 cells,
radioactive ligand binding assay and detection of a covalent reaction
cDNAs, transfection into HEK 293 cells, radioactive ligand binding
assay and detection of a covalent reaction are detailed elsewhere
[19,20]. Basically the latter included (a) incubation of membranes
expressing wild type or mutant receptors with the reactive compound,
(b) extensive washing of the membranes in order to remove non-re-
acted agent, and (c) a radioactive ligand binding assay to determine
residual binding. No covalent reaction would result in 100% residual
binding/0% reaction, and 100% covalent reaction would result in 0%
residual binding/100% reaction. As the efficiency of membrane recov-
ery during the whole procedure was found to be quite variable and dif-
fered between two experiments up to 30%, we assumed covalent
reaction when residual binding was below 70% of the controls. In all
experiments, each single sample was measured in triplicate.
2.3. Expression and functional characterization in Xenopus oocytes and
modification of receptor function by the Imid-NCS compound
cRNA synthesis, Xenopus laevis oocyte injection and electrophysio-
logical experiments have been previously described [19–23]. Covalent
modification of a1N102Cb2c2receptors by Imid-NCS compound was
tested as follows: after obtaining a reproducible response to the appli-
cation of GABA, the Imid-NCS compound freshly diluted to 1 lM in
perfusion medium was applied for 3 min. Maximal final DMSO con-
centration was 0.1% and did not affect the response to GABA in con-
trol experiments. This treatment was followed by several GABA
applications in intervals of 4 min to reach a steady level. Subsequently,
1 lM diazepam was co-applied with GABA to investigate a covalent
*Corresponding author. Fax: +41 31 631 3737.
E-mail address: email@example.com (E. Sigel).
0014-5793/$32.00 ? 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 581 (2007) 4718–4722
reaction with Imid-NCS compound. The irreversible effect was then
calculated as current potentiation after treatment with Imid-NCS com-
pound divided by current potentiation without treatment.
3.1. Binding properties of the cysteine-reactive compounds and
The –Cl atom in diazepam and the –N3group in Ro15-4513
were replaced with a –NCS group (Fig. 1) such as to create
NCS and Imid-NCS compounds, respectively. The –C atom
of the –NCS group is reactive with cysteine residues. Both sub-
stances were able to displace [3H] flunitrazepam or [3H] Ro15-
1788, respectively from wild type a1b2c2receptors with an
affinity of 3 lM and 10 nM, respectively [19,20]. Thus, the
chemically reactive molecules retained affinity for the benzodi-
The neighboring a1F99, a1F100, a1N102 and a1G103 resi-
dues of a1H101, were individually mutated to cysteine. These
mutations did not compromise [3H] Ro15-1788 binding. All
four mutated receptors as well as a1H101b2c2  bound
[3H] Ro15-1788 with an estimated affinity between 0.6 and
6.1 nM (data not shown).
3.2. Irreversible reaction of the NCS and Imid-NCS compounds
with mutant receptors at the binding level
The receptors were exposed to 50 lM NCS compound
(Fig. 2A) or 100 nM Imid-NCS compound (Fig. 2B) sepa-
rately, and subsequently non-covalently bound reactive ligand
was removed by extensive washing. The 500-fold difference in
used concentrations takes into account the apparent affinities
of these ligands for the binding site. The compounds are ex-
pected to first reversibly occupy their binding site, then upon
proper apposition of the –SH group of the cysteine of the mu-
tant receptor with the –C atom of the –NCS group from the
reactive agent, a covalent bond between ligand and receptor
is formed. Fig. 2 shows residual binding of a1b2c2receptors
and mutant receptors. From these data, it may be calculated
that a1H101Cb2c2and a1N102Cb2c2receptors reacted cova-
lently with 50 lM NCS compound to an extent of 83 ± 3%
(n = 3) and 53 ± 10% (n = 3) (Fig. 2A), respectively and with
100 nM Imid-NCS compound to an extent of 87 ± 5% 
and 44 ± 7% (n = 3; Fig. 2B). a1F99Cb2c2receptors showed
in average 64% residual binding after exposure to NCS com-
pound, which is near the 70% threshold defined in the method
section. As this mutation led to abnormal allosteric properties
towards benzodiazepines (see below), this covalent reaction
was not further investigated.
3.3. Consequences of the mutations a1F99C and a1N102C on
allosteric effects of benzodiazepines
We investigated the functional consequences of the mutation
to cysteine on a1F99Cb2c2 and a1N102Cb2c2 receptors ex-
pressed in Xenopus oocytes. In a1b2c2 receptors, diazepam
allosterically potentiates currents elicited by GABA to a max-
imum potentiation of about 225% and an EC50 of about
100 nM , Ro15-1788 acts as an antagonist and Ro15-4513
as a partial negative allosteric modulator . In a1F99Cb2c2
mutant receptors, 1 lM diazepam inhibited the GABA-elicited
current by 15 ± 6% and Ro15-1788 by 33 ± 4% (n = 3 each;
data not shown). This mutation thus severely affects allosteric
properties of benzodiazepines.
Fig. 3 shows the allosteric properties of a1N102Cb2c2mu-
tant receptors. Diazepam showed an EC50of 366 ± 33 nM,
about 4 times higher as compared to a1b2c2receptors, with a
maximum potentiation of 226 ± 69% (n = 4). Ro15-1788 and
Ro15-4513 had small stimulatory effects with a maximum
potentiation of 21 ± 8% (n = 4) and 18 ± 9% (n = 4), respec-
tively. The concentration–response curve of NCS compound
ranging from 30 nM to 30 lM did not lead to saturation
(n = 3). We can only conclude that the apparent affinity is
higher than 10 lM. The Imid-NCS compound potentiated
the GABA-elicited current with an apparent affinity of
16.7 ± 0.8 nM and a maximum potentiation of 63 ± 5%
Fig. 1. Chemical structure of the reactive ligands used. Top, diazepam
and NCS compound. Bottom, Ro15-1788, Ro15-4513 and Imid-NCS
Fig. 2. Residual binding after irreversible reaction of NCS and Imid-
NCS compounds with wild type and mutant GABAAreceptors. (A)
shows results obtained with 50 lM NCS compound and (B) with
100 nM Imid-NCS compound. Receptors were individually exposed to
one of the reactive compounds for 1 h on ice, the membranes were
extensively washed, and residual binding was subsequently determined
using [3H] Ro15-1788 as radioactive ligand. Data were expressed in
percent of an internal control where the reactive compound was
absent. Results are shown as means ± S.D. for individual experiment
where each measurement was carried-out in triplicates. The experi-
ments were performed three times when a covalent reaction was
suspected. As explained in Section 2, a covalent reaction was assumed
to take place if residual binding was <70% (indicated by the dashed
bar). Results on a1b2c2and a1H101Cb2c2receptors are from  for
the NCS compound and  for the Imid-NCS compound.
K.R. Tan et al. / FEBS Letters 581 (2007) 4718–4722
(n = 4). Please note that current potentiation by NCS and
Imid-NCS compounds is partly due to an irreversible reaction
(Fig. 3). These data should be compared with those of
a1H101Cb2c2 receptors , Ro15-1788 and Ro15-4513
showed an EC50of 30 ± 6 and 17 ± 3 nM, respectively and
exhibited small stimulatory effects amounting to 22 ± 11 and
40 ± 2%, respectively (n = 3 for each). The apparent affinity
of Imid-NCS compound was 17 ± 1 nM with a maximum
potentiation to about 90 ± 7% (n = 3). Thus, a1N102Cb2c2
and a1H101Cb2c2receptors showed similar properties towards
Ro15-1788, Ro15-4513 and Imid-NCS compound.
3.4. Irreversible reaction of the Imid-NCS compound to
a1N102Cb2c2receptors at the functional level
a1N102Cb2c2receptors were exposed for 5 min to 20 lM or
50 lM NCS compound. This treatment did neither result in a
stable change of the current amplitude elicited by GABA nor
in a decrease of the effect by 1 lM diazepam (data not shown).
This has to be compared to the results obtained on a1H101b2c2
receptors where exposure to 1 lM NCS compound for 5 s led
to covalent reaction of 25% of the receptors [19,24].
Fig. 4 shows the consequences of exposure of a1N102Cb2c2
receptors to 1 lM Imid-NCS compound for 3 min. This treat-
ment resulted initially in a large potentiation that upon re-
moval of the non-covalently bound Imid-NCS compound
relaxed to 32 ± 6% (n = 3). When 1 lM diazepam was subse-
quently co-applied to GABA, a further increase of the current
amplitude was detected to 112 ± 28% (n = 3). This effect
amounting to about 80% potentiation should be compared
with the one elicited by 1 lM diazepam co-applied with GABA
to oocytes not exposed to Imid-NCS compound that
amounted to 190 ± 38% (n = 6). Thus, potentiation by diaze-
pam is reduced by about 58% after treatment with Imid-
NCS compound. These results demonstrate that 1 lM Imid-
a1N102Cb2c2receptors, making the corresponding benzodiaz-
epine binding sites inaccessible to other benzodiazepine ligands
such as diazepam. Similar experiments carried out with
a1H101b2c2receptors showed that after covalent reaction with
Imid-NCS compound current potentiation by 10 lM zolpidem
was reduced by more than 90% .
compoundreacts covalently withpart of the
3.5. Comparison of the reaction rates of a1H101Cb2c2and
a1N102Cb2c2receptors with the NCS and the Imid-NCS
compounds at the functional level
Reaction rates of a1H101Cb2c2and a1N102Cb2c2receptors
with diazepam- and imidazobenzodiazepine-derivatives at the
functional level will be compared in the following. Exposure
of a1H101Cb2c2receptors to 1 lM NCS compound for 5 s
led to about 25% of the maximum level of the covalent reac-
tion [19,24], whereas no reaction was observed when 50 lM
NCS compound was applied for 5 min to a1N102Cb2c2recep-
tors (see above). This failure to detect covalent reaction at the
functional level is presumably due to a lower affinity of NCS
compound to a1N102Cb2c2receptors at room temperature.
Together with the binding data, this indicates that upon occu-
pancy by a diazepam derivative, a1H101C and a1N102C are
both oriented towards the reactive atom of the bound ligand,
although a1N102C is less accessible or more distant than
Application of about 400 nM Imid-NCS compound for
1 min to a1H101Cb2c2receptors led to 50% of the maximum
level of the covalent reaction . A similar level was reached
in a1N102Cb2c2receptors within 3 min of 1 lM Imid-NCS
exposure (see above). Therefore, the rate of the covalent reac-
a1N102Cb2c2 receptors. Cumulative concentration–response curves
shown for diazepam (closed circle, n = 4), Ro15-1788 (closed square,
n = 4), Ro15-4513 (closed triangle, n = 4) NCS compound (open circle,
n = 3) and Imid-NCS compound (open triangle, n = 4). GABA was
used at a concentration of 4 lM, which elicited EC2?5.
3. Functional modulationbybenzodiazepine ligandsof
Fig. 4. Functional analysis of the action of Imid-NCS compound on
a1N102Cb2c2 receptors. A representative experiment is shown.
a1N102Cb2c2 receptors functionally expressed in Xenopus oocytes
were exposed to GABA (4 lM, EC2?5, closed circles) every 4 min
before and after exposure to 1 lM Imid-NCS compound for 3 min
(arrow). The response elicited by GABA initially was increased then
decreased over wash out period until a steady level was reached.
Subsequently, 1 lM diazepam was co-applied with GABA (open
circle) and an increase in the amplitude of the GABA-elicited current
was recorded. This increase is smaller to the one obtained in oocytes
not treated with Imid-NCS compound (open square).
K.R. Tan et al. / FEBS Letters 581 (2007) 4718–4722
tion by Imid-NCS compound with a1H101C is at least three
times faster as compared to a1N102C. This assumes a similar
affinity of the Imid-NCS compound to a1H101Cb2c2 and
a1N102Cb2c2receptors. In fact, half maximal stimulation by
the Imid-NCS compound was observed at about 17 nM in
each receptor type (Fig. 3, this paper; ).
The residue a1H101 of the a1b2c2 GABAA receptor is
important for the binding of benzodiazepines [25–27] and
has been shown to directly interact with diazepam and imi-
dazobenzodiazepines [19,20,24]. This residue is located in loop
A of the a1subunit. We were interested in the conformation of
the loop A and focused on four residues neighboring a1H101:
a1F99, a1F100, a1N102 and a1G103. We performed proximity-
accelerated chemical reaction where those four residues were
individually mutated to cysteine and incubated with a diaze-
pam- (NCS compound) or an imidazobenzodiazepine- (Imid-
NCS compound) cysteine reactive derivative. Covalent reac-
tion of the two partners is evidence for apposition of the two
reactive groups. Such results provide information on the orien-
tation of the side chain of a residue respective to the ligand.
The a1H101Cb2c2receptors have been shown earlier to react
covalently with both of the reactive agents [19,20]. We show
here that at the binding level both NCS and Imid-NCS com-
pounds react irreversibly with a1N102Cb2c2receptors, though
to a lower extent than a1H101Cb2c2receptors [19,20]. In addi-
tion, the NCS compound might weakly react with a1F99Cb2c2
receptors. Since allosteric properties were compromised in
these receptors, this reaction was not further investigated.
a1F100Cb2c2and a1G103Cb2c2receptors exhibited no cova-
lent reaction with both ligands at the binding level. Whether
or not this indicates that these residues point away from the
binding pocket is not clear. Negative findings could also arise
if the mutual orientation is unfavorable for a covalent reac-
We also showed covalent reaction of a1N102Cb2c2receptors
with the Imid-NCS compound at the functional level. Relative
reaction rates of a1H101Cb2c2 and a1N102Cb2c2 receptors
with the NCS and the Imid-NCS compounds at the functional
level were estimated. It may be noted that in the receptor con-
formation stabilized by imidazobenzodiazepines, a1H101C
and a1N102C are more similarly positioned to the reactive
atom of the ligand than in the conformation stabilized by diaz-
Our observations are relevant for a modeling of loop A con-
tributing to the benzodiazepine binding site.
Acknowledgements: We thank Dr. V. Niggli for carefully reading the
manuscript. This work was supported by the Swiss National Founda-
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