On the Benzodiazepine Binding Pocket in GABAAReceptors*
Received for publication, October 16, 2003, and in revised form, November 11, 2003
Published, JBC Papers in Press, November 11, 2003, DOI 10.1074/jbc.M311371200
Dmytro Berezhnoy‡, Yves Nyfeler‡§, Anne Gonthier¶, Herve ´ Schwob¶, Maurice Goeldner¶,
and Erwin Sigel‡?
From the ‡Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland and the ¶Laboratoire de Chimie
Bioorganique, Unite ´ Mixte de Recherche 7514 CNRS, Universite ´ Louis Pasteur, Strasbourg 67401, Illkirch Cedex, France
Benzodiazepines are used for their sedative/hypnotic,
anxiolytic, muscle relaxant, and anticonvulsive effects.
They exert their actions through a specific high affinity
binding site on the major inhibitory neurotransmitter
receptor, the ?-aminobutyric acid, type A (GABAA) re-
ceptor channel, where they act as positive allosteric
modulators. To start to elucidate the relative position-
ing of benzodiazepine binding site ligands in their bind-
ing pocket, GABAAreceptor residues thought to reside
in the site were individually mutated to cysteine and
combined with benzodiazepine analogs carrying sub-
stituents reactive to cysteine. Direct apposition of such
reactive partners is expected to lead to an irreversible
site-directed reaction. We describe here the covalent
interaction of ?1H101C with a reactive group attached
to the C-7 position of diazepam. This interaction was
studied at the level of radioactive ligand binding and at
the functional level using electrophysiological methods.
Covalent reaction occurs concomitantly with occupancy
of the binding pocket. It stabilizes the receptor in its
allosterically stimulated conformation. Covalent modi-
fication is not observed in wild type receptors or when
using mutated ?1H101C-containing receptors in combi-
nation with the reactive ligand pre-reacted with a sulf-
hydryl group, and the modification rate is reduced by
the binding site ligand Ro15-1788. We present in addi-
tion evidence that ?2Ala-79 is probably located in the
access pathway of the ligand to its binding pocket.
mitter receptors in the mammalian brain. They are hetero-
meric protein complexes consisting of five subunits, which are
arranged pseudo-symmetrically around a central Cl?-selective
ion channel (1).
Initially, a GABA/benzodiazepine-binding protein has been
purified (2), and later two cDNAs thought to represent the
receptor have been cloned (3). A variety of subunit isoforms
have been cloned since then, leading to a multiplicity of recep-
tor subtypes (1, 4–9). The major receptor isoform of the GABAA
receptor in mammalian brain probably consists of ?1, ?2, and ?2
1receptors are the major inhibitory neurotrans-
subunits (1, 4, 10–12). The ? subunit has been shown to be
required for functional modulation of the receptor channels by
benzodiazepines (13, 14). Different approaches have indicated
a 2?:2?:1? subunit stoichiometry for this receptor (15–20). The
receptor channel is modulated by numerous drugs (21), includ-
ing compounds acting at the benzodiazepine binding site. Ben-
zodiazepines belong to the classic ligands of this site and exert
their anxiolytic, sedative, muscle relaxant, and anticonvulsive
action by positive allosteric modulation of different isoforms of
the GABAAreceptor channel. An antagonist acting at this site
is also in clinical use (22), while negative allosteric modulators
such as methyl 6,7-dimethoxy-4-ethyl-?-carboline-3-carboxyl-
ate are investigational tools.
Amino acid residues His-101, Tyr-159, Gly-200, Thr-206, and
Tyr-209 on the ?1subunit, and Phe-77, Ala-79, Thr-81, and
Met-130 on the ?2subunit have been suggested to be part of, or
close to the binding pocket for the ligands of the benzodiazepine
binding site (23–34). The region around ?2Phe-77 has been
shown to assume ?-sheet structure and undergo conforma-
tional changes upon channel gating (34). Many of the men-
tioned amino acid residues are homologous to amino acid res-
idues on the ?1and ?2subunits that take part in the formation
of the binding site for the channel agonist GABA or are located
close to them (35–42). The channel agonist and allosteric mod-
ulators acting at the benzodiazepine site are thus binding to
pseudo-symmetric structures (29, 43). The abovementioned
residues lining either the benzodiazepine binding site or the
GABA binding site are all homologous to residues suggested to
form the binding site of acetylcholine on the nicotinic acetyl-
choline receptor (43). The recently crystallized acetylcholine-
binding protein, which shows a weak homology to the extracel-
lular part of the GABAAreceptor (44), allowed a first structural
insight into the ligand binding domain through homology
Many studies (e.g. Refs. 46–49) have been undertaken with
the aim to characterize spatial properties of the benzodiazepine
binding pocket. These studies used either in vivo effects or
chloride flux experiments in combination with radioligand
binding studies on brain membranes of a large number of
structurally related compounds. Derived models for the bind-
ing pocket are complex and suggest distinct but partially over-
lapping binding sites for ligands differing in their allosteric
effect, but a consensus view failed to emerge. A drawback of
brain studies is the heterogeneity of GABAAreceptors.
It is obviously important to map all the amino acid residues
participating in the formation of the benzodiazepine pocket
relative to the ligands of this site in a recombinant receptor.
Initial approaches have indicated that the pending phenyl res-
idue of classic benzodiazepines may be located close to
?2Phe-77 (50) and ?1His-101 (51).
In pioneering work Karlin and Akabas (for review see Ref.
52) introduced site specific mutation to cysteine in combination
* This work was supported by Swiss National Science Foundation
Grant 3100-064789.01/1. The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Max Planck Institute of Immunobiology, D-79108
? To whom correspondence should be addressed. Tel.: 41-31-632-3281;
Fax: 41-31-632-4992; E-mail: email@example.com.
1The abbreviations used are: GABAA, ?-aminobutyric acid, type A;
NCS compound, 7-isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodi-
azepin-2-one; Cl compound, 7-nitro-5-phenyl-3-chloro-1,3-dihydro-2H-
1,4-benzodiazepin-2-one; CMV, cytomegalovirus.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 279, No. 5, Issue of January 30, pp. 3160–3168, 2004
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
with nonspecific cysteine-reactive agents for the study of pro-
teins. Recently, a novel technique to elucidate relative position
of a ligand in its binding pocket has been successfully applied
in several cases (53–59). The technique has been described in
detail (60). In this approach, receptors in which residues
thought to reside in the binding pocket are individually mu-
tated to cysteine and then combined with binding site ligands
carrying substituents reactive to cysteine. Direct apposition of
such reactive substituents with a cysteine residue is expected
to lead to a covalent reaction. Given a series of controls, such
engineered site-directed reactions provide reliable information
on the orientation of a ligand within its binding site.
We applied here this novel approach to the benzodiazepine
binding site. We show that this strategy works in the present
case and describe the covalent interaction of ?1H101C with a
reactive group attached to the C-atom in diazepam normally
carrying a Cl atom. We show in addition that ?2Ala-79 is most
probably located in the access pathway of the ligand to its
Synthesis of the Reactive Substance
Thiophosgen (63 ?l, 0.828 mmol, 2 eq.) was slowly added to a stirred
solution of 7-amino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-
one(110 mg, 0.414 mmol) and sodium hydrogenocarbonate (70 mg,
0.828 mmol, 2 eq.) in 10 ml of an aqueous solution of tetrahydrofuran
(50%). The resulting mixture was stirred 15 min at room temperature.
40 ml of 5% aqueous solution of NaHCO3was added to the reaction
mixture, and after evaporation of the tetrahydrofuran, the aqueous
layer was extracted with CH2Cl2. the organic layer was washed with 5%
aqueous solution of NaHCO3, dried over Na2SO4, and evaporated under
reduced pressure. The residue was purified by chromatography on silica
gel eluted by heptane/ethyl acetate 1/1. NMR1H (300 MHz-CDCl3): ? ?
3.41 (s, 3H), 3.75 (d, J ? 10.8 Hz, 1H), 4.83 (d, J ? 10.8 Hz, 1H), 7.14
(d, J ? 2.4 Hz, 1H), 7.26–7.60 (m, 7H). IR (KBr): ? ? 2104 cm?1, s
N-Chlorosuccinimide (2.5 g, 187 mmol, 55 eq.) in portions of 500 mg was
added over 2 days to a solution of nitrazepam (1 g, 3.39 mmol) in CCl4.
A catalytic amount of azoisobutyro nitrile was added, and the solution
was heated to reflux (2 days). The solvent was removed under reduced
pressure, and the residue was dissolved in Et2O. The solid N-chloro-
succinimide in excess was removed by filtration, and the Et2O was
evaporated under reduced pressure. The resulting residue was purified
by chromatography on silica gel eluted by heptane/AcOEt 7/3, giving
22% of the desired Cl compound. NMR1H (200 MHz-CDCl3): ? ? 3.57
(s, 3H), 5.63 (s, 1H), 6.70 (s, 1H), 7.35–8.51 (m, 8H).
Construction of Receptor Subunits
The cDNAs coding for the ?1, ?2, and ?2S subunits of the rat GABAA
receptor channel have been described elsewhere (61–63). The mutant
subunits ?1H101C, ?2T73C, ?2D75C, ?2A79C, and ?2T81C were pre-
pared using the QuikChangeTMmutagenesis kit (Stratagene). For cell
transfection, the cDNAs were subcloned into the polylinker of pBC/
CMV (64). This expression vector allows high level expression of a
foreign gene under control of the cytomegalovirus promoter. The ?
subunit was cloned into the EcoRI, and the ? and ? subunits were
subcloned into the SmaI site of the polylinker by standard techniques.
Transfection of Recombinant GABAAReceptor in Cultured Cells
The cells were maintained in minimum essential medium (Invitro-
gen) supplemented with 10% fetal calf serum, 2 mM glutamine, 50
units/ml penicillin, and 50 ?g/ml streptomycin by standard cell culture
techniques. Equal amounts (total of 20 ?g of DNA/90-mm dish) of
plasmids coding for GABAAreceptor subunits were transfected into
human embryonic kidney 293 cells (ATCC CRL 1573) by the calcium
phosphate precipitation method (65). After overnight incubation, the
cells were washed twice with serum-free medium and refed with
Approximately 60 h after transfection the cells were harvested by
washing with ice-cold phosphate-buffered saline, pH 7.4, and centri-
fuged at 560 ? g. The buffer containined 10 mM potassium phosphate,
100 mM KCl, 0.1 mM K-EDTA, pH 7.4. Cells were homogenized by
sonication in the presence of 10 ?M phenylmethylsulfonyl fluoride and
1 mM EDTA. Membranes were collected by three centrifugation-resus-
pension cycles (100,000 ? g for 20 min) and then used for ligand binding
or stored at ?20 °C.
Membranes were resuspended in the buffer mentioned above using a
tip sonifier. Resupended cell membranes were incubated in a total
volume of 0.1–0.4 ml for 90 min on ice in the presence of [3H]Ro15-1788
(78.6 Ci/mmol, PerkinElmer Life Sciences) or [3H]flunitrazepam (71–84
Ci/mmol, PerkinElmer Life Sciences) and various concentrations of
competing ligands. In the case of displacement studies using NCS
compound this compound was present for 20–30 min. Membranes
(5–80 ?g of protein/filter) were collected by rapid filtration on GF/C
filters presoaked in 0.3% polyethylenimine. After three washing steps
with 5 ml of buffer, the filter-retained radioactivity was determined by
liquid scintillation counting. Nonspecific binding was determined in the
presence of 100 ?M Ro15-1788 or 100 ?M flunitrazepam, respectively.
Data were fitted by using a nonlinear least-squares method to the
equations, B(c) ? Bmax/(1 ? (Kd/c)n), for binding curves, and B(c) ?
Bmax/(1 ? (c/IC50)n), for displacement curves with a single component,
where c is the concentration of ligand, B is binding, Bmaxis maximal
binding, Kdis the dissociation constant, and n is the Hill coefficient.
IC50values were converted to Kivalues according to the Cheng-Prusoff
equation (66). Protein concentration was determined with the BCA
protein assay kit (Pierce) with bovine serum albumin as standard.
Wash-out Procedure for Reactive Substances
NCS compound was dissolved in dioxane at a concentration of 20 mM.
A stock solution was kept at ?20 °C and used at most for 2 months. Cl
compound was freshly dissolved in Me2SO at a concentration of 5 mM.
Final dilutions were prepared immediately before the experiment. We
estimated that ?25% of the NCS compound was reacted in buffer alone
within 1 h. Membranes were incubated with different NCS compound in
total volume of 0.2 ml for 30–60 min on ice. Maximal final solvent
concentration during this incubation was 1%. Subsequently, 1.8 ml of
ice-cold buffer were added and the sample was centrifuged at 14,000 ?
gmaxat 2 °C for 30 min. The supernatant was removed, and 2 ml of
ice-cold buffer was added to the pellet, followed by sonication. Centrif-
ugation was performed again, and the whole procedure repeated. After
the third centrifugation the supernatant was removed leaving ?50 ?l in
the tube, and 0.31 ml of ice-cold buffer was added. After sonication a
binding assay was performed in a total volume of 0.4 ml. During this
procedure 30–70% of the protein was lost. Controls containing no reac-
tive substance allowed standardization in each experiment and were set
to 100% binding. In some cases, NCS compound was reacted with free
cysteine to inactivate it. For this purpose 100 ?M of NCS compound was
preincubated for 1 h together with 100 mM cysteine.
Expression in Xenopus Oocytes
Capped cRNAs were synthesized (Ambion, Austin, TX) from the
linearized pCMV vectors containing the different subunits, respec-
tively. A poly-A tail of about 400 residues was added to each transcript
using yeast poly-A polymerase (United States Biologicals, Cleveland,
OH). The concentration of the cRNA was quantified on a formaldehyde
gel using Radiant Red stain (Bio-Rad) for visualization of the RNA and
known concentrations of RNA ladder (Invitrogen) as standard on the
same gel. cRNA combinations were precipitated in ethanol/isoamylal-
cohol 19:1 and stored at ?20 °C. For injection, the alcohol was removed
and the cRNAs were dissolved in water. Oocytes were injected with 50
nl of the cRNA solution. The combination of wild type or mutated ?1, ?2,
and ?2subunits was expressed at 10 nM:10 nM:50 nM (67). The injected
oocytes were incubated in modified Barth’s solution (10 mM HEPES, pH
7.5, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.34 mM
Ca(NO3)2, 0.41 mM CaCl2, 100 units/ml penicillin, 100 ?g/ml strepto-
mycin) at 18 °C for at least 24 h before the measurements. Xenopus
laevis oocytes were prepared, injected, and defoliculated as described
previously (14, 68).
Two-electrode Voltage Clamp
Electrophysiological experiments were performed by the two-elec-
trode voltage clamp method at a holding potential of ?80 mV. The
perfusion medium contained 90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM
CaCl2, and 5 mM Na-HEPES (pH 7.4). To quantify GABA sensitivity,
agonist concentrations between 0.1 and 10,000 ?M were applied for 20 s
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Orientation of Benzodiazepines in Their Binding Pocket