Retrochalcone derivatives are positive allosteric modulators at synaptic and extrasynaptic GABAA receptors in vitro

Article abstract of DOI:10.1111/j.1476-5381.2010.01142.x

BACKGROUND AND PURPOSE Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABA_A receptors (GABA _ARs). We previously reported that trans-6,4′- dimethoxyretrochalcone (Rc-OMe), a hydrolytic derivative of the corresponding flavylium salt, displayed nanomolar affinity for the benzodiazepine binding site of GABA_ARs. Here, we evaluate the functional modulations of Rc-OMe, along with two other synthetic derivatives trans-6-bromo-4′- methoxyretrochalcone (Rc-Br) and 4,3′-dimethoxychalcone (Ch-OMe) on GABA_ARs. EXPERIMENTAL APPROACH Whole-cell patch-clamp recordings were made to determine the effects of these derivatives on GABA_ARs expressed in HEK-293 cells and in hippocampal CA1 pyramidal and thalamic neurones from rat brain. KEY RESULTS Rc-OMe strongly potentiated GABA-evoked currents at recombinant α _1-4β₂γ_2s and α ₄β₃I receptors but much less at α₁β₂ and α₄β₃. Rc-Br and Ch-OMe potentiated GABA-evoked currents at α₁β ₂γ_2s. The potentiation by Rc-OMe was only reduced at α₁H101Rβ₂γ_2s and α₁β₂N265Sγ_2s, mutations known to abolish the potentiation by diazepam and loreclezole respectively. The modulation of Rc-OMe and pentobarbital as well as by Rc-OMe and the neurosteroid 3α,21-dihydroxy-5α-pregnan-20-one was supra-additive. Rc-OMe modulation exhibited no apparent voltage-dependence, but was markedly dependent on GABA concentration. In neurones, Rc-Br slowed the decay of spontaneous inhibitory postsynaptic currents and both Rc-OMe and Rc-Br positively modulated synaptic and extrasynaptic diazepam-insensitive GABA_ARs. CONCLUSIONS AND IMPLICATIONS The trans-retrochalcones are powerful positive allosteric modulators of synaptic and extrasynaptic GABA_ARs. These novel modulators act through an original mode, thus making them putative drug candidates in the treatment of GABA_A-related disorders in vivo.

Full text of DOI:10.1111/j.1476-5381.2010.01142.x

DOI:10.1111/j.1476-5381.2010.01142.x
www.brjpharmacol.org
British Journal of
Pharmacology
BJP
Correspondence
RESEARCH PAPERbph_1142 1326..1339
Thomas Grutter, Laboratoire de
Biophysicochimie des Récepteurs
Canaux, UMR 7199 CNRS,
Conception et Application de
Molécules Bioactives, Faculté
de Pharmacie, Université de
Strasbourg, 67400 Illkirch,
France. E-mail:
Retrochalcone derivatives
are positive allosteric
modulators at synaptic
and extrasynaptic GABAA
receptors in vitro
grutter@bioorga.u-strasbg.fr
----------------------------------------------------------------
This work was supported by ANR
(06-0050-01) and CNRS (PIME) to
T. G., and by Grant-in-Aid for
Scientific Research of Japan
(18077009 to J. N., 21600019
to H. I.). R. J. is a recipient of
fellowship from the China
Scholarship Council.
----------------------------------------------------------------
Ruotian Jiang1, Akiko Miyamoto2, Adeline Martz1, Alexandre Specht3,
Hitoshi Ishibashi2, Marie Kueny-Stotz4, Stefan Chassaing4,
Raymond Brouillard4, Lia Prado de Carvalho1, Maurice Goeldner3,
Junichi Nabekura2, Mogens Nielsen5 and Thomas Grutter1
Keywords
GABAA ion channels;
trans-retrochalcones; allosteric
modulators; flavylium salts
----------------------------------------------------------------
1Laboratoire de Biophysicochimie des Récepteurs Canaux, UMR 7199 CNRS, Conception et
Application de Molécules Bioactives, Faculté de Pharmacie, Université de Strasbourg, Illkirch,
France, 2Department of Developmental Physiology, National Institute for Physiological Sciences,
Okazaki, Japan, 3Laboratoire de Chimie Bioorganique, UMR 7199 CNRS, Conception et
Application de Molécules Bioactives, Faculté de Pharmacie, Université de Strasbourg, Illkirch,
France, 4Laboratoire de Chimie des Polyphénols, UMR 7177 CNRS, Institut Le Bel, Université de
Strasbourg, Strasbourg, France, and 5University of Copenhagen, Faculty of Pharmaceutical
Sciences, Copenhagen, Denmark
Received
29 June 2010
Revised
10 September 2010
Accepted
29 October 2010
BACKGROUND AND PURPOSE
Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABAA receptors (GABAARs). We
previously reported that trans-6,4′-dimethoxyretrochalcone (Rc-OMe), a hydrolytic derivative of the corresponding flavylium
salt, displayed nanomolar affinity for the benzodiazepine binding site of GABAARs. Here, we evaluate the functional
modulations of Rc-OMe, along with two other synthetic derivatives trans-6-bromo-4′-methoxyretrochalcone (Rc-Br)
and 4,3′-dimethoxychalcone (Ch-OMe) on GABAARs.
EXPERIMENTAL APPROACH
Whole-cell patch-clamp recordings were made to determine the effects of these derivatives on GABAARs expressed in HEK-293
cells and in hippocampal CA1 pyramidal and thalamic neurones from rat brain.
KEY RESULTS
Rc-OMe strongly potentiated GABA-evoked currents at recombinant a1–4b2g2s and a4b3d receptors but much less at a1b2 and
a4b3. Rc-Br and Ch-OMe potentiated GABA-evoked currents at a1b2g2s. The potentiation by Rc-OMe was only reduced at
a1H101Rb2g2s and a1b2N265Sg2s, mutations known to abolish the potentiation by diazepam and loreclezole respectively. The
modulation of Rc-OMe and pentobarbital as well as by Rc-OMe and the neurosteroid 3a,21-dihydroxy-5a-pregnan-20-one
was supra-additive. Rc-OMe modulation exhibited no apparent voltage-dependence, but was markedly dependent on GABA
concentration. In neurones, Rc-Br slowed the decay of spontaneous inhibitory postsynaptic currents and both Rc-OMe and
Rc-Br positively modulated synaptic and extrasynaptic diazepam-insensitive GABAARs.
CONCLUSIONS AND IMPLICATIONS
The trans-retrochalcones are powerful positive allosteric modulators of synaptic and extrasynaptic GABAARs. These novel
modulators act through an original mode, thus making them putative drug candidates in the treatment of GABAA-related
disorders in vivo.
© 2011 The Authors
British Journal of Pharmacology © 2011 The British Pharmacological Society
1326 British Journal of Pharmacology (2011) 162 1326–1339

Retrochalcones modulation at GABAA receptors
BJP
Abbreviations
BDZ, benzodiazepine; Ch-OMe, 4,3′-dimethoxychalcone; GABAAR, g-aminobutyric acid type A receptor; HEK, human
embryonic kidney; IPSCs, inhibitory postsynaptic currents; Rc-Br, trans-6-bromo-4′-methoxyretrochalcone; Rc-OMe,
trans-6,4′-dimethoxyretrochalcone; THDOC, 3a,21-dihydroxy-5a-pregnan-20-one; THIP,
4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one; wt, wild type
Introduction
A
9
OH
B
H2O/H+
O+
C
A
B
A
The g-aminobutyric acid type A (GABAA) receptor is the main
inhibitory ligand-gated chloride channel in the brain. The
receptor is the target for a wide range of important therapeutic
agents, including benzodiazepines (BDZs), general anaesthet-
ics and neurosteroids (D’Hulst et al., 2009; Olsen and Sieghart,
2009). GABAA receptors are comprised of a heteropentameric
assembly of distinct subunits, for which the most prevalent
form mediating the majority of fast synaptic inhibition in the
adult mammalian brain consists of an abg subunit combina-
tion (Fritschy et al., 1992; Somogyi et al., 1996). On the other
hand, receptor subtypes comprised of abd subunits that play a
key role in mediating tonic inhibition (Semyanov et al., 2004;
Glykys and Mody, 2007) are extrasynaptically located and are
also targets for endogenous neuroactive steroids (Belelli and
Lambert , 2005) and ethanol (Jia et al., 2008).
O
Flavylium salt
trans-retrochalcone
B
MeOH
NaOH (30%)
R2
O
O
R2
OMe
RT 4h
ρ = 60-70%
+
R1
R1
O
OMe
R1 = OMe, R2 = OH
R1 = OMe, R2 = H
R1 = Br, R2 = OH
Rc-OMe:
Ch-OMe:
Rc-Br:
diazepam
Ch-OMe Rc-Br
10 μM
Rc-OMe
10 μM
C
GABA
1 μM
10 μM
10 μM
Flavonoids are natural substances found in vascular
plants (Harborne and Williams, 2000) and display a wide
range of biological activities from anxiolytic (Griebel et al.,
1999) to anti-inflammatory and analgesic effects (Heidari
et al., 2009). It has been known since the late 1980s that this
family of compounds acts at GABAA receptors (Nielsen et al.,
1988). The ability of these compounds to compete with radio-
labelled BDZs in rat and bovine brain tissues and to exhibit
anxiolytic activity in rodents, suggests that these compounds
mediate their activities through the BDZ site at GABAA recep-
tors. However, subsequent studies have demonstrated that
the compounds modulate GABAA receptors at a site indepen-
dent of the high-affinity classical BDZ binding site (Hall et al.,
2004; Hansen et al., 2005; Fernandez et al., 2008), indicating
that this new family of compounds may exert their action
through a novel, yet unidentified, binding site.
We have recently found that a series of flavylium salts also
display binding displacement of the radiolabelled BDZ ligand
Ro15-1788 in rat cortical membranes in vitro (Kueny-Stotz
et al., 2008). More precisely, it is not the flavylium salts per se
that exhibit binding activities but their corresponding hydro-
lytic trans-retrochalcones products, through an in vitro chemi-
cal transformation (Kueny-Stotz et al., 2008) (Figure 1A). One
of these substituted trans-retrochalcones, namely trans-6,4′-
dimethoxyretrochalcone (Rc-OMe; see Figure 1B for chemical
structure), displays nanomolar affinity for the BDZ binding
site of brain GABAA receptors (Kueny-Stotz et al., 2008),
leading to the attractive hypothesis that flavylium salts could
be used as convenient precursors of trans-retrochalcones
when dissolved in neutral buffers, resulting in a possible
delayed action at GABAA receptors in vivo.
500 pA
2 s
Figure 1
Rc-OMe, Rc-Br and Ch-OMe are positive modulators at recombinant
a1b2g2S receptor. (A) Hydrolysis of flavylium derivatives leads to the
corresponding trans-retrochalcones (Kueny-Stotz et al., 2008). (B)
Condensation step for synthesis of Rc-OMe, Rc-Br and Ch-OMe. (C)
Examples of current traces showing potentiations of GABA-elicited
currents induced by Rc-OMe, Rc-Br, Ch-OMe and diazepam at
recombinant a1b2g2S receptors expressed in HEK-293 cells. Currents
were recorded in the same cell separated by ~30 s washout.
Ch-OMe, 4,3′-dimethoxychalcone; HEK, human embryonic
kidney; Rc-Br, trans-6-bromo-4′-methoxyretrochalcone; Rc-OMe,
trans-6,4′-dimethoxyretrochalcone.
BDZs but also from the pentobarbital, 3a,21-dihydroxy-5a-
pregnan-20-one (THDOC) and loreclezole modulatory sites.
Both the strong dependence of their effects on g-subunit and
GABA concentration together with their sensitivity to extra-
synaptic GABAA receptors reveal an original action mode for
these trans-retrochalcones. Our results are therefore helpful in
understanding the molecular mechanism underlying the
modulation by the trans-retrochalcones of brain GABAA
receptors.
In this paper, we extend the characterization of Rc-OMe
and two other substituted derivatives at GABAA receptors
expressed in human embryonic kidney (HEK)-293 cells and in
neurones from thalamus and hippocampus in vitro. We report
that the trans-retrochalcones positively modulate GABAA
receptors at a novel site distinct not only from the classical
Methods
Chemicals
Rc-OMe, trans-6-bromo-4′-methoxyretrochalcone (Rc-Br) and
4,3′-dimethoxychalcone (Ch-OMe) were synthesized as
British Journal of Pharmacology (2011) 162 1326–1339 1327

R Jiang et al.
BJP
described next. Other drugs were purchased from Sigma (St
Louis, MO, USA). All the products were stored in aliquots at
-20°C. Final concentration of dimethyl sulphoxide, if
present, never exceeded 0.3% throughout experiments per-
formed in this study, and this concentration had no effect on
GABA-elicited currents.
ensure the incorporation of the g (or d) subunit to the recep-
tor, HEK-293 cells were transfected with 0.3 mg cDNAs in a
1:1:10 ratio of a:b:g (or d) (Boileau et al., 2002) using calcium
phosphate precipitation. A green fluorescent protein cDNA
construct (0.3 mg) was added to identify cells that were effec-
tively transfected. Cells were washed 1 day after transfection
with fresh medium and used within 24–72 h.
Synthesis of Rc-OMe, Rc-Br and Ch-OMe
An aqueous solution of sodium hydroxide (60%, 25 mL) was
added to a solution of 2-hydroxy-4-methoxy-benzaldehyde
(5 mM; for Rc-OMe), 3-methoxy-benzaldehyde (5 mM; for
Ch-OMe), or 2-hydroxy-4-bromo-benzaldehyde (5 mM; for
Rc-Br) and 4-methoxyacetophenone in methanol (25 mL).
The reaction mixture was stirred for 4 h using a mechanical
stirrer. The mixture was then poured into ice-cold hydrochlo-
ric acid (pH adjusted to 2). The solid obtained was filtered,
dissolved in dichloromethane (150 mL) and washed with a
saturated aqueous solution of sodium bicarbonate. The
organic layer was then collected, dried, and evaporated to
dryness. The residue obtained was crystallized from metha-
nol. Yield was 60–70%.
Preparations of hippocampal CA1 pyramidal
neurones and thalamic neurones
For hippocampal CA1 pyramidal neurones, Wistar rats, at
postnatal day (P) 12–18, were decapitated under pentobar-
bital sodium anaesthesia (100 mg·kg-1, i.p.). For thalamic
neurones, 3–5 week-old Wistar rats were used. Coronal brain
slices containing hippocampus or ventrobasal thalamus at a
thickness of 400 mm were prepared by use of a microslicer
(VT-1000S; Leica, Nussloch, Germany). Slices were kept in a
control incubation solution (see next) saturated with 95% O2
and 5% CO2 at room temperature (22–24°C) for at least 1 h
before mechanical dissociation. Slices were then transferred
into a 35 mm culture dish (Primaria 3801; Becton Dickinson,
Rutherford, NJ, USA) containing the standard external solu-
tion (see next), and the hippocampal CA1 or ventrobasal
thalamus regions were identified under a binocular micro-
scope. Details of the mechanical dissociation procedure used
to isolate single neurones with adherent and functional
presynaptic boutons have been given previously (Rhee et al.,
1999; Akaike and Moorhouse, 2003). Briefly, a fire-polished
glass micropipette was placed on the surface of the brain slice,
and the tip of the pipette was vibrated horizontally at
30–60 Hz for about 2 min using a piezoelectric manipulator
(CELL ISOLATOR, K.T. Lab, Saitama, Japan). Slices were
removed and the mechanically dissociated neurones allowed
to settle and adhere to the bottom of the dish for at least
10 min before recordings commenced.
1
Rc-OMe: RMN H (300 MHz, CD3OD, 25°C): d = 3.78 (s,
3
3
3H), 3.88 (s, 3H), 6.80 (d, 1H, J = 8.8 Hz), 6.86 (dd, 1H, J =
8.8 Hz, 4J = 2.9 Hz), 7.04 (m, 2H), 7.20 (d, 1H, 4J = 2.9 Hz),
7.78 (d, 1H, 3J = 15.7 Hz), 8.06 (m, 2H), 8.07 (d, 1H, 3J =
15.7 Hz) ppm. RMN 13C (75 MHz, CD3OD, 25°C): d = 54.6,
54.9, 112.2, 113.6, 116.6, 118.3, 121.1, 122.0, 130.6, 130.9,
140.1, 151.6, 153.0, 163.8, 190.1 ppm.
Rc-Br: RMN 1H (400 MHz, CD3OD, 25°C): d = 3.90 (s, 3H),
6.84 (d, 1H, 3J = 8.8 Hz), 7.06 (d, 2H, 3J = 9.2 Hz), 7.35 (dd, 1H,
4
4
3J = 8.8 Hz, J = 2.4 Hz), 7.82 (d, 1H, J = 2.4 Hz), 7.83 (d, 1H,
3J = 16.0 Hz), 7.99 (d, 1H, 3J = 15.6 Hz), 8.09 (d, 2H, 3J =
8.8 Hz) ppm. RMN 13C (100 MHz, CD3OD, 25°C): d = 54.1,
112.6, 115.1, 119.0, 123.7, 125.5, 126.6, 132.1, 132.4, 135.2,
139.8, 157.9, 165.4, 191.2 ppm.
Ch-OMe: RMN 1H (400 MHz, CDCl3, 25°C): d = 3.88 (s,
3
3H), 3.91 (s, 3H), 6.97 (m, 1H), 7.00 (d, 2H, J = 8.8 Hz), 7.18
Electrophysiology of HEK-293 cells
(m, 1H), 7.27 (m, 1H), 7.35 (m, 1H), 7.54 (d, 1H, 3J = 16.0 Hz),
7.78 (d, 1H, 3J = 15.6 Hz), 8.06 (d, 1H, 3J = 8.8 Hz) ppm. RMN
13C (100 MHz, CDCl3, 25°C): d = 55.5, 55.5, 113.4, 113.9,
116.1, 121.0, 122.2, 129.9, 130.8, 131.1, 136.5, 143.9, 160.0,
163.5, 188.7 ppm.
Currents were recorded using the whole-cell configuration of
the patch-clamp technique only from fluorescent cells. Cells
were maintained at a holding potential of -60 mV except for
the voltage-dependence experiments. Patch pipettes (3–5 MW)
contained (mM): 140 KCl, 5 MgCl2, 5 EGTA, 10 HEPES, pH 7.3.
External solution contained (mM): 140 NaCl, 2.8 KCl, 2 CaCl2,
2 MgCl2, 10 glucose, 10 HEPES, pH 7.3. In these conditions, the
theoretical Nernst equilibrium potential of Cl- current is
-0.1 mV. The solutions were delivered through three parallel
tubes placed immediately above the cell. These tubes were
horizontally displaced with the aid of a computer-driven
system (SF 77A Perfusion fast step, Warner, Hamden, CT, USA)
that ensures solution exchange in 5–10 ms. GABA and/or
GABA plus modulators were applied briefly (2–3 s) with a
washout period of at least 30 s between applications.
Mutagenesis
pSN3 plasmids containing the cDNA encoding all the human
GABAA subunits used in this study were generously provided
by Marianne L. Jensen, Neurosearch, Denmark. Site-directed
mutagenesis was carried out using the QuickChange® II Site-
directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and
mutation was confirmed by DNA sequencing.
Cell culture and transfection in
HEK-293 cells
Electrophysiology of hippocampal CA1
HEK-293 cells were cultured in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum (Invit-
rogen, Carlsbad, CA, USA), 1X GlutaMax, 100 U·mL-1
penicillin and 100 mg·mL-1 streptomycin (Invitrogen).
Trypsin-treated cells were seeded onto glass coverslips in
35 mm dishes pretreated with poly-L-Lysine (Sigma) 1 day
before transfection and incubated at 37°C with 5% CO2. To
pyramidal neurones and thalamic neurones
All electrical measurements were performed using the con-
ventional whole-cell patch recording with Axopatch 200B
amplifier (Molecular Devices, Sunnyvale, CA, USA) at room
temperature. The neurones were voltage clamped at a holding
potential (VH) of -65 mV. Membrane potentials were cor-
1328 British Journal of Pharmacology (2011) 162 1326–1339

Retrochalcones modulation at GABAA receptors
BJP
rected by -5 mV to compensate for the patch pipette-bath
liquid junction potential. Patch pipettes were made by a
PC-10 microelectrode puller (Narishige, Tokyo, Japan) from
capillary glass with an inner filament (GD-1.5; Narishige).
The resistance of the recording pipettes filled with internal
solution (see next) was 5–7 MW. Isolated neurones were
viewed under phase contrast on an inverted microscope (IX-
71; Olympus, Tokyo, Japan). Current and voltage were con-
tinuously monitored on an oscilloscope (VC-6725; Hitachi,
Tokyo, Japan). Membrane currents were filtered at 2 kHz,
digitized at 10 kHz and stored on a personal computer
equipped with pClamp 8.2 (Molecular Devices, Sunnyvale,
CA, USA). The ionic composition of the control incubation
solution consisted of (mM): 124 NaCl, 5 KCl, 1.3 KH2PO4, 24
NaHCO3, 2.4 CaCl2, 1.3 MgCl2 and 10 glucose saturated with
95% O2 and 5% CO2. The standard external solution used for
recordings from isolated neurones contained (mM): 150
NaCl, 2.5 KCl, 1.0 MgCl2, 2.0 CaCl2, 10 glucose, 10 HEPES,
and was adjusted to a pH of 7.4 with Tris-base. The standard
external solution routinely contained 10 mM 6-cyano-7-
nitroquinozaline-2, 3-dione disodium, 20 mM DL-2-amino-5-
phosphonovaleric acid to block ionotropic glutamatergic
currents. Tetrodotoxin (0.3 mM) was also added to the record-
ing solution to block the burst activity of spontaneous
inhibitory post-synaptic currents (IPSCs) (Inada et al., 2010).
The internal (patch-pipette) solution for the whole-cell
patch recording contained (mM): 90 CsCl, 55
tion, [D] was the drug concentration; and nH was the Hill
coefficient. Spontaneous IPSCs were analysed using Mini-
Analysis program (Synaptosoft, Leonia, NJ, USA). Only single
IPSC events were visually selected for analysing the ampli-
tude and decay kinetics. Data are expressed as mean Ϯ SEM.
Statistical differences were determined using Student’s two-
tailed t-test. For multiple groups, one-way ANOVA with Tukey’s
multiple comparison post hoc test was used.
Results
Synthesis of trans-retrochalcones
The retrochalcones Rc-OMe and Rc-Br were prepared through
the base-catalyzed Claisen–Schmidt condensation of
p-methoxy acetophenone with substituted o-OH benzalde-
hydes while the chalcone Ch-OMe used m-OMe benzalde-
hyde for the condensation. (Stirrett et al., 2008) (Figure 1B).
The synthesized (retro)chalcones are
positive allosteric modulators of
a1b2g2s GABAA receptors
Recombinant a1b2g2s GABAA receptors were expressed in HEK-
293 cells and the whole-cell configuration of the patch-clamp
technique was used to evaluate the action of the three deriva-
tives. GABA EC50 value was determined (Table 1). The effect of
each of the three compounds (10 mM) on the GABA (1 mM)-
evoked currents was determined. As depicted in Figure 1C, all
three derivatives potentiated, although differently, GABA-
evoked currents. The trans-retrochalcones Rc-OMe and Rc-Br
exhibited higher efficacy than diazepam (10 mM), a classical
BDZ allosteric modulator of GABAA receptors (Figure 1C). By
contrast, Ch-OMe potentiated GABA-evoked currents simi-
larly to diazepam (Figure 1C). Overall, these results indicate
that the trans-retrochalcones are strong positive allosteric
modulators of the a1b2g2s GABAA receptor. Because the
methoxy-derivative potentiated the GABA response approxi-
mately to the same extent as the bromo-derivative, we
decided to further study these two molecules either separately
or together.
Cs-methanesulphonate, 10 HEPES,
2 EGTA, 4 lidocaine
N-ethyl chloride, 4 ATP-Mg, 0.4 GTP-Na, and was adjusted to
a pH of 7.3 with Tris-base. Voltage ramps from a VH of -65 mV
to +15 mV for 0.5 s were applied before and during the appli-
cation of GABA. To obtain current-voltage relationships, the
ramp current measured before the GABA response was sub-
tracted from the current obtained during the response.
Data analysis
Amplitudes of currents induced by exogenously applied
GABA or GABA plus modulators were measured at the peaks
of the responses. The modulatory effect on GABA or 4,5,6,7-
tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) response
was defined as potentiation (fold) = (Iagonist+modulator/Iagonist) – 1,
where Iagonist was the agonist-evoked current and Iagonist+modulator
was the current evoked by the co-application of agonist plus
modulator. Concentration-response relationships for agonist
as well as for agonist plus modulator were constructed, and
the Hill equation was fitted (IGOR PRO 5.03) to the data
according to:
Subunit dependence of potentiations
induced by Rc-OMe
We further characterized the action of Rc-OMe at recombi-
nant a1b2g2s, a2b2g2s, a3b2g2s and a4b2g2s GABAA receptors to
establish that the potentiation induced by this compound is
dependent on the a-subunit; the a1b2 combination was also
included to evaluate the contribution of the g-subunit for
Rc-OMe-induced potentiation. These receptors were func-
tionally expressed in HEK-293 cells (Table 1) and GABA EC~2
concentration was determined for each subunit combination.
The concentration-response relationship was then con-
structed for Rc-OMe-induced potentiations of an EC~2 GABA-
elicited current at each subunit combination (Figure 2B).
Rc-OMe was used up to 100 mM because of its limited solu-
bility in buffers.
I
1
=
nH
Imax
EC50 ⎞
[D]
⎛
1+
⎜
⎝
⎟
⎠
where I and Imax were the peak current to a given concentra-
tion of the drug and the maximum current, respectively; EC50
was the concentration of the drug giving half maximum
response; [D] was the drug concentration; and nH was the Hill
coefficient. The potentiations were also fitted to the previ-
ously mentioned Hill equation, but in which I and Imax were
the potentiation elicited by a given concentration of the drug
and the maximum potentiation, respectively; EC50 was the
concentration of the drug giving half maximum potentia-
Rc-OMe potentiated GABA-elicited currents at a1b2g2s,
a2b2g2s and a3b2g2s (Figure 2A,B), all in a concentration-
dependent manner. EC50 values were 17.7 Ϯ 3.0 (n = 11)
and 7.6 Ϯ 1.6 mM (n = 4) for a1b2g2s and a2b2g2s, respectively,
British Journal of Pharmacology (2011) 162 1326–1339 1329

R Jiang et al.
BJP
Table 1
Functional parameters for Rc-OMe modulation at recombinant wild-type receptors and mutants
GABA
Rc-OMe
(in the presence
of 30 mM Rc-OMe)
(in the presence
of EC~2 GABA)
GABA
Receptor
a1b2g2S
EC50, mM
nH
n
5
5
5
6
4
5
6
6
4
EC50, mM
12.1 Ϯ 4.3b
N.D.
nH
n
EC50, mM
nH
Fold maximal potentiation
n
23.1 Ϯ 3.3
1.5 Ϯ 0.1
0.7 Ϯ 0.1b
5
17.7 Ϯ 3.0 1.6 Ϯ 0.1 19.6 Ϯ 2.2 (at 30 mM)
7.6 Ϯ 1.6 1.4 Ϯ 0.2 13.6 Ϯ 2.3 (at 30 mM)
11
4
a2b2g2S
62.3 Ϯ 12.8 1.7 Ϯ 0.1
115.8 Ϯ 24.7 1.6 Ϯ 0.3
a3b2g2S
N.D.
N.D.
8.1 Ϯ 3.4 3.8 Ϯ 1.0 6.2 Ϯ 1.4 (at 30 mM)
N.D. 2.1 Ϯ 0.3 (at 10 mM)
7.4 Ϯ 0.9 1.5 Ϯ 0.2 10.4 Ϯ 2.0a (at 100 mM)
15.2 Ϯ 1.2 (at 100 mM)
5
a4b2g2S
13.4 Ϯ 1.3
3.9 Ϯ 0.9
57.0 Ϯ 11.6a 1.3 Ϯ 0.1
1.5 Ϯ 0.1
1.5 Ϯ 0.2
33.4 Ϯ 15.5
N.D.
185.2 Ϯ 111.8 0.6 Ϯ 0.1b
0.9 Ϯ 0.1b
6
5
5
a1b2
7
a1H101Rb2g2S
a1b2N265Sg2S
a4b3d
3
30.1 Ϯ 4.9
3.8 Ϯ 1.0
1.7 Ϯ 0.4
1.9 Ϯ 0.2
1.1 Ϯ 0.2
1.3 Ϯ 0.2
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
7.8 Ϯ 0.7a (at 30 mM)
6
N.D.
N.D.
a4b3
aValues are significantly different from that of a1b2g2S, P < 0.05 (Student’s unpaired t-test).
bValues are significantly different from that determined in the absence of Rc-OMe, P < 0.05 (Student’s paired t-test).
Rc-OMe, trans-6,4′-dimethoxyretrochalcone; N.D., not determined.
but for a3b2g2s, EC50 could not be determined due to a lack of
Rc-OMe exerts its potentiation effect mainly
saturation up to 100 mM (Figure 2B). At a1b2g2s, because of the
bell-shaped curve, the EC50 value might be an underestimate
of the true value (Figure 2B). The maximal potentiations
reached 19.6 Ϯ 2.2-, 13.6 Ϯ 2.3- and 15.2 Ϯ 1.2-fold at 30, 30
and 100 mM, respectively, at a1b2g2s, a2b2g2s and a3b2g2s (n =
4–11) (Table 1). In the control experiments, diazepam (3 mM)
potentiated GABA-elicited currents at these receptors (n =
4–11), suggesting the successful incorporation of the
g-subunit into these expressed receptors (exemplified for
a1b2g2s in Figure 2A).
through a site different from that of the
classical BDZs
We further investigated the challenging hypothesis that
Rc-OMe binds to
a site independent of that of the
classical BDZs by performing the following additional
experiments.
Firstly, Ro 15–1788, an antagonist that binds to the BDZ
binding site with nanomolar affinity, was used to inhibit
Rc-OMe-induced potentiation at a1b2g2s. Ro 15–1788 (10 mM)
only reduced by 34.4 Ϯ 3.3% the potentiation induced by
10 mM Rc-OMe (n = 7) (Figure 3A), whereas in control experi-
ments Ro 15–1788 abolished the potentiation induced by
3 mM diazepam (data not shown). We also verified that 10 mM
Ro 15–1788 did not by itself potentiate the GABA response
(108 Ϯ 8% of control, n = 6).
Secondly, if Rc-OMe and diazepam act through two, topo-
logically different, binding sites, then one can expect an
additive effect of the two drugs. Indeed, co-stimulation of the
GABA-elicited current with 1 mM Rc-OMe plus 3 mM diaz-
epam, a concentration that saturates the high-affinity BZD
binding site, resulted in a 2.3 Ϯ 0.7-fold potentiation, close to
the sum of the potentiations recorded on the same cell by
1 mM Rc-OMe (1.1 Ϯ 0.3-fold) and 3 mM diazepam (1.7 Ϯ
0.4-fold) applied separately (n = 5) (Figure 3B). This near-
additive effect further strongly supports the notion that
the main action site of Rc-OMe is different from that of
diazepam.
Unexpectedly, Rc-OMe potentiated GABA-elicited cur-
rents at a4b2g2s with a 6.2 Ϯ 1.4-fold maximal potentiation
observed at 30 mM (n
response curve was also
=
5), and the concentration-
a bell-shaped curve (Figure 2
and Table 1). Diazepam (3 mM), as expected, did not
potentiate GABA-elicited currents at this classical BDZ
insensitive receptor (n = 5) (Figure 2A). Taken together,
the observed potentiating extent was a1b2g2s > a2b2g2s
a3b2g2s > a4b2g2s.
ª
Rc-OMe by itself (up to 100 mM) evoked only tiny currents
in the cells expressing each of these four combinations
(<8.3% of the maximal GABA-evoked currents in all cases,
data not shown), indicating a very poor GABAA receptor
agonist action for Rc-OMe.
The maximal potentiation at a1b2 receptors was only 2.1
Ϯ 0.3-fold (at 10 mM, n = 7), highly reduced compared with
that at a1b2g2s (19.6 Ϯ 2.2-fold, n = 11), suggesting that the
potentiation induced by Rc-OMe depends strongly on the
presence of the g-subunit (Figure 2).
Moreover, a rebound current was observed upon washout
after co-application of GABA and Rc-OMe (100 mM)
(Figure 2), indicating that at high concentration Rc-OMe may
act as an open channel blocker at a1b2. Diazepam (3 mM), as
expected, did not potentiate GABA-elicited currents at this
BDZ-insensitive receptor (n = 7) (Figure 2A).
Finally, we introduced H101R mutation in the a1 subunit,
a mutation known to abolish the high-affinity binding of
classical BDZs (Wieland et al., 1992; Rudolph et al., 1999).
Mutant a1H101Rb2g2s was functionally expressed in HEK-293
cells (Table 1) and, as expected, was not responsive to 3 mM
diazepam (data not shown). By contrast, Rc-OMe still poten-
tiated EC~2 GABA-elicted currents (EC50 = 7.4 Ϯ 0.9 mM and
1330 British Journal of Pharmacology (2011) 162 1326–1339

Retrochalcones modulation at GABAA receptors
BJP
A
A
∗∗∗
GABA
100
α1β2γ2S
Rc-OMe, μM
10 30 100
diazepam, 3 μM
1
3
50
GABA
+ Rc-OMe
+ Ro15-1788
0
100 pA
+ Rc-OMe
1 s
2 nA
2 s
α4β2γ2S
GABA
B
∗
+Rc-OMe
+diazepam
GABA
4
∗
200 pA
2 s
α1β2
2
GABA
300 pA
2 s
0
400 pA
2 s
B
30
20
10
0
C
α β γ2S
α1β2γ
30
α2β2γ2S
α3β2γ2S
α1β2γ2S
2S
α41β22
20
10
0
α1H101Rβ2γ2S
0.1
1
10
Rc-OMe, μM
100
0.1
1
10
Rc-OMe, μM
100
Figure 2
Figure 3
Rc-OMe-induced potentiations of GABA-elicited currents at recom-
binant a1b2g2S, a2b2g2S, a3b2g2S, a4b2g2S and a1b2 receptors. (A)
Examples of current traces showing the effects induced by different
concentrations of Rc-OMe on EC~2 GABA-elicited currents at a1b2g2S,
a4b2g2S and a1b2. Also shown are the control experiments carried out
in the same cells with diazepam (3 mM). (B) Concentration-response
curves for Rc-OMe-induced potentiations of EC~2 GABA currents at
a1b2g2S, a2b2g2S, a3b2g2S, a4b2g2S and a1b2 receptors (EC~2 concentra-
tions are 1, 4, 6, 1 and 0.15 mM respectively). Data (data points at
100 mM for a1b2g2S and a4b2g2S are not included) are fitted to the Hill
equation (in red) except for a1b2 and a3b2g2S receptors. Data points
and error bars in this figure and all other figures represent mean Ϯ
SEM. Rc-OMe, trans-6,4′-dimethoxyretrochalcone.
Rc-OMe-induced potentiation is mediated through a site indepen-
dent of that of the classical BDZs. (A) Left, examples of current traces
showing the potentiation induced by Rc-OMe (10 mM) (black trace)
or by Rc-OMe (10 mM) plus Ro 15–1788 (10 mM) (grey trace); the
GABA-elicited current (1 mM) is also shown. Right, summary of inhi-
bition induced by the presence of Ro 15–1788 (n = 7), control was
set to 100%. ***P < 0.0001 (Student’s paired t-test). (B) Left,
examples of current traces showing potentiations of GABA-evoked
currents (1 mM) induced by Rc-OMe (1 mM) or diazepam (3 mM), or
by diazepam (3 mM) plus Rc-OMe (1 mM). Right, summary of addi-
tive potentiation induced by Rc-OMe plus diazepam (n = 5). *P <
0.05 (Student’s paired t-test). (C) Concentration-response curve for
Rc-OMe-induced potentiations of EC~2 GABA currents (3 mM) at
a1H101Rb2g2S (n = 3). Data were fitted to the Hill equation. Data
and fitted curve for wt receptor were taken from Figure 2B.
BDZ, benzodiazepine; Rc-OMe, trans-6,4′-dimethoxyretrochalcone;
wt, wild type.
maximal potentiation = 10.4 Ϯ 2.0-fold at 100 mM, n = 3) in
these mutants, although less efficiently than at wild-type (wt)
receptors (Figure 3C). Interestingly, a high concentration of
Rc-OMe (100 mM) did not induce an inhibitory effect with
this mutant, an effect previously observed with the wt recep-
tor. These results suggest that the absence of the high-affinity
BDZ binding site has moderate impact on the potentiations
induced by Rc-OMe.
Taken together, these three sets of experiments clearly
demonstrate that Rc-OMe exerts its potentiation effect
mainly through a site different from that of the classical BDZ
binding site.
British Journal of Pharmacology (2011) 162 1326–1339 1331

R Jiang et al.
BJP
∗∗
∗∗
∗∗
∗∗
60
40
20
0
GABA
GABA
A
+Rc-OMe
+Rc-OMe
+THDOC
+PB
+Rc-OMe
+THDOC
+Rc-OMe
+PB
500 pA
1 s
B
C
∗∗
PB
THDOC
4
2
20
10
0
+Rc-OMe
100 pA
+Rc-OMe
0
2 s
Figure 4
Rc-OMe-induced potentiation is mediated through a site independent of those of pentobarbital (PB), 3a,21-dihydroxy-5a-pregnan-20-one
(THDOC) and loreclezole. (A) Left, examples of current traces showing the supra-additive potentiation of GABA-evoked currents (1 mM) by
Rc-OMe (10 mM) plus PB (100 mM) or by Rc-OMe (10 mM) plus THDOC (3 mM) at a1b2g2S. Right, summary of supra-additive potentiation induced
by Rc-OMe plus PB (n = 8) or by Rc-OMe plus THDOC (n = 7). **P < 0.01 (Student’s paired t-test). (B) Left, examples of traces of the potentiation
of PB (100 mM)-evoked current or of THDOC (3 mM)-evoked current induced by Rc-OMe (10 mM). Right, summary of potentiations of PB-evoked
currents (n = 5) or of THDOC (3 mM)-evoked currents (n = 5) induced by Rc-OMe at a1b2g2S. (C) Summary of the potentiation of EC~2 GABA-elicited
currents (1.5 mM) induced by Rc-OMe (30 mM) at the mutant a1b2N265Sg2s in the absence and presence of Ro 15–1788 (10 mM) (n = 4–6) as
compared with that obtained at wt (data for wt are taken from Figure 2B, n = 13). **P < 0.01 (Student’s unpaired t-test). Rc-OMe, trans-6,4′-
dimethoxyretrochalcone; wt, wild type.
et al., 2006). These supra-additive rather than competitive
effects strongly suggest that the site of action of Rc-OMe for
mediating its potentiation effects at GABAA receptors is dif-
ferent from those of PB and THDOC.
Rc-OMe does not mediate its action through
pentobarbital- and neurosteroid-binding sites
To gain further insights into the molecular mechanism
underlying potentiations induced by Rc-OMe, we adopted
the concept of additivity to distinguish the action site of
Rc-OMe from those of pentobarbital (PB) and THDOC, which
are classified as a general anaesthetic and neurosteroid,
respectively, both acting as positive modulators of GABAA
receptors.
At a1b2g2s, co-stimulation of GABA-elicited currents by
Rc-OMe (10 mM) and PB (100 mM) resulted in a supra-additive
effect of a 43.5 Ϯ 7.3-fold potentiation, while in the same cell
Rc-OMe and PB exerted a 5.8 Ϯ 1.0-fold and a 20.8 Ϯ 3.4-fold
In HEK-293 cells expressing a1b2g2s, either 100 mM PB or
3 mM THDOC by itself evoked Cl- currents (Figure 4B), con-
firming their agonist actions at these concentrations (Thomp-
son et al., 1996; Hosie et al., 2006), and these currents were
also potentiated by 10 mM Rc-OMe (1.5 Ϯ 0.3-fold and 3.1 Ϯ
0.4-fold, respectively, for PB and THDOC, n = 5) (Figure 4B).
We thus suggest that these currents elicited by PB or THDOC,
which were potentiated by Rc-OMe, together with GABA-
elicited currents, which were potentiated by Rc-OMe plus PB
or THDOC (see Figure 4A), contribute to the previously men-
potentiation (n = 8) respectively (Figure 4A).
tioned supra-additive currents.
This supra-additive effect was also observed with
Rc-OMe (10 mM) and THDOC (3 mM), leading to a 38.7 Ϯ
8.2-fold potentation while Rc-OMe and THDOC exerted a
Potentiation by Rc-OMe is largely reduced but
still present at the mutant a1b2N265Sg2s
5.8 Ϯ 1.0-fold and a 21.4 Ϯ 5.9-fold potentiation (n = 7)
respectively (Figure 4A). PB 100 mM and 3 mM THDOC were
chosen because these concentrations induce their maximal
potentiation effect at a1b2g2s (Thompson et al., 1996; Hosie
The potentiation of GABA-elicited currents induced by lore-
clezole is abolished in the point mutant a1b2N265Sg2s
1332 British Journal of Pharmacology (2011) 162 1326–1339

Retrochalcones modulation at GABAA receptors
BJP
(Wingrove et al., 1994). This mutant was functionally
expressed in HEK-293 cells and Rc-OMe (30 mM) was applied
in the presence of EC~2 GABA concentration (Table 1). The
potentiation induced by diazepam (3 mM) was not affected
(data not shown), whereas a 7.8 Ϯ 0.7-fold (n = 6) poten-
tiation induced by Rc-OMe remained strong but was
reduced by 60.2% compared with that in the wt receptor
(Figure 4C). Surprisingly, this residual potentiation was not
affected by the presence of 10 mM Ro 15–1788 (7.8 Ϯ 0.5-
fold, n = 4) (Figure 4C), contrasting to its 30% inhibition at
the wt receptor.
desensitization rates of GABA-evoked currents at high GABA
concentrations.
Rc-OMe and Rc-Br are potent and efficacious
modulators of native BDZ-sensitive GABAA
receptors expressed in hippocampal CA1
pyramidal neurones
We further investigated the modulatory effects induced by
the trans-retrochalcones at native GABAA receptors in neu-
rones. In acutely isolated hippocampal CA1 pyramidal neu-
rones, GABA (1 mM) consistently evoked an inward current.
This GABA response is enhanced by diazepam (0.5 mM) (data
not shown), suggesting the involvement of g-subunit- con-
taining GABAA receptors. The GABA response was concentra-
tion dependent and the EC50 and Hill coefficient were 3.5 Ϯ
0.4 mM and 1.9 Ϯ 0.2 respectively (n = 6) (Figure S1A,D). The
current induced by GABA (1 mM) was markedly potentiated
by Rc-Br (3 mM) (1.6 Ϯ 0.5-fold, n = 8) (Figure 6A) and the
response to GABA gradually recovered after washout of Rc-Br.
In addition, application of Rc-Br enhanced the GABA-
induced current, but significantly less than the first applica-
tion (by 60.8 Ϯ 4.7%, n = 8) (Figure 6B). Thus, only one
application of Rc-Br plus GABA was made at each recorded
neurone to construct the concentration-response curve
(Figure 6C). Curve fitting to the pooled data, according to the
Hill equation, yielded an EC50 value and Hill coefficient of
0.7 mM and 2.0 (n = 5–6) respectively. Rc-OMe also enhanced
GABA-elicited currents (Figure 6C) with an EC50 value and
Hill coefficient of 1.1 mM and 2.6 (n = 4–6) respectively.
The modulatory effects of Rc-OMe at
recombinant GABAA receptors are dependent
on GABA concentration
In HEK-293 cells expressing a1b2g2s or a4b2g2s receptors,
GABA dose-response curves were constructed in the absence
and presence of Rc-OMe respectively. The presence of
Rc-OMe (30 mM) profoundly modified the GABA dose-
response curve (Figure 5A), mainly by decreasing the Hill
coefficient with a slight decrease of the EC50 value (Table 1).
The decrease of the Hill coefficient was also observed for
a4b2g2s receptors (Figure 5B), but the EC50 values were not
affected (Table 1). Furthermore, at a high GABA concentra-
tion (i.e. ՆEC50), Rc-OMe enhanced the apparent desensiti-
zation rates of GABA-evoked currents (Figure 5) at both
a1b2g2s and a4b2g2s receptors. Overall, these data show
that Rc-OMe potentiates GABA-elicited currents only at low
GABA concentrations, but enhances the apparent
A
α1β2γ2S
1.0
300
1
3
10
30
GABA
GABA + 30 μM Rc-OMe
0.5
0.0
2 nA
2 s
1
10
100 1000
0.1
B
GABA, μM
α4β2γ2S
300
1
3
10
30
1.0
0.5
0.0
GABA
GABA + 30 μM Rc-OMe
2 nA
2 s
1
10
100 1000
0.1
GABA, μM
Figure 5
The effects induced by Rc-OMe at recombinant GABAA receptors are dependent on GABAconcentration. Left, examples of current traces evoked
by different concentrations of GABA in the absence and presence of Rc-OMe at a1b2g2S (A) and a4b2g2S (B). Right, GABA concentration-response
curves in the absence and presence of Rc-OMe (30 mM) at a1b2g2S (n = 5) (A) and a4b2g2S (n = 6) (B). Data were fitted to the Hill equation. Rc-OMe,
trans-6,4′-dimethoxyretrochalcone.
British Journal of Pharmacology (2011) 162 1326–1339 1333

R Jiang et al.
BJP
A
Rc-Br
Rc-Br
GABA
100 pA
15 s
4 min
6 min
2 min
10 min
12 min
0 min
8 min
14 min
B
C
∗∗
Rc-Br
Rc-OMe
2.0
2.0
1.0
0
1.0
0
2nd
1st
10
0.1
1.0
Rc Compounds, μM
Figure 6
Effects of Rc-Br and Rc-OMe on GABA-induced currents in acutely isolated hippocampal CA1 pyramidal neurones. (A) Examples of
a
current trace showing the effect of Rc-Br on the current evoked by GABA. (B) The second application of Rc-Br produced a
smaller potentiation (n = 8). **P < 0.01 (Student’s paired t-test). Current amplitude in the presence of Rc-Br was normalized to that obtained
just before Rc-Br application. (C) Concentration-response relationships for the potentiation of GABA-elicited current induced by
Rc-Br and Rc-OMe (n
= 4–6). Data were fitted to the Hill equation. Rc-Br, trans-6-bromo-4′-methoxyretrochalcone; Rc-OMe,
trans-6,4′-dimethoxyretrochalcone.
Ϯ
1.6 mV and -15.5
Ϯ 2.3 mV (n = 7) respectively
Potentiation of GABA-elicited currents
(Figure S2B).
induced by Rc-OMe or Rc-Br is not
These results suggest that the potentiation of GABA-
elicited currents induced by the two trans-retrochalcones is
not voltage dependent.
voltage dependent
We determined the I-V curve for the GABA-elicited current
(1 mM) in the absence and presence of Rc-OMe (10 mM) in
HEK-293 cells expressing a1b2g2s receptors (Figure S2A).
According to the I-V curve, the potentiation of GABA-elicited
currents induced by Rc-OMe did not appear to be voltage
dependent. The reversal potential was 2.7 Ϯ 2.9 mV in the
absence of Rc-OMe, and 3.8 Ϯ 3.8 mV in its presence (n = 3).
The I-V curve for the GABA-elicited current (3 mM) in
the absence and presence of Rc-Br (3 mM) was determined in
hippocampal CA1 pyramidal neurones using a voltage-ramp
protocol. Similar to that in the recombinant system with
Rc-OMe, no voltage dependence was observed. The reversal
potential in the absence and presence of Rc-Br was -11.1
Rc-Br slowed the decay of IPSCs
Because our dissociated preparations show spontaneous
IPSCs (Akaike and Moorhouse, 2003), the effect of Rc-Br on
spontaneous IPSCs was also analysed. As shown in Figure 7,
Rc-Br (3 mM) significantly slowed the decay of IPSCs. The
mean half-life (t50) of IPSC in the absence and presence of
Rc-Br was 16.5 Ϯ 0.9 ms and 29.8 Ϯ 1.7 ms respectively (n =
5). On the other hand, Rc-Br had no significant effect on the
mean amplitude of the IPSC (n = 5).
1334 British Journal of Pharmacology (2011) 162 1326–1339

Retrochalcones modulation at GABAA receptors
BJP
observed in reticular thalamic neurones which express no
extrasynaptic receptors (Gibbs et al., 1996). A low concentra-
tion of THIP (1 mM) was used to selectively activate the
d-subunit-containing GABA receptors in the VB neurones
(Belelli et al., 2005; Cope et al., 2005; Jia et al., 2005). THIP
(1 mM) elicited a substantial inward current, which was fully
blocked by 2 mM SR-95531, a competitive antagonist of the
GABAA receptor (Figure 8A) but was insensitive to 0.5 mM
diazepam (Figure 8C). Although GABAA receptor antagonists
have been reported to produce a shift of the holding currents
in VB thalamic slice preparations (Cope et al., 2005; Jia et al.,
2005), SR-95531 failed to induce detectable changes in the
holding currents in our dissociated neurones (Figure 8A). This
was because the isolated single neurones had no ambient
GABA, which is needed for activation of the extrasynaptic
receptors. Markedly, the THIP-elicited current was potenti-
ated by 3 mM Rc-Br (1.9 Ϯ 0.2-fold) and 3 mM Rc-OMe (2.1 Ϯ
0.2-fold) (Figure 8B,C), and these potentiations were not
affected by the presence of 20 mM Ro 15–1788 (2.4 Ϯ 0.3-fold
for Rc-OMe and 3.3 Ϯ 0.7-fold for Rc-Br, n = 5–8) (Figure 8C).
Decreasing the THIP concentration to 0.1 mM still activated
inward currents, and Rc-OMe potentiated these currents by
7.1 Ϯ 1.5 fold (n = 5; Figure S3).
To confirm these observations from the VB thalamic neu-
rones, a4b3d and a4b3 receptors were expressed in HEK-293
cells (Table 1). Compared with a4b3 receptors, incorporation
of the d-subunit into functional a4b3d receptors significantly
reduced the extent of the inhibition induced by zinc (44 Ϯ
6% inhibition for a4b3, n = 7; 12 Ϯ 6% for a4b3d, n = 6, see
Figure 8D,E), in agreement with previous studies (Storustovu
and Ebert, 2006), but strongly increased potentiation of
0.3 mM GABA-evoked responses (corresponding to EC~6 for
a4b3d and EC~9 for a4b3) induced by 30 mM Rc-OMe (7.5 Ϯ
2.6-fold for a4b3d, n = 6; 1.1 Ϯ 0.4-fold for a4b3, n = 6) or 30 mM
Rc-Br (8.6 Ϯ 2.6-fold for a4b3d, n = 6; 1.3 Ϯ 0.5-fold for a4b3,
n = 6, Figure 8D,E). We finally checked that these GABA-
evoked responses were virtually insensitive to 3 mM diazepam
(120 Ϯ 4% of control, n = 4). Overall, these data support those
obtained in the neurones and strongly suggest that the ret-
rochalcone derivatives are positive allosteric modulators at
diazepam-insensitive d-containing GABAA receptors.
B
A
C
20 pA
120 ms
20 pA
120 ms
D
80
60
40
20
Control
Rc-Br
20 pA
50 ms
0
Control
Rc-Br
E
∗∗
40
30
20
10
0
Control
Rc-Br
Figure 7
Effect of Rc-Br on spontaneous IPSCs in acutely isolated hippocampal
CA1 pyramidal neurones. (A) Examples of current traces showing
control IPSCs in the absence of Rc-Br. (B) Spontaneous IPSCs
in the presence of Rc-Br (3 mM). (C) Average IPSC in the
absence (dashed line, average from 102 events) and presence
(solid line, average from 61 events) of Rc-Br. (D, E) Summary
for amplitude (D) and half-time of IPSC (E). **P < 0.01 (Student’s
paired t-test). IPSC, inhibitory postsynaptic current; Rc-Br,
trans-6-bromo-4′-methoxyretrochalcone.
Rc-Br and Rc-OMe are also positive
allosteric modulators of extrasynaptic
diazepam-insensitive GABAA receptors
expressed in ventrobasal thalamic neurones
and of a4b3d receptors expressed in
Discussion and conclusions
In this study, two trans-retrochalcones (Rc-OMe and Rc-Br)
and a chalcone (Ch-OMe) derivative that were initially
shown to act at GABAA receptors (Kueny-Stotz et al., 2008)
were synthesized and demonstrated to stimulate GABA-
evoked currents. It should be noted that the two trans-
retrochalcones differ chemically from the chalcone by the
presence of the hydroxyl group at position 9 of the aromatic
ring A (Figure 1A). The two trans-retrochalcones were found
more effective than the chalcone (Ch-OMe) at potentiating
GABA-evoked currents at a1b2g2s receptors and consequently
were further studied.
The two trans-retrochalcones’ modulation of the GABAA
receptor reveals an original action mode: (i) a novel site of
action different from those of BDZs, pentobarbital, THDOC
and loreclezole; (ii) strong dependence on the presence of the
g-subunit; (iii) high dependence on GABA concentration at
HEK-293 cells
We finally evaluated the modulatory effects of Rc-Br and
Rc-OMe at extrasynaptic diazepam insensitive (possibly
d-subunit containing) GABAA receptors expressed in ven-
trobasal (VB) thalamic neurones. These neurones have syn-
aptic (a1b2g2) and extrasynaptic (a4b2d) GABAA receptors
(Pirker et al., 2000; Belelli et al., 2005; Peden et al., 2008). The
extrasynaptic GABAA receptors are pharmacologically and
functionally distinct from their synaptic counterparts (Belelli
et al., 2009).
The GABAA receptors of the isolated neurones were acti-
vated by low concentrations (<1 mM) of GABA (Figure S1B,D).
This high sensitivity is consistent with the properties of the
a4b2d receptors (Jia et al., 2005) but distinct from those
British Journal of Pharmacology (2011) 162 1326–1339 1335

R Jiang et al.
BJP
A
B
C
Rc-OMe
3 μM
Rc-Br
3 μM
THIP
1 μM
∗∗
THIP
1 μM
THIP
1 μM
SR95531 2 μM
4
3
2
∗∗
∗∗
∗∗
1
0
50 pA
20 s
30 pA
15 s
100 pA
15 s
D
E
Rc-Br
30 μM
Rc-OMe
30 μM
Zinc
1 μM
α4β3δ vs α4β3
GABA
0.3 μM
α4β3δ
20
60
∗∗
Rc-Br
Rc-OMe
∗
∗
10
5
40
20
0
α4β3
0
200 pA
5 s
Figure 8
Rc-Br and Rc-OMe modulation of d-containing receptors expressed in ventrobasal (VB) thalamic neurones and in HEK-293 cells. (A) Examples of
traces showing the THIP-elicited current that can be blocked by SR95531 (2 mM) in VB thalamic neurones. (B) Examples of traces showing the
potentiation of THIP-elicited current induced by Rc-Br or by Rc-OMe in VB thalamic neurones. (C) Summary of the experiments shown in (A and
B) (n = 5–8). **P < 0.01 (compared with vehicle group, ANOVA test). (D) Examples of traces showing the modulation of GABA-elicited current
by zinc, Rc-OMe and Rc-Br at a4b3 and a4b3d receptors expressed in HEK-293 cells. For each subtype expressed, currents were recorded
in the same cell, separated by ~30 s washout. (E) Summary of the experiments shown in (D). *P < 0.05, **P < 0.01 (Student’s unpaired
t-test). HEK, human embryonic kidney; Rc-Br, trans-6-bromo-4′-methoxyretrochalcone; Rc-OMe, trans-6,4′-dimethoxyretrochalcone; THIP,
4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one.
recombinant synaptic GABAA receptors; and (iv) positive
modulation of d-containing GABAA receptors. To our knowl-
edge, this is the first time that molecules belonging to the
flavonoid family have been shown to act at extrasynaptic
d-containing GABAA receptors.
exist at the GABAA receptors (Johnston, 1996), and cross-talk
between these sites has often been reported (Ueno et al.,
1997; Williams and Akabas, 2000; Sigel et al., 2001). Supra-
additive modulations suggest that the site of action of
Rc-OMe is independent to of those of PB and THDOC that
mediate both agonist and potentiating actions. Nevertheless,
the potentiation induced by Rc-OMe was reduced by 50–60%
in a mutation made either in the classical BDZ binding site
located at the extracellular domain or in the loreclezole
binding site located in the transmembrane domain. These
two sites are topologically distinct, making it unlikely that
one retrochalcone molecule binds simultaneously to both
sites. A simple explanation for the observed inhibitions is
that both the BDZ and loreclezole sites contribute directly,
but independently, to Rc-OMe potentiation. However, if this
hypothesis was true, the specific BDZ antagonist Ro 17–1588
would have further inhibited the potentiation induced by
The binding site
Rc-OMe mainly acts as positive modulator, but at a high
concentration (100 mM) it also acts as a very poor agonist as
well as an open channel blocker. It is thus possible that
complex molecular mechanisms could account for Rc-OMe
actions and multiple binding sites may exist for mediating
the different actions at the GABAA receptors. The following
part focuses on the site mediating its main action: a strong
potentiation effect.
At least 11 distinct binding sites, including agonist recog-
nition sites as well as allosteric sites, have been proposed to
1336 British Journal of Pharmacology (2011) 162 1326–1339

Retrochalcones modulation at GABAA receptors
BJP
Rc-OMe at a1b2N265Sg2s, which was not the case. Another
plausible hypothesis is that Rc-OMe binds to a new site,
which is somehow allosterically coupled to those of BDZ and
loreclezole. This hypothesis is supported by a previous study
which showed that binding of diazepam or Ro 17–1588
induces conformational changes at the level of the trans-
membrane domain (Williams and Akabas, 2000). Neverthe-
less, it is also possible that the BDZ and loreclezole binding
sites contribute to the Rc-OMe site through a partly overlap-
ping area.
Given that the trans-retrochalcones share a common
chemical scaffold with other flavonoids, including flavones
and flavans, which have also been demonstrated to modulate
GABAA receptors at a site independent of the high-affinity
classical BDZ binding site (Hall et al., 2004, 2005; Fernandez
et al., 2008), it is conceivable that the trans-retrochalcones
could share a common binding core with these flavone and
flavan derivatives. Nevertheless, it should be noted that the
structure-pharmacology relationships are complex for fla-
vonoids acting at the GABAA receptors. Although synthetic
flavan-3-ol derivatives have also been reported to act as posi-
tive modulators at GABAA receptors, they displayed little
selectivity between a1b2 and a1b2g2s receptors (Fernandez
et al., 2008), whereas Rc-OMe clearly distinguishes a1b2g2s
from a1b2 receptors. In addition, other flavonoids, like
chrysin or apigenin, failed to potentiate but rather antago-
nized a1b2g2s receptors (Goutman et al., 2003). In the present
study, our data suggest that a retrochalcone skeleton (due to
the presence of the hydroxyl group at position 9 of the
aromatic ring A) has more potential than a chalcone skeleton
for development as a new modulator of GABAA receptors. All
these data, including the present study, suggest that within
the flavonoids’ scaffold different types of GABAA modulators
can be achieved by subtle chemical modifications.
The finding that Rc-OMe only had a weak potentiating
effect at the a1b2 receptor suggests the g-subunit has a major
role in its modulating action. The subunit stoichiometry of
abg receptors has been established as 2a:2b:1g (Baumann
et al., 2002; Boileau et al., 2005), whereas that of ab receptors
is either 3a:2b (Boileau et al., 2005) or 2a:3b (Baumann et al.,
2001; Gonzales et al., 2008). Our data suggest that the
replacement of an a or a b-subunit by a g-subunit has a
dramatic effect on the potentiation induced by Rc-OMe.
Thus, to precisely identify the binding site of the trans-
retrochalcones, chimera constructions carrying the g-subunit
and either the a- or the b-subunit would be particularly
useful. On the other hand, potentiation observed at a4b3d
receptor suggests that the g-subunit may not be indispensable
for the potentiation by the trans-retrochalcones, raising the
possibility that the d-subunit could also contribute, directly
or indirectly, to the trans-retrochalcones’ binding site.
2010) as well as a volatile anaesthetic, sevoflurane (Wu et al.,
1996). The amphiphiles, including Triton X-100, octyl-b-
glucoside and docosahexaenoic acid, act as potentiators at
low GABA concentrations, whereas they act as inhibitors at
high concentrations. It has been suggested that the effects of
these amphiphiles might be partly non-specific, as they could
be due to a decrease in bilayer stiffness and increase in elas-
ticity. It is unlikely that the trans-retrochalcones share a
common mechanism with these amphiphiles that underlies
the GABA dependence, because the actions of the trans-
retrochalcones are not voltage dependent and the molecules
themselves are not charged. More experiments such as single
channel analysis and kinetic studies may be useful to address
this issue.
The high efficacy of the potentiation induced by the trans-
retrochalcones at very low GABA concentrations may have
potential therapeutic implications. One may envisage that
when applied to receptors displaying low GABA sensitivity, as
observed for some epileptic mutants for which mutations
cause channel-gating defects, such compounds will exert
their potentiating effects in such a way that they will activate
the receptor as efficiently as if the receptor displays a gain-
of-function phenotype.
Overall, we showed that the trans-retrochalcones are pow-
erful positive allosteric modulators at the synaptic and extra-
synaptic GABAA receptors with original pharmacological
properties. Our data are helpful in understanding the
structure-pharmacology relationships for the flavonoid and
in developing novel modulators acting on GABAA receptors
by referring to the retrochalcone scaffold. The fact that the
trans-retrochalcones, including their flavylium salts precur-
sors, involve a family displaying a wide range of biological
activities reinforced by the novel pharmacological properties
shown in this study, indicates we should perform behaviour
tests in vivo, in the search for putative therapeutic candidates.
Acknowledgement
We are grateful to Marianne L. Jensen, Neurosearch, Denmark
for generously providing the GABAA plasmids.
Conflict of interest
None.
References
Akaike N, Moorhouse AJ (2003). Techniques: applications of the
nerve-bouton preparation in neuropharmacology. Trends
Pharmacol Sci 24: 44–47.
The dependence on GABA concentration
Another
pharmacological
property
of
the
trans-
retrochalcones is that the potentiation is highly dependent
on GABA concentration. The potentiation was very effica-
cious at EC~2 and null at EC>50. At a high GABA concentration,
the apparent desensitization rates of currents were also accel-
erated in the presence of Rc-OMe. This strong GABA depen-
dence has already been observed for other compounds acting
at GABAA receptors such as some amphiphiles (Chisari et al.,
Baumann SW, Baur R, Sigel E (2001). Subunit arrangement of
gamma-aminobutyric acid type A receptors. J Biol Chem 276:
36275–36280.
Baumann SW, Baur R, Sigel E (2002). Forced subunit assembly in
alpha1beta2gamma2 GABAA receptors. Insight into the absolute
arrangement. J Biol Chem 277: 46020–46025.
British Journal of Pharmacology (2011) 162 1326–1339 1337

R Jiang et al.
BJP
Belelli D, Lambert JJ (2005). Neurosteroids: endogenous regulators
of the GABA(A) receptor. Nat Rev Neurosci 6: 565–575.
Hall BJ, Chebib M, Hanrahan JR, Johnston GA (2005).
6-Methylflavanone, a more efficacious positive allosteric modulator
of gamma-aminobutyric acid (GABA) action at human recombinant
alpha2beta2gamma2L than at alpha1beta2gamma2L and
alpha1beta2 GABA(A) receptors expressed in Xenopus oocytes.
Eur J Pharmacol 512: 97–104.
Belelli D, Peden DR, Rosahl TW, Wafford KA, Lambert JJ (2005).
Extrasynaptic GABAA receptors of thalamocortical neurons: a
molecular target for hypnotics. J Neurosci 25: 11513–11520.
Belelli D, Harrison NL, Maguire J, Macdonald RL, Walker MC,
Cope DW (2009). Extrasynaptic GABAA receptors: form,
pharmacology, and function. J Neurosci 29: 12757–12763.
Hansen RS, Paulsen I, Davies M (2005). Determinants of
amentoflavone interaction at the GABA(A) receptor. Eur J
Pharmacol 519: 199–207.
Boileau AJ, Baur R, Sharkey LM, Sigel E, Czajkowski C (2002). The
relative amount of cRNA coding for gamma2 subunits affects
stimulation by benzodiazepines in GABA(A) receptors expressed in
Xenopus oocytes. Neuropharmacology 43: 695–700.
Harborne JB, Williams CA (2000). Advances in flavonoid research
since 1992. Phytochemistry 55: 481–504.
Heidari MR, Foroumadi A, Amirabadi A, Samzadeh-Kermani A,
Azimzadeh BS, Eskandarizadeh A (2009). Evaluation of
Boileau AJ, Pearce RA, Czajkowski C (2005). Tandem subunits
effectively constrain GABAA receptor stoichiometry and recapitulate
receptor kinetics but are insensitive to GABAA receptor-associated
protein. J Neurosci 25: 11219–11230.
anti-inflammatory and analgesic activity of a novel rigid 3,
4-dihydroxy chalcone in mice. Ann N Y Acad Sci 1171: 399–406.
Hosie AM, Wilkins ME, Silva HM, Smart TG (2006). Endogenous
neurosteroids regulate GABAA receptors through two discrete
transmembrane sites. Nature 444: 486–489.
Chisari M, Shu HJ, Taylor A, Steinbach JH, Zorumski CF,
Mennerick S (2010). Structurally diverse amphiphiles exhibit
biphasic modulation of GABAA receptors: similarities and
differences with neurosteroid actions. Br J Pharmacol 160: 130–141.
Inada H, Maejima T, Nakahata Y, Yamaguchi J, Nabekura J,
Ishibashi H (2010). Endocannabinoids contribute to metabotropic
glutamate receptor-mediated inhibition of GABA release onto
hippocampal CA3 pyramidal neurons in an isolated neuron/bouton
preparation. Neuroscience 165: 1377–1389.
Cope DW, Hughes SW, Crunelli V (2005). GABAA
receptor-mediated tonic inhibition in thalamic neurons. J Neurosci
25: 11553–11563.
Jia F, Pignataro L, Schofield CM, Yue M, Harrison NL, Goldstein PA
(2005). An extrasynaptic GABAA receptor mediates tonic inhibition
in thalamic VB neurons. J Neurophysiol 94: 4491–4501.
D’Hulst C, Atack JR, Kooy RF (2009). The complexity of the GABAA
receptor shapes unique pharmacological profiles. Drug Discov
Today 14: 866–875.
Jia F, Chandra D, Homanics GE, Harrison NL (2008). Ethanol
modulates synaptic and extrasynaptic GABAA receptors in the
thalamus. J Pharmacol Exp Ther 326: 475–482.
Fernandez SP, Mewett KN, Hanrahan JR, Chebib M, Johnston GA
(2008). Flavan-3-ol derivatives are positive modulators of GABA(A)
receptors with higher efficacy for the alpha(2) subtype and
anxiolytic action in mice. Neuropharmacology 55: 900–907.
Johnston GA (1996). GABAA receptor pharmacology. Pharmacol
Ther 69: 173–198.
Fritschy JM, Benke D, Mertens S, Oertel WH, Bachi T, Mohler H
(1992). Five subtypes of type A gamma-aminobutyric acid receptors
identified in neurons by double and triple immunofluorescence
staining with subunit-specific antibodies. Proc Natl Acad Sci USA
89: 6726–6730.
Kueny-Stotz M, Chassaing S, Brouillard R, Nielsen M, Goeldner M
(2008). Flavylium salts as in vitro precursors of potent ligands to
brain GABA-A receptors. Bioorg Med Chem Lett 18: 4864–4867.
Nielsen M, Frokjaer S, Braestrup C (1988). High affinity of the
naturally-occurring biflavonoid, amentoflavon, to brain
benzodiazepine receptors in vitro. Biochem Pharmacol 37:
3285–3287.
Gibbs JW, 3rd, Schroder GB, Coulter DA (1996). GABAA receptor
function in developing rat thalamic reticular neurons: whole cell
recordings of GABA-mediated currents and modulation by
clonazepam. J Neurophysiol 76: 2568–2579.
Olsen RW, Sieghart W (2009). GABA A receptors: subtypes provide
diversity of function and pharmacology. Neuropharmacology 56:
141–148.
Glykys J, Mody I (2007). Activation of GABAA receptors: views from
outside the synaptic cleft. Neuron 56: 763–770.
Peden DR, Petitjean CM, Herd MB, Durakoglugil MS, Rosahl TW,
Wafford K et al. (2008). Developmental maturation of synaptic and
extrasynaptic GABAA receptors in mouse thalamic ventrobasal
neurones. J Physiol 586: 965–987.
Gonzales EB, Bell-Horner CL, Dibas MI, Huang RQ, Dillon GH
(2008). Stoichiometric analysis of the TM2 6′ phenylalanine
mutation on desensitization in alpha1beta2 and
alpha1beta2gamma2 GABA A receptors. Neurosci Lett 431:
184–189.
Pirker S, Schwarzer C, Wieselthaler A, Sieghart W, Sperk G (2000).
GABA(A) receptors: immunocytochemical distribution of 13
subunits in the adult rat brain. Neuroscience 101: 815–850.
Goutman JD, Waxemberg MD, Donate-Oliver F, Pomata PE,
Calvo DJ (2003). Flavonoid modulation of ionic currents mediated
by GABA(A) and GABA(C) receptors. Eur J Pharmacol 461: 79–87.
Rhee JS, Ishibashi H, Akaike N (1999). Calcium channels in the
GABAergic presynaptic nerve terminals projecting to meynert
neurons of the rat. J Neurochem 72: 800–807.
Griebel G, Perrault G, Tan S, Schoemaker H, Sanger DJ (1999).
Pharmacological studies on synthetic flavonoids: comparison with
diazepam. Neuropharmacology 38: 965–977.
Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM
et al. (1999). Benzodiazepine actions mediated by specific
gamma-aminobutyric acid(A) receptor subtypes. Nature 401:
796–800.
Hall BJ, Chebib M, Hanrahan JR, Johnston GA (2004).
Flumazenil-independent positive modulation of
gamma-aminobutyric acid action by 6-methylflavone at human
recombinant alpha1beta2gamma2L and alpha1beta2 GABAA
receptors. Eur J Pharmacol 491: 1–8.
Semyanov A, Walker MC, Kullmann DM, Silver RA (2004).
Tonically active GABA A receptors: modulating gain and
maintaining the tone. Trends Neurosci 27: 262–269.
1338 British Journal of Pharmacology (2011) 162 1326–1339

Retrochalcones modulation at GABAA receptors
BJP
Sigel E, Baur R, Furtmueller R, Razet R, Dodd RH, Sieghart W
(2001). Differential cross talk of ROD compounds with the
benzodiazepine binding site. Mol Pharmacol 59: 1470–1477.
Supporting information
Additional Supporting Information may be found in the
online version of this article:
Somogyi P, Fritschy JM, Benke D, Roberts JD, Sieghart W (1996).
The gamma 2 subunit of the GABAA receptor is concentrated in
synaptic junctions containing the alpha 1 and beta 2/3 subunits in
hippocampus, cerebellum and globus pallidus. Neuropharmacology
35: 1425–1444.
Figure S1 GABAA receptor-mediated currents in hippocam-
pal CA1 pyramidal and ventrobasal thalamic neurones. (A)
Example of the current traces evoked by various concentra-
tions of GABA in the hippocampal CA1 pyramidal neurone.
(B) Example of the current traces of GABA-induced current in
ventrobasal thalamic neurone. (C) THIP-induced currents in
ventrobasal thalamic neurone. (D) Concentration-response
relationship for GABA and THIP (n = 6). Data are fitted to the
Hill equation.
Figure S2 Potentiations of GABA-elicited currents induced
by Rc-OMe is not voltage dependent. (A) Left, examples of
current traces showing the potentiation of GABA-elicited
currents (1 mM) by Rc-OMe (10 mM) at +60 mV and -60 mV
at recombinant a1b2g2s receptors expressed in HEK-293 cells.
Right, I–V curve for GABA-elicited currents (1 mM) in the
absence and presence of Rc-OMe (n = 3). (B) Left, represen-
tative current traces with voltage ramps from a VH of
-65 mV to +15 mV for 0.5 s in the absence and presence of
Rc-Br (3 mM). Right, baseline-subtracted current–voltage
curves for the GABA-induced current shown in the left
(n = 7).
Stirrett KL, Ferreras JA, Jayaprakash V, Sinha BN, Ren T, Quadri LE
(2008). Small molecules with structural similarities to siderophores
as novel antimicrobials against Mycobacterium tuberculosis and
Yersinia pestis. Bioorg Med Chem Lett 18: 2662–2668.
Storustovu SI, Ebert B (2006). Pharmacological characterization of
agonists at delta-containing GABAA receptors: functional selectivity
for extrasynaptic receptors is dependent on the absence of
gamma2. J Pharmacol Exp Ther 316: 1351–1359.
Thompson SA, Whiting PJ, Wafford KA (1996). Barbiturate
interactions at the human GABAA receptor: dependence on
receptor subunit combination. Br J Pharmacol 117: 521–527.
Ueno S, Bracamontes J, Zorumski C, Weiss DS, Steinbach JH (1997).
Bicuculline and gabazine are allosteric inhibitors of channel
opening of the GABAA receptor. J Neurosci 17: 625–634.
Wieland HA, Luddens H, Seeburg PH (1992). A single histidine in
GABAA receptors is essential for benzodiazepine agonist binding. J
Biol Chem 267: 1426–1429.
Figure S3 Representive traces showing the potentiation
induced by Rc-OMe of current elicited by 0.1 mM THIP in VB
thalamic neurones. The-all-points distribution was fitted by
the sum of two Gaussians, and the peak of these fits was used
to estimate current amplitude.
Williams DB, Akabas MH (2000). Benzodiazepines induce a
conformational change in the region of the gamma-aminobutyric
acid type A receptor alpha(1)-subunit M3 membrane-spanning
segment. Mol Pharmacol 58: 1129–1136.
Wingrove PB, Wafford KA, Bain C, Whiting PJ (1994). The
modulatory action of loreclezole at the gamma-aminobutyric acid
type A receptor is determined by a single amino acid in the beta 2
and beta 3 subunit. Proc Natl Acad Sci USA 91: 4569–4573.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author for
the article.
Wu J, Harata N, Akaike N (1996). Potentiation by sevoflurane of the
gamma-aminobutyric acid-induced chloride current in acutely
dissociated CA1 pyramidal neurones from rat hippocampus. Br J
Pharmacol 119: 1013–1021.
British Journal of Pharmacology (2011) 162 1326–1339 1339