0022-3565/97/2832-0488$00.
00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:488 –493, 1997
[3H]RY 80: A High-Affinity, Selective Ligand for g-Aminobutyric
AcidA Receptors Containing Alpha-5 Subunits1, 2
PHIL SKOLNICK, RONA J. HU, CHRISTINE M. COOK, STEPHEN D. HURT, JOSEPH D. TROMETER, RUIYAN LIU,
QI HUANG and JAMES M. COOK
Laboratory of Neuroscience, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
Maryland (R.J.H., C.M.C., P.S.), New England Nuclear, Boston, Massachusetts (S.D.H., J.D.T.), and Department of Chemistry, University of
Wisconsin-Milwaukee, Milwaukee, Wisconsin (R.L., Q.H., J.M.C.)
Accepted for publication July 29, 1997
ABSTRACT
The radiochemical synthesis and pharmacological properties
are described of [3H]RY 80 (ethyl-8-acetylene-5,6-dihydro-5-
methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carbox-
ylate, [ethyl-3H]). This compound is one of a series of 8-substi-
tuted imidazobenzodiazepines that exhibits both high affinity
and selectivity for g-aminobutyric acid (GABA)A receptors con-
taining alpha-5 subunits. Saturable, high-affinity (Kd ;0.7 nM)
binding of [3H]RY 80 was observed in hippocampal mem-
branes. The maximum number (Bmax) of [3H]RY 80 binding sites
was ;18% of that obtained with [3H]flunitrazepam, a radioli-
gand that labels all “diazepam-sensitive” GABAA receptors.
This value is consistent with previous estimates (10 –20%) of
the proportion of rat hippocampal GABAA receptors containing
alpha-5 subunits determined by immunoprecipitation with se-
lective antibodies and competition experiments using an alpha-
GABAA receptors possess multiple, allosterically linked
modulatory sites that are loci for drug action (reviewed in
Skolnick and Paul, 1988; Johnston, 1996). However, from a
therapeutic and drug development perspective, benzodiaz-
epine binding sites are perhaps the most important. Thus,
benzodiazepine binding sites mediate the principal therapeu-
tic actions of 1,4-benzodiazepines (e.g., diazepam and fluraz-
epam) as well as a large group of structurally unrelated
molecules including imidazopyridines (e.g., zolpidem), cyclo-
pyrrolones (e.g., zopiclone) and b-carbolines (e.g., abecarnil).
GABAA receptors are a heterogeneous family of ligand-
gated ion channels that may be assembled from at least 15
structurally related subunits (alpha, beta, gamma, delta and
rho) (reviewed in Stephenson, 1995). Immunochemical stud-
Received for publication March 19, 1997.
1
Portions of this work were presented at the 35th Annual Meeting of the
American College of Neuropsychpharmacology, December 9 –13, 1996, San
Juan, PR.
2
This work was supported in part by a predoctoral fellowship from the
American Society for Pharmacology and Experimental Therapeutics (C.M.C.)
and NIMH Grant MH-46851 (J.M.C.). R.J.S. is a PRAT Fellow, NIGMS.
ABBREVIATIONS: GABA, g-aminobutyric acid; DMCM, methyl-6,7-dimethoxy-4-ethyl-b-carboline-3-carboxylate; HEK, human embryonic kid-
ney.
488
Vol. 283, No. 2
Printed in U.S.A.
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5-selective ligand. In recombinant GABAA receptors composed
of alpha-5 beta-3 gamma-2 subunits, the Kd of [3H]RY 80 (;0.5
nM) was consistent with the value obtained in hippocampus,
whereas the Bmax value was not significantly different from that
obtained with [3H]flunitrazepam. The potencies of several ben-
zodiazepine site ligands to inhibit [3H]RY 80 binding to hip-
pocampal membranes were in agreement with the values ob-
tained in recombinant (alpha-5 beta-3 gamma-2) GABAA
receptors. [3H]RY 80 was used both in a “GABA shift” assay to
correctly predict the in vivo actions of a novel, alpha-5-selective
ligand and to characterize a population of GABAA receptors
containing alpha-5 subunits in neonatal rat cortex. These find-
ings demonstrate that [3H]RY 80 can be used as a radioligand
to examine the properties of GABAA receptors containing al-
pha-5 subunits.
ies have demonstrated that GABAA receptors most often
exist as ternary complexes composed of alpha, beta, and
gamma subunits (; DeBlas, 1996; Fritschy and Mohler, 1993)
arranged as pentamers (Nayeem et al., 1994). Although
GABAA receptor subunit stoichiometry remains controver-
sial (Backus et al., 1993; Chang et al., 1996; Tretter et al.,
1997), both the affinities and efficacies of drugs acting at this
family of ligand-gated ion channels (including benzodiaz-
epine site ligands) appear to be defined by subunit composi-
tion. For example, studies in recombinant GABAA receptors
have shown that the alpha subunit is a primary determinant
of ligand affinity at benzodiazepine binding sites (Hading-
ham et al., 1993; Lüddens et al., 1990; Pritchett and Seeburg,
1990), with the gamma subunit playing a smaller, albeit
significant role for some ligands (Benke et al., 1996; Lüddens
et al., 1994). Ligand efficacy at benzodiazepine binding sites
appears to be determined primarily by the gamma subunit
(Ducic et al., 1993; von Blankenfeld et al., 1990; Wafford et
al., 1993). These studies in recombinant receptors have pro-
vided valuable insights that explain many aspects of the
1997
pharmacological heterogeneity of wild-type GABAA recep-
tors, which has been recognized for almost 20 years (Klepner
et al., 1979; Lippa et al., 1982; Young et al., 1981).
With few exceptions (e.g., GABAA receptors in the cerebel-
lum containing alpha-6 subunits, the so-called “diazepam-
insensitive GABAA receptors”; Gunnersen et al., 1996; Lüd-
dens et al., 1990; Wong et al., 1995), it is inherently more
difficult to study the pharmacological properties of benzodi-
azepine site ligands at specific subpopulations of wild-type
compared with recombinant GABAA receptors. This is due, in
part, to a remarkable receptor heterogeneity present at the
cellular level (Fritschy and Mohler, 1995; McKernan and
Whiting, 1996; Wisden et al., 1992) and the paucity of high-
affinity, selective ligands capable of discriminating among
these receptor subpopulations. Based on the ;10-fold selec-
tivity of Ro 15– 4513 for recombinant GABAA receptors con-
taining alpha-5 subunits (compared with receptors contain-
ing alpha-1, alpha-2 or alpha-3 subunits); Hadingham et al.,
1993; Lüddens et al., 1994), a series of novel 8-substituted
imidazobenzodiazepines (Liu et al., 1995, 1996) were pre-
pared in an attempt to increase this selectivity. Several of
these compounds exhibited both high-affinity (Ki ;0.4 –5 nM)
and selectivity (up to 75-fold) for recombinant GABAA recep-
tors containing alpha-5 subunits. Moreover, these imidazo-
benzodiazepines inhibited [3H]flunitrazepam binding to rat
hippocampal membranes (that are relatively enriched in
GABAA receptors containing alpha-5 subunits; McKernan et
al., 1991a) with the characteristics of high-affinity, subtype-
selective ligands (Liu et al., 1996). The present study de-
scribes the radiochemical synthesis and pharmacological
properties of one member of this series, [3H]RY 80 (ethyl-8-
acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]
benzodiazepine-3-carboxylate, [ethyl-3H]). The results ob-
tained in both wild-type and recombinant GABAA receptors
indicate that [3H]RY 80 is a useful radioligand for studying
specific receptor populations containing alpha-5 subunits.
Materials and Methods
Cell culture and transfection. HEK 293 cells (American Type
Culture Collection, Rockville, MD) were maintained at 37° in 5% CO2
as previously described (Gunnersen et al., 1996). Cells were trans-
fected with cDNAs for the rat alpha-5, beta-3 and gamma-2 (8, 8 and
5 mg of DNA/10-cm2 dish containing ;4 3 106 cells) subunits by
calcium phosphate precipitation (Gorman et al., 1990). The cells were
harvested ;48 hr later, and a washed membrane suspension was
prepared as previously described (Gunnersen et al., 1996). These
membrane suspensions were stored at ;70° until assayed. The be-
ta-3 and gamma-2s cDNAs were subcloned into pCDNA1 and
pcDNA3 vectors, respectively (Gunnersen et al., 1996; Harris et al.,
1995). The alpha-5 cDNA (the gift of Dr. H. Lüddens, University of
Mainz) was subcloned from a BlueScript to a CMV vector by stan-
dard techniques.
Tissue preparation. Adult male and juvenile (6–8 days postpar-
tum; both sexes) Sprague-Dawley rats (Taconic Farms, German-
town, NY) were killed by decapitation. The brains were rapidly
removed and placed in beakers containing ice-cold 50 mM Tris-
citrate buffer, pH 7.8. After dissection, the tissues were disrupted in
50 volumes of ice-cold Tris-citrate buffer using a Polytron (20 sec;
setting 6–7) (Brinkmann Instruments, Westbury, NY). The homoge-
nates were centrifuged at 20,000 3 g (4°C) for 20 min. The superna-
tants were discarded and the pellets resuspended in an equal volume
of buffer and recentrifuged. This “washing” procedure was repeated
A Novel Radioligand for GABAA Receptors 489
a total of five times. Tissue suspensions were frozen on solid CO2 and
stored at 270°C until assayed.
Radioligand binding. Studies in recombinant receptors were
performed in a final volume of 1 ml consisting of: tissue suspension
(;0.2 mg of protein), 0.2 M NaCl, [3H]RY 80 or flunitrazepam and 50
mM Tris-citrate buffer, pH 7.8, to volume. For studies in wild-type
receptors (from adult hippocampus and juvenile cerebral cortex), the
volume of membrane suspension was varied to yield between 0.02
and 0.1 mg of protein/assay. In competition experiments, 50 ml of
buffer was replaced by drugs and/or GABA (30 mM); the concentra-
tion of [3H]RY 80 routinely used in competition experiments was
;0.5 to 0.6 nM. Nonspecific binding was defined with Ro 15–1788 (10
mM). In pilot experiments to optimize incubation conditions, specific
binding of [3H]RY 80 was obtained at a range of temperatures (4°,
25° and 37°), with the optimum ratio of specific and nonspecific
binding achieved at 4°. Under these assay conditions, [3H]RY 80 (0.8
nM) binding to hippocampal membranes reached equilibrium by 30
min and was maintained for $2 hr. Samples were routinely har-
vested at 2 hr. Assays (4°C) were terminated after 2 hr by rapid
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filtration (Brandel M-48R, Gaithersburg, MD) through GF/B filters
followed by two 5-ml washes with ice-cold 50 mM Tris-citrate buffer.
Radioactivity retained by the filters was measured in an LS 6500
liquid scintillation counter (Beckman Instruments, Palo Alto, CA).
Data were analyzed with GraphPAD InPlot 4 (GraphPAD Software,
San Diego, CA). Protein concentrations were determined using the
BCA protein assay reagent (Pierce, Rockford, IL).
In vivo studies. Adult, male NIH/Swiss mice (;30 g) were in-
jected (0.1 ml i.p.) with graded doses of QHII-066 (7-acetyleno-1,3-
dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) or vehicle
(10% diluted Emulphor/90% saline). Mice were placed in individual
plastic cages and administered either RY-24 [t-butyl-8-acetylene-5,6-
dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-
carboxylate; 20 mg/kg i.p.) or DMCM (7.5 mg/kg i.p.) 10 min. later.
Animals were observed (10 min) for the presence of tonic and clonic
convulsions (Liu et al., 1996).
Synthesis of [3H]RY 80. 8-Acetylene-5,6-dihydro-5-methyl-6-
oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylic acid (15 mg;
0.05 mmol) was added to a 10-ml round-bottom flask and dissolved in
N, N9-dimethylformamide (DMF) (1 ml). After the addition of 0.2 M
lithium hydroxide (0.25 ml), the reaction was stirred at room tem-
perature for 1 hr and then heated (70°C) for 30 min. The reaction
mixture was cooled to room temperature, and the solvents were
removed under reduced pressure. The residue was taken up in DMF
(1 ml) and transferred to a 5-ml tritiation flask. [3H]Ethyl iodide (0.1
mmol) was added to this mixture, and the reaction was stirred at
70°C for 16 hr (fig. 1). The labiles were removed with ethanol, and
the residue was taken up in 10 ml of ethanol. Thin-layer chromatog-
raphy (ethyl acetate/hexanes 10:1) of the crude reaction mixture
revealed two major components with RF values of ;0.4 (RY 80) and
;0.1 (presumed to be the quaternary salt of RY 80). The crude
reaction mixture was purified by high performance liquid chroma-
tography on a Zorbax RX-C8 analytical column with a mobile phase
of 1% triethylammonium acetate (pH 4.0)/acetonitrile (75:25) at a
flow rate of 1 ml/min. The material corresponding to product (detect-
ed by UV absorbance at 274 nm) had a retention time of ;25 min.
The solvents were removed with multiple ethanol azeotropes, and
Fig. 1. Radiochemical preparation of [3H]RY 80.
490 Skolnick et al. Vol. 283

the residue was taken up in 50 ml of ethanol. The specific activity of
[ethyl-3H]RY 80 was 55.4 Ci/mmol, as determined by FAB mass
spectroscopy. The radiochemical purity of [3H]RY 80 was 99%, as
determined by high performance liquid chromatography.
Materials. [3H]Flunitrazepam (specific activity, 85.8 Ci/mmol)
was purchased from Dupont-New England Nuclear (Boston, MA).
3-Carbomethoxy-b-carboline, QHII-066 (the 7-acetyleno-congener of
diazepam; Huang et al., 1996), RY-24 (the t-butyl ester congener of
RY 80; Liu et al., 1996) and 8-acetylene-5,6-dihydro-5-methyl-6-oxo-
4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate were synthe-
sized at the University of Wisconsin-Milwaukee. Ro 15–1788 was
donated by Hoffmann-LaRoche (Nutley, NJ). Zolpidem was the gift
of Synthelabo (Laboratoire Experimental Recherche Synthelabo;
Paris, France). DMCM was purchased from Research Biochemicals
(Natick, MA). All other reagents and chemicals were obtained from
standard commercial sources.
Results
ceptors containing alpha-5 beta-3 gamma-2 receptors
(Graham et al., 1996; Lüddens et al., 1994; Pritchett and Seeburg,
1990) did not produce a concentration-dependent inhibition of
[3H]RY 80 binding to either recombinant alpha-5 beta-3 gamma-2
receptors (fig. 3A) or hippocampal membranes (fig. 3B). In con-
trast, the potency of an alpha-5-selective ligand, RY-24 (the t-butyl
ester congener of RY 80) (Liu et al., 1995), to inhibit [3H]RY 80
binding was similar in recombinant receptors and hippocampal
membranes (IC50, 0.95 6 0.22 and 0.82 6 0.13 nM, respectively)
(fig. 3). The potency of QHII-066 (the 7-acetyleno congener of
diazepam), which exhibits a moderate selectivity for recombinant
alpha-5-containing receptors (Huang et al., 1996), was similar in
recombinant alpha-5 beta-3 gamma-2 receptors and hippocampal
membranes (IC50, 41 6 5 vs. 56 6 10 nM, respectively). GABA
increased the potency of QHII-066 to inhibit [3H]RY 80 binding by
;2.5–3-fold in both preparations (IC50, 17 6 3 vs. 18 6 7 nM,
respectively) (fig. 3).

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3 Anticonvulsant actions of QHII-066. The anticonvul-
[ H]RY 80 binding to recombinant alpha-5 beta-3 gam-
ma-2 receptors and hippocampal membranes. Saturable,
high-affinity (Kd, 0.53 6 0.09 nM) binding of [3H]RY 80 was ob-
served in membranes prepared from HEK 293 cells transfected
with cDNAs encoding alpha-5 beta-3 gamma-2 subunits (fig. 2A).
The maximum number of binding sites (Bmax) obtained with
[3H]RY 80 (173 6 9 fmol/mg of protein) was not significantly
different from the values obtained using [3H]flunitrazepam (190 6
12 fmol/mg of protein; Kd, 1.3 6 0.18 nM) (fig. 2A). The apparent
affinity of [3H]RY 80 in hippocampal membranes (Kd, 0.686.04
nM vs. 1.5 6 0.12 nM for [3H]flunitrazepam) was comparable to
that obtained in recombinant receptors, whereas the Bmax value
was ;17.6% of that obtained with [3H]flunitrazepam (302 6 21
fmol/mg of protein vs. 1712 6 156 fmol/mg of protein, respectively)
(fig. 2B). In identically prepared cerebellar membranes, saturable
binding of [3H]RY 80 was not observed using radioligand concen-
trations of #20 nM (data not shown). To confirm that the receptor
population labeled by [3H]RY 80 corresponds to GABAA receptors
containing alpha-5 subunits, the effects of several ligands with
well defined characteristics at these receptors were examined.
Zolpidem, which binds with low (mM) affinity to recombinant re-
sant actions of QHII-066 were examined because GABA in-
creased the potency of this compound in vitro (i.e., a positive
“GABA-shift”) (fig. 3). Consistent with previous findings (Liu
et al., 1996), parenteral administration of DMCM (7.5 mg/kg)
and RY-24 (20 mg/kg) produced tonic and clonic convulsions
in 100% and 80% of mice, respectively. Higher doses of RY-24
did not result in a greater percentage of animals exhibiting
convulsions (Liu et al., 1996; and data not shown). QHII-066
reduced both RY-24- and DMCM-induced convulsions in a
dose-dependent manner with ED50 values of ;0.6 and ;2.9
mg/kg, respectively (fig. 4)
[3H]RY 80 binding to neonatal rat cortex. High-affin-
ity (Kd, 0.81 6 0.25 nM), saturable (Bmax, 464 6 104 fmol/mg
of protein) binding of [3H]RY 80 was obtained in cortical
membranes prepared from 6- to 8-day-old rat pups (fig. 5).
This binding was zolpidem insensitive and inhibited by
RY-24 in a concentration-dependent fashion (IC50, 2.2 6 0.4
nM) (fig. 5, inset). The Bmax value obtained with [3H]RY 80
was ;31% of the value obtained with [3H]flunitrazepam
(1461 6 317 fmol/mg of protein; Kd, 1.2 6 0.1 nM).

Fig. 2. Saturation studies of: [3H]RY 80 binding to (A) recombinant alpha-5 beta-3 gamma-2 receptors and (B) hippocampal membranes HEK 293
cells were transiently transfected with cDNAs encoding rat alpha-5 beta-3 gamma-2 subunits. The cells were harvested ;48 hr after transfection
and membranes prepared as described. Hippocampal membranes were prepared as described. A, Representative saturation isotherm of [3H]RY
80 (F) and [3H]flunitrazepam (f). The Kd and Bmax values in these experiments were 0.43 nM and 182 fmol/mg of protein and 1.3 nM and 183
fmol/mg of protein for [3H]RY 80 and [3H]flunitrazepam, respectively. In pilot experiments, the binding of [3H]RY 80 to cerebellar membranes was
not saturable using radioligand concentrations of #20 nM (data not shown). B, In this representative experiment, the Kd and Bmax values were 0.71
nM and 314 fmol/mg of protein and 1.2 nM and 1707 fmol/mg of protein for [3H]RY 80 and [3H]flunitrazepam, respectively. Insets, Rosenthal
(Scatchard) analysis of the respective saturation isotherms in (A) recombinant tissue and (B) hippocampal membranes. The radioligand
concentrations ranged from ;0.12 to 3.7 nM and ;0.5 to 12.6 nM for [3H]RY 80 and [3H]flunitrazepam, respectively. These experiments were
repeated three or more times (see Results) with [3H]RY 80 and [3H]flunitrazepam.
1997 A Novel Radioligand for GABAA Receptors 491

Fig. 3. Inhibition of [3H]RY 80 binding to (A) recombinant alpha-5 beta-3 gamma-2 receptors and (B) hippocampal membranes: [3H]RY 80 binding
to recombinant and hippocampal membranes was assayed as described. These are representative experiments repeated at least three times. ‚,
RY-24; F, QHII-066 1 GABA; E, QHII-066; p, zolpidem. The x axes depicts the log of concentrations used. [3H]RY 80 was used at a concentration
of ;0.5 nM in these experiments. The IC50 values (in nM) in recombinant and hippocampal membranes were 0.83 and 1.03 for RY-24, 34 and 53

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for QHII-066 and 14.3 and 20.9 for QHII-066 1 GABA, respectively. The IC50 values for zolpidem could not be estimated. Consistent with the
described inverse agonist properties of RY 80 demonstrated both in vitro and in vivo (Liu et al., 1995, 1996), GABA (30 mM) inhibited [3H]RY 80
binding by 48.7 6 4.1% and 41 6 1.8% in recombinant receptors and hippocampal membranes, respectively. The IC50 values calculated for
QHII-066 in the presence of GABA reflect this inhibitory effect of GABA. The Hill slopes in recombinant and hippocampal membranes were 0.84 6
0.1 and 0.72 6 0.04 for RY-24, 0.99 6 0.03 and 0.84 6 0.1 for QHII-066 and 1.05 6 0.05 and 0.79 6 0.12 for QHII-066 1 GABA, respectively.
These experiments were repeated at least three times (see Results).

Discussion indicate the rodent hippocampus is relatively enriched in this

The high-affinity and selectivity of several novel imidazo-
benzodiazepines for recombinant GABAA receptors contain-
ing alpha-5 subunits (Liu et al., 1995) suggest that a radio-
labeled form of one (or more) of these compounds could be
used to examine the pharmacological properties of the corre-
sponding wild-type receptors. Although GABAA receptors
containing alpha-5 subunits are minor constituents of the
total GABAA receptor pool, both in situ hybridization
(Khrestchatisky et al., 1989; Wisden et al., 1992) and immu-
nochemical studies (Endo and Olsen, 1993; McKernan et al.,
1991a, 1991b; Mertens et al., 1993; Thompson et al., 1992)
Fig. 4. Anticonvulsant actions of QHII-066. Mice were injected (0.1 ml
i.p.) with varying doses of QHII-066 or vehicle (5–20 mice/dose). At 10
min later, the mice were injected with RY-24 (20 mg/kg i.p.) or DMCM
(7.5 mg/kg i.p.) and observed (10 min) for the presence of convulsions.
RY-24 and DMCM produced a maximum of 80% (16 of 20) and 100%
(10 of 10) convulsions, respectively. The inability of RY-24 to produce
convulsions in 100% of the mice is consistent with previous data (Liu et
al., 1996). The ED50 values of QHII-066 were 0.6 and 2.9 mg/kg vs.
RY-24 and DMCM, respectively.
subunit compared with other brain regions. The feasibility of
selectively labeling this receptor subpopulation in hippocam-
pus was supported by competition studies with RY-24, the
t-butyl ester congener of RY 80. Thus, RY-24 inhibition of
[3H]flunitrazepam binding to hippocampal membranes is
best fit to a two-site competition curve, with the high-affinity
component (IC50 ; 0.6 nM) representing 16 6 4% of the sites
labeled by [3H]flunitrazepam (Liu et al., 1996). This high-
affinity of RY-24 is consistent with both the value obtained in
recombinant receptors composed of alpha-5 beta-3 gamma-2
subunits (Liu et al., 1995) and the proportion of these high-
affinity sites corresponds to the values obtained by immuno-
precipitation with alpha-5 subunit-specific antibodies in rat
hippocampus (McKernan et al., 1991a; Mertens et al., 1993).
Although other imidazobenzodiazepines in this series exhibit
a greater selectivity for recombinant GABAA receptors bear-
ing alpha-5 subunits than RY 80 (#;75-fold compared with
60-fold for RY 80; Liu et al., 1995, 1996), this compound was
the simplest to prepare in its radiolabeled form (fig. 1).
The binding of [3H]RY 80 to recombinant alpha-5 beta-3
gamma-2 receptors was saturable (fig. 2A), with a Kd value
(0.53 6 0.09 nM) comparable to the Ki value (;0.5 nM)
obtained in recombinant human receptors composed of al-
pha-5 beta-3 gamma-2 subunits (Liu et al., 1995). Moreover,
the Bmax value obtained with [3H]RY 80 was not significantly
different from the value obtained with [3H]flunitrazepam,
indicating that both radioligands label the same receptor
populations (fig. 2B). Although saturable, high-affinity bind-
ing (Kd, 0.69 6 0.07 nM) of [3H]RY 80 was also detected in
hippocampal membranes, the Bmax value was only ;18% of
the value obtained with [3H]flunitrazepam, a radioligand
thought to label all “diazepam-sensitive” GABAA receptor
isoforms. The fraction of hippocampal GABAA receptors la-
beled by [3H]RY 80 is consistent with the values obtained by
both immunoprecipitation with alpha-5-selective antibodies
(;15–16%) (Mertens et al., 1993; McKernan et al., 1991a)
and competition studies using the alpha-5-selective ligand
492 Skolnick et al.
Fig. 5. [3H]RY 80 binding to neonatal rat cortex demonstrates a comparison with [3H]flunitrazepam. Cortical membranes were prepared from 6-
to 8-day-old rat pups as described in the text. In this representative experiment, (A) the Kd and Bmax values for RY 80 (F) and flunitrazepam (f)
were 0.62 and 1.5 nM and 322 and 1160 fmol/mg of protein, respectively. B, Rosenthal (Scatchard) plot of the saturation isotherm represented
in A. C, [3H]RY 80 binding (0.75 nM) was insensitive to zolpidem (E) but potently inhibited (IC50 ; 2.9 nM) by RY-24 (L). The abscissae are in log
units. These experiments were repeated three times.
RY-24 (16%) (Liu et al., 1996). In contrast, saturable binding
of [3H]RY 80 (at concentrations of #20 nM) was not observed
in cerebellar membranes (fig. 2, legend), an observation con-
sistent with both the low expression of alpha-5 subunits in
this brain region (McKernan et al., 1991b; Wisden et al.,
1992) and the selectivity of RY 80 for recombinant GABAA
receptors bearing this subunit (Liu et al., 1995).
Zolpidem binds with very low (mM) affinity to both recom-
binant GABAA receptors containing alpha-5 subunits
(Pritchett and Seeburg, 1990; Hadingham et al., 1993 Lüd-
dens et al., 1994; fig. 3A) and hippocampal GABAA receptors
that have been immunoprecipitated with alpha-5-selective
antibodies (Mertens et al., 1993; McKernan et al., 1991a).
Thus, the inability of zolpidem to significantly reduce [3H]RY
80 binding in hippocampal membranes (fig. 3B) is consistent
with the hypothesis that this radioligand selectively labels
GABAA receptors bearing alpha-5 subunits. This hypothesis
is also consistent with the agreement in potency of RY-24 (an
alpha-5-selective ligand) to inhibit [3H]RY 80 binding to hip-
pocampal membranes (fig. 3B) and recombinant GABAA re-
ceptors with alpha-5 subunits (fig. 3A and Liu et al., 1995)
and to inhibit a component of [3H]flunitrazepam binding to
hippocampal membranes representing ;16% of the total re-
ceptor pool (Liu et al., 1996).
The ability of GABA to modulate the affinity of benzodiaz-
epine site ligands (the “GABA shift”) remains a robust neu-
rochemical measure of efficacy. GABA shift assays tradition-
ally use brain membranes (Skolnick et al., 1982) containing
heterogeneous receptor populations. The resulting values
thus represent an average efficacy because this measure is
dependent on subunit composition (Graham et al., 1996; von
Blankenfeld et al., 1990). To determine whether ligand effi-
cacy could be correctly predicted in a subpopulation of hip-
pocampal GABAA receptors using [3H]RY 80, we examined
the effect of GABA on QHII-066, the 7-acetyleno congener of
diazepam. This compound was recently reported to bind with
moderate ($7-fold) selectivity to recombinant alpha-5 beta-3
gamma-2 receptors compared with isoforms containing other
alpha subunits (Huang et al., 1996). GABA produced a ;2.7-
fold increase in the potency of QHII-066 in both hippocampal
membranes and recombinant alpha-5 beta-3 gamma-2 recep-
tors (fig. 3). If the positive GABA shift obtained with QHII-
066 in hippocampal membranes is predictive of in vivo effi-
cacy, then this compound should exhibit some of the
pharmacological properties common to other benzodiazepine
site agonists. To test this hypothesis, we examined the anti-
convulsant properties of QHII-066. This measure was se-
Vol. 283
lected because several of the alpha-5-selective imidazobenzo-
diazepines, such as RY-24, are inverse agonists at alpha-5
Downloaded from jpet.aspetjournals.org at ASPET Journals on March 6, 2016
beta-3 gamma-2 receptors expressed in Xenopus oocytes (Liu
et al., 1995) and are convulsant in mice (Liu et al., 1996). As
predicted from its efficacy in hippocampal membranes, QHII-
066 blocked RY-24 induced convulsions in a dose-dependent
fashion and was ;5-fold less potent against DMCM-induced
convulsions (fig. 4). These observations indicate that [3H]RY
80 may be useful in evaluating ligand efficacies at wild-type
GABAA receptors bearing alpha-5 subunits. Although more
speculative, the higher potency of QHII-066 in blocking
RY-24 compared with DMCM-induced convulsions suggests
its anticonvulsant properties may be related to an action at
GABAA receptors containing alpha-5 subunits.
In situ hybridization studies have shown mRNA encoding
the alpha-5 subunit is relatively abundant in the neonatal
rat brain (Laurie et al., 1992). This expression diminishes
substantially during development, and in the adult brain,
mRNA encoding the alpha-5 subunit is relatively abundant
only in the hippocampus (Wisden et al., 1992). In contrast,
either undetectable (McKernan et al., 1991b) or very low
levels (Sieghart et al., 1993) of the corresponding protein
have been detected with subunit-specific antibodies. Based
on a comparison of the Bmax values obtained with [3H]RY 80
and flunitrazepam in cortical membranes from 6- to 8-day-
old rat pups, GABAA receptors bearing alpha-5 subunits
represent ;31% of the receptor pool (Results and fig. 5).
Although it could be argued that [3H]RY 80 is labeling other
GABAA receptor isoforms in the neonatal cortex, its Kd value
(0.81 6 0.25 nM) is similar to that obtained in both adult
hippocampus and recombinant receptors (fig. 2). Moreover,
[3H]RY 80 binding to juvenile cortex is zolpidem-insensitive
and potently inhibited by the alpha-5-selective ligand RY-24
(fig. 5, inset). The apparent discrepancy between the low
levels of alpha-5 immunoreactive protein relative to both an
abundance of mRNA encoding this subunit and the Bmax
value estimated with [3H]RY 80 may be attributable to the
extensive glycosylation of alpha-5 subunits (Sieghart et al.,
1993) that may interfere with the antigen-antibody reaction
in neonatal brain.
In summary, [3H]RY 80 appears to label specific popula-
tions of GABAA receptors containing an alpha-5 subunit and
may be used in much the same manner as [3H]zolpidem to
study receptor populations bearing alpha-1 subunits (De-
Vaud and Morrow, 1994). As such, [3H]RY 80 may be used to
evaluate the potency and efficacy of compounds at wild-type
GABAA receptors containing alpha-5 subunits, as a radioli-
1997
gand for autoradiographic studies and as a probe for exam-
ining these receptors after physiological and pharmacological
manipulations.
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Send reprint requests to: Dr. P. Skolnick, Chief, Laboratory of Neuro-
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