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Vol. 63, Issue 2, 289-296, February 2003
Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin (A.M.K., J.S.-C., D.A.W., C.C.); Department of Pharmacology, Emory University, Atlanta, Georgia (J.A.T.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Several structural subclasses of ligands bind to the benzodiazepine
(BZD) binding site of the GABAA receptor. Previous studies from this laboratory have suggested that imidazobenzodiazepines (i-BZDs, e.g., Ro 15-1788) require domains in the BZD
binding site for high-affinity binding that are distinct from the
requirements of classic BZDs (e.g., flunitrazepam). Here, we
used systematic mutagenesis and the substituted cysteine accessibility
method to map the recognition domain of i-BZDs near two
residues implicated in BZD binding, 
2A79 and
2T81. Both classic BZDs and i-BZDs protect cysteines substituted at 2A79 and
2T81 from covalent modification, suggesting that these
ligands may occupy common volumetric spaces during binding. However,
the binding of i-BZDs is more sensitive to mutations at
2A79 than classic BZDs or BZDs that lack a 3'-imidazo
substituent (e.g., midazolam). The effect that 2A79
mutagenesis has on the binding affinities of a series of structurally
rigid i-BZDs is related to the volume of the 3'-imidazo substituents. Furthermore, larger amino acid side chains introduced at
2A79 cause correspondingly larger decreases in the
binding affinities of i-BZDs with bulky 3' substituents.
These data are consistent with a model in which 2A79
lines a subsite within the BZD binding pocket that accommodates the 3'
substituent of i-BZDs. In agreement with our
experimental data, computer-assisted docking of Ro 15-4513 into a
molecular model of the BZD binding site positions the 3'-imidazo
substituent of Ro 15-4513 near 2A79.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Benzodiazepines
(BZDs) are therapeutic agents commonly used in the treatment of
anxiety, sleeplessness, and epilepsy (Doble and Martin, 1996
). BZDs
exert their anxiolytic, hypnotic, and anticonvulsant effects by
interacting with a unique modulatory site on the
GABAA receptor, the main effector of neuronal
inhibition within the central nervous system (Hevers and Lüddens,
1998). The BZD binding site is on the extracellular surface of the
GABAA receptor at an interface formed by the 
and subunits (Smith and Olsen, 1995; Sigel and Buhr, 1997). Several
studies have identified residues on both the subunit (Duncalfe et
al., 1996; Amin et al., 1997; Buhr et al., 1997b; Davies et al., 1998;
Schaerer et al., 1998; Renard et al., 1999; Davies et al., 2001) and
the subunit (Buhr and Sigel, 1997; Buhr et al., 1997a; Wingrove et al., 1997; Kucken et al., 2000) that mediate high-affinity BZD binding;
however, the specific interactions between individual amino acids and
BZD ligands and the orientation of BZDs within the recognition site
remain unclear (for review, see He et al., 2001).
The structures of BZD binding site ligands are quite diverse. Classic
BZDs, such as flunitrazepam and flurazepam, possess a common
1,4-benzodiazepine nucleus with a 5-phenyl substituent (Fig.
1; Sternbach, 1979). A different class of
BZD ligands possesses both a 5-phenyl substituent and an imidazo ring
substituted at positions 1 and 2 of the diazepine nucleus (e.g.,
midazolam; Fig. 1). In contrast, BZDs such as Ro 15-4513 and Ro 15-1788 possess the imidazo ring but lack the 5-phenyl substituent (Fig. 1).
Our research has sought to identify specific domains of the
2 subunit that are important for binding
different structural classes of BZDs and to establish how each class of
ligand is oriented within the BZD binding site.
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Imidazobenzodiazepines (i-BZDs), such as Ro 15-4513 and Ro
15-1788, seem to possess structural requirements for binding that are
distinct from classic BZDs. Previously, we demonstrated that mutation
of 2A79 had a larger effect on the binding
affinities of Ro 15-4513 and Ro 15-1788 than on the classic BZD ligand
flunitrazepam (Kucken et al., 2000). Additionally, Ro15-1788 as well as
the classic BZD flurazepam impeded the covalent modification of a cysteine substituted at 2A79, whereas
modification of 2T81C was significantly
impeded by Ro15-1788 but not by flurazepam (Teissére and
Czajkowski, 2001). Based on these data, we hypothesized that 2A79 and 2T81 line
part of an i-BZD subsite of the BZD binding site.
In this article, we extend these studies and further test our
hypothesis. The binding affinities of seven different BZD ligands were
measured after systematic mutation of 2A79. In
addition, we examined the ability of several BZD ligands with different structures and functional efficacies to slow the rate of covalent modification of 2A79C and
2T81C. Our studies indicate that
2A79 and 2T81
contribute to a subsite of the BZD binding pocket that accommodates the
3' substituent of the i-BZD imidazo ring (see Fig. 1). Using
the recently crystallized molluscan acetylcholine binding protein
(AChBP) (Brejc et al., 2001) as a structural template, we modeled the
BZD binding site of the GABAA receptor and
describe in part the three-dimensional relationship between
i-BZD ligands and the 2 subunit of
the GABAA receptor.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mutagenesis.
Rat cDNAs encoding 1,
2, and 2S subunits
were used for all molecular cloning, radioligand binding, and
functional studies. Site-directed mutagenesis of
2A79 and 2T81 was
carried out using recombinant oligonucleotides and the polymerase chain
reaction. For radioligand binding, amino acid mutations were made using a myc 9E10 epitope-tagged 2 subunit
as the template. The presence of the epitope had no detectable effect
on ligand recognition or on expression of the
2 subunit (Kucken et al., 2000). Wild-type and
mutant subunits were subcloned into pCEP4 (Boileau et al., 1998) for
transient expression in human embryonic kidney (HEK) 293 cells
(American Type Culture Collection, Manassas, VA) or into pGH19 (Liman
et al., 1992; Robertson et al., 1996) for expression in Xenopus
laevis oocytes. All 2 mutants were
verified by restriction enzyme analysis and double-strand DNA sequencing.
Transient Transfection and Radioligand Binding.
HEK 293 cells were transiently transfected with 1,
2, and 2myc or
2myc mutant subunits using a standard CaHPO4 precipitation method (Kucken et al.,
2000). Cells were harvested 48 h after transfection, and membrane
homogenates were prepared as described previously (Kucken et al.,
2000). Membrane homogenates (100 µg) were incubated at room
temperature with [3H]flunitrazepam at
sub-KD concentrations for wild-type or
mutant receptors and 8 to 11 concentrations of unlabeled ligand in a final volume of 250 µl. Data were fit using the equation:
Y = Bmax/[1 + (X/IC50)], where Y is the
specifically bound disintegrations per minute,
Bmax is the maximal binding,
X is the concentration of unlabeled ligand, and
IC50 is the concentration of unlabeled ligand
that reduces the maximal specific binding by 50% (GraphPad Software,
San Diego, CA). KI values were
calculated using the equation: KI = IC50/(1 + [radioligand]/KD) (Cheng and
Prusoff, 1973; Chou, 1974), where KD
refers to the equilibrium dissociation constant of the radioligand. The
use of this equation assumes that ligand binding follows the law of
mass action, is competitive, and that the data reflect one-site binding
with no cooperativity. KI values were
obtained from at least three independent experiments, each with
triplicate determinations. [3H]Flunitrazepam
(85 Ci/mmol) was obtained from PerkinElmer Life Sciences
(Boston, MA). Nonradioactive BZDs were obtained from Hoffman-La Roche
(Nutley, NJ) or RBI/Sigma (Natick, MA).
Expression in Oocytes and Electrophysiology.
Capped cRNAs
encoding the 1, 2,
2, 2A79C, or
2T81C subunits in pGH19 were transcribed in
vitro using the mMessage mMachine T7 kit (Ambion, Austin, TX). Oocytes
were harvested from X. laevis and injected within 24 h
with 27 nl of cRNA (10-200 pg/nl/subunit) in the ratio 1:1:10
(//; Boileau et al., 2002). Expressed receptors were
functionally assayed using two-electrode voltage clamp
(Vhold = 
80 mV, room temp) as described
previously (Teissére and Czajkowski, 2001). Working
concentrations of GABA and BZD ligands were made up in ND96 oocyte
perfusion solution (96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2, and 5 mM HEPES, pH 7.2). In all electrophysiological experiments, both
GABA-activated current (IGABA) and
flurazepam-mediated potentiation of GABA-activated current
(IGABA + flurazepam) were
measured. Flurazepam-mediated potentiation of
IGABA was defined as [(IGABA + flurazepam/IGABA) 1] × 100. Rates of sulfhydryl-specific covalent modification of
122A79C or 122T81C
receptors by methanethiosulfonate (MTS) reagents were determined using
the following protocol: 1) flurazepam potentiation of
IGABA was measured by applying 1 µM GABA and
then applying 1 µM GABA + 1 µM flurazepam (corresponding to
~EC50 concentration of GABA and
~EC80 concentration of flurazepam); 2) the
oocyte was washed for 3 min in ND96 buffer; and 3) flurazepam
potentiation of IGABA was measured again. This
protocol was repeated until flurazepam potentiation of
IGABA changed by less than 5%. After potentiation stabilized, the rate of MTS reaction was measured by
applying a subsaturating 5 s application of an MTS reagent 30 s after determination of IGABA and
IGABA + flurazepam. Applications of the MTS
reagent were repeated until flurazepam potentiation of
IGABA no longer decreased.
2A79C-containing receptors were reacted with
200 µM N-biotinylaminoethyl MTS (MTSEA-Biotin; Biotium,
Hayward, CA) and 2T81C-containing receptors
were reacted with 20 µM N-biotinylcaproylaminoethyl
CAP MTS (MTSEA-Biotin-CAP; Biotium) as described previously
(Teissére and Czajkowski, 2001).
kt, where A is the initial
potentiation, k is the pseudo-first-order rate constant of
the reaction, and t is the time in seconds (GraphPad). The
derived pseudo-first-order rate constant was converted into a
second-order rate constant (k2,
M1s1) by dividing by
the concentration of MTS reagent used to correct for the concentration
dependence of this effect (Pascual and Karlin, 1998). To verify the
accuracy of the protocol, some of the rate determinations were
conducted at two different concentrations of MTS-reagent.
The ability of different BZDs to slow the rate of MTS modification of
2A79C and 2T81C was
assayed by coapplying a BZD with the MTS-reagent during the rate
determinations. The following BZDs were tested (all applied at
~EC95 concentrations): flurazepam, Ro 15-1788, Ro 15-4513, midazolam, Ro 40-6129, and Ro 41-3380. In these
experiments, flurazepam potentiation of IGABA was
stabilized before measuring the rate of MTS modification as follows: 1)
1 µM GABA and 1 µM GABA + 1 µM flurazepam were applied to an
oocyte; 2) the oocyte was treated with an EC95
concentration of BZD and then washed for 3 min in ND96; and 3)
flurazepam potentiation of IGABA was measured
again using 1 µM GABA and 1 µM GABA + 1 µM flurazepam. This
protocol was repeated until IGABA and
IGABA + flurazepam changed by less than 10% and
demonstrated that the wash time was sufficient to wash out the test
EC95 concentration of BZD. In some cases, after
treating the oocytes with MTS-reagent in the presence of a BZD,
receptors were re-exposed to the same concentration of MTS-reagent
alone to demonstrate that a maximal decrease in flurazepam potentiation
of IGABA could still be obtained.
Homology Modeling of the BZD Binding Site.
The mature
protein sequences of the rat 1 and
2 subunits were modeled by comparison with the
deduced three-dimensional structure of a subunit of the AChBP (Brejc et
al., 2001). The crystal structure of the AChBP was downloaded from the
Protein Data Bank (PDB code 1I9B) and loaded into Swiss Protein
Databank Viewer (http://ca.expasy.org/spdbv). The mature
1 protein sequence from T12 to I222 and the
mature 2 protein sequence from D26 to M233
were aligned with the AChBP primary amino acid sequence (Cromer et al.,
2002) and threaded onto the AChBP tertiary structure using the
"Interactive Magic Fit" function of Swiss Protein Databank Viewer.
The threaded subunits were imported into SYBYL (Tripos, Inc., St.
Louis, MO) and energy minimized (< 0.5 kcal/Å). The first 100 iterations were carried out using Simplex minimization (Press et al.,
1988) followed by 1000 iterations using the Powell conjugate gradient
method (Powell, 1977). An
1/2 BZD binding site
interface was assembled by overlaying the monomeric subunits on the
AChBP scaffold and the resulting structure was imported into SYBYL and
energy minimized. Docking of Ro 15-4513 was performed using AutoDock
3.0 (Morris et al., 1998). The ligand started out in an arbitrary
conformation, orientation, and position and the docking simulation was
carried out using a Lamarckian genetic algorithm (Morris et al., 1998).
AutoDock 3.0, like other docking programs, treats the receptor protein as a fixed target; thus, in the final docked structure, the binding site residue side-chains have not moved.
Statistics. Data were analyzed by one-way analysis of variance, applying the Dunnett post-test for significance of differences between treatments (GraphPad). Comparisons used log(KI) or log(k2) values for the analysis.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The Effect of Systematic 2A79 Mutagenesis on
i-BZD Binding Affinity.
Ten point mutations were
made at 2A79 to evaluate the contribution of
this residue to BZD ligand affinity
(2A79
Gly, Ser, Cys, Glu, Gln, Leu, Phe,
Tyr, Arg, Trp). These residues were chosen to represent a range of
amino acid properties (i.e., size, charge, hydrophobicity). Wild-type
(122) or mutant
(122-mutant) receptors were expressed in HEK 293 cells and the binding affinities (KI) of flunitrazepam, Ro 15-4513 and
Ro 15-1788 were measured by displacement of
[3H]flunitrazepam. Wild-type receptors bound
flunitrazepam, Ro 15-4513, and Ro 15-1788 with
KI values of 8.9, 3.9, and 3.5 nM,
respectively (Table 1). Only 3 of the 10 mutations at 2A79 significantly altered
flunitrazepam affinity. The 2A79R, -C, and -Q
mutations reduced flunitrazepam affinity 3-, 5-, and 9-fold,
respectively. In contrast, 9 of the 10 mutations at
2A79 significantly reduced the binding
affinities of Ro 15-4513 and Ro 15-1788 (Table 1). For Ro 15-4513, the
decreases in affinity ranged from 6-(A79S) to 93-fold (A79F). For Ro
15-1788, the decreases ranged from 3-(A79S) to 21-fold (A79Y). Because
mutations at 2A79, in general, had larger
affects on the binding affinities of i-BZDs than on
flunitrazepam affinity, we hypothesized that
2A79 lines a subsite of the BZD binding pocket
important for i-BZD binding. Furthermore, the results implied that the chemical elements that are unique to i-BZDs
(i.e., the imidazo ring and/or the 3' substituent) are probably near 2A79.
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2A79 mutation, midazolam binding affinity
was examined. Midazolam contains an imidazo ring that is similar to Ro
15-4513 and Ro 15-1788, but it does not possess a 3' substituent (see
Fig. 1). The KI of midazolam was
determined for several 2A79 mutant receptors (2A79G, -C, -E, -L, -R, and -Y). In general,
the effects 2A79 mutation had on midazolam
affinity mirrored those observed for flunitrazepam rather than Ro
15-4513 or Ro 15-1788 (Table 1). For example, the
2A79G, -E, -L, and -Y mutations altered the affinities of midazolam and flunitrazepam less than 4.5-fold yet decreased Ro 15-4513 affinity between 12- and 52-fold. The
2A79R mutation did not significantly alter Ro
15-4513 or Ro 15-1788 affinities, yet flunitrazepam and midazolam
affinities were significantly decreased 3- and 8-fold, respectively.
These results suggest that the sensitivity of Ro 15-4513 and Ro 15-1788 binding to 2A79 mutation is probably caused by
the 3'-imidazo substituent of these ligands.
To examine the potential spatial relationship between
2A79 and the 3' substituent of the
i-BZD imidazo ring, the binding affinities of three
additional i-BZDs (Ro 40-6129, Ro 41-0639, Ro 41-3380) were
measured. Like Ro 15-4513 and Ro 15-1788, these compounds each possess
a 3'-imidazo substituent. In contrast to the 3' substituents of Ro
15-4513 and Ro 15-1788, which are relatively flexible polar esters, the
3' substituents of Ro 40-6129, Ro 41-0639, and Ro 41-3380 are rigid,
hydrophobic alkynes. Because of their rigid nature, and the observation
that Ro 40-6129, Ro 41-0639, Ro 41-3380 only differ from each other in
the volume of their 3' substituents (45.4 Å3,
60.7 Å3, and 109.6 Å3,
respectively), these compounds were ideal for testing the effect of 3'
substituent size on i-BZD binding affinity after mutation of
2A79.
The ability of Ro 40-6129, Ro 41-0639, and Ro 41-3380 to displace
the binding of [3H]flunitrazepam in wild type,
2A79C-, 2A79E-,
2A79L-, 2A79Y-, and
2A79R-containing receptors was measured (Fig.
2, Table 2). All three
ligands bound to wild-type GABAA receptors with
high affinity. All of the mutations significantly reduced, by 10-fold or more, the binding affinity of Ro 41-3380, which possesses the largest 3' imidazo substituent. Three of the five mutations
significantly reduced the affinities of Ro 41-0639 and Ro 40-6129, which possess progressively smaller 3' substituents (Table 2).
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2A79 was increased, we observed
correspondingly larger decreases in Ro 41-3380 binding affinity. Taken
together, these data are consistent with a model in which
2A79 lines a subsite within the BZD binding pocket that accommodates the 3' substituent of i-BZD
ligands.
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Ability of i-BZDs to Slow Sulfhydryl Modification of
2A79C and 2T81C.
We compared the
ability of flurazepam (positive modulator), midazolam (positive
modulator), Ro 40-6129 (zero modulator), Ro 41-3380 (positive
modulator), Ro 15-1788 (zero modulator) and Ro 15-4513 (negative
modulator) to impede covalent modification of
2A79C and 2T81C. BZD
ligands with different structures and functional efficacies were
used to determine whether either of these properties influenced the
ability of the ligand to slow the rate of covalent modification of
cysteines substituted at 2A79 and
2T81. The rate of methanethiolsulfonate (MTS)
modification of an engineered cysteine depends on several factors: 1)
the permeability of the pathway to the substituted cysteine; 2) the
electrostatic potential in the binding site and along the pathway; 3)
the ionization of the sulfhydryl group; and 4) local steric
restrictions. All of the ligands significantly slowed the rates at
which MTSEA-Biotin and MTSEA-Biotin-CAP reacted with
2A79C and 2T81C,
respectively (Fig. 4, Table
3).
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2A79C was slowed by the presence of both
flurazepam and Ro 15-1788, whereas modification of
2T81C seemed to be slowed only by Ro 15-1788 (Teissére and Czajkowski, 2001). Although the presence of
flurazepam showed a clear trend in slowing the rate of MTSEA-Biotin-CAP modification of 2T81C (29%), the rate was not
statistically significant. Here, the inclusion of additional data
demonstrated that flurazepam significantly slowed (39%) the rate of
covalent modification of 2T81C (Fig. 4, Table
3). Because differences in BZD functional efficacy did not alter the
ability of the ligands to protect 2A79C or
2T81C from sulfhydryl modification, it is
likely the protection is caused by a direct steric block of the
substituted cysteines and not caused by allosteric conformational
changes in the protein that accompany BZD binding. Differences in
ligand structure also did not affect the ability of the ligands to
protect either 2A79C or
2T81C from sulfhydryl modification. The
results indicate that both classic BZDs and i-BZDs, when
bound, lie topologically close to 2A79 and
2T81.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The side-chains of 2F77,
2A79 and 2T81 are
located adjacent to each other on a -strand within the BZD binding
site (Teissére and Czajkowski, 2001). Mutagenesis of
2F77 affects the binding affinity of both
classic BZDs and i-BZDs (Buhr et al., 1997a; Sigel et al.,
1998), whereas mutagenesis of 2A79 decreases
the binding affinity of i-BZDs more than classic BZDs (Table
1) indicating that different residues within the binding pocket are
important for stabilizing classic BZD and i-BZD binding.
Both classic BZDs and i-BZDs, however, slow covalent
modification of 2A79 and
2T81 (Fig. 4, Table 3). Thus, although classic
BZDs do not require 2A79 for high affinity
binding, when bound they are located close enough to
2A79C and 2T81C to
sterically interfere with the covalent addition of a sulfhydryl reagent
at these positions.
Orientation of i-BZDs in the BZD Binding Site.
Based on our experimental data, we propose that
2A79 and 2T81 line a
region in which the 3'-imidazo substituent of i-BZDs is
positioned. Several lines of evidence support this model of i-BZD
orientation. Overall, 2A79 mutations disrupt
i-BZD binding to a greater extent than classic BZD binding
which suggests that structural elements unique to i-BZDs
(and not common elements, such as the fused diazepine nucleus) are
positioned near 2A79 within the BZD
recognition site. The effect of 2A79
mutagenesis on the binding affinities of structurally rigid
i-BZDs (Ro 41-3380 > Ro 40-0639 > Ro 41-6129) is
related to the volume of each 3' substituent (109.6 Å3, 60.7 Å3 and 45.4 Å3, respectively; Fig. 3). Furthermore, larger
amino acid side chains introduced at 2A79
cause correspondingly larger decreases in the binding affinities of
i-BZDs with bulky 3' substituents. These data can be
explained by a model in which the addition of bulky side chains at
position 79 decreases the volume of the binding site pocket and hinders
occupation of the site by ligands bearing large 3' substituents, such
as Ro 41-3380. A possible contradiction to this model is that the
2A79R mutation did not disrupt the binding
affinities of Ro 15-4513 or Ro 15-1788, which also possess fairly large
3' substituents (82.8 Å3). However, a favorable
interaction between the arginine side chain and the ester group of
these compounds could overcome potential steric interference. It is
also possible that the arginine side chain could interact with
neighboring residues or with the peptide backbone to minimize its
impact on the binding of these ligands.
2T81 resulted in small but significant
decreases in i-BZD binding affinities (Kucken et al., 2000).
In this study, mutation of 2T81 to cysteine
significantly decreased the binding of the i-BZDs, Ro
40-6129, Ro 41-0639 and Ro 41-3380 between 3 and 8 fold (Table 2).
Mutation of 2F77 to tyrosine has no affect on
Ro 40-6129, Ro 41-0639 and Ro 41-3380 binding (Sigel et al., 1998).
Taken together, these data, in addition to the result that i-BZDs protect 2A79C and
2T81C from covalent modification, support the
conclusion that 2A79 and
g2T81 line a region of the BZD binding pocket in
which the 3'-imidazo substituent of i-BZDs is positioned.
Our data also suggest that there is a size limit to what can be
accommodated within the binding cavity. Consistent with this idea,
large substitutions at the 3' position of i-BZDs are
not tolerated. For example, as the size of the ester group
increases from
CO2CH2CH3
(Ro 15-1788) to
CO2CH2C(CH3)3,
binding affinity is reduced 100-fold (Wong et al., 1993). Mutations at
2A79 affect Ro 15-4513 binding to a greater
extent than Ro 15-1788 (Table 1). One explanation of these results is
that even though the 3' substituents of Ro 15-4513 and Ro 15-1788 are
the same size, the overall length of Ro 15-4513, from the 7-azide group
to the end of the 3'ester substituent, is longer than Ro 15-1788 (Fig. 1).
Interestingly, i-BZDs with small 3' substituents (e.g.,
CO2CH3) also have decreased
binding affinities (Wong et al., 1993), suggesting that there may be an
optimal size relationship between the 3' substituent and the binding
site. Mutation of 2A79 to a smaller residue
(e.g., glycine) as well as to larger residues (e.g., phenylalanine,
tyrosine) significantly decreases Ro 15-4513 and Ro 15-1788 binding
affinities (Table 1) and suggests that size of the side chain at this
position influences i-BZD binding affinity. Other amino acid
properties, such as hydrophobicity, aromaticity, charge, and H-bonding
capability, did not correlate with the decreases in i-BZD
binding affinity measured after 2A79 mutagenesis.
Homology Model of the BZD Binding Site.
Much of our
experimental data were completed before the publication of the
molluscan AChBP crystal structure (Brejc et al., 2001). To help
facilitate discussion of our results and to provide additional support
for our model of i-BZD orientation, we homology-modeled the
benzodiazepine binding site using the structure of the AChBP as a
template (Fig. 5). It is important to
recognize that the model is only a static picture of the BZD binding
site captured in an indeterminate state. The homology model is based on
the structure of the AChBP crystallized in the presence of the putative agonist HEPES. Because AChBP binds acetylcholine with high affinity, it
has been hypothesized that this structure may represent receptor in
either an open or desensitized state (Brejc et al., 2001).
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2 subunit from T73 to T81 forms a strand
(Teissére and Czajkowski, 2001). Our secondary structure
prediction agrees with the crystal structure of the AChBP, where
residues aligned with this region of the GABAA
receptor form part of a strand (AChBP, 2). Many of the residues
previously identified by mutagenesis, the substituted cysteine
accessibility method (1Y159,
1T206, 1Y209,
2Y58, 2F77,
2A79, and 2T81), and
photoaffinity labeling (1H101) as contributing
to the BZD binding site (Duncalfe et al., 1996; Amin et al., 1997; Buhr
et al., 1997a,b; Davies et al., 1998; Kucken et al., 2000;
Teissére and Czajkowski, 2001) are positioned at the
1/2 subunit interface
and define a cavity that probably forms the BZD-binding site (Fig. 5A).
In the absence of a crystal structure of the
GABAA receptor bound with a BZD ligand, it is
difficult to identify which residues directly contact a ligand and to
predict how the ligands are oriented in the binding pocket. Ro 15-4513 can be used as a photoaffinity label for the BZD binding site of the
GABAA receptor (Mohler et al., 1984; Sieghart et
al., 1987). The azide group at the 7 position of the fused diazepine
ring (Fig. 1) is the photoreactive moiety and, when exposed to UV
light, the aryl azide undergoes ring-expansion and subsequent bond
formation with nearby nucleophilic groups (Hermanson, 1996). Studies
using both bovine brain and recombinant GABAA
receptors have established that Ro 15-4513 photoincorporates into the
subunit in a region that lies between G103 and the C terminus
(Davies et al., 1996; Duncalfe and Dunn, 1996). Davies and Dunn (1998)
mapped a region of incorporation to the extracellular loop between
transmembrane regions two and three. Recently, however, a site of Ro
15-4513 incorporation was mapped to a tyrosine residue equivalent to
rat 1Y209 (Sawyer et al., 2002). Combining our data with this finding, we envision that Ro 15-4513 spans the binding
site between 1Y209 and
2A79, with the azide substituent facing the
1 subunit and the 3'-imidazo substituent
facing the 2 subunit. In agreement with our
experimental data, computational docking of Ro 15-4513 into the BZD
binding site positions the 3'-imidazo substituent of Ro 15-4513 near
2A79 (Fig. 5, B and C). The docking of Ro
15-1788 resulted in the same positioning of the 3'-imidazo substituent.
Although our data clearly define the orientation of i-BZDs
within the binding site, how the binding of i-BZDs promote
local movements within the binding site that are coupled to changes in
GABA binding and/or GABA activation of the channel remains unknown. The
size of the 3' substituent alone does not seem to predict
i-BZD efficacy. Ro 15-4513, which possesses a fairly large 3' substituent (82.8Å3) is a BZD
inverse-agonist, Ro 15-1788 (82.8 Å3) is a BZD
antagonist, Ro 40-6129 (45.4 Å3) is a BZD
antagonist, Ro 41-0639 (60.7 Å3) is a weak BZD
partial agonist, and Ro 41-3380 (109.6 Å3) is a
BZD agonist. According to allosteric theory, modulators that bind to a
receptor protein exert their effects by initiating an allosteric
transition in the protein that indirectly modifies the conformation of
the agonist binding site (Changeux and Edelstein, 1998). It is likely
that functional coupling between the BZD and GABA binding sites is
accompanied by structural rearrangements in the receptor protein that
change the apparent affinity of both sites. Previously, we demonstrated
that GABA binding and/or GABA-mediated receptor activation causes
structural rearrangements in the BZD binding site that can be detected
at A79 (Teissére and Czajkowski, 2001). We speculate that BZD
binding also promotes structural rearrangements within the BZD binding
site and that these local changes determine how a particular BZD will
modulate the GABA response. Because the therapeutic value of BZDs
depends on their efficacy, identification of the residues mediating
these local movements is an important goal for future studies.
| |
Acknowledgments |
|---|
We thank Lisa M. Sharkey for assistance in rate of modification experiments, Lisa A. Wheeler for assistance in binding assays, Eric Wise for aid in the design of loop structures, and Dr. J. Glen Newell for invaluable discussion and critical reading of this manuscript.
| |
Footnotes |
|---|
Received August 7, 2002; Accepted October 22, 2002
This work was supported in part by National Institutes of Mental Health/National Research Service Award grant MH12966 (to J.A.T.) and National Institutes of Neurological Disorders and Stroke Grant NS34727 (to C.C.).
A.M.K. and J.A.T. contributed equally to this work.
Address correspondence to: Cynthia Czajkowski, Ph.D., Dept. of Physiology, University of Wisconsin-Madison, 1300 University Ave., 197 MSC, Madison, WI 53706. E-mail: czajkowski{at}physiology.wisc.edu
| |
Abbreviations |
|---|
BZD, benzodiazepine;
i-BZD, imidazobenzodiazepine;
AChBP, acetylcholine binding protein;
HEK, human
embryonic kidney;
MTS, methanethiosulfonate;
MTSEA-Biotin, N-biotinaminoethyl methanethiosulfonate;
MTSEA-Biotin-CAP, N-biotincaproylaminoethyl
methanethiosulfonate;
IGABA, GABA-gated Cl
current.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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subunit are crucial for benzodiazepine binding and allosteric modulation of -aminobutyric acidA receptors.
Mol Pharmacol
51:
833-841 subunit of 1
2
2 GABAA receptors drastically alter the affinity for ligands of the benzodiazepine binding site.
J Biol Chem
272:
11799-118041 subunit of -aminobutyric acidA receptors influence affinities for benzodiazepine binding site ligands.
Mol Pharmacol
52:
676-6822 subunit of the -aminobutyric acid type A receptors results in altered benzodiazepine binding specificity.
Proc Natl Acad Sci USA
94:
8824-88291 subunit reveals a domain that affects sensitivity to GABA and benzodiazepine site ligands.
J Neurochem
79:
55-62[CrossRef][Medline].-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the subunit.
J Biol Chem
271:
9209-92142 subunit of the -aminobutyric acidA receptor.
Mol Pharmacol
57:
932-939 subunit.
J Gen Physiol
111:
717-739-aminobutyric acid type A receptor conferring subtype selectivity for benzodiazepine site ligands.
J Biol Chem
274:
13370-13374 subunit.
Soc Neurosci Abstr
27:
591.15.-aminobutyric acid type A receptor subunit residues photolabeled by the imidazobenzodiazepine [3H]Ro15-4513.
J Biol Chem
277:
50036-500451 subunit of GABAA receptors affects the interaction with selected benzodiazepine binding site ligands.
Eur J Pharmacol
354:
283-287[CrossRef][Medline].122 -aminobutyric acidA receptors: relative orientation of ligands and amino acid side chains.
Mol Pharmacol
54:
1097-1105-strand in the 2 subunit lines the benzodiazepine binding site of the GABAA receptor: structural rearrangements detected during channel gating.
J Neurosci
21:
4977-4986 subunit of the -aminobutyric acidA receptor that determine ligand binding and modulation at the benzodiazepine binding site.
Mol Pharmacol
52:
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) and
) receptors is
shown. The displacing ligands differ only in the size of their
respective 3'-imidazo substituents. Values are mean ± S.E.M. of
triplicate determinations and were fit by nonlinear regression
analysis. KI values are reported in Table
), Ro 41-0639 (60.7, 
), and Ro 41-3380 (109.6, 
, MTS + 1 µM Ro 41-3380). Data points are mean ± S.D. from
at least three independent experiments. The calculated second-order
rate constants for the MTS reaction are presented in Table 