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INTRODUCTION |
The
-aminobutyric acid type A
(GABAA)1 receptor
is the major inhibitory ligand-gated ion channel in the mammalian brain
(reviewed in Ref. 1). This chloride channel is a heteropentameric
complex composed of different subunits that exist in many isoforms,
1-6,
1-3,
1-3,
,
, and
1-2. The
1
2
2 combination is thought
to be the most abundant subtype of this receptor family found in the
adult brain (2) and corresponds to the first pharmacological GABAA receptor subtype clearly identified
(3).
Many classes of drugs interact with this receptor, including the
benzodiazepines (BZ), which are the most widely used tranquilizers. The
BZ binding site is located between the
and
subunits (4). In
addition to BZs, other chemically distinct classes of molecules (5, 6)
interact with this binding site, including the triazolopyridazines (CL218872), the
-carbolines, and the imidazopyridines (zolpidem, saripidem).
Mapping the BZ binding site by alanine scanning mutagenesis (7) has led
to the identification of histidine 101 of the
1 subunit
(
1-His101) as a major component of the
binding site interacting either with the nitrogen present in the core
structure of most BZ site ligands (8) or with a phenyl moiety close to
the core structure of these ligands (9). Several other amino acids
surround the BZ binding site. For instance,
1Y159S and
1Y209S substitutions affect diazepam-mediated
potentiation of the GABAA receptor, and these mutants also
abolish [3H]Ro15-1788 binding to the receptor (10). The
1Y161A,
1T206A, and
2F77A
substitutions result in a 3-fold increase in potentiation by diazepam
(11, 12).
An inherent drawback of site-directed mutagenesis for the study of
receptor ligand interactions is the difficulty in determining whether
the amino acid substitution introduced produced its effect directly at
the level of the ligand binding site. It is still theoretically
possible that the mutation could induce an allosteric conformational
change of the receptor itself that indirectly affects ligand binding
properties. One way of minimizing this problem is to compare
GABAA receptor affinities for subtype-selective ligands
before and after mutation of the subunit showing the lowest affinity
for the ligand and to take into account only those mutations yielding
increases in ligand affinity as indicators for the involvement of the
mutated amino acid at the ligand binding site. Such studies have
identified
1-Thr162,
1-Gly200,
1-Val211,
2-Phe77, and
2-Met130 by comparison of the
1 and
3 subunits (13), the
1 and
6 subunits (14), or the
2 and
3 subunits (12, 15). For the present study, the ability of zolpidem to strongly discriminate between
1
2
2 and
5
2
2 GABAA
receptor subtypes (16) was used to characterize mutated
5 subunits in an attempt to identify those amino acids
imparting the preference of zolpidem for the
1 subunit
compared with
5. By swapping the domain responsible for
zolpidem interaction from the
5 subunit with that of the
1 subunit and then serially replacing the amino acids in
this
5 sequence, we exhaustively searched for amino
acids contributing to high affinity for zolpidem. This approach
pinpointed three amino acid substitutions that are sufficient to confer
an
1-like zolpidem affinity on the
5 subunit.
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EXPERIMENTAL PROCEDURES |
Nomenclature--
Amino acids are numbered in
1
(rat sequence) with the putative signal peptide (25 amino acids)
removed and in
3 and
5 by an alignment
with their equivalent positions in
1.
Compounds and Biological Materials--
Compounds were obtained
from the following sources: zolpidem, saripidem (Synthélabo
Recherche); CL218872 (American Cyanamide); and
[3H]flumazenil (83.2 Ci/mmol) (NEN Life Science
Products). Cloned cDNAs encoding the
1,
5,
2, and
2 subunits of
rat GABAA receptor were supplied by Prof. P. H. Seeburg (University of Heidelberg) and were subcloned into expression
vectors as described previously (17).
Mutagenesis--
Chimeras were generated using the Higuchi
method (18). 50-Mer polymerase chain reaction primers, overlapping in
the chimeric area with 15 pairing nucleotides in 5' and 3', were used
to generate chimeric polymerase chain reaction products to be subcloned
into the BsmBI and EcoRI sites of the
5 cDNA. Site-directed mutagenesis was obtained with
the unique site elimination method (19). Selection primer was designed
to disrupt a ScaI site in the ampicillin resistance. Mutagenic primer was always designed with at least 10 bases before and
after the mutated codons, ending with a G or C. For mutant
5m10, two mutagenic primers were used. Plasmids were
purified by ion exchange chromatography (Qiagen). All mutants were
checked by sequencing.
Protein
Expression--
x
2
2
combinations of rat GABAA receptor subunits (where
x is
1,
5, or any chimera
between the two) were transiently transfected in a 1:4:0.2 ratio using
the Chen and Okayama procedure (20) on HEK293 cells seeded at 3 × 106/100-mm dish in minimum essential medium (Life
Technologies, Inc.). Cells were collected 2 days post-transfection for
binding assays. For electrophysiological assays, cells were transfected
with the Fugene reagent (Boehringer Mannheim) according to the
manufacturer's protocol.
[3H]Flumazenil Equilibrium Binding
Assays--
Binding assays were performed as described previously
(17). Briefly, 50 µg of membrane preparation from the transfected cells were incubated with different concentrations of
[3H]flumazenil for the equilibrium saturation analysis.
For competitive inhibition experiments, 50 µg of membrane preparation
of the transfected cells were incubated with
[3H]flumazenil at a concentration of 4 nM in
the presence of various concentrations of the competing ligand. The
concentration of ligand that produced half-maximal inhibition of
[3H]flumazenil binding (IC50 value) was
established by nonlinear regression analysis using Origin 3.0 software
(Microcal Inc.) for each of the GABAA receptor combinations
and a Ki value deduced using the Cheng and Prusoff
equation (21).
Electrophysiology--
We used the whole cell configuration of
the patch-clamp technique with symmetrical chloride conditions (pipette
solution composition was 140 mM CsCl, 1 mM
MgCl2, 1 mM CaCl2, 11 mM EGTA, 10 mM Hepes/Tris-OH, pH 7.2, 4 mM Na2ATP; external solution was 147 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 10 mM Hepes/Tris-OH, pH 7.4). Currents were recorded using an
Axopatch 1D amplifier (Axon Instrument) connected to a personal
computer driven by the Clampex7 software. For EC50
determinations, the nonlinear curve fitting routine of the Origin
software (Microcal Software Inc.) was used.
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RESULTS |
Binding Properties of
5 versus
1
Subunit-containing Receptors--
The
1 or
5 subunits were co-expressed together with
2 and
2 in HEK 293 cells, and the
parameters of [3H]flumazenil binding to membrane
preparations of these transfected cells were evaluated in equilibrium
binding assays. Transfected receptors displayed a similar affinity for
several benzodiazepine site ligands such as flumazenil but differed
remarkably in their affinity for zolpidem and CL218872 (Table
I). CL218872 is 3-10 times more
selective for
1- than for
5-containing
subtypes, and zolpidem is over 300 times more selective (Table I) (17, 22, 23).
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Table I
Zolpidem and CL218872 affinity for GABAA receptors with
x 2 2 combinations
Kd values (flumazenil) were assessed by
[3H]flumazenil binding equilibrium saturation analyses, and
Ki values (zolpidem, CL218872) were determined using
[3H]flumazenil competitive inhibition experiments.
Ki values represent the mean ± S.D. of three
independent experiments.
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Binding Properties of Chimeras--
Three chimeras
(
5C1,
5C2, and
5C3) of
1 and
5 were constructed in the putative
extracellular N-terminal domain (Fig. 1).
The
5C1 subunit corresponds to an
5
subunit in which the 198-204 region (GTENIST) is replaced by the
corresponding amino acids of
1 (DSGIVQS). The
5C2 subunit has an additional 172-176 region (NGSTK) of
5 replaced by the corresponding amino acids of
1 (REPAR). The
5C3 subunit has an
additional 162-164 region (TRA) of
5 replaced by the
corresponding amino acids of
1 (PNS). Each of these
chimeras was transfected with
2 and
2
subunits in HEK293 cells, and the affinity of the corresponding
combination for [3H]flumazenil was determined. Both wild
type and chimeric receptors displayed similar high affinity for
[3H]flumazenil (Table II),
demonstrating that the pharmacological profile of this BZ antagonist at
its binding site was not affected by these mutations.

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Fig. 1.
Alignment of partial protein sequence of
1 (top
lane) and 5
subunits (bottom lane). Regions
replaced in chimeras C1 to C3 are lettered in black, and the
three 1 amino acids essential for zolpidem binding are
enclosed in a gray circle.
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Table II
Mutant receptor affinity for zolpidem
Chimeric subunits were constructed by exchanging various parts of the
N-terminal extracellular domain between 5 and 1.
Single amino acid substitutions were realized by exchanging various
amino acids of the N-terminal extracellular domain between 5
and 1. Kd values (flumazenil) were
assessed by [3H]flumazenil binding equilibrium saturation
analyses, and Ki values (zolpidem) were assessed
using [3H]flumazenil competitive inhibition experiments.
Values represent the mean ± S.D. of three independent experiments
for the chimeras and two independent experiments for the mutants
m1-m7. For m1-m7, the Kd value for
[3H]flumazenil was assumed to be the same as that exhibited
by chimera 5C1. Amino acids underlined are the same as in
1 sequence.
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The
5C1
2
2 receptor showed
a Ki value of 244 ± 93 nM for
zolpidem, representing an increase in affinity of at least 100-fold for
zolpidem compared with that of the wild type
5
2
2. The
Ki values of
5C2
2
2 and
5C3
2
2 for zolpidem were 210 ± 52 and 99.2 ± 53.1 nM, respectively. The
5C3
2
2 combination exhibited an affinity for zolpidem close to that exhibited by the
1
2
2 combination.
Regions 162-164 and 198-204 of
1 are therefore of
crucial importance for zolpidem affinity. To determine the precise
amino acids involved in high affinity zolpidem binding, combinations of
amino acids were then substituted between
5 and
1 in the 162-164 and 198-204 regions of
5.
Single Amino Acid Substitutions (Table II)--
Seven amino acids
are different from
1 in the 198-204 region of
5. Each combination of six amino acids in this region
was substituted by their corresponding residues found in
1. In other terms, mutants
5m1 to
5m7 correspond to chimera
5C1 except for
a single residue in the 198-204 region that is conserved as in the
wild-type
5.
The various
5 mutants in combination with
2 and
2 retained their capacity to bind
[3H]flumazenil with the exception of mutant
5m4, which we chose not to analyze further. Mutants
5m3 and
5m7 lost part of their affinity
for zolpidem compared with
5C1; the amino acids
differing in these mutants compared with
5C1 were
Gly200 and Ser204, respectively. To confirm
that these amino acids were indeed responsible for zolpidem affinity,
an
5m9 (E200G/T204S) that incorporates these two amino
acids in
5 was constructed (Table I). The
5m9 subunit in combination with
2 and
2 had a zolpidem affinity of 829 ± 197 nM, nearly the same as chimera
5C1. This result pinpoints the determinant roles that
1-Gly200 and
1-Ser204 play in zolpidem binding.
The contribution of amino acids 162-164 of
1 is shown
by chimera
5C3, whose affinity for zolpidem could be
explained by a P162T mutation (14). The
5m10
(P162T/E200G/T204S) mutant that integrates both E200G and T204S and
this new substitution was constructed (Table I). The resultant
5m10
2
2 combination had an
affinity of 105 ± 17 nM for zolpidem, corresponding
to more than a 100-fold increase in affinity for this compound compared with that of
5
2
2. A small
3-fold difference of affinity for zolpidem remained compared with that
of
1
2
2, but no other
substitution appeared obvious that might account for this subtle change.
CL218872 Binding (Table I)--
CL218872 displayed a 3-fold
selectivity for the
1
2
2
compared with the
5
2
2
subunit combination (Ki values of 137 ± 25 and
333 ± 17 nM, respectively). The affinities of the
5m8
2
2,
5m9
2
2, and
5m10
2
2 combinations for
CL218872 were examined in [3H]flumazenil competitive
inhibition experiments to check if the same amino acids involved in
high affinity binding for zolpidem would also explain the more limited
subtype selectivity observed with CL218872. Mutant
5m8
(E200G) gave a Ki value of 157 ± 23 nM for CL218872. Also,
5m10 (P162T, E200G,
T204S) gave a Ki value of 127 ± 9 nM for CL218872, again an affinity close to that displayed
by
1
2
2 for this
compound. In contrast,
5m9 (E200G/T204S)
exhibited a significantly lower affinity for CL218872 with a
Ki value of 447 ± 62 nM.
Functional Characterization of
5m10
2
2
Receptor--
Agonists of the BZ site allosterically increase the
apparent affinity of GABAA receptors for GABA (5) and
thereby potentiate chloride currents obtained with submaximal GABA
concentrations. Zolpidem has been shown to maximally potentiate
GABA-induced currents at 1 µM by as much as 384% at
1
2
2 receptors stably
expressed in HEK293 cells, but 10 µM zolpidem is without
effect at
5
2
2 receptors
(17). To study the functional relevance of the three mutations
introduced into the
5 subunit, a whole cell patch clamp analysis of HEK cells cotransfected with
5m10
2
2 was performed. We
wanted to verify not only the responsiveness of the mutant receptor to
GABA but also check whether the gain in affinity for zolpidem observed
in this mutated receptor translated into a potentiation of GABA-induced
currents. The EC50 value for GABA measured on two cells was
4.2 ± 0.4 µM, a value that compares well with those of 4.5 and 4.2 µM observed for recombinant
1
2
2 and
5
2
2 receptors, respectively (24). As shown in Fig. 2,
both diazepam and zolpidem potentiated the Cl
current
induced by GABA at the mutated
5m10
2
2 receptor. The mean
potentiation values recorded from six cells were 108 ± 28 and
139 ± 31% for 1 µM of zolpidem and diazepam,
respectively. The EC50 value of zolpidem (measured on four
cells) was 192 ± 5.2 nM.

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Fig. 2.
Whole cell recording of HEK293 cells
transfected with the
5m10 2 2
mutant. Currents were recorded using a whole cell protocol upon
application of GABA (1 µM; black
bars) and subsequently GABA in the presence of diazepam
(open bars) or zolpidem (hatched
bars).
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DISCUSSION |
All mutations in the
5 subunit were designed to
substitute
5 residues by amino acids in their equivalent
positions in
1. None of these mutations (except mutant
5m4) affected the affinity of the resultant recombinant
GABAA receptor for [3H]flumazenil, the ligand
used as a probe (see Table II). Moreover, all
5 mutants
in this study (except mutant of
5m4) showed an increase in
affinity for zolpidem compared with that of the wild type
5 subunit. This gain of function strategy strongly
suggests that the amino acids identified are involved in zolpidem
binding as opposed to an indirect effect resulting from a
conformational modification. The only exception is mutant
5m4, which unexpectedly conferred no
[3H]flumazenil binding on transfected cells; we currently
do not know with this particular mutant whether the
5m4
polypeptide is destabilized or if an
5m4
2
2 heteromeric receptor
is indeed formed but with conformational change impeding
[3H]flumazenil binding.
Among benzodiazepine site ligands, zolpidem has a strong selectivity
toward
1-containing GABAA receptors. A
chimera constructed by Wieland and Lüddens (14) comprising the
N-terminal part of
6 up to amino acid 158 (with R101H)
together with the rest of the protein exchanged for the corresponding
fragment of
1 showed an excellent affinity for zolpidem
(Ki = 125 ± 50 nM). The zolpidem
binding site on
1 could thus be expected to be localized
principally between
1-Ala160 and the first
putative transmembrane domain of
1 (amino acids 226-246). Three segments differ significantly between
1
and
5 in this region of the protein: 162-164, 172-176,
and 198-204 of
5. We thus generated three chimeras by
substituting these
5 segments by their
1 counterpart.
The
5C1
2
2 combination gave
a Ki value of 244 ± 93 nM for
zolpidem. This represented a 100-fold increase in affinity compared
with the wild-type
5-containing combination, which might correlate with the establishment of an additional hydrogen acceptor interaction (2-3 kcal/mol). Previous data (13, 14, 25) have suggested
that the
1-Gly200 contained in this region
is of some importance for zolpidem binding. However, Gly200
is replaced by a glutamic acid in
3 and
5, but
3 nevertheless retains a medium
affinity for zolpidem (23). Thus, Gly200 by itself is not
sufficient to explain the low affinity of
5-containing receptors for zolpidem. As such, a combination of amino acid
substitutions in the 198-204 region of
5 could play an
additional role, permitting further increases in zolpidem affinity to
be attained. The
5C1 chimera includes this combination
of amino acids and, in addition, others whose presence did not affect
the parameters of zolpidem binding.
Rather than testing all possible combinations, seven mutants were
generated, each having six of the 198-204 amino acids substituted with
their corresponding
1 counterparts. Two mutants
(
5m3 and
5m7) did not confer an affinity
for zolpidem equivalent to that of chimera
5C1 (Table
II). The two amino acids that were unchanged in these mutants revealed
certain substitutions necessary to explain chimera
5C1
properties. The generation of
5m9 confirmed that two
amino acids changes (E200G/T204S) are also sufficient to confer
5 with high affinity zolpidem binding properties (Table
I).
The
5C2
2
2 combination
exhibited a Ki value of 210 ± 52 nM for zolpidem, indicating that amino acids 172-176 play no significant role in zolpidem selectivity. The
5C3
2
2 combination gave a
Ki value of 99.2 ± 53.1 nM for
zolpidem, indicating that amino acids 162-164 are situated at the high
affinity zolpidem binding site. The mutant
5m10
(P162T/E200G/T204S) confirmed our assumption (based on results from
Ref. 14) that
5-Pro162 could have limited
influence on zolpidem affinity.
Alignment of
1 and
5 sequences reveals
that 115 amino acids are different between
1 and
5. Given this difference, our present data highlight the
finding that only three single amino acid substitutions are sufficient
and necessary to confer an
1-like affinity for zolpidem
on
5-containing receptors. Additional evidence discussed
below may favor the hypothesis that
5P162T and
5E200G are involved in conformational changes and
5T204S is implicated in hydrogen bonding between the
hydroxyl group of the serine side chain and an hydrogen acceptor site
in zolpidem (Table II).
Serine
1-Ser204 Interacts with the
Carbonyl Group of Zolpidem (Fig.
3A)--
Serine
1-Ser204 is essential for zolpidem affinity
as observed when comparing mutant
5m7 with
5C1. Since serine residues can form hydrogen bonds with
proton acceptor sites,
1-Ser204 could be
involved in such an interaction with zolpidem. We suggest that this
tandem interaction occurs via the carbonyl group of the acetamide side
chain (Fig. 3A), and other experimental evidence also seems
to support this hypothesis. Thus, NMR conformational studies on
zolpidem and another imidazopyridine, saripidem, has revealed that
zolpidem and saripidem differ in the nature and flexibility of their
side chains (26). That study reported that zolpidem can only adopt one
specific conformation, whereas saripidem has two conformational states,
one similar to that of zolpidem and a second in which the amidomethyl
side chain of saripidem is on another plane (26). Interestingly,
saripidem displays far less selectivity than zolpidem between
1 and
5. These observations suggest that
the common conformations of zolpidem and saripidem are appropriate for
maximal affinity with
1, and the second conformation observed only for saripidem corresponds to a different orientation of
the carbonyl that permits accommodation of the
5
pocket.

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Fig. 3.
Interpretation of mutagenesis results and
modeling of the BZ binding pocket interacting with zolpidem
(A) or CL218872 (B).
Pro162 causes displacement of a hydrophobic area.
Gly200 also changes the conformation of the surrounding
amino acid chain, and Ser204 in particular interacts with
zolpidem (A). Solid lines represent
the 1 binding pocket, and dashed
lines represent the 5 binding pocket.
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The conformation of the carbonyl group present in the zolpidem molecule
indicates that this carbonyl might well interact with Ser204. If this were the case, the carbonyl of saripidem
could interact with the Ser204 of
1 in the
conformation common to zolpidem and saripidem, whereas with
5 the alternative conformational state of saripidem
would interact with another acceptor site (for instance
Ser203 or Thr204).
The carbonyl of zolpidem may thus be a major determinant accounting for
the high selectivity of this compound for
1- compared with
5-containing combinations. Moreover, we suggest
that this interaction might be with
1-Ser204, in which case this serine would be
an essential component of the binding site that predetermines
1
2
2 benzodiazepine subtype selectivity.
Glycine
1-Gly200 Has a Conformational
Role (Fig. 3)--
Glycine is known to confer conformational freedom
to peptidic chains. A glycine residue located close to amino acid
position 200 is a feature common to all
subunits. Nonetheless, this
residue occurs at slightly different positions on these subunits,
thereby possibly contributing to different conformational states of
their BZ binding sites. The
1-Gly200 amino
acid may be in a position that orients the pocket favorably for
zolpidem binding. Since the equivalent glycine residue occurs 2 amino
acids upstream in
3 and
5, this could
probably alter the BZ binding pocket of these subunits unfavorably for
high affinity zolpidem binding. Ser204 could be
inappropriately oriented to interact optimally with the zolpidem
carbonyl group. This could explain why
3 combinations have intermediate affinity (compared with
1 and
5) for zolpidem,
3 bearing
Ser204 and not Gly200. This hypothesis would
correlate with the fact that
5 and
3 (in
which glycine residues occur at equivalent positions) exhibit similar
affinity for CL218872 (23).
On the basis of this potential conformational role of
1-Gly200, one can speculate on reasons
behind the loss of flumazenil binding observed with the mutant
5m4. Since the mutant
5m4 contains an
asparagine from
5 adjacent to Gly200,
instead of being 3 amino acids away from the glycine as in
5, the close proximity of this asparagine to the glycine
residue might disrupt the BZ binding site.
Proline
5-Pro162 Has a Conformational
Role (Fig. 3)--
The increase in affinity for zolpidem obtained with
the
5P162T mutation (
5m10 compared with
5m9) is approximately 5-fold (Table I). Although this
cannot be explained by an additional hydrogen bond, it is known that
proline induces turns and secondary structures. We can thus assume that
this P162T mutation is responsible for a local reorganization of the
zolpidem binding site, presumably allowing a better hydrophobic
interaction with the pyridine heterocycle of zolpidem. Two tyrosines
(
1-Tyr159 and
1-Tyr161) closely situated to this proline
have also been reported to be implicated in benzodiazepine binding (10,
27), one affecting potentiation of GABA by benzodiazepines, the other
affecting flumazenil affinity. These amino acids may interact with
zolpidem, but, since they are present on
1 and
5, substitution mutagenesis would not help to resolve
this point. In the case of CL218872 (Table I), it appears that
Ser204 has a negative effect (mutant
5m9) on
the affinity for CL218872 when the P162 is present. This effect
disappears in the presence of Thr162 (mutant
5m10), thus suggesting that Pro162 may
somewhat narrow the BZ binding pocket. As such, although no clear cut
conclusion can be made regarding the involvement of
1-Thr162 in the BZ binding site, it would
appear that
5-Pro162 alters the pocket configuration.
Key Residues Implicated in CL218872 Binding (Table I, Fig.
3B)--
CL218872, which displays a 3-fold selectivity for
1 compared with
3 and
5,
does not possess a proton acceptor comparable with the carbonyl
function of zolpidem. Nevertheless, CL218872 could be expected to
occupy the BZ binding pocket with a certain degree of similarity to
that displayed by zolpidem, since
1-Gly200
is known to affect the interaction of the BZ binding site with CL218872
(13, 14, 25). Mutant
5m8 (E200G), indeed, conferred high
affinity for CL218872, but the presence of Ser204 somehow
affected affinity for CL218872 in mutant
5m9 (E200G, T204S), and the receptor affinity for CL218872 was again restored in
mutant
5m10 with the P162T mutation. The
5 mutants behaved as if Gly200 was essential
for high affinity toward CL218872, and as if the presence of
Pro162 (mutant
5m9) would change the binding
pocket such that Ser204 interfered with the binding of this
compound. This confirms that
1-Thr162 and
1-Ser204 are situated close to the CL218872
binding pocket even if these residues may not interact directly with
this compound.
Conclusions and Perspectives (Fig. 3)--
Both
and
subunits contribute to the BZ site on GABAA receptors (4),
and it is generally assumed that the binding site is located at the
/
interface (4). The present study takes into account gain of
affinity mutations incorporated into
5 to confer an
1-like affinity for some benzodiazepine site ligands. These data may be associated with previous mutagenesis studies on
and
subunits, mainly the identification of
1-His101 (7) and
2-Phe77 or
2-Met130 (12, 15). The
2-Phe77 substitution has the property to
alter the affinity for different benzodiazepine site ligands or for
only zolpidem depending on the amino acid residue substituted (15).
Based upon this and the current findings of this report, we can now
propose a new molecular model of the
1-selective high
affinity binding site occupied by zolpidem and in part by CL218872. We
suggest that two regions of the binding pocket interact with proton
acceptor sites of the ligand, one being
1-His101 interacting with the nitrogen atom
of the imidazole ring and the other
1-Ser204
interacting with the carbonyl group of zolpidem. In addition, two
sterically-defined hydrophobic regions of
1 would
interact with aromatic moieties of ligands. One hydrophobic region
would be located near
1-Thr162, where it
would interact with the heterocycle of zolpidem. The second hydrophobic
interaction might occur between
2-Phe77 or
2-Met130 and zolpidem; this possibly takes
place through the phenyl in position 2 of the heterocycle of zolpidem,
given that CL218872, which does not possess an equivalent chemical
group, is not affected by changes on these amino acid residues (12,
15).
The specific moieties of zolpidem fitting into the binding pocket
should be confirmed with analogs of zolpidem, whose affinities for our
mutants would need to be tested in light of this new model. The three
amino acids identified in this study are essential components inherent
to the high affinity binding site of
1
subunit-containing receptors that explain the selectivity of this
binding pocket for
1-specific ligands. The
identification of similar residues in other
subunits should help
delineate other GABAA receptor subtype-specific binding
sites and aid in the design of subtype-specific ligands for these
subunits. The availability of such compounds is clearly of crucial
importance in dissecting out the role of the different
GABAA receptors in various GABAergic neuronal circuits in
the brain.