(Received for publication, November 5, 1996, and in revised form, January 27, 1997)
From the Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland
Recombinant 1
2
2
-aminobutyric acid
type A (GABAA) receptors were functionally expressed
in Xenopus oocytes. Upon the mutation F77L, diazepam and Ro
15-1788 retained the ability to interact with the benzodiazepine
binding site, but zolpidem lost this ability. To quantify these data,
radioligand binding experiments were performed using membrane
preparations of transiently transfected human embryonic kidney 293 cells. The amino acid
77, phenylalanine, was also mutated to
tyrosine, tryptophan, and isoleucine. Although there was little effect
on Ro 15-1788 binding upon mutation to tyrosine, the loss in affinity
for diazepam was from 12 to 2,720 nM. The change to
leucine, in contrast, resulted in little change in the diazepam
affinity, whereas there was a strongly reduced affinity for zolpidem
from 17 to 4,870 nM and for methyl
6,7-dimethoxy-4-ethyl-
-carboline-3-carboxylate (DMCM) from 1.9 to
1,780 nM, respectively. The change to tryptophan resulted
in two-phasic displacement curves, and only about 50% of the
[3H]flunitrazepam binding could be displaced by zolpidem,
DMCM, and Ro 15-1788, respectively, whereas midazolam and diazepam
still resulted in 100% displacement, indicating the presence of two sites upon expression of this mutant receptor. Functional expression in
Xenopus oocytes showed that all mutant channels displayed a comparatively small change (<4.3-fold) in their apparent agonist affinity and that these channels could still be functionally modulated by ligands of the benzodiazepine binding site. We conclude that subtle
changes in
F77 drastically affect benzodiazepine pharmacology and
that this residue probably interacts directly with most ligands of the
benzodiazepine binding site and therefore defines part of the
benzodiazepine binding pocket.
GABAA1 receptors are the major ion channels in mammalian brain conferring neuronal inhibition. Two subunits have initially been purified (1), and their coding DNA has been cloned (2). Later, a total of 15 mammalian subunits have been cloned (for reviews, see Refs. 3-7). They are homologous to subunits of the nicotinic acetylcholine receptor, of the glycine receptor, and the serotonin type 3 receptor, and it is assumed that the natural receptor is a pentameric protein (8).
The GABAA receptor is the site of action of benzodiazepines
and related compounds (Fig. 1; for review, see Ref. 6).
There is a widespread use of some benzodiazepines for their anxiolytic, sedative, muscle relaxant, and anticonvulsive properties, and the
structural determinants underlying benzodiazepine action are of
interest. Clinically used, sedative benzodiazepines act as positive
allosteric modulators of the receptor. The 1 subunit has been
described as the major subunit that is photoaffinity labeled by
[3H]flunitrazepam (9), and one major labeled amino acid
has been identified (10). In agreement with these observations,
in vitro binding studies identified several amino acid
residues in
1,
3,
4, and
6 to be involved in benzodiazepine
binding (11-14). In addition, a
subunit is required for functional
modulation of the channels by benzodiazepines (15, 16). Point mutations in the
2 subunit also affect the benzodiazepine pharmacology (17,
18) in these functional experiments. The view that
subunits are
essential for the formation of the benzodiazepine binding site was
recently confirmed with
2-less mice (19). Thus,
and
subunits
are both thought to contribute to the benzodiazepine binding site, but
its structural localization remains to be described.
The imidazopyridine zolpidem selectively binds to 1-containing
GABAA receptors and is able to displace diazepam there
(20). The
2 and
3 subunits confer intermediate zolpidem affinity
and the
5 subunit very low affinity to triple subunit combinations
x
2
2 (21-23). That the
subunit may indeed
contribute to the zolpidem site is substantiated by the observation
that zolpidem displays very low affinity to
3, in contrast to
2-containing receptors (24, 25).
Recently, we have identified the amino acid side chains 1 161 and
1 206, and
2 77 as important for increased stimulatory effects of
diazepam in
1
2
2 receptor channels expressed in
Xenopus oocytes at the functional level (18). Interestingly,
F77L resulted in an almost complete loss of zolpidem
effects. We attempted to understand this phenomenon better. We report
drastic consequences of four different point mutations of the wild type
phenylalanine in the
2 subunit for ligand specificity of the
benzodiazepine binding site. Some binding data indicate the presence of
two benzodiazepine binding sites. A probable way of interaction of the
wild type channel with different ligands is discussed. Our work
represents an initial step leading to a rational drug design based on
the identification of structural determinants on the receptor
protein.
The cDNAs coding for
the 1,
2, and
2S subunits of the rat GABAA
receptor channel have been described elsewhere (26-28). The mutant
F77L has a phenylalanine to leucine substitution at position 77 of the mature peptide and has been described before (18,
29). The mutant subunits
F77Y,
F77I, and
F77W were prepared using the QuikChangeTM
mutagenesis kit (Stratagene). For cell transfection, the cDNAs were
subcloned into the polylinker of pBC/CMV (30). This expression vector
allows high level expression of a foreign gene under control of the
cytomegalovirus promoter. The
subunit was cloned into the
EcoRI, and the
and
subunits were subcloned into the
SmaI site of the polylinker by standard techniques.
Xenopus
laevis oocytes were prepared, injected, defoliculated, and
currents recorded as described (18, 31). Briefly, oocytes were injected
with 50 nl of capped, polyadenylated cRNA dissolved in 5 mM
K-HEPES (pH 6.8). This solution contained the transcripts coding for
each of the different subunits at a concentration of 100 nM
(calculated from the UV absorption) to allow injection of about
stoichiometric amounts. Electrophysiological experiments were performed
by the two-electrode voltage-clamp method at a holding potential of
80 mV. To quantify GABA sensitivity, agonist concentrations between
0.03 and 10,000 µM were applied for 20 s, and a
washout period of 4-15 min was allowed to ensure full recovery from
desensitization. Current responses were fitted to the Hill equation:
I = Imax/(1 + (EC50/[A])n, where I
is the current amplitude at a given concentration of GABA
(A), Imax is the maximum current
amplitude, EC50 is the concentration of agonist yielding
half-maximal current amplitudes, and n is the Hill
coefficient. Allosteric potentiation via the benzodiazepine site was
measured at a GABA concentration eliciting 5-15% of the maximal GABA
current amplitude by coapplication of GABA and the drugs acting at the
benzodiazepine binding site. Ro 15-1788, in contrast to the other
ligands of the benzodiazepine binding site used here, developed its
effects not instantaneously. Therefore, it was preapplied for 30 s
to ensure a rapid onset of action upon perfusion with GABA. Unless
mentioned otherwise, oocytes were only exposed to a single drug in
addition to GABA, to avoid contamination, and the perfusion system was
cleaned by washing with dimethyl sulfoxide for the same reason.
The cells were maintained in minimum essential medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin by standard cell culture techniques. Equal amounts (total of 20 µg of DNA/90-mm dish) of plasmids coding for GABAA receptor subunits were transfected into human embryonic kidney 293 cells (ATCC CRL 1573) by the calcium phosphate precipitation method (32). After overnight incubation, the cells were washed twice with serum-free medium and refed with medium. Wild type receptor and triple subunit combinations expressed well as indicated by the respective Bmax of 1.1-6.0 pmol binding sites/mg of protein, with an average of 2.9 ± 1.4 pmol, as determined by radioligand binding assays. An exception was the tryptophan mutant, which resulted in the expression of 0.59-0.64 pmol binding sites/mg of protein.
Membrane PreparationApproximately 60 h after
transfection the cells were harvested by washing with ice-cold
phosphate-buffered saline (pH 7.0) and centrifuged at 150 × g. Cells were washed with buffer containing 10 mM potassium phosphate, 100 mM KCl, 0.1 mM K-EDTA (pH 7.4). Cells were homogenized by sonication in
the presence of 10 µM phenylmethylsulfonyl fluoride and 1 mM EDTA. Membranes were collected by three
centrifugation-resuspension cycles (100,000 × g for 20 min) and then used for ligand binding or stored at 20 °C.
Resuspended cell membranes (0.2-0.5 ml) were incubated for 90 min on ice in the presence of [3H]Ro 15-1788 (87 Ci/mmol, DuPont NEN) or [3H]flunitrazepam (86 Ci/mmol, DuPont NEN) and various concentations of competing ligands. Membranes (20-80 µg of protein/filter) were collected by rapid filtration on GF/C filters presoaked in 0.3% polyethyleneimine. After three washing steps with 4 ml of buffer, the filter-retained radioactivity was determined by liquid scintillation counting. Nonspecific binding was determined in the presence of 10 µM Ro 15-1788 or 10 µM flunitrazepam, respectively. Data were fitted by using a nonlinear least squares method to the equation B(c) = Bmax · c/(Kd + c) for binding curves and B(c) = Bmax/(1 + (c/IC50)n) for displacement curves with a single component, where c is the concentration of ligand; B, binding; Bmax, maximal binding; Kd, dissociation constant; and n, Hill coefficient. Displacement curves IC50 values were converted to Ki values according to the Cheng-Prusoff equation (33). In the case of the tryptophan mutant, for some ligands assuming two components yielded the better fit. Protein concentration was determined with the Bio-Rad protein assay kit with bovine serum albumin as standard.
The dual subunit combination 1
2 or triple subunit
combinations
1
2
2 were functionally expressed in
Xenopus oocytes and characterized using electrophysiological
techniques. Diazepam displays approximately a 3-fold enhancement of the
stimulation of GABA currents after the point mutation F77L in the
subunit, whereas zolpidem almost lost the ability to affect GABA
currents (18; Table I). The effects of all compounds
tested were independent of the size of the current amplitude expressed
from a given subunit combination or the time point after injection of
the oocytes with the corresponding cRNAs.
|
It was interesting to know whether zolpidem fails to bind mutated
channels or whether the zolpidem still binds and only fails to
stimulate GABA currents in these channels. To answer this question, we
performed competition experiments and tried to counteract the stimulatory effects of 1 µM diazepam with increasing
amounts of zolpidem or of the antagonist Ro 15-1788 in cumulative
dose-response curves, respectively (data not shown). 1 µm Ro
15-1788 did not alter GABA responses in mutant and wild type channels.
In Fig. 2A it is shown that 1 µM Ro 15-1788 can completely reverse the stimulatory
effect of diazepam. In contrast to this observation, the effect of 0.3 µM diazepam cannot be reduced by 3 µM
zolpidem (Fig. 2B and Table I). This lower concentration of
diazepam was chosen from a dose-response curve for the stimulation of
GABA currents by diazepam and results in about 70% of the maximal
stimulation in mutant channels (18). Based on our experiments, we
conclude that Ro 15-1788 can but zolpidem cannot compete with diazepam in mutant F77L channels.
Point Mutation of
These electrophysiological experiments resulted in a good
description of functional effects, but only in a qualitative assessment of the interaction with ligands of the benzodiazepine binding site with
the GABAA receptor. A quantitative description of the binding was obtained using equilibrium binding of [3H]Ro
15-1788 to membrane preparations of human embryonic kidney cells
transiently transfected with 1
2
2 GABAA receptor
cDNAs or transfected with the triple combination containing the
mutant
F77L subunit. Binding occurred in each case to a
single class of binding sites (Fig. 3A). The
affinity for Ro 15-1788 was reduced about 28-fold in the mutant
channels compared with the wild type combination (Table
II). However, it should be noted that the affinity for
this ligand was still relatively high (17 nM). The binding specificities of wild type and mutant receptors were analyzed for a
number of other modulatory drugs acting at the benzodiazepine binding
site. Data are summarized in Table II. The affinity for the
non-benzodiazepine ligands tested was most affected, displaying Ki values above 1 µM. In contrast,
diazepam retained medium affinity (about an 8-fold decrease). Most
remarkably, the affinity of the
-carboline DMCM was decreased
940-fold for the mutant receptor (Fig. 3B and Table II). The
affinities for zolpidem and Cl 218872 were reduced 325- and 130-fold,
respectively. Best fit was obtained assuming a Hill coefficient of 1.0 in all cases. Thus, all drugs tested displaced [3H]Ro
15-1788 apparently by binding to a single, noncooperative site and
displaced the radioactive ligand to the same extent as high
concentrations of nonradioactive Ro 15-1788.
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Further point mutants were then prepared and
studied in the same way. Fig. 4 shows a similar
experiment comparing the binding of [3H]Ro 15-1788 to
wild type receptors and receptors containing the mutated
F77Y subunit. Although there was very little effect on Ro 15-1788 affinity (Fig. 4A), the displacement of
[3H]Ro 15-1788 by diazepam (Fig. 4B) was
drastically affected, showing a 230-fold decrease in diazepam affinity
(Fig. 4B and Table II). Interestingly, the same mutation
resulted in an about a 7- and 3-fold increase in Cl 218872 and zolpidem
affinity, respectively (Table II). Besides the mentioned ligands,
midazolam, flunitrazepam, and DMCM were also characterized in the same
way, and the results are summarized in Table II. All curves were fitted
satisfactorily assuming a Hill coefficient of 1.0.
[3H]Ro 15-1788 displayed no specific binding after expression of the isoleucine mutant-containing triple subunit combination. Using [3H]flunitrazepam, we obtained evidence for the presence of a single type of binding site with almost the same affinity as wild type receptors (Table II). Comparison of receptors containing isoleucine and leucine instead of the wild type phenylalanine surprisingly shows little difference in apparent diazepam affinity, whereas there is 74-fold difference in Ro 15-1788 affinity and a 2,020-fold difference to wild type receptors (Table II). Other non-benzodiazepines displayed no measurable affinity to the isoleucine-containing mutant. The loss in DMCM affinity of >5,000-fold for the change from wild type to the isoleucine mutant is the largest effect observed here (Table II). Again, all curves were fitted satisfactorily assuming a Hill coefficient of 1.0.
Point Mutation[3H]Ro 15-1788 displayed again no specific binding after expression of this triple subunit combination. Saturation binding curves using [3H]flunitrazepam revealed at least a 6-fold reduced affinity compared with wild type receptors, with a Kd of 18 nM. Displacement curves were in this case measured at 6 nM. As indicated under "Materials and Methods," this mutant resulted in about 20% of the normally observed expression. A tryptophan residue in this position may interfere somewhat with the biosynthesis or assembly in this expression system.
Whereas midazolam and diazepam displaced radioactive flunitrazepam to
the same extent as did nonradioactive flunitrazepam, zolpidem, Ro
15-1788, and DMCM displaced only 40-60%. The affinity of zolpidem for
this partial displacement was about the same as in the wild type, and
the affinities for DMCM and Ro 15-1788 were strongly decreased (Table
II). The displacement curve for diazepam was fitted with a Hill
coefficient of 0.64, which may indicate the presence of more than one
site having a slightly different affinity for diazepam. Cl 218872 almost lost the ability to bind to the mutated receptor (Table II). The
displacement curves for diazepam, zolpidem, and Cl 218872 are
illustrated in Fig. 5. Obviously, there are two binding
sites with different properties present. The implications of this
heterogeneous behavior is discussed further below.
Functional Properties of Mutant Channels
Triple subunit
combinations were expressed functionally in
Xenopus oocytes. All mutants analyzed expressed
GABA-activated chloride currents of maximal current amplitude
comparable to those of wild type channels. Only a small decrease
(factor of 1.6-4.3) in the apparent GABA affinity (EC50)
was observed between wild type and mutant channels (Table
III). For the leucine mutant about a 5-fold reduced
agonist affinity has been described before (29). GABA currents by wild
type and mutant channels were stimulated by coapplication of 0.3 µM diazepam together with 1-3 µM GABA, indicating that all mutants studied here retain allosteric coupling to
the agonist site. As expected for this concentration of diazepam, receptors containing the tyrosine substitution displayed a
reduced response to diazepam but retained strong stimulation by 1 µM zolpidem (Table III).
|
This study demonstrates that a single amino acid in position 77 of
the mature 2 subunit of
1
2
2 GABAA receptors
drastically affects the pharmacology of benzodiazepine site ligands.
Interestingly, this residue is directly homologous to
1F64, which
has been implied in agonist binding (29, 34). In the following we
discuss results of a functional study, of binding experiments, and of
displacement of binding by several compounds representative for the
various classes of benzodiazepine site ligands, for four different
mutations in this position.
Expression in Xenopus oocytes of the
F77L mutant together with
1 and
2 subunits results
in ion channels displaying a 3-fold increased stimulatory effect of
diazepam compared with wild type channels, whereas the effect by
zolpidem nearly disappears. Functional competition experiments indicate
that upon mutation the antagonist Ro 15-1788 competes with diazepam in
the mutated channel, but zolpidem has lost this ability. Thus, zolpidem
apparently fails to bind to mutated channels, whereas Ro 15-1788 still
seems to bind to these channels.
This interpretation is directly supported by binding
experiments to membrane preparations of transiently transfected cells. Although the affinity for zolpidem is reduced about 325-fold in the
above mutant, the affinity for the antagonist Ro 15-1788 is reduced
only 28-fold. From these data, it becomes clear that the concentrations
of the ligands used in functional competition experiments were too low
to expect displacement of diazepam in the case of zolpidem.
Interestingly, the affinity for the -carboline DMCM was almost 3 orders of magnitude lower than in wild type channels, therefore
displaying the largest effect after mutation of
F77 to leucine. Of
all the ligands tested, the affinity to diazepam was the least
affected. Its binding affinity was reduced only about 8-fold, and it
retained a medium affinity. Preliminary functional data show that 1 µM flunitrazepam similar to diazepam increases potentiation of GABA-induced currents and therefore indicate that the
mutant channel still has a considerable affinity for this benzodiazepine. This suggestion was also confirmed with binding studies. Interestingly, it appears that the affinities of all benzodiazepines tested are still well below 100 nM after
mutation to leucine, whereas the affinities of non-benzodiazepines are much lower (µM range).
At the level of binding experiments the leucine mutant
was compared with the isoleucine mutant. Although both mutant receptors displayed an affinity toward flunitrazepam similar to that of the wild
type, a small decrease in diazepam affinity was observed. However, the
affinity for Ro 15-1788 was affected much more strongly in the
isoleucine (2,020-fold) than in the leucine (28-fold) mutant. Thus, a
simple shift of a methyl group by one carbon atom leads to a 74-fold
change. It would be interesting to study the alanine mutant to see
whether this is the result of steric hindrance. The 1 subunit has an
isoleucine residue in the position homologous to 77 in the
2 subunit
and also has no measurable affinity for Ro 15-1788 and DMCM; but it
still displays relatively high affinity for flunitrazepam. This
behavior is reminiscent of our isoleucine mutation. It would be
interesting to change the isoleucine in
1 into a phenylalanine and
study the consequences for the Ro 15-1788 affinity.
The phenylalanine in position 77 of the subunit
was also replaced by a more bulky amino acid. The change to tyrosine,
i.e. the introduction of an additional hydroxyl group, left
the affinity for Ro 15-1788 almost unaffected and even increased the
affinities for zolpidem and Cl 218872, whereas the affinities for
diazepam, flunitrazepam, midazolam, and DMCM were drastically
decreased. This indicates that an entity in the latter, but not in the
former compounds is not tolerated by the substituted aromatic ring.
Furthermore, the interaction of
77 with classical benzodiazepines
and the antagonist Ro 15-1788 is different.
Replacement of phenylalanine by tryptophan yielded
binding data that could be interpreted as the presence of two binding
sites for ligands of the benzodiazepine site on a receptor pentamer (see "Results"). This finding was surprising, as there was no indication from our binding data on other mutants for a positive or
negative cooperativity between two sites. The properties of the two
binding sites are very different from each other. If a 2 subunit
always takes part in the formation of a benzodiazepine binding site and
there is only one drug binding site/
subunit interface, this
would indicate the presence of two
2 subunits in a receptor
pentamer. Alternatively, the alteration at the
2 subunit containing
site would have to be communicated by allosteric mechanisms to the
2
subunit-independent site.
An alternative explanation for our data would be the existence of a second pool of receptors with different binding properties. It is however intriguing that the relative abundance of the two populations was about 1:1 in all experiments. On a functional level, after expression of the mutant channels in Xenopus oocytes, no evidence was obtained pointing to a heterogeneity of this site. Namely, cumulative dose-response curves of the stimulation of GABA currents by diazepam did not point to the presence of multiple sites (data not shown).
The presence of more than one binding site on a receptor has also been postulated on the basis of an altered rate of dissociation by the same or other ligands in cerebellum (35). However, as shown by Prinz and Striessnig (36), this cannot be taken as conclusive evidence for the presence of multiple sites. The best indication for the presence of multiple sites has been provided by photoaffinity labeling with [3H]flunitrazepam of brain extracts, where it has been shown that covalent binding of one molecule destroys about four sites for reversible binding (37, 38). The presence of more than one binding site on a receptor for negative allosteric modulators is also suggested from the two-phasic response to these agents in functional measurements of receptors expressed in Xenopus oocytes (39).
Functional Properties of the Mutant ChannelsAfter functional expression in Xenopus oocytes all mutant receptors formed channels with a slightly (1.6-4.3-fold) reduced apparent affinity for the agonist GABA. The agonist binding site is allosterically coupled to the benzodiazepine binding site. In view of the relatively small changes in the apparent binding affinities for the agonist and the huge changes in the observed binding affinities for ligands of the benzodiazepine binding site, we think it more likely that the latter site is affected directly. All mutated channels were also stimulated by ligands of the benzodiazepine binding site, indicating that the allosteric interaction between this site and the agonist site was preserved.
Interaction of Ligands of the Benzodiazepine Binding Sites with the ReceptorIt is clear that there are large effects of the
substituent present in position 77 of the 2 subunit for the
specificity of the benzodiazepine binding site. Obviously, the
non-benzodiazepines prefer an aromatic substituent in position 77, and
addition to the phenyl ring of a hydroxyl group even increases the
affinity for zolpidem and Cl 218872. Exchange of the aromatic ring by
smaller side chains results in much larger effects for
non-benzodiazepines (130->5,000) than for diazepam, flunitrazepam, and
midazolam (1.5-8.3). Remarkably, the loss in affinity of mutant
receptors is always different between this group of three
benzodiazepines and Ro 15-1788. We suggest that
77 interacts
directly with benzodiazepine binding site ligands. Obviously,
different ligands interact in a different fashion with the wild type
phenyl residue, but it is of course unlikely that chemically distinct
ligands have identical interactions with the receptor site. As both
types of ligand compete for binding, their respective binding sites
have to overlap.
Small chemical changes in position 77 of the subunit have huge effects on the benzodiazepine pharmacology. The
analysis of the molecular design of the benzodiazepine binding sites
should help in the understanding of how these ligands bind to the
GABAA receptor. Such an analysis may provide a rational
basis for drug design. The phenylalanine identified here appears to be
a key determinant for the activity of zolpidem, DMCM, Cl 218872, and the binding of the antagonist Ro 15-1788 and must be very close to or
part of the diazepam binding site of GABAA receptors.
We are grateful to Professor H. Reuter, in whose institute this work was carried out, for continuous encouragement.