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Vol. 57, Issue 3, 419-426, March 2000
-Aminobutyric
Acid (GABA) Binding at GABAB Receptors: Involvement of
Serine 269 of the GABABR1 Subunit
Centre Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique de Pharmacologie-Endocrinologie, UPR 9023-Centre National de la Recherche Scientifique, Montpellier, France (T.G., L.P., C.J., I.B., J.-P.P.); and TA Nervous system, Novartis Pharma AG, Basel, Switzerland (S.U., J.M., B.M., J.H., W.F., B.B., K.K.).
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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The 
-aminobutyric acid (GABA) receptor type B (GABABR)
is constituted of at least two homologous proteins, GABABR1
and GABABR2. These proteins share sequence and structural
similarity with metabotropic glutamate and Ca2+-sensing
receptors, both of which are sensitive to Ca2+. Using rat
brain membranes, we report here that the affinity of GABA and
3-aminopropylphosphinic acid for the GABABR receptor is
decreased by a factor >10 in the absence of Ca2+. Such a
large effect of Ca2+ is not observed with baclofen or the
antagonists CGP64213 and CGP56999A. In contrast to baclofen, the
potency of GABA in stimulating GTPS binding in rat brain membranes
is also decreased by a factor >10 upon Ca2+ removal. The
potency for Ca2+ in regulating GABA affinity was 37 µM.
In cells expressing GABABR1, the potency of GABA, but not
of baclofen, in displacing bound 125I-CGP64213 was
similarly decreased in the absence of Ca2+. To identify
residues that are responsible for the Ca2+ effect, the
pharmacological profile and the Ca2+ sensitivity of a
series of GABABR1 mutants were examined. The mutation of
Ser269 into Ala was found to decrease the affinity of GABA, but not of
baclofen, and the GABA affinity was found not to be affected upon
Ca2+ removal. Finally, the effect of Ca2+ on
the GABAB receptor function is no longer observed in cells coexpressing this GABABR1-S269A mutant and the wild-type
GABABR2. Taken together, these results show that Ser269,
which is conserved in the GABABR1 protein from
Caenorhabditis elegans to mammals, is critical for the
Ca2+-effect on the heteromeric GABAB receptor.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The
neurotransmitter -aminobutyric acid (GABA) activates both ionotropic
receptors (the GABAA and
GABAC receptors) and G protein-coupled
metabotropic receptors (the GABAB receptors). GABAA and GABAC receptors
are permeable to Cl
ions and mediate, in most
cases, fast inhibition of the postsynaptic neurons.
GABAB receptors mostly couple to pertussis
toxin-sensitive G proteins (Gi or Go) and accordingly inhibit the
activity of adenylyl cyclase and voltage-gated
Ca2+-channels or activate G protein-regulated
inwardly rectifying K+-channels (Kir3) (for
reviews, see Kerr and Ong, 1995
; Bettler et al., 1998). When located on
the postsynaptic element, the GABAB receptors
inhibit neuronal excitability (Kerr and Ong, 1995; Bettler et al.,
1998). GABAB receptors are also found on
glutamatergic or GABA-ergic terminals, where they inhibit
neurotransmitter release. GABAB receptors are
widely expressed in the central nervous system, both in brain and
spinal cord. Accordingly, these receptors play important physiological
roles in pain transmission, absence epilepsy, and drug addiction (Kerr
and Ong, 1995; Bettler et al., 1998).
Two GABAB receptor proteins have been identified:
GABABR1 (Kaupmann et al., 1997) and
GABABR2 (Jones et al., 1998; Kaupmann et al.,
1998b; White et al., 1998; Kuner et al., 1999; Ng et al., 1999).
Neither receptor was found to efficiently couple to G proteins when
expressed alone (Kaupmann et al., 1997, 1998a,b; Martin et al., 1999).
Indeed, GABAB receptors that efficiently activate pertussis-toxin-sensitive G proteins were only obtained in cells coexpressing GABABR1 and
GABABR2 (Jones et al., 1998; Kaupmann et al.,
1998b; White et al., 1998; Kuner et al., 1999; Ng et al., 1999).
Moreover, these two receptors were found to associate at least by their
carboxyl-terminal sequence (White et al., 1998; Kuner et al., 1999),
further suggesting that GABAB receptors function in a heteromeric form. Both GABABR1 and R2
proteins share sequence similarity with family 3 G protein-coupled
receptors (GPCRs) (Bockaert and Pin, 1999), which include metabotropic
glutamate receptors (mGluRs), Ca2+-sensing
receptors (CaSR) and putative pheromone and taste receptors. Like all
family 3 GPCRs, GABAB receptors possess a large
amino-terminal domain structurally related to some bacterial
periplasmic proteins, where ligands bind (Galvez et al., 1999;
Malitschek et al., 1999). This amino-terminal domain is attached to a
heptahelical transmembrane domain that constitutes the G
protein-activating domain, as demonstrated for mGluRs (Pin et al.,
1994; Gomeza et al., 1996a).
Because GABAB receptors share sequence similarity
with mGluRs and CaSRs, two types of receptors sensitive to
Ca2+ ions (Brown and Hebert, 1997; Kubo et al.,
1998; Saunders et al., 1998), we examined a possible regulation of
GABAB receptors by Ca2+
ions. We found that Ca2+ affects the affinity of
various GABAB ligands differently and identified
an amino acid residue in GABABR1 that is critical
for this effect.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
GABA was obtained from Sigma (L'Isle d'Abeau,
France). 125I-CGP64213 was synthesized from
ethyl-(1,1-diethoxyethyl)phosphonate as described elsewhere (Froestl et
al., 1995) and labeled to a specific radioactivity of >2000 Ci/mmol
(ANAWA AG, Wangen, Switzerland). All the other ligands were synthesized
in-house (Froestl et al., 1995). Serum, culture media, and other
solutions used for cell culture were from Life Technologies, Inc.
(Cergy Pontoise, France).
Culture and Transfection of 293 Cells.
Human embryonic
kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's
medium (Life Technologies, Cergy Pontoise, France) supplemented with
10% fetal calf serum and transfected by electroporation as described
previously (Gomeza et al., 1996b). Electroporation was carried out in a
total volume of 300 µl with 10 µg of carrier DNA, plasmid DNA
containing the wild-type or mutated GABABR1 (1 µg) (Galvez et al., 1999), GABABR2 (1 µg)
(Kaupmann et al., 1998a), or Gqi9 (1 µg) (Conklin et al., 1993) and
10 million cells.
Ligand Binding Assay.
Rat brain membranes were prepared as
described previously (Olpe et al., 1990) and stored at 
70°C.
Membranes of HEK 293 cells were prepared as follows: 24 h after
transfection, the cells were transferred into serum-free Dulbecco's
modified Eagle's medium. Forty-eight hours after transfection, the
cells were washed and homogenized in Tris-Krebs buffer [20 mM Tris-Cl,
pH 7.4, 118 mM NaCl, 5.6 mM glucose, 1.2 mM
KH2PO4, 1.2 mM
MgSO4, 4.7 mM KCl, 1.8 mM
CaCl2 (omitted for calcium-free experiments)]
and centrifuged for 20 min at 40,000g. The pellet was
resuspended in Tris-Krebs buffer and stored at 80°C. For ligand
competition assays, thawed membranes (10 µg of protein) were
incubated with 0.1 nM 125I-CGP64213 in the
absence or presence of unlabeled ligands at different concentrations,
as indicated on the figures. This concentration of
125I-CGP64213 is about 10 times lower than the
affinity of this radioligand on the native or
GABABR1 receptor, such that the
IC50 values measured for all unlabeled compounds
are not significantly different from their affinity. The nonspecific
binding was determined using 10 mM GABA or 100 nM CGP54626A. The
incubation was terminated by filtration through GF/C Whatman glass
fiber filters (Whatman International Ltd., Maidstone, England).
GTP35S Assay.
Rat brain synaptic membranes
were prepared as described previously (Olpe et al., 1990) and stored in
70°C. To perform this assay on recombinant receptors, membranes
were prepared from Chinese hamster ovary K1 cells (American Type
Culture Collection, Manassas, VA) stably transfected with
GABABR1b and GABABR2 cDNAs.
Human GABABR1b and rat
GABABR2 constructs [R1 in pcDNA3.1, Invitrogen (San Diego, CA); R2 in pC1-neo (Promega, Madison, WI) were
cotransfected (1:1 ratio of plasmids) using the Superfect transfection
system from Qiagen AG (Basel, Switzerland). Stably transfected
cell clones were selected by double selection with 1 mg/ml geneticin
and 250 µg/ml zeocin. Membranes were thawed using an ice-water bath
and centrifuged at 4°C for 15 min at 20,000g. After
addition of 1.2 ml of ice-cold water, the pellet was shaken, incubated
on ice for 1 h, and centrifuged again. Afterward, the pellet was
homogenized in guanosine 5'-O-(3-thiotriphosphate) (GTPS)
buffer (50 mM Tris-Cl, pH 7.7, 10 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl) using a glass/Teflon homogenizer. Assay mixtures
including membranes (20-30 µg) and test substances were prepared in
Packard-Pico-Plates 96 (300-µl volume), supplemented with 30 µM GDP
(Sigma) (10 µM in experiments conducted with recombinant receptors)
and incubated for 30 min at room temperature. When experiments were
conducted in the presence of Ca2+,
CaCl2 was added at a concentration of 2.0 mM
(final free Ca2+ concentration, 1.8 mM). The main
incubation started by the addition of
[35S]GTPS (Amersham Paisley, UK) to a
final concentration of 0.2 nM (0.3 nM in experiments conducted with
recombinant receptors). Nonspecific binding was measured in the
presence of 10 µM unlabeled GTPS. After 40 min incubation at room
temperature, the amount of bound [35S]GTPS
was assessed by standard procedures such as vacuum filtration through
GF/B filters followed by liquid scintillation counting or scintillation
proximity assay using 1.5 mg of wheat germ agglutinin-coated scintillation proximity assay beads (Amersham) per 250 µl of assay mixture.
Determination of Inositol Phosphate Accumulation. Determination of Inositol phosphate (IP) accumulation in transfected cells was performed after labeling the cells overnight with myo-[3H]inositol (23.4 Ci/mol; NEN, Les Ullis, France). The stimulation was conducted for 30 min in a medium containing 10 mM LiCl and the indicated concentration of agonist. Results are expressed as the amount of IP produced over the radioactivity present in the membranes.
Data Analysis.
The curves were fitted with either GraphPAD
Prism software (GraphPAD Software, San Diego, CA) or Kaleidagraph
software (Abelbeck/Synergy Software, Reading, PA) using the equation
y = {(ymax ymin) / [1 + (×/EC50)nH]) + ymin}, where EC50 is the concentration of the
compound necessary to obtain 50% of the maximal effect and
nH is the Hill coefficient.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Ca2+ Differently Regulates the Affinity of Several
Compounds on Native GABAB Receptors.
The effect of
Ca2+ removal on the affinity of several ligands
interacting with the GABAB receptor was examined
in rat brain membranes using 125I-CGP64213 as
radioligand. This compound has previously been reported to selectively
label GABABR1 proteins in the mammalian brain
(Kaupmann et al., 1997). As shown in Table
1, the potencies of cold CGP64213 and of
CGP56999A, another antagonist, were only slightly affected upon removal
of Ca2+ (1 mM EGTA added). A more pronounced effect of
Ca2+ was, however, observed with the other antagonist
CGP54626A. In contrast, the potency of the agonists GABA and
APPA were decreased by a factor of more than 10 (Table 1). This effect
is not a general effect for all GABABR agonists
because the potency of baclofen was not affected by
Ca2+ removal (Table 1). This indicates that
Ca2+ is necessary for high-affinity binding of
some, but not all, compounds interacting with native
GABABR1. Because of the low concentration of
125I-CGP64213 used (1/10 of its
Kd value), the IC50
values measured for unlabeled compounds are very near their
Ki (affinity) values.
|
S
binding in a dose-dependent manner (Table
2). In the absence of
Ca2+, the EC50 values for
GABA and APPA were increased by a factor 80 whereas that of baclofen
was only slightly affected (Fig. 1). When
the Log(EC50) values for GABA determined at
various Ca2+ concentrations were plotted against
the Ca2+ concentration, an
EC50 value of 37 ± 5 µM was determined
for Ca2+ (Fig. 1a, inset).
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Effect of Ca2+ on the Recombinant GABAB
Receptors.
Because 125I-CGP64213 has been
reported to bind on the GABABR1 subunit but not
on the GABABR2 subunit (Kaupmann et al., 1998a), we first examined the effect of Ca2+ on the
recombinant GABABR1a subunit expressed alone in
HEK 293 cells. As shown in Table 1, the affinities of CGP64213,
CGP56999A, and baclofen were not affected by removal of
Ca2+, but those of GABA and APPA were largely
decreased (Fig. 2 and Table 1). Similar
data were obtained with the GABABR1b splice variant, which lacks the 120 amino-terminal residues that correspond to
the sushi domains of GABABR1a (data not shown)
(Kaupmann et al., 1997; Hawrot et al., 1998).
|
S binding on membranes prepared from
cells stably expressing both GABABR1 and
GABABR2 subunits (Table 2). On these membranes, the effect of baclofen was found to be unaffected by
Ca2+ removal (Table 2). The shift in GABA and
APPA potencies observed upon removal of Ca2+ was,
however, found to be lower on the recombinant receptor than on the
native receptors (Table 2).
Effect of Point Mutations in GABABR1 on the
Ca2+-Effect.
Because Ca2+
affects both the potency measured in a functional assay and the
affinity of GABA and APPA, it is likely that Ca2+
acts directly on the ligand-binding suppress domain of the
GABABR1 protein. We therefore first examined
whether the effect of Ca2+ could still be
observed on a truncated GABABR1 receptor protein that corresponds to the LBP-like domain attached to the plasma membrane
by only one transmembrane segment. We previously reported that such a
truncated receptor still binds 125I-CGP64213
(Malitschek et al., 1999). On such a truncated protein, the affinity of
GABA, but not of baclofen, was also decreased in the absence of
Ca2+ (data not shown), indicating that
Ca2+ interacts on the LBP-like domain of the
GABABR1 protein.
). These two serine residues are conserved in
GABABR1a (Ser247 and Ser246, respectively) (Fig.
3), and using molecular modeling and
mutagenesis, we showed that Ser246 is probably directly involved in the
binding of GABA (Galvez et al., 1999). One would therefore
predict that Ser247 could be responsible for the
Ca2+ effect in GABABR1a. In
addition to Ser247, at least two other serine residues (Ser268 and
Ser269) are also near Ser246 in our three-dimensional model of the
GABABR1a binding site. Interestingly, Ser269 of
GABABR1a aligned with a serine residue of the
CaSR that plays an important role for its activation by
Ca2+ (Bräuner-Osborne et al., 1999) (see
Fig. 3).
|
) (Table
3). Moreover, Ca2+
removal from the incubation buffer resulted in a decreased affinity for
GABA in GABABR1a-S247A and
GABABR1a-S268A mutants but not in the
GABABR1a-S269A mutant (Fig.
4 and Table 3), further indicating that
Ser269 plays a critical role in the Ca2+ effect.
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Ser269 of the GABABR1 Receptor Subunit Plays a Critical
Role for the Ca2+-Effect on the Heteromeric
GABAB Receptor.
The effect of the mutation of Ser269
in GABABR1a was examined on the functional
activity of the heteromeric
GABABR1/GABABR2 receptor.
To that aim, GABABR1a and
GABABR2 were coexpressed in HEK 293 cells
together with the chimeric G protein 
-subunit Gqi9 [Gq in which
nine residues of the carboxyl-terminus have been replaced by those of
Gi (Conklin et al., 1993)]. Under these conditions, the
GABAB receptor was found to stimulate
phospholipase C (PLC), and display pharmacological properties similar
to those measured using other functional assays (Franek et al., 1999). Using this assay, and as observed with other functional readouts, GABA
and baclofen display a similar potency in the presence of 1 mM
Ca2+ (Fig. 5 and
Table 3). In the absence of extracellular Ca2+,
GABA was 10 times less potent than baclofen (Fig. 5a and Table 3), as
observed with the native receptor or the recombinant heteromeric receptor using the GTPS binding assay (Table 2).
|
S binding (see above), it
probably resulted from a Ca2+ requirement in a
step downstream of the G protein activation by the
GABAB receptor. Indeed,
Ca2+ is known to have a positive effect on PLC
activity (Cockcroft and Thomas, 1992) such that the removal of
Ca2+ from the external milieu can affect the
coupling efficacy of a receptor to PLC, leading to a decrease in both
potency and efficacy of agonists. In agreement with this proposal, the
maximal effect not only of baclofen but also of GABA or APPA was
decreased by 2- to 3-fold (Fig. 5A), indicating it is a general feature
of the PLC cascade activation upon stimulation of the receptors by any
agonist. Moreover, the effect of Ca2+ on the
potency of baclofen in stimulating IP formation occurred in a
concentration range (30-100 µM) different from that affecting the
potency of GABA (1-30 µM) in this assay. According to this additional effect of Ca2+ on the PLC activation,
the effect of Ca2+ on the GABA potency on
wild-type and mutated receptors was always compared with that of baclofen.
GABA and baclofen still activated in a dose-dependent manner the
heteromeric GABAB receptors, constituted of the
wild-type GABABR2 and either the
GABABR1a-S247A or
GABABR1a-S268A mutant. These two agonists
displayed a similar affinity on these two mutant receptors in the
presence of Ca2+, whereas GABA became much less
potent than baclofen in the absence of Ca2+
(Table 3). These data are very similar to those obtained with the
wild-type receptor, further indicating that these two serine residues
are not involved in the Ca2+-sensitivity of the
GABA activation of the heteromeric receptor.
In cells expressing the GABABR1a-S269A mutant and
GABABR2, we noticed a decrease by about a factor
10 of the GABA potency but no change in the EC50
value for baclofen compared with the wild-type receptor (Fig. 5 and
Table 3). Interestingly, this difference in the GABA potency mirrors
the change in potency of GABA observed on the wild-type receptor upon
Ca2+ removal. In addition, the relative potency
of GABA over that of baclofen on cells expressing
GABABR1a-S269A and GABABR2
remained unchanged in the absence of external
Ca2+ (Fig. 5b and Table 3). This shows that the
GABABR1a-S269A/GABABR2 heteromer is no longer sensitive to Ca2+.
In brain membranes, we noticed that the removal of
Ca2+ largely decreased not only the affinity of
GABA, but also that of APPA. If Ser269 is playing a critical role for
the Ca2+ effect on the
GABAB receptor, not only the affinity of GABA, but also that of APPA should be affected by this mutation. As shown in
Table 4, this is exactly what we
observed. Moreover, this mutation largely decreased the effect of
Ca2+ on the affinity of APPA (Table 4).
Surprisingly, whereas the affinity of CGP54626A and CGP56999A on either
wild-type or recombinant receptors is slightly decreased upon
Ca2+ removal (Table 1), the mutation of Ser269
into alanine resulted in a large decrease in the affinity of CGP54626A
but not of CGP56999A (Table 4). This revealed that the effect of the
mutation of Ser269 on the affinity of CGP54626A cannot be simply
explained by the suppression of the action of
Ca2+ on the GABABR1
subunit.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The present data show that Ca2+ is necessary for the high affinity of GABA and APPA on native and recombinant GABAB receptors. Surprisingly, the action of baclofen on this receptor does not require Ca2+ ions. Such a specific decrease in GABA and APPA affinities on wild-type receptor observed upon Ca2+ removal was mimicked by mutating the Ser269 of GABABR1a into Ala. Moreover, Ca2+ removal has no further effect on the GABA and APPA affinities on this mutant receptor. These data demonstrate that Ser269 in the GABABR1 subunit is responsible for the Ca2+ effect on the heteromeric GABAB receptor. In agreement with an important role for Ser269 in GABABR1, this specific residue is conserved in both the Caenorhabditis elegans and Drosophila melanogaster GABABR1 receptors (Fig. 3). In contrast, Ser247 and Ser268, which we show are not important for the Ca2+-effect on GABABR1 are not conserved in the C. elegans and D. melanogaster sequences (Fig. 3).
Comparison with CaSR and mGluRs.
Within the different members
of the family 3 GPCRs, Ca2+ has been shown to
regulate the activity of the CaSR and of some mGluRs. In the case of
the CaSR, Ca2+ seems to be sufficient to activate
the receptor (Brown et al., 1993; Hammerland et al., 1999). In the case
of mGluR1, mGluR3, and mGluR5, Kubo et al. proposed that
Ca2+ also directly activates these receptors
(Kubo et al., 1998). However, other authors did not observe such a
direct activation of mGluR1 by Ca2+; rather, they
found that Ca2+ increases the potency of
glutamate to activate the receptor (Saunders et al., 1998). These later
observation are not in contrast to those reported by Kubo et al.,
because it is possible that in Kubo's experiments, the extracellular
glutamate concentration was too low to activate the receptor in the
absence of Ca2+, but became effective upon
addition of Ca2+. Ca2+
seems, therefore, to exert a similar action on some mGluRs and GABAB receptors acting as an allosteric regulator
and increasing the potency of some agonists. However, the apparent
affinity of Ca2+ on mGluRs and
GABAB receptors is quite different, being in the millimolar range for mGluRs (Kubo et al., 1998; Saunders et al., 1998)
and around 37 µM for GABAB receptors,
suggesting that Ca2+ may not interact with these
two receptor types in the same way.
Possible Mode of Action of Ca2+ on
GABABR1.
Ca2+ has also been
shown to act on the extracellular leucine/isoleucine/valine binding
protein (LIVBP)-like domain of the CaSR (Hammerland et al., 1999).
Recently, Ser170 has been reported to play a critical role for the
action of Ca2+ (Bräuner-Osborne et al.,
1999). Interestingly, this residue aligns with Ser269 of
GABABR1a (Fig. 3). However, the difference in
potencies of Ca2+ on these two receptors (37 µM
on GABABR and 3 mM on CaSR), plus the observation
that Ca2+ is sufficient to activate CaSR but not
GABABR, suggest that Ca2+
does not act similarly on these two receptors.
). This residue is Ser166 in mGluR1, which is replaced by an Asp in
mGluR2 (see Fig. 3). Ser166 is very close to the glutamate binding
site, because the -carboxylic and -amino groups of glutamate have
been proposed to interact with Ser165 and Thr188, respectively (O'Hara
et al., 1993) (Fig. 3). In GABABR1a, Ser246
aligns with Ser165 of mGluR1 and has been reported to play a critical
role for the binding of GABA and other GABAB
ligands (Galvez et al., 1999). These two residues align with
Ser79 of LIVBP (Fig. 3), which forms a hydrogen bound with the
-carboxylic group of leucine in the bacterial protein (Sack et al.,
1989), suggesting that the glutamate and GABA binding sites in mGluRs
and GABABR1a may be similar to that of the
leucine binding site in LIVBP. Although Ser166 of mGluR1 is conserved
in both mammalian GABABR1a (Ser247) and
GABABR2 (Ser137), this residue is not involved in
the Ca2+ effect on the
GABAB receptor. Indeed, as summarized above, our data indicate that Ser269 of GABABR1a is
responsible for the Ca2+ effect. Ser269 of
GABABR1a aligns with Thr188 of mGluR1 and Thr102 of LIVBP (Fig. 3). In mGluR1, Thr188 has been shown to interact with
glutamate (O'Hara et al., 1993), and in LIVBP, Thr102 forms a hydrogen
bond with both the -amino and -carboxylic groups of leucine (Sack
et al., 1989). Because GABA lacks the -amino group common to both
leucine and glutamate, two possible modes of action of
Ca2+ on the GABAB receptor
can be proposed. Ca2+ could compensate the lack
of the -amino group of GABA, interacting with Ser269 and allowing a
better positioning of the carboxylic group of GABA for its proper
interaction with Ser246. Alternatively, Ca2+ may
not interact directly with Ser269 but may affect the position of its OH
group, allowing the formation of an additional hydrogen bond with the
carboxylic group of GABA. Further experiments are required to clarify
the mode of action of Ca2+ on the
GABAB receptor.
The observation that baclofen does not require
Ca2+ for high-affinity binding on
GABABR1 may be explained in two ways. One
possibility is that, because of the additional chlorophenyl group of
this molecule, baclofen does not interact the same way as GABA in
GABABR1a. Alternatively, the presence of the
chlorophenyl group may not allow the binding of
Ca2+ on the receptor.
Possible Physiopathological Relevance of the
Ca2+-Effect on GABAB Receptors.
The
potency of Ca2+ on both CaSR and mGluRs is in the
millimolar range (Brown and Hebert, 1997; Kubo et al., 1998);
therefore, it is in the range of the Ca2+
concentrations found in blood and in cerebrospinal fluid. Accordingly, the Ca2+-sensing function of both CaSR and mGluRs
may play important physiological roles. In the case of the CaSR, this
has been pointed out by identifying many point mutations in the CaSR
gene that affect the potency of Ca2+ to activate
this receptor, and that are associated with genetic diseases affecting
the regulation of calcemia (Pollak et al., 1993; Pollak et al., 1994;
Brown and Hebert, 1997). In the case of the GABAB
receptor, we found that the EC50 value for
Ca2+ is 37 µM, such that a significant shift of
the GABA dose-response curve can only be detected when the
extracellular Ca2+ concentration was lowered to
100 µM. In the brain, such low extracellular Ca2+ concentrations can be reached only under
particular conditions, such as activation of glutamate receptors,
especially those of the NMDA type (Pumain et al., 1983; Pumain and
Heinemann, 1985; Pumain et al., 1987), or after epileptic seizure
(Pumain et al., 1983; Heinemann et al., 1986; Armand et al., 1995). If
this occurs in the vicinity of the GABAB
receptors, it will result in a decreased activity of
GABAB receptors, thereby preventing this receptor from being activated by GABA. Because GABAB
receptors regulate both glutamatergic and GABA-ergic synaptic
transmission, it is difficult to predict whether such an effect will
facilitate or inhibit the development of the seizure.
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Acknowledgments |
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We thank Dr. Joëll Bockaert for constant support during this work and Audrey Boncompain and Karin Hofstetter for technical help.
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Footnotes |
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Received July 30, 1999; Accepted October 20, 1999
This work was supported by grants from the "Action Incitative Physique et Chimie du vivant" (PCV97-115), the European Community Biomed2 (BMH4-CT96-0228) and Biotech2 (BIO4-CT96-0049) programs, the Fondation pour la Recherche Médicale, Novartis Pharma (Basel, Switzerland) and the association Retina France (all to J.-P.P.)
Send reprint requests to: Dr. Thierry Galvez, CCIPE-Centre National de la Recherche Scientifique-UPR9023, Laboratoire des Mécanismes Moléculaires des Communications Cellulaires, 141 rue de la Cardonille, F-34094 Montpellier Cedex 5, France. E-mail: galvez{at}ccipe.montp.inserm.fr
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Abbreviations |
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GABA, -aminobutyric acid;
GPCR, G
protein-coupled receptor;
mGluR, metabotropic glutamate receptor;
CaSR, Ca2+-sensing receptor;
HEK, human embryonic kidney;
GTPS, guanosine 5'-O-(3-thiotriphosphate);
IP, inositol phosphates;
APPA, 3-aminopropylphosphinic acid;
LBP, leucine
binding protein;
PLC, phospholipase C;
LIVBP, leucine/isoleucine/valine
binding protein.
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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to that of Gi.
Nature (Lond)
363:
274-276[Medline]. subunit C-termini.
Neuropharmacology
38:
1657-1666[Medline].15, G16 and chimeric Gq/i proteins: Characterization of new antagonists.
Mol Pharmacol
50:
923-930[Abstract]. metabotropic glutamate receptor-mediated signaling.
Neuropharmacology
37:
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), 1 µM (
) 3 µM (
), 10 µM (
), 30 µM (
)
100 µM (
), 1000 µM (
). Data are means ± S.E.M. of a triplicate determination from a typical experiment. Inset
in a: plot of the EC50 value for GABA as a function of
Ca2+ concentration.
strands in
italic, and 
