Abstract
The compounds CGP7930 [2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol] and its close analog CGP13501 were identified as positive modulators of γ-aminobutyric acidB (GABAB) receptor function. They potentiate GABA-stimulated guanosine 5′-O-(3-[35S]thiotriphosphate) (GTPγ[35S]) binding to membranes from a GABAB(1b/2) expressing Chinese hamster ovary (CHO) cell line at low micromolar concentrations and are ineffective in the absence of GABA. The structurally related compounds propofol and malonoben are inactive. Similar effects of CGP7930 are seen in a GTPγ[35S] binding assay using a native GABAB receptor preparation (rat brain membranes). Receptor selectivity is demonstrated because no modulation of glutamate-induced GTPγ[35S] binding is seen in a CHO cell line expressing the metabotropic glutamate receptor subtype 2. Dose-response curves with GABA in the presence of different fixed concentrations of CGP7930 reveal an increase of both the potency and maximal efficacy of GABA at the GABAB(1b/2) heteromer. Radioligand binding studies show that CGP7930 increases the affinity of agonists but acts at a site different from the agonist binding site. Agonist affinity is not modulated by CGP7930 at homomeric GABAB(1b) receptors. In addition to GTPγ[35S] binding, we show that CGP7930 also has modulatory effects in cellular assays such as GABAB receptor-mediated activation of inwardly rectifying potassium channels in Xenopus laevis oocytes and Ca2+ signaling in human embryonic kidney 293 cells. Furthermore, we show that CGP7930 enhances the inhibitory effect ofl-baclofen on the oscillatory activity of cultured cortical neurons. This first demonstration of positive allosteric modulation at GABAB receptors may represent a novel means of therapeutic interference with the GABA-ergic system.
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. It activates two classes of receptors: ionotropic, chloride-permeable GABAA receptors and metabotropic GABAB receptors. The structure and function of GABAB receptors have been reviewed extensively (Bettler et al., 1998; Marshall et al., 1999; Bowery and Enna, 2000; Couve et al., 2000; Jones et al., 2000; Kuriyama et al., 2000; Marshall, 2000). The GABAB receptor is a member of the “family 3” G-protein-coupled receptors (GPCRs) (reviewed in Couve et al., 2000), which also comprises metabotropic glutamate receptors (mGluRs), the calcium-sensing receptor, and a group of mammalian vomeronasal and candidate taste receptors (Hoon et al., 1999). Like the other members of this family, the GABAB receptor has a molecular structure that is characterized by its seven transmembrane-spanning domains and a large extracellular N-terminal ligand binding domain related to periplasmic bacterial amino acid binding proteins. GABAB receptors modulate the activity of inwardly rectifying potassium channels and high voltage-activated calcium channels. Furthermore, they also inhibit adenylate cyclase activity in native and recombinant systems. By these mechanisms, they act post- and presynaptically to inhibit neuronal excitability and neurotransmitter release, respectively. A thorough molecular investigation of GABAB receptors was initiated by the cloning of a first receptor protein GABAB(1), which exists in two N-terminal splice variants, 1a and 1b (Kaupmann et al., 1997). Unexpectedly, however, heterologous expression of GABAB(1) receptor protein has not made possible the measurement of robust functional responses. This finding has remained unexplained until the discovery that the formation of heterodimeric assemblies between GABAB(1) and a novel GABAB(2) protein is a prerequisite to form functional GABAB receptors (Jones et al., 1998;Kaupmann et al., 1998; White et al., 1998; Kuner et al., 1999).
Allosteric modulation of GABAB and some mGluR receptors by calcium has been described previously (Kubo et al., 1998;Saunders et al., 1998; Wise et al., 1999; Galvez et al., 2000a). The calcium sensing receptor is, in turn, allosterically activated by amino acids (Conigrave et al., 2000). Furthermore, noncompetitive inhibitors of “group I” mGluRs acting at a site distinct from the agonist binding site have also been found (for reviews, see Pin et al., 1999;Spooren et al., 2001). However, no allosteric modulation of GABAB receptor activity by low-molecular-weight organic compounds has been observed to date. This study describes two molecules with such effects, CGP13501 and CGP7930 (Fig.1). Positive allosteric modulators act synergistically with an agonist, but have no intrinsic efficacy on their own. Thus, they act only when and where the endogenous agonist is present and thus have more physiological effects than pure agonists, which activate receptors independently of synaptic activity. Therefore, positive allosteric modulators are expected to have a better side effect profile than conventional agonists and thus are of considerable therapeutic interest.
Materials and Methods
Stable Transfection and Culture of CHO Cell Clones.
Chinese hamster ovary K1 (CHO-K1) cells were stably transfected with GABAB(1b) and GABAB(2)cDNAs. Human GABAB(1b) [in pcDNA3.1, (Invitrogen, Carlsbad, CA)] and rat GABAB(2)[in pC1-neo (Promega, Madison, WI)] constructs were cotransfected (1:1 ratio of plasmids) using the Superfect transfection system from QIAGEN AG (Basel, Switzerland). Stably transfected cell clones were selected and cultured in Dulbecco's modified eagle medium (glutamine-free Dulbecco's modified Eagle's medium; Invitrogen) supplemented with 10% fetal calf serum, 20 μg/mll-proline, 400 μg/ml l-glutamine, 1 mg/ml geneticin, 250 μg/ml zeocin. The cells were grown to 80 to 90% confluence in 14-cm cell culture dishes. For some specificity experiments, a CHO cell line stably expressing the mGluR2 metabotropic glutamate receptor (Flor et al., 1995) was also used.
Preparation of Membranes from CHO Cells.
The culture dishes were washed twice with ice-cold HEPES buffer, pH 7.4. Buffer was added and the cells were scraped off. Crude membranes from several dishes were collected in a 50-ml tube and centrifuged at 4°C for 20 min at 15,000 rpm in an SS34 rotor (Sorvall, Newton, CT). The pellet was resuspended in buffer and homogenized using a glass-glass homogenizer (10 strokes). Afterward, the suspension was centrifuged (18,000 rpm, 30 min, 4°C), and the pellet was resuspended in a small volume of buffer and homogenized again (20 strokes). Aliquots were frozen in liquid nitrogen and stored at −80°C. On the day of the experiment, the frozen membranes were thawed and then centrifuged for 10 min at 15,000 rpm and 4°C. The pellet was resuspended in 1 ml of ice-cold distilled water and incubated for 1 h on ice. After a further centrifugation as before, the final pellet was resuspended in the appropriate amount of assay buffer (see below).
Preparation of Rat Brain Membranes for Native Receptor Assays.
Membranes from rat brain cortex were prepared as described in detail earlier (Olpe et al., 1990).
GTPγ[35S] Assay.
The composition of the assay mixtures [in a final volume of 250 μl in 96-well, clear-bottomed microtiter Isoplates (PerkinElmer Wallac, Turku, Finland)] was as follows: 50 mM Tris-HCl buffer, pH 7.7, 10 mM MgCl2, 0.2 mM EGTA, 2 mM CaCl2, 100 mM NaCl, 10 μM guanosine 5′-diphosphate (30 μM with rat cortical membranes; Sigma Chemical, Buchs, Switzerland), 50 μl of the membrane suspension described above (approximately 10–20 μg of protein), 1.5 mg of wheat germ agglutinin-coated SPA beads (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK), 0.3 nM [35S]GTPγS (∼1000 Ci/mmol, stabilized solution; Amersham Pharmacia Biotech), and the test compounds (agonists and/or modulators) at the appropriate concentrations. Nonspecific binding was measured in the presence of unlabeled GTPγS (Sigma) in excess (10 μM). The samples were incubated at room temperature for 60 min before the SPA beads were sedimented by centrifugation at 2600 rpm for 10 min. The plates were then counted in a Wallac 1450 MicroBeta liquid scintillation counter. For data analysis, nonspecific binding was subtracted from all the other values; the effects of GABA and modulators were expressed relative to basal activity, measured in the absence of agonist. Concentration-response curves were analyzed by nonlinear regression. Prism 3.0 software (GraphPad Software, San Diego, CA) was used for all data calculations.
Radioligand Binding Experiments.
The protocols for measuring the binding of the radioligands [3H]CGP62349 (a competitive antagonist) and [3H]APPA ([3H]CGP27492, an agonist ligand) were based essentially on methods described previously (Olpe et al., 1990; Hall et al., 1995; Bittiger et al., 1996). The [3H]CGP62349 binding assay was performed in the SPA format; in the [3H]APPA binding assay, bound and free radioligand were separated by centrifugation. Saturation and displacement curves were analyzed by nonlinear curve fitting to the appropriate models and using Prism 3.0 software.
Measurement of Change in Intracellular Calcium Concentration by Fluorometry.
For measurement of changes in intracellular calcium concentrations, HEK293 cells were transiently transfected with GABAB(1b/2a). All transfections included Gαqo5 to couple GABABreceptors to phospholipase C (Franek et al., 1999) and were made as described in detail previously (Pagano et al., 2001). Transfected HEK293 cells were plated into poly-d-lysine coated 96-well plates (BD Biosciences, San Jose, CA). Twenty-four to seventy-two hours after transfection, cells were loaded for 45 min with 2 μM fluo-4 AM (Molecular Probes, Eugene, OR) in HBSS (Invitrogen) containing 50 μM probenecid (Sigma). Plates were washed twice in the incubation buffer (HBSS) and transferred to a fluorescence imaging plate reader (FLIPR; Molecular Devices, Sunnyvale, CA). Fluorescence was measured at room temperature for 3 min after the addition of CGP7930 to check for agonistic effects of the compound. A second recording period of 3 min was initiated 10 min after the start of the first measurement. CGP7930 was present from the start, and 1 μM GABA in HBSS was added at 20 s after the start of the second reading. Relative fluorescence changes over baseline (ΔF/F) were determined. Concentration-response curves were recorded with three to eight wells per concentration and experiment; the data were pooled and fitted using Igor Pro (Wavemetrics, Lake Oswego, OR) with a logistic equation using weighted nonlinear regression.
FLIPR Experiments on Neuronal Networks.
Primary cultures of cortical neurons were prepared from embryonic day 16 to 18 Sprague-Dawley rats (Wang and Gruenstein, 1997). Dissociated cells were plated on poly-l-lysine coated plates and incubated at 37°C in 5% CO2 for 7 to 10 days. About 15 min before experiments, the culture medium was removed and cells were loaded with 2 μM fluo-4 AM in HBSS supplemented with 10 mM HEPES, pH adjusted to 7.4. After loading, cells were washed twice in the incubation medium (HBSS without Mg2+) and then transferred to the fluorescence reader. Fluorescence was measured at room temperature and at a sampling rate of 0.5 Hz. Drugs were dissolved in HBSS without Mg2+ and added to the cultures during recording. Oscillations were analyzed using IgorPro by peak detection and calculation of the ratio of peak frequencies before and after compound addition.
Oocyte Electrophysiology.
Experiments were performed as described earlier (Lingenhoehl et al., 1999). Briefly, lobes of oocytes were removed surgically from anesthetized (1.2 g/l MS222) femaleXenopus laevis frogs. Oocytes were separated and defolliculated and injected with 10 to 50 ng of rat GABAB(1a) (or GABAB(1b)) together with GABAB(2) and rat Kir3.1, 3.2, and 3.4 coding mRNAs and incubated at 18°C for 3 to 8 days. Two-electrode voltage clamp recordings were done with electrodes filled with 3 M KCl. Oocytes were continuously perfused with normal frog Ringer solution (115 mM NaCl, 10 mM HEPES, 2.5 mM KCl, 1.8 mM CaCl2, pH 7.2) or high-potassium Ringer solution (90 mM KCl, 27.5 mM NaCl, 10 mM HEPES, 1.8 mM CaCl2, pH 7.2). Recordings were performed at a clamp potential of −70 mV. To test the positive modulatory activity of CGP7930, the compound was applied with ascending concentrations and a fixed GABA concentration.
Chemicals.
CGP7930 and CGP13501 were synthesized in house. Propofol and malonoben were from Tocris Cookson Ltd. (Bristol, UK). Stock solutions of these compounds were prepared in dimethyl sulfoxide and subsequently diluted in the respective assay buffers. The final concentrations of dimethyl sulfoxide in the various assays usually did not exceed 0.3% and did not interfere with the measured parameters. [3H]APPA ([3H]CGP27429, 50 Ci/mMol) and [3H]CGP62349 (85 Ci/mMol) were obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO).
Results
CGP7930 and CGP13501 Positively Modulate Recombinant and Native GABAB Receptor Activity in a GTPγ[35S] Binding Assay.
The stimulation of GTPγ[35S] binding is a widely used functional assay for GPCRs. GABA stimulated GTPγ[35S] binding in membranes from CHO cells stably expressing GABAB(1b/2). The maximal stimulation obtained with a saturating concentration (100 μM) corresponded to a 2- to 3-fold of the basal activity measured in the absence of an agonist (Fig. 2). This effect of GABA was mediated via GABAB receptors, because it was blocked by the competitive GABAB receptor antagonist CGP56999A (Fig. 2C) and it was not observed in membranes from CHO cells that had not been transfected with GABAB receptor cDNA (not shown). The compounds CGP7930 and its aldehyde analog CGP13501 (Fig. 1) were found to substantially increase the effects of different GABA concentrations (Fig. 2, A and B). Similarly, CGP7930 increased the agonistic effect ofl-baclofen (not shown). The compounds propofol and malonoben (Fig. 1), which are closely related chemically, had no such effects (Fig. 2, A and B). Propofol (2.5 μM and 25 μM) also did not antagonize the effects of CGP7930 (2.5 μM and 25 μM, not shown). CGP7930 and CGP13501 produced little or no stimulation of GTPγ[35S] binding in the absence of GABA or when the effect of GABA was blocked by a competitive antagonist (Fig.2C). They also did not potentiate glutamate-induced GTPγ[35S] binding in membranes from CHO cells expressing the mGluR2 metabotropic glutamate receptor (Fig.3).
To characterize the positive modulatory effects in more detail, further experiments were performed with the more active compound CGP7930. To evaluate whether CGP7930 also acts on native GABAB receptors, GTPγ[35S] binding studies on membranes from rat brain cortex were performed. The addition of GABA to this preparation stimulated GTPγ[35S] binding; the stimulation could be inhibited by well-established GABAB receptor antagonists (data not shown). The effect of GABA was again potentiated by CGP7930 (Fig.4, bottom). Concentration-response curves, established with native or recombinant receptor preparations at fixed concentrations of GABA (1 μM and 20 μM), revealed EC50 values for CGP7930 in the low micromolar range (Fig. 4, Table 1). The EC50 value for CGP7930 was similar for recombinant and native receptors (Table 1).
GABAB Receptor Modulation Is Mediated via an Increase in Both Agonist Affinity and Efficacy.
Concentration-response curves for GABA at different fixed concentrations of CGP7930 revealed a dual mechanism of recombinant GABAB receptor modulation, involving an increase of agonist potency as well as of maximal efficacy (Fig.5, Table2).
An increase of agonist affinity induced by CGP7930 also became apparent in radioligand binding assays. In saturation experiments with the agonist radioligand [3H]APPA, labeling native GABAB receptors in rat cortical membranes, 30 μM CGP7930 produced an increase in affinity, without a change in theB max value (Fig.6, top; Table3). The displacement of [3H]CGP62349 (a competitive antagonist) from recombinant GABAB receptors by GABA revealed a more complex situation (Fig. 6, bottom). When membranes from cells expressing the GABAB(1b/2) heterodimer were used, the displacement curves were biphasic, with a minor high-affinity component and a major low-affinity component (Fig. 6, bottom; Table4). The modulator CGP7930 increased the affinity of GABA for the minor component, whereas the remaining part of the displacement curve was unchanged in the presence or absence of this compound. The biphasicity of these curves was presumably caused by the presence of overexpressed monomeric GABAB(1b)subunits in addition to GABAB(1b/2) heterodimers (see the discussion section). In fact, in membranes from cells expressing the GABAB(1b) subunit alone, only a single component with low affinity for GABA was detected, which was not influenced by CGP7930. The inset in the bottom panel of Fig. 6 also shows that CGP7930 did not displace the competitive antagonist radioligand directly from the agonist recognition site on the GABAB receptor.
Positive Modulation of GABAB Receptors by CGP7930 Does not Differentiate GABAB(1) Splice Variants 1a and 1b.
The effects of CGP7930 on the regulation of inwardly rectifying potassium channels via GABAB receptors inXenopus laevis oocytes are shown in Fig.7. Exposure of the oocytes to a high potassium (90 mM) Ringer solution elicited an inward current that was reversibly amplified in the presence of GABA. The effect of a low concentration (0.3 μM) of GABA was increased in the presence of CGP7930; the current traces obtained during the preincubation with CGP7930 clearly show that this compound had no effect on its own. The positive modulation produced by CGP7930 was observed with both GABAB receptor subunit combinations, GABAB(1a/2) and GABAB(1b/2). The effect of CGP7930 was reversible, because upon washout, the peak size was near control levels after about 15 min (data not shown). The EC50value of CGP7930 in this assay was approximately 1 μM, similar to the values obtained in GTPγ[35S] experiments with recombinant receptors.
Effects of CGP7930 on GABAB Receptors in Intact HEK293 Cells.
HEK293 cells were transiently transfected with GABAB(1/2) and the Gαqo5G-protein subunit. CGP7930 concentration-dependently increased a transient Ca2+ signal induced by 1 μM GABA (Fig. 8). The pEC50value for CGP7930 in this assay was 5 ± 0.04. CGP7930, up to 30 μM, added during the preincubation phase, elicited no calcium signal on its own.
CGP7930 Reduces Calcium Oscillations in Rat Cortical Neuron Primary Cultures.
Dissociated rat cortical neurons in primary culture form synaptically connected networks. Removal of Mg2+from the incubation medium elicits synchronized calcium oscillations in these neurons (Fig. 9A; Wang and Gruenstein, 1997). The GABAB receptor agonistl-baclofen (3 μM) reduced the firing frequency in this neuronal network (Fig. 9B), an effect that was reversed by the competitive antagonist CGP54626A (Fig. 9C). At a low concentration (0.3 μM), at which it had no effect on its own, CGP7930 increased the effect of 3 μM l-baclofen (Fig. 9, D and E).
Discussion
This study describes for the first time the identification of low-molecular-weight organic compounds that act as positive allosteric modulators at GABAB receptors in a native environment (rat brain membranes, neuronal cultures) or in recombinant expression systems (stably or transiently transfected mammalian cell lines, X. laevis oocytes).
The compound CGP7930, structurally close to the general anesthetic propofol, and its aldehyde analog CGP13501 potentiated GABA-induced signals in a functional receptor test (GTPγ[35S] binding), using membranes from CHO cells stably expressing the GABAB receptor (Fig.2). The findings that these signals exceeded the response elicited by a maximally active concentration of GABA alone and that these two compounds did not stimulate GTPγ[35S] binding in the absence of GABA to any relevant extent clearly show that they acted as positive modulators, without intrinsic agonistic activity. In GTPγ[35S] experiments, but not in the other assays, a very marginal effect was seen with the modulators alone (Fig.2C) that might, however, be due to a small constitutive activity of a part of the receptor population. The modulatory effects of CGP7930 and CGP13501 were GABAB receptor selective because they were not observed in the same host cells (CHO-K1) expressing mGluR2, which couples to the same G-proteins (Go/Gi) as the GABAB receptor (Fig. 3). This finding also strongly suggests that the modulators affect the GABAB receptor itself, rather than the G-protein or the membrane. Moreover, chemical specificity of these effects is also indicated by the fact that the two structurally related compounds malonoben and propofol were without effect in this assay. This is interesting insofar as propofol acts as a general anesthetic by a mechanism that involves positive modulation at the ionotropic GABAA receptor (Hales and Lambert, 1991). Propofol differs from the two active compounds in that it has two isopropyl- instead of t-butyl substituents in positions 2 and 6 and lacks a further side chain in position 4. This side chain apparently has to fulfill relatively stringent structural requirements, because a rather small difference between CGP7930 and CGP13501 (an alcohol instead of an aldehyde function in the terminal position of the side chain) conferred a more pronounced modulatory activity to the former compound. Also, malonoben with its more different side chain was completely inactive in our experiments.
Concentration-response curves established with CGP7930 in the presence of fixed GABA concentrations revealed micromolar potencies (EC50 values) at recombinant and native GABAB receptors (Fig. 4). On the other hand, when concentration-response curves were measured for GABA at different fixed concentrations of CGP7930 (Fig. 5), it became evident that the modulator simultaneously increased the potency and the maximal activity of GABA. Such dual effects are unusual for allosteric enhancers at GPCRs and ionotropic receptors. For example, benzodiazepines modulate GABAA receptors by enhancing GABA responses only at subsaturating, not at maximally active, GABA concentrations (Choi et al., 1981). Similarly, brucine and some analogs thereof act as allosteric muscarinic receptor modulators by increasing agonist potency in radioligand binding and functional assays without affecting the maximal response (e.g., in GTPγ[35S] experiments) (Lazareno et al., 1998; Birdsall et al., 1999). Whereas these effects can be described by a ternary allosteric model in which both the primary and allosteric ligands simultaneously bind to the receptor and reciprocally modulate their respective affinities, the situation with our GABAB receptor modulators is obviously more complex. The recently described extension of the two-state model of receptor activation (Hall, 2000) accounts for the allosteric effects of compounds that, like CGP7930, affect not only the affinity but also the intrinsic efficacy of agonists. On the other hand, the interpretation that the augmentation of the maximal stimulation obtained in our GTPγ[35S] experiments reflects an increase in receptor number can be ruled out by the finding that the B max value in our [3H]APPA binding experiments remained unchanged in the presence of CGP7930. Also, the effects found with CGP7930 are clearly different from those described for saponin, which increases not only the maximal level of stimulation (presumably via a nonreceptor mechanism) but, unlike CGP7930, also the baseline values in GTPγ[35S] assays (Cohen et al., 1996).
An increase of agonist potency would presumably be related to a concurrent increase in affinity, which should be detectable in radioligand binding assays. When we displaced the antagonist radioligand [3H]CGP62349 with GABA, biphasic inhibition curves were obtained in membranes from CHO cells expressing GABAB(1b/2) heterodimers (Fig. 6, Table 4). Only the minor high-affinity component was influenced by CGP7930. The two phases of the displacement curves could well be caused by the presence of both receptors coupled to and uncoupled from G-proteins, as is known for other GPCRs. On the other hand, they could also indicate that in the stably transfected CHO GABAB(1b/2) cell line used, the GABAB(1) subunit is strongly overexpressed and exists to a large extent in a monomeric form. In fact, the IC50 value for the predominant low-affinity component was similar to that found in experiments using cell membranes containing the GABAB(1b) subunit only, which was also not modulated by CGP7930. It is known that GABA and competitive antagonists bind to the GABAB(1)subunit and that agonist affinities are higher in GABAB(1/2) heterodimers compared with GABAB(1) monomers (Kaupmann et al., 1997, 1998;White et al., 1998). On the other hand, saturation curves with native receptors using the agonist [3H]APPA, which preferentially detects the high affinity agonist site on the heteromer GABAB(1/2), revealed a clear increase in ligand affinity induced by the modulator (Fig. 6). It seems therefore that CGP7930 can exert its modulatory action only in GABAB(1/2) heterodimeric receptor assemblies, not in the GABAB(1) subunit alone, implying that CGP7930 either acts via GABAB(2) (by binding to this subunit) or at least needs the presence of GABAB(2) to be able to exert its effect.
Positive allosteric modulation of GABAB receptor activity was not only demonstrated in membrane preparations, but also in more complex cellular assay systems. In X. laevis oocytes injected with mRNA for the GABAB receptor and for inwardly rectifying (Kir 3) potassium channels, CGP7930 potentiated the effect of GABA on potassium currents (Fig. 7). These experiments were carried out with both the GABAB(1a/2) and GABAB(1b/2) subunit combinations, and similar effects were seen in both cases. Thus, the modulatory effect of CGP7930 was independent of the splice variant of the GABAB(1) subunit of the GABAB receptor. In transiently transfected cell lines, GABAB receptors induce a calcium signal when they are coexpressed with an appropriate chimeric G-protein, enabling them to couple to the phospholipase C pathway (Bräuner-Osborne and Krogsgaard-Larsen, 1999; Franek et al., 1999; Pagano et al., 2001; Wood et al., 2000). The increase of intracellular calcium concentrations elicited by the addition of GABA to HEK293 cells transiently transfected with GABAB receptors and the chimeric G-protein Gαqo5 was again potentiated by CGP7930 in a concentration-dependent fashion (Fig. 8), whereas CGP7930 on its own did not produce an increase of intracellular calcium.
At the next level of complexity, the modulatory effects of CGP7930 were confirmed in a test system representing a neuronal network. Dissociated rat cortical neurons in primary culture produce synchronized calcium oscillations in low extracellular Mg2+ (Wang and Gruenstein, 1997; Fig. 9), resulting from the interplay of spontaneous depolarizations of inhibitory and excitatory neurons. The GABAB receptor agonist l-baclofen reduced the frequency of these oscillations, an effect that was again potentiated by CGP7930 (Fig. 9).
It is well known that GABAB receptors are positively modulated by calcium ions in an allosteric fashion (Wise et al., 1999; Galvez et al., 2000a). However, the following findings show that this action of calcium is of a different nature and occurs via another site than the modulation by the compounds described in this study: in GTPγ[35S] stimulation experiments, Ca2+ increases the affinity of GABA without influencing its maximal effect (Wise et al., 1999; Galvez et al., 2000a). CGP7930 positively modulates agonism produced by GABA andl-baclofen, whereas Ca2+ enhances only the potency of GABA, not that of baclofen (Galvez et al., 2000a). Also, in contrast to CGP7930, calcium enhances the affinity of GABA as a displacer of an antagonist radioligand in membranes from CHO cells expressing the GABAB(1) subunit only (Galvez et al., 2000a). Furthermore, our experiments were conducted in the presence of a saturating concentration of calcium; therefore, the effects observed with CGP7930 and CGP13501 were additive with those of calcium ions.
In summary, we have shown that CGP7930 and CGP13501 act as positive allosteric modulators of GABAB receptor function. The allosteric nature of the effects of these compounds is supported by three main findings: first, they have no relevant agonistic effect when applied without GABA; second, the maximal stimulation of GTPγ[35S] binding in the presence of these compounds exceeds the effect of a saturating concentration of GABA alone (i.e., the modulators act synergistically with GABA); finally, the compound CGP7930 does not bind to the agonist recognition site of the GABAB receptor. As discussed above, our radioligand binding studies show that the presence of the GABAB(2) subunit is necessary for the positive modulation. At present, it is unclear whether CGP7930 binds to GABAB(1) or GABAB(2) or even acts at the interface between the two subunits. All agonist and antagonist ligands known so far bind to the GABAB(1) subunit (Kaupmann et al., 1997;Malitschek et al., 1999; Galvez et al., 2000b). On the other hand, not only is GABAB(2) responsible for the targeting of the GABAB receptor to the cell surface (Pagano et al., 2001), its extracellular domain also enhances agonist affinity at GABAB(1) and is necessary for agonist activation of the receptor (Galvez et al., 2001). Thus, it seems that GABAB(1) serves as the orthosteric ligand binding subunit and GABAB(2) as an allosteric subunit, through which positive modulators might well act. Alternatively, it is also conceivable that such modulators might bind to the transmembrane domain of one or both GABAB receptor subunits, as has been demonstrated for noncompetitive mGluR antagonists (Pagano et al., 2000). To address this question, studies with site-directed mutagenesis on individual GABAB receptor subunits will be needed.
Acknowledgments
We thank Dr. R. Kuhn for critically reading the manuscript, Dr. P. Flor for providing cells expressing mGluR2, and R. Brom, M. Erb, C. Lampert, M. Horvath, D. Ristig, and V. Schuler for their excellent technical assistance.
Footnotes
- Received May 15, 2001.
- Accepted July 20, 2001.
Abbreviations
- GABA
- γ-aminobutyric acid
- GPCR
- G-protein-coupled receptor
- mGluR
- metabotropic glutamate receptor
- CHO
- Chinese hamster ovary
- GTPγS
- guanosine 5′-O-(3-thiotriphosphate)
- SPA
- scintillation proximity assay
- CGP7930
- 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol
- APPA
- 3-aminopropylphosphinic acid
- HEK
- human embryonic kidney
- HBSS
- Hanks' balanced salt solution
- FLIPR
- fluorescence imaging plate reader
- The American Society for Pharmacology and Experimental Therapeutics