Advertisement
Research ArticleArticle

Novel Plant Substances Acting as β Subunit Isoform-Selective Positive Allosteric Modulators of GABAA Receptors

Roland Baur, Urs Simmen, Martin Senn, Urs Séquin and Erwin Sigel
Molecular Pharmacology September 2005, 68 (3) 787-792; DOI: https://doi.org/10.1124/mol.105.011882

Abstract

GABAA receptors are modulated by a large variety of compounds. A common chemical characteristic of most of these modulators is that they contain a cyclic entity. Three linear molecules of a polyacetylene structure were isolated from the East African medicinal plant Cussonia zimmermannii Harms and shown to allosterically stimulate GABAA receptors. Stimulation was not abolished by the absence of the γ2 subunit, the benzodiazepine antagonist Ro15-1788 (8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester), or the point mutation β2N265S that abolishes effects by loreclezole. At a concentration of 30 μM, the substances by themselves elicited only tiny currents. Maximal stimulation at α1β2γ2 amounted to 110 to 450% for the three substances, and half-maximal stimulation was observed at concentrations of 1 to 2 μM. Stimulation was subunit composition-dependent and was for the substance MS-1, α1β2γ2 ≈ α1β2 ≈ α3β2γ2 > α2β2γ2 > α5β2γ2 ≈ α1β3γ2 ≈ α6β2γ2 > α1β1γ2, for MS-2 α1β2γ2 ≈ α3β2γ2 ≈ α1β2 > α2β2γ2 ≈ α6β2γ2 ≈ α5β2γ2 > α1β1γ2, and for MS-4, α1β2γ2 ≈ α1β2 ≈ α5β2γ2 ≈ α3β2γ2 ≈ α2β2γ2 > α6β2γ2 ≫ α1β1γ2. Maximal stimulation by MS-1 was 450% at α1β2γ2, 80% at α1β1γ2, and 150% at α1β3γ2. MS-1 was thus specific for receptors containing the β2 subunit. The reversal potential was unaffected by 10 μM MS-1, whereas apparent picrotoxin affinity for current inhibition was increased approximately 3-fold. In summary, these positive allosteric modulators of GABAA receptors of plant origin have a novel unusual chemical structure and act at a site independent of that of benzodiazepines and loreclezole.

GABA is the major inhibitory neurotransmitter in the mammalian central nervous system. It acts at three types of receptors, the G-protein-coupled GABAB receptor, and the GABAA and GABAC receptors, which both constitute ion channels. Two subunits of the GABAA receptor have initially been purified (Sigel et al., 1983), and their coding DNA has been cloned (Schofield et al., 1987). Numerous subunits have since been cloned (for review, see Macdonald and Olsen, 1994; Rabow et al., 1995; Barnard et al., 1998). These subunits show homology to subunits of the nicotinic acetylcholine receptors, the glycine receptor, and the 5HT3 receptor. The GABAA receptors are heteromeric protein complexes consisting of five subunits that are arranged around a central Cl--selective channel (Macdonald and Olsen, 1994). The major receptor isoform of the GABAA receptor in the brain presumably consists of α1, β2, and γ2 subunits (Laurie et al., 1992; Benke et al., 1994; Macdonald and Olsen, 1994; Rabow et al., 1995; McKernan and Whiting, 1996; Barnard et al., 1998). Different approaches have indicated a 2α:2β:1γ subunit stoichiometry for this receptor (Backus et al., 1993; Chang et al., 1996; Tretter et al., 1997; Farrar et al., 1999; Baumann et al., 2001, 2002).

The GABAA receptor is the site of action of many modulatory compounds, among them the benzodiazepines (for review, see Sieghart, 1995). Both binding sites, those for the channel agonist GABA and those for benzodiazepines, are thought to be located at subunit interfaces in a homologous position (for review, see Galzi and Changeux, 1994; Sigel and Buhr, 1997). Most of the allosteric modulators contain a ring in their chemical structure.

Cussonia zimmermannii Harms belongs to the genus Cussonia of the family Araliaceae. It occurs in Kenya and Tanzania and grows in lowland rain forests, lowland dry evergreen forests, and woodlands at altitudes of 0 to 400 m (Tennant, 1968). The marrow of the stem and branches is eaten to treat epilepsy, and a decoction of the root is taken as a remedy for labor pain (Haerdi, 1964). In addition, an infusion of the leaves is used as a wash for people suffering from fever or ague and a decoction of the roots is taken as a remedy for gonorrhea (Kokwaro, 1976).

Herein, we describe three potent positive allosteric modulators of GABAA receptors with a linear polyacetylene structure isolated from the plant C. zimmermannii Harms. They are shown to act at a site independent of both the benzodiazepine and the loreclezole site. These compounds display a most interesting and unprecedented subunit specificity.

Materials and Methods

Substances. MS-1, MS-2, and MS-4 were isolated from the East African medicinal plant C. zimmermannii Harms. The methods of isolation and structure determination will be described elsewhere.

[3H]Flunitrazepam Binding. Cortex material derived from rats (stem RORO; RCC Ltd., Basel, Switzerland) was homogenized on ice with 50 mM Tris/HCl, pH 7.4, 120 mM NaCl, and 5 mM KCl using a Polytron homogenizer (Kinematica, Basel, Switzerland). The homogenate was centrifuged at 31,000g for 10 min at 4°C. The pellet was resuspended with 50 mM Tris/HCl, pH 7.4, and centrifuged as described above for a total of three washing steps. The membrane fraction was stored at -80°C. For the binding of [3H]flunitrazepam (final concentration, 1 nM; Amersham Biosciences Inc., Piscataway, NJ), 200 mg of membrane protein (BCA protein assay) was used. Nonspecific binding was determined in the presence of 10 μM diazepam. Binding equilibrium was reached within 1 h at room temperature, and binding assays were terminated after this time by rapid filtration using GF/C filters (Whatman, Maidstone, UK). Filters were washed three times with ice-cold Tris/HCl, pH 7.4, buffer. Radioactivity on filters was determined by liquid scintillation counting (Tri-Carb 2100TR; PerkinElmer Life and Analytical Sciences, Boston, MA). Results are given as the mean ± S.E.M. of two to four individual experiments performed in triplicate.

Construction of Receptor Subunits. The cDNAs coding for the α1, β2, and γ2S(γ2) subunits of the rat GABAA receptor channel have been described elsewhere (Lolait et al., 1989; Malherbe et al., 1990a,b). For cell transfection, the cDNAs were subcloned into the polylinker of pBC/CMV (Bertocci et al., 1991). This expression vector allows high-level expression of a foreign gene under control of the cytomegalovirus promoter. The cDNAs coding for α2, α3, α5, α6, β1, and β3 were prepared similarly. α Subunits were cloned into the EcoRI, and the β subunits were subcloned into the SmaI site of the polylinker by standard techniques.

Expression in Xenopus laevis Oocytes. Capped cRNAs were synthesized (Ambion, Austin, TX) from the linearized pBC/CMV vectors containing wild-type α1, α2, α3, α5, α6, β1, β2, β3, and γ2, respectively. A poly(A) tail of ∼400 residues was added to each transcript using yeast poly(A) polymerase (U.S. Biochemical Corp., Cleveland, OH). The concentration of the cRNA was quantified on a formaldehyde gel using Radiant Red stain (Bio-Rad, Hercules, CA) for visualization of the RNA and known concentrations of RNA ladder (Invitrogen, Carlsbad, CA) as standard on the same gel. cRNA combinations in nuclease-free water were stored at -80°C. Isolation of oocytes from the frogs, culturing of the oocytes, injection of cRNA, and defolliculation were performed as described previously (Sigel, 1987; Sigel et al., 1990). Oocytes were injected with 50 nl of the cRNA solution. The combination of wild-type α1 and β2 subunits was expressed at 75 and 75 nM, and the combination of wild-type αx, βx, and γ2 subunits was expressed at 10, 10, and 50 nM (Boileau et al., 2002). For control purposes, cRNA coding for a voltage-gated sodium channel (Kuhn and Greeff, 1999) was used at a concentration of 40 nM. The injected oocytes were incubated in modified Barth's solution [10 mM HEPES, pH 7.5, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.34 mM Ca(NO3)2, 0.41 mM CaCl2, 100 U/ml penicillin, and 100 μg/ml streptomycin] at 18°C for at least a day before the measurements.

Two-Electrode Voltage-Clamp Measurements. All measurements were done in medium containing 90 mM NaCl, 1 mM MgCl2, 1 mM KCl, 1 mM CaCl2, and 5 mM HEPES, pH 7.4, at a holding potential of -80 mV. To quantify stimulation by MS compounds, agonist concentrations eliciting ∼5% of the maximal current amplitude were applied alone or in combination with increasing concentrations of MS compounds between 0.03 and 30 μM for 20 s, and a washout period of 4 min was allowed to ensure full recovery from desensitization. The stimulation was then calculated as Stimulation = [(Iafter MS/Ibefore MS) - 1] × 100%. Stimulation was fitted to the Hill equation: I = Imax/1 + (EC50/A)nH, where I is the current amplitude at a given concentration of MS compound A, Imax is the current amplitude at maximal stimulation, EC50 is the concentration of MS compound yielding half-maximal current amplitudes, and nH is the Hill coefficient. GABA-evoked currents (at ∼10% of the maximal current amplitude) were inhibited by varying concentrations of picrotoxin. Inhibition curves for picrotoxin were fitted with the equation I(c) = I(0)/(1 + (IC50/c), where I(0) is the control current in the absence of picrotoxin standardized to 100%, I(c) is the relative current amplitude, c is the concentration of picrotoxin, and IC50 is the concentration of picrotoxin causing 50% inhibition of the current. Voltage-dependent sodium currents were determined by a potential jump from a holding potential of -100 to -15 mV.

Data are given as the mean ± S.E.M. (number of experiments for at least two batches of oocytes). The perfusion system was cleaned between drug applications by washing with 100% dimethyl sulfoxide (DMSO) to avoid contamination. The stock solution of MS compounds was 40 mM in DMSO. The final concentration of DMSO in the medium was always adjusted to 0.5%. These concentrations of DMSO did not by themselves significantly affect GABA-elicited currents. Currents were measured using a modified OC-725 amplifier (Warner Instruments, Hamden, CT) in combination with an XY-recorder or digitized using a MacLab/200 (ADInstruments, Oxfordshire, UK).

Fig. 1.
Fig. 1.

Structures of the three polyacetylene compounds.

Results

Three Polyacetylene Compounds Stimulate [3H]Flunitrazepam Binding

The compounds were named MS compounds, and their structures are shown in Fig. 1. All three compounds are linear, polyunsaturated with two triple bonds, and contain an acetate group. These compounds were isolated from the plant C. zimmermannii Harms. Elucidation of the chemical structures was performed using UV spectroscopy, infrared spectroscopy, 1H NMR, 13C NMR, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, correlated spectroscopy, electron ionization mass spectroscopy, fast atom bombardment mass spectrometry, and high-resolution electron ionization mass spectrometry (manuscript in preparation). In traditional local medicine, this plant is used, among many other purposes, for the treatment of epilepsy. Within a screening approach, the plant was shown to contain components able to allosterically interact with the benzodiazepine-binding site on GABAA receptors. The property to stimulate the binding of [3H]flunitrazepam was then used to isolate three substances. Figure 2 shows the dose-dependent stimulation of [3H]flunitrazepam binding of MS-1, MS-2, and MS-4.

Electrophysiological Studies

Positive Allosteric Modulation. Functional effects of MS compounds were investigated in electrophysiological studies at recombinant GABAA receptors expressed in X. laevis oocytes. Because of solubility, the MS compounds were only used up to a concentration of 30 μM. GABA was always used at concentrations eliciting 2 to 6% of the maximal current amplitude in the corresponding GABAA receptor type. At α1β2γ2, each MS compound by itself (30 mM) elicited tiny currents amounting to <0.1% of the maximal current elicited by GABA, but all of the compounds exhibited a potent positive allosteric modulatory effect by enhancing the GABA-stimulated current at α1β2γ2. This concentration-dependent stimulation is documented for MS-1 at α1β2γ2 with a GABA concentration of 7 μM (Fig. 3). Maximum stimulation at α1β2γ2 was achieved with ∼10 μM MS-1. We also tested whether 10 μM MS-1 stimulated near-maximal currents elicited by GABA. In the presence of 500 μM GABA, 10 μM MS-1 did not significantly affect the current amplitudes [97.7 ± 1.5% of the control (n = 3)]. Figure 4 shows the effect of 10 μM MS-1 on the GABA concentration dependence of the current. In the absence of MS-1, the Ka for GABA and the Hill coefficients were 24 ± 5 μM (n = 4) and 1.5 ± 0.1 (n = 4), respectively. In the presence of 10 μM MS-1, the concentration response curve was less steep and characterized by Ka for GABA of 21 ± 3 μM (n = 4) and a Hill coefficient of 1.1 ± 0.1 (n = 4). Stimulation by 10 μM MS-1 at low concentrations of GABA amounts to only ∼2.3-fold compared with the value of 4-fold expected from the data shown below. The reason for this discrepancy is not known, but it may be due to the repetitive application of 10 μM MS-1 in these experiments.

Fig. 2.
Fig. 2.

Stimulation of [3H]flunitrazepam binding by MS-1, MS-2, and MS-4. Specific binding stimulated by MS-1 (▪), MS-2 (○), and MS-4 (▵) is shown relative to control binding in the absence of drugs. Data are expressed as the mean ± S.E.M. of two to four individual experiments.

Subunit Specificity of MS-1.Figure 5A shows an averaged concentration response curve of this type of experiment for α1β2γ2. Maximal stimulation is ∼450%, and half-maximal stimulation was observed at a concentration (EC50) of ∼1.5 mM. Replacement of α1 in this subunit combination by other α subunit isoforms such as α2, α3, α5, or α6 had little effect on EC50, which varied between 0.6 and 1.0 μM, but had in some cases a drastic effect on the maximal stimulation (Fig. 5A). The extent of stimulation was α1β2γ2 ≈ α3β2γ2 > α2β2γ2 > α5β2γ2 ≈ α6β2γ2. Figure 5B shows the effect of the β subunit isoform and the lack of effect upon omitting γ2 from α1β2γ2. Replacing β2 in α1β2γ2 by β1 or β3 drastically reduced maximal stimulation from 450% to 80 and 150%, respectively. Introducing the point mutation β2N265S that is known to strongly reduce stimulatory effects by loreclezole (Wingrove et al., 1994) into α1β2γ2 had only a relatively weak effect in this case, reducing maximal stimulation by approximately one third (Fig. 5B).

Fig. 3.
Fig. 3.

Concentration dependence of allosteric stimulation by MS-1 at α1β2γ2 GABAA receptors. Recombinant α1β2γ2 GABAA receptors expressed in X. laevis oocytes and exposed to either 7 mM were GABA alone or in combination with increasing concentrations of MS-1. The experiment was repeated twice on oocytes of two different batches with similar results.

Fig. 4.
Fig. 4.

Effect of MS-1 on the GABA concentration dependence. A, recombinant α1β2γ2 GABAA receptors were expressed in X. laevis oocytes and exposed to increasing GABA concentrations either alone (•) or in combination with 10 μM MS-1 (○). Data are given as the mean ± S.E.M. (up to four oocytes from two different batches).

Subunit Specificity of MS-2 and MS-4. Figures 6 and 7 show subunit specificities of MS-2 and MS-4, respectively. Again, the replacement of α1 in α1β2γ2 by other α subunit isoforms such as α2, α3, α5, or α6 had little effect on EC50, which varied between 0.8 and 1.3 μM for MS-2 and 1.4 and 3.5 μM for MS-4, but had in some cases a drastic effect on the maximal stimulation (Figs. 6 and 7). Maximal stimulation was ∼300% for MS-2 and ∼110% for MS-4. The following specificity in this respect was observed for MS-2, α1β2γ2 ≈ α3β2γ2 ≈ α1β2 > α2β2γ2 ≈ α6β2γ2 ≈ α5β2γ2 > α1β1γ2, and for MS-4, α1β2γ2 ≈ α1β2 ≈ α5β2γ2 ≈ α3β2γ2 ≈ α2β2γ2 > α6β2γ2 ≫ α1β1γ2.

Lack of Inhibition by the Benzodiazepine Antagonist Ro15-1788

We tested whether MS-1 would act at a known site on the GABAA receptor. Currents elicited by 2 to 7 μM GABA were stimulated by 10 μM MS-1 (377 ± 43%; n = 7). If 1 μM benzodiazepine antagonist Ro15-1788 was coapplied, stimulation would not be significantly altered with 391 ± 90% (n = 3) (Fig. 8). In control experiments, the current stimulation by 1 μM diazepam was completely abolished in the presence of 1 μM Ro15-1788 (Fig. 8). In contrast to Ro15-1788, the antagonist of the ROD site, ROD178B (Sigel et al., 2001), at a concentration of 100 μM, significantly reduced stimulation to 134 ± 9% (n = 3).

Costimulation by MS-1 and Diazepam

The above observations indicate independent sites of action of MS-1 and diazepam. Even if this were the case, whether the two drugs act additively cannot be predicted. Currents were elicited by 2 to 7 μM GABA followed by coapplication with 1 μM diazepam and subsequently the combination of 1 μM diazepam and 10 μM MS-1. As shown in Fig. 8, diazepam induced a stimulation of 244 ± 12% (n = 8) and the combination of diazepam and MS-1 induced a stimulation of 871 ± 152% (n = 4), demonstrating an additive effect of the two drugs.

Effect of MS-1 on the Apparent Affinity of Picrotoxin

Currents elicited by 3 or 4 μM GABA were inhibited by increasing concentrations of the channel pore blocker picrotoxin. The half-maximal concentration of picrotoxin for current inhibition was 2.6 ± 0.4 μM (n = 3) in the absence and 1.0 ± 0.1 μM (n = 3) in the presence of 10 μM MS-1. MS-1 increased apparent picrotoxin affinity ∼2.6-fold (Fig. 9).

Effect of MS-1 on the Apparent Affinity of Bicuculline

GABA-induced currents amounting to ∼20% of the maximal current amplitude were inhibited by increasing concentrations of the competitive GABA antagonist bicuculline. The half-maximal concentration of bicuculline for current inhibition was 1.3 ± 0.1 μM (n = 4) in the absence of 10 μM MS-1 and nonsignificantly increased to 2.3 ± 0.4 μM(n = 4) in the presence of 10 μM MS-1 (data not shown).

Fig. 5.
Fig. 5.

Subunit isoform specificity of MS-1. Recombinant α1β2γ2 (•), α2β2γ2 (○), α3β2γ2 (▪), α5β2γ2 (□), and α6β2γ2 (▴) (A) and α1β2γ2 (•), α1β2 (▵), α1β2N265Sγ2 (×), α1β3γ2 (+), and α1β1γ2 (♦) (B) GABAA receptors were expressed in X. laevis oocytes and exposed to either GABA alone or in combination with increasing concentrations of MS-1. Data are given as the mean ± S.E.M. (up to three oocytes from two different batches).

Ion Selectivity Is Maintained and Stimulation Is Independent of Potential

The reversal potential and the potential dependence of the current elicited by GABA were both not altered in the presence of 10 μM MS-1. The reversal potential was -29 ± 1 mV (n = 3) in the absence of 10 μM MS-1 and was not altered in its presence (data not shown).

Fig. 6.
Fig. 6.

Subunit isoform specificity of MS-2. Recombinant α1β2γ2 (•), α1β2 (▵), α2β2γ2 (○), α3β2γ2 (▪), α5β2γ2 (□), α6β2γ2 (▴), and α1β1γ2 (♦) GABAA receptors were expressed in X. laevis oocytes and exposed to either GABA alone or in combination with increasing concentrations of MS-2. Data are given as the mean ± S.E.M. (up to three oocytes from two different batches).

Specificity of the MS Compounds

Because the structure of the MS compounds suggests interaction with the lipid bilayer, they could in principle non-specifically interact with any membrane protein. Even if their specificity for receptors containing the β2 subunit argued for a specific effect, we were concerned with this possibility. Therefore, we tested the effects of MS-1 on the rat brain voltage-gated sodium channel IIA. We chose concentrations of MS-1 of 1, 5, and 30 μM, which should be compared with the concentrations eliciting half-maximal effects (200% stimulation) at α1β2γ2 GABAA receptors (0.6-1.5 μM). 1, 5, and 30 μM MS-1 weakly stimulated peak sodium currents elicited by a voltage jump from -100 to -15 mV by 8.8 ± 2.5% (n = 3), 3.8 ± 2.5% (n = 4), and 6.6 ± 3.6% (n = 3), respectively (data not shown). In contrast, currents induced by a voltage jump from -60 to -15 mV were inhibited and inhibition amounted to 20.3 ± 3.1% (n = 3), 11.0 ± 2.6% (n = 4), and 15.6 ± 2.6% (n = 3), respectively. With both potential protocols and all three concentrations of MS-1 tested, voltage-dependent inactivation was slowed down to a similar extent. For example, inactivation after a pulse from -100 to -15 mV was fitted with mono-exponential function characterized by the time constant t. This time constant was slightly increased by 1 μM MS-1 from 5.6 ± 0.3 ms (n = 3) in its absence to 9.3 ± 0.4 ms (n = 3) in its presence. In summary, the effects of MS-1 on the voltage-gated sodium channel are small, arguing again against a nonspecific membrane effect.

Fig. 7.
Fig. 7.

Subunit isoform specificity of MS-4. Recombinant α1β2γ2 (•), α1β2 (▵), α2β2γ2 (○), α3β2γ2 (▪), α5β2γ2 (□), α6β2γ2 (▴), and α1β1γ2 (♦) GABAA receptors were expressed in X. laevis oocytes and exposed to either GABA alone or in combination with increasing concentrations of MS-4. Data are given as the mean ± S.E.M. (up to three oocytes from two different batches).

Discussion

In this study, we showed that three substances isolated from C. zimmermannii Harms act as potent positive allosteric modulators at GABAA receptors with a half-maximal stimulation at a concentration of 0.6 to 3.5 μM and a maximal stimulation of 110 to 450%. The threshold of stimulation was below 0.1 μM. The substances did not open the channels themselves, even at high concentrations, and the reversal potential was not affected. This, together with data shown in Fig. 4, suggest an action on channel gating.

Fig. 8.
Fig. 8.

Lack of inhibition by the benzodiazepine antagonist Ro15-1788 and additive stimulation by diazepam and MS-1. Recombinant α1β2γ2 GABAA receptors were exposed to one of the following: 1) GABA in combination with 10 μM MS-1 or 1 μM diazepam; 2) GABA in combination with 10 μM MS-1 and 1 μM Ro15-1788, 100 μM ROD178B, or 1 μM diazepam; or 3) GABA in combination with 1 μM diazepam and 1 μM Ro15-1788. Data are given as the mean ± S.E.M. (up to three oocytes from two different batches).

All three substances are of a polyacetylene structure (Fig. 1). The only report known to us on a connection between polyacetylenes and the GABAA receptor deals with cicutoxin isolated from water hemlock and analogs (Uwai et al., 2000). Cicutoxin has been shown to displace the channel blocker [3H]EBOB (PerkinElmer Life and Analytical Sciences) from its binding site with an IC50 of 0.5 μM. No functional studies were performed, except it was shown that the compound was able to kill mice with a LD50 of ∼3 mg/kg. Another compound, virol A, was shown to have approximately half of the potency in both cases. Virol A was later shown to inhibit currents elicited by GABA in acutely dissociated rat hippocampal CA1 neurons with an IC50 of ∼1 μM (Uwai et al., 2001). Most remarkably, the compounds described here are linear molecules. The majority of positive allosteric modulators of GABAA receptors contain at least one cyclic entity. Other linear compounds affecting GABAA receptors are unsaturated fatty acids, including docosahexaenoic acid (DHA) (Nabekura et al., 1998). At low concentrations ≤1 μM, DHA stimulates GABA responses up to 20% in a γ subunit-dependent way. At higher concentrations, DHA inhibits GABA responses in a γ subunit-independent manner.

Fig. 9.
Fig. 9.

MS-1 affects inhibition by picrotoxin. Recombinant α1β2γ2 GABAA receptors were exposed to either GABA alone (•) or in combination with 10 μM MS-1 (○). Currents were inhibited by increasing concentrations of picrotoxin. Data are given as the mean ± S.E.M. (three oocytes from two different batches).

The three novel compounds showed unique subunit selectivity profiles. Omitting the γ subunit from α1β2γ2 did not affect the extent of stimulation. In contrast, the β subunit present seems to strongly influence stimulation. Replacing β2 with β1 reduced maximal stimulation more than 4-fold. Replacing β2 with β3 reduced maximal stimulation approximately 3-fold. Thus, MS-1 has specificity for receptors containing the β2 subunit. Introducing the point mutation into β2, N265S, which is known to strongly reduce stimulatory effects by loreclezole (Wingrove et al., 1994), had only a relatively weak effect, reducing maximal stimulation by ∼35%. This indicates that the site for MS compounds is probably different from that for loreclezole. This conclusion is enforced by the fact that loreclezole shows a different β subunit specificity, eliciting large simulation at receptors containing both the β2 and β3 subunits and only a small stimulation at receptors containing the β1 subunit (Wafford et al., 1994). The α subunit isoform present in αxβ2γ2 profoundly affected the extent of stimulation. For all three studied compounds, strongest modulation was seen in α1β2γ2, whereas only a weak stimulation was observed in α1β1γ2. The precise sequence for the extent of stimulation differed for the three compounds, α1β2γ2 ≈ α1β2 ≈ α3β2γ2 > α2β2γ2 > α5β2γ2 ≈ α1β3γ2 ≈ α6β2γ2 > α1β1γ2 for MS-1, α1β2γ2 ≈ α3β2γ2 ≈ α1β2 > α2β2γ2 ≈ α6β2γ2 ≈ α5β2γ2 > α1β1γ2 for MS-2, and α1β2γ2 ≈ α1β2 ≈ α5β2γ2 ≈ α3β2γ2 ≈ α2β2γ2 > α6β2γ2 ≫ α1β1γ2 for MS-4. The precise sequence of specificity is different for the three compounds, but for all compounds, action at α3β2γ2 is similar to action at α1β2γ2.

The relative lack of effects on the voltage-gated sodium channel together with the observed GABAA receptor subunit specificity argues against nonspecific perturbance of the membrane by the present linear hydrophobic molecules. It would be interesting to know where these novel compounds have their site of action in GABAA receptors. The independence of the stimulation from the presence of the γ subunit and its resistance to the benzodiazepine antagonist Ro15-1788 clearly document a site of action different from the benzodiazepine-binding site. The β subunit specificity and the relative lack of effect of the mutation β2N265S argues for a site of action different from loreclezole. The α subunit specificity differs from that of classic benzodiazepines, which fail to affect α6β2γ2 receptors.

In summary, we have described novel positive allosteric modulators of GABAA receptors belonging to the polyacetylenes. The pharmacological characterization is at the in vitro level, and the suitability of the compounds for therapeutic applications needs to be shown. Because chemical synthesis seems feasible, in vivo experiments are within reach.

Acknowledgments

We thank Dr. V. Niggli for carefully reading the manuscript and K. Tan and N. Boulineau for X. laevis surgery.

Footnotes

  • This work was supported by the Swiss National Science Foundation Grants 3100A0-105372/1 and 200020-100003/1.

  • ABBREVIATIONS: DMSO, dimethyl sulfoxide; MS-1, 1-ethyl-6-hydroxypentadeca-2,4-dinyl acetate; MS-2, 6-hydroxy-1-vinylpentadeca-2,4-dinyl acetate; MS-4, (6S)-16-acetoxy-6-hydroxy-1-vinylhexadeca-2,4-diynyl acetate; EBOB, ethynylbicycloorthobenzoate; DHA, docosahexaenoic acid; Ro 15-1788, 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester.

  • Received February 8, 2005.
  • Accepted June 6, 2005.

References

  1. ↵
    Backus KH, Arigoni M, Drescher U, Scheurer L, Malherbe P, Möhler H, and Benson JA (1993) Stoichiometry of a recombinant GABAA receptor deduced from mutation-induced rectification. Neuroreport 5: 285-288.
    OpenUrlCrossRefPubMed
  2. ↵
    Barnard EA, Skolnick P, Olsen RW, Möhler H, Sieghart W, Biggio G, Braestrup C, Bateson AN, and Langer SZ (1998) International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acid A receptors: classification on the basis of subunit structure and receptor function. Pharmacol Rev 50: 291-313.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Baumann SW, Baur R, and Sigel E (2001) Subunit arrangement of γ-aminobutyric acid, type A receptors. J Biol Chem. 276: 36275-36280.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Baumann SW, Baur R, and Sigel E (2002) Forced subunit assembly in α1β2γ2 GABAA receptors. Insight into the absolute arrangement. J Biol Chem 277: 46020-46025.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Benke D, Fritschy JM, Trzeciak A, Bannwarth W, and Möhler H (1994) Distribution, prevalence and drug binding profile of gamma-aminobutyric acid type A receptor subtypes differing in the β-subunit variant. J Biol Chem 269: 27100-27107.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Bertocci B, Miggiano V, Da Prada M, Dembic Z, Lahm H-W, and Malherbe P (1991) Human catechol-O-methyltransferase: cloning and expression of the membrane-associated form. Proc Natl Acad Sci USA 88: 1416-1420.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Boileau AJ, Baur R, Sharkey LM, Sigel E, and Czajkowski C (2002) The relative amount of cRNA coding for γ2 subunits affects stimulation by benzodiazepines in GABAA receptors expressed in Xenopus oocytes. Neuropharmacology 43: 695-700.
    OpenUrlCrossRefPubMed
  8. ↵
    Chang Y, Wang R, Barot S, and Weiss DS (1996) Stoichiometry of a recombinant GABAA receptor. J Neurosci 16: 5415-5424.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Farrar SJ, Whiting PJ, Bonnert TP, and McKernan RM (1999) Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer. J Biol Chem 274: 10100-10104.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Galzi J-L and Changeux JP (1994) Neurotransmitter-gated ion channels as unconventional allosteric proteins. Curr Opin Struct Biol 4: 554-565.
    OpenUrlCrossRef
  11. ↵
    Haerdi F (1964) Afrikanische Heilpflanzen—plantes médicinales africaines. Die Eingeborenen-Heilpflanzen des Ulanga-Distriktes Tanganjikas (Ostafrika). Acta Trop 8 Suppl: 1-278.
  12. ↵
    Kokwaro JO (1976) Medicinal Plants of East Africa. East African Literature Bureau, Kampala, Uganda.
  13. ↵
    Kuhn FJ and Greeff NG (1999) Movement of voltage sensor S4 in domain 4 is tightly coupled to sodium channel fast inactivation and gating charge immobilization. J Gen Physiol 114: 167-183.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Laurie DJ, Seeburg PH, and Wisden W (1992) The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J Neurosci 12: 1063-1076.
    OpenUrlAbstract
  15. ↵
    Lolait SJ, O'Carroll AM, Kusano K, Muller JM, Brownstein MJ, and Mahan LC (1989) Cloning and expression of a novel rat GABAA receptor. FEBS Lett 246: 145-148.
    OpenUrlCrossRefPubMed
  16. ↵
    Macdonald RL and Olsen RW (1994) GABAA receptor channels. Annu Rev Neurosci 17: 569-602.
    OpenUrlCrossRefPubMed
  17. ↵
    Malherbe P, Draguhn A, Multhaup G, Beyreuther K, and Mohler H (1990a) GABAA-receptor expressed from rat brain a- and b-subunit cDNAs displays potentiation by benzodiazepine receptor ligands. Brain Res Mol Brain Res 8: 199-208.
    OpenUrlCrossRefPubMed
  18. ↵
    Malherbe P, Sigel E, Baur R, Persohn E, Richards JG, and Mohler H (1990b) Functional characteristics and sites of gene expression of the α1, β1, γ2-isoform of the rat GABAA receptor. J Neurosci 10: 2330-2337.
    OpenUrlAbstract
  19. ↵
    McKernan RM and Whiting PJ (1996) Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci 19: 139-143.
    OpenUrlCrossRefPubMed
  20. ↵
    Nabekura J, Noguchi K, Witt MR, Nielsen M, and Akaike N (1998) Functional modulation of human recombinant gamma-aminobutyric acid type A receptor by docosahexaenoic acid. J Biol Chem 273: 11056-11061.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Rabow LE, Russek SJ, and Farb DH (1995) From ion currents to genomic analysis: recent advances in GABAA receptor research. Synapse 21: 189-274.
    OpenUrlCrossRefPubMed
  22. ↵
    Schofield PR, Darlison MG, Fujita N, Burt DR, Stephenson FA, Rodriguez H, Rhee LM, Ramachandran J, Reale V, Glencorse TA, et al. (1987) Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor superfamily. Nature (Lond) 328: 221-227.
    OpenUrlCrossRefPubMed
  23. ↵
    Sieghart W (1995) Structure and pharmacology of gamma-aminobutyric acid A receptor subtypes. Pharmacol Rev 47: 181-233.
    OpenUrlPubMed
  24. ↵
    Sigel E (1987) Properties of single sodium channels translated by Xenopus oocytes after injection with messenger ribonucleic acid. J Physiol (Lond) 386: 73-90.
    OpenUrlPubMed
  25. ↵
    Sigel E, Baur R, Trube G, Mohler H, and Malherbe P (1990) The effect of subunit composition of rat brain GABAA receptors on channel function. Neuron 5: 703-711.
    OpenUrlCrossRefPubMed
  26. ↵
    Sigel E, Baur R, Furtmueller R, Razet R, Dodd RH, and Sieghart W (2001) Differential cross talk of ROD compounds with the benzodiazepine binding site. Mol Pharmacol 59: 1470-1477.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Sigel E and Buhr A (1997) The benzodiazepine binding site of GABAA receptors. Trends Pharmacol Sci 18: 425-429.
    OpenUrlCrossRefPubMed
  28. ↵
    Sigel E, Stephenson FA, Mamalaki C, and Barnard EA (1983) A γ-aminobutyric acid/benzodiazepine receptor complex from bovine cerebral cortex. J Biol Chem 258: 6965-6971.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Tennant JR, Milne-Redhead E, and Polhill RM (1968) Flora of tropical East Africa. Araliaceae. Crown Agents for Overseas Governments and Administrations, London
  30. ↵
    Tretter V, Ehya N, Fuchs K, and Sieghart W (1997) Stoichiometry and assembly of a recombinant GABAA receptor subtype. J Neurosci 17: 2728-2737.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Uwai K, Ohashi K, Takaya Y, Ohta T, Tadano T, Kisara K, Shibusawa K, Sakakibara R, and Oshima Y (2000) Exploring the structural basis of neurotoxicity in C(17)-polyacetylenes isolated from water hemlock. J Med Chem 43: 4508-4515.
    OpenUrlCrossRefPubMed
  32. ↵
    Uwai K, Ohashi K, Takaya Y, Oshima Y, Furukawa K, Yamagata K, Omura T, and Okuyama S (2001) Virol A, a toxic trans-polyacetylenic alcohol of Cicuta virosa, selectively inhibits the GABA-induced Cl- current in acutely dissociated rat hippocampal CA1 neurons. Brain Res 889: 174-180.
    OpenUrlCrossRefPubMed
  33. ↵
    Wafford KA, Bain CJ, Quirk K, McKernan RM, Wingrove PB, Whiting PJ, and Kemp JA (1994) A novel allosteric modulatory site on the GABAA receptor β subunit. Neuron 12: 775-782.
    OpenUrlCrossRefPubMed
  34. ↵
    Wingrove PB, Wafford KA, Bain C, and Whiting PJ (1994) The modulatory action of loreclezole at the gamma-aminobutyric acid type A receptor is determined by a single amino acid in the β2 and β3 subunit. Proc Natl Acad Sci USA 91: 4569-4573.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

Novel Plant Substances Acting as β Subunit Isoform-Selective Positive Allosteric Modulators of GABAA Receptors

Roland Baur, Urs Simmen, Martin Senn, Urs Séquin and Erwin Sigel
Molecular Pharmacology September 1, 2005, 68 (3) 787-792; DOI: https://doi.org/10.1124/mol.105.011882
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement