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Subunit Isoform-Selective Positive Allosteric Modulators of GABAA Receptors
Department of Pharmacology, University of Bern, Bern, Switzerland (R.B., E.S.); and Departments of Pharmacy (U.Si.) and Chemistry (M.S., U.Sé.), University of Basel, Basel, Switzerland
Received for publication February 8, 2005.
Accepted for publication June 6, 2005.
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Abstract |
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2 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 
2N265S that abolishes effects by loreclezole. At a concentration of 30 µM, the substances by themselves elicited only tiny currents. Maximal stimulation at 
122 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, 122 
12 322 > 222 > 522 132 622 > 112, for MS-2 122 322 12 > 222 622 522 > 112, and for MS-4, 122 12 522 322 222 > 622 >> 112. Maximal stimulation by MS-1 was 450% at 122, 80% at 112, and 150% at 132. MS-1 was thus specific for receptors containing the 2 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.
), 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 1, 2, and 2 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 2:2:1 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.
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Materials and Methods |
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[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 1, 2, and 2S(2) 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 2, 3, 5, 6, 1, and 3 were prepared similarly. Subunits were cloned into the EcoRI, and the 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 1, 2, 3, 5, 6, 1, 2, 3, and 2, 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 1 and 2 subunits was expressed at 75 and 75 nM, and the combination of wild-type x, x, and 2 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] x 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).
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122, 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 122. This concentration-dependent stimulation is documented for MS-1 at 122 with a GABA concentration of 7 µM (Fig. 3). Maximum stimulation at 122 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.
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Subunit Specificity of MS-1. Figure 5A shows an averaged concentration response curve of this type of experiment for 122. Maximal stimulation is 450%, and half-maximal stimulation was observed at a concentration (EC50) of 1.5 mM. Replacement of 1 in this subunit combination by other subunit isoforms such as 2, 3, 5, or 6 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 122 322 > 222 > 522 622. Figure 5B shows the effect of the subunit isoform and the lack of effect upon omitting 2 from 122. Replacing 2 in 122 by 1 or 3 drastically reduced maximal stimulation from 450% to 80 and 150%, respectively. Introducing the point mutation 2N265S that is known to strongly reduce stimulatory effects by loreclezole (Wingrove et al., 1994) into 122 had only a relatively weak effect in this case, reducing maximal stimulation by approximately one third (Fig. 5B).
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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 1 in 122 by other subunit isoforms such as 2, 3, 5, or 6 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, 122 322 12 > 222 622 522 > 112, and for MS-4, 122 12 522 322 222 > 622 >> 112.
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), at a concentration of 100 µM, significantly reduced stimulation to 134 ± 9% (n = 3).
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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).
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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).
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).
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 2 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 122 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.
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Discussion |
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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 subunit-dependent way. At higher concentrations, DHA inhibits GABA responses in a subunit-independent manner.
The three novel compounds showed unique subunit selectivity profiles. Omitting the subunit from 122 did not affect the extent of stimulation. In contrast, the subunit present seems to strongly influence stimulation. Replacing 2 with 1 reduced maximal stimulation more than 4-fold. Replacing 2 with 3 reduced maximal stimulation approximately 3-fold. Thus, MS-1 has specificity for receptors containing the 2 subunit. Introducing the point mutation into 2, 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 subunit specificity, eliciting large simulation at receptors containing both the 2 and 3 subunits and only a small stimulation at receptors containing the 1 subunit (Wafford et al., 1994). The subunit isoform present in x22 profoundly affected the extent of stimulation. For all three studied compounds, strongest modulation was seen in 122, whereas only a weak stimulation was observed in 112. The precise sequence for the extent of stimulation differed for the three compounds, 122 12 322 > 222 > 522 132 622 > 112 for MS-1, 122 322 12 > 222 622 522 > 112 for MS-2, and 122 12 522 322 222 > 622 >> 112 for MS-4. The precise sequence of specificity is different for the three compounds, but for all compounds, action at 322 is similar to action at 122.
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 subunit and its resistance to the benzodiazepine antagonist Ro15-1788 clearly document a site of action different from the benzodiazepine-binding site. The subunit specificity and the relative lack of effect of the mutation 2N265S argues for a site of action different from loreclezole. The subunit specificity differs from that of classic benzodiazepines, which fail to affect 622 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.
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Acknowledgements |
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Footnotes |
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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.
Address correspondence to: Dr. Erwin Sigel, Department of Pharmacology, Friedbuehlstrasse 49, CH-3010 Bern, Switzerland. E-mail: erwin.sigel{at}pki.unibe.ch
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  R. W. Olsen and W. Sieghart International Union of Pharmacology. LXX. Subtypes of {gamma}-Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Composition, Pharmacology, and Function. Update Pharmacol. Rev., September 1, 2008; 60(3): 243 - 260. [Abstract] [Full Text] [PDF]
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), 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.
) or in combination with 10 µM MS-1 (
), and 
) (A) and 
) (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).
