Pharmacological Properties of GABAA Receptors Containing 1 Subunits

GABAA receptors composed of 1, 2, 1 subunits are expressed in only a few areas of the brain and thus represent interesting drug targets. The pharmacological properties of this receptor subtype, however, are largely unknown. In the present study, we expressed 1 2 1-GABAA receptors in Xenopus laevis oocytes and analyzed their modulation by 21 ligands from 12 structural classes making use of the two-microelectrode voltage-clamp method and a fast perfusion system. Modulation of GABA-induced chloride currents (IGABA) was studied at GABA concentrations eliciting 5 to 10% of the maximal response. Triazolam, clotiazepam, midazolam, 2-(4-methoxyphenyl)-2,3,5,6,7,8,9,10-octahydro-cyclohepta-(b)pyrazolo[4,3d]pyridin-3-one (CGS 20625), 2-(4-chlorophenyl)-pyrazolo[4,3c]quinolin-3-one (CGS 9896), diazepam, zolpidem, and bretazenil at 1 M concentrations were able to significantly ( 20%) enhance IGABA in 1 2 1 receptors. Methyl-6,7-dimethoxy-4-ethyl-carboline-3-carboxylate, 3-methyl-6-[3-trifluoromethyl-phenyl]-1,2,4-triazolo[4,3-b]pyridazine (Cl 218,872), clobazam, flumazenil, 5-(6-ethyl-7-methoxy-5-methylimidazo[1,2a]pyrimidin-2-yl)-3-methyl-[1,2,4]-oxadiazole (Ru 33203), 2-phenyl-4-(3-ethyl-piperidinyl)-quinoline (PK 9084), flurazepam, ethyl-7-methoxy-11,12,13,13a-tetrahydro-9-oxo-9H-imidazo[1,5a]pyrrolo[2,1-c] [1,4]benzodiazepine-1-carboxylate (L-655,708), 2-(6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)-4methyl-thiazole (Ru 33356), and 6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)phenylmethanone (Ru 32698) (1 M each) had no significant effect, and flunitrazepam and 2-phenyl4-(4-ethyl-piperidinyl)-quinoline (PK 8165) inhibited IGABA. The most potent compounds triazolam, clotiazepam, midazolam, and CGS 20625 were investigated in more detail on 1 2 1 and 1 2 2S receptors. The potency and efficiency of these compounds for modulating IGABA was smaller for 1 2 1 than for 1 2 2S receptors, and their effects on 1 2 1 could not be blocked by flumazenil. CGS 20625 displayed the highest efficiency by enhancing at 100 M IGABA ( 1 2 2) by 775 17% versus 526 14% IGABA ( 1 2 1) and 157 17% IGABA ( 1 2) (p 0.05). These data provide new insight into the pharmacological properties of GABAA receptors containing 1 subunits and may aid in the design of specific ligands for this receptor subtype. GABA is the principal inhibitory neurotransmitter in the mammalian brain. It mediates fast synaptic inhibition by interaction with the GABAA receptor. GABAA receptors are ligandgated ion channels that are modulated by a large number of clinically relevant drugs such as benzodiazepines (BZs), barbiturates, neurosteroids, and anesthetics (Sieghart, 1995). They are assembled from individual subunits forming a pentameric structure. Nineteen isoforms of mammalian GABAA receptor subunits have been cloned: 1–6, 1–3, 1–3, , , p, 1–3, and (Barnard et al., 1998; Simon et al., 2004). The major receptor subtype of the GABAA receptor in adults consists of 1, 2, and 2 subunits, and the most likely stoichiometry is two subunits, two subunits, and one subunit (Sieghart and Sperk, 2002). This work was supported by the Austrian Science Fund (Fonds zur Förderung der wissenschaftlichen Forschung) grant P12649-MED (to S.H.). S.K. and I.B. contributed equally to this work. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.017236. ABBREVIATIONS: CGS 20625, 2-(4-methoxyphenyl)-2,3,5,6,7,8,9,10-octahydro-cyclohepta-(b)pyrazolo[4,3-d]pyridin-3-one; CGS 9896, 2-(4-chlorophenyl)-pyrazolo[4,3-c]quinolin-3-one; IGABA, GABA-induced chloride currents; DMCM, methyl-6,7-dimethoxy-4-ethyl-carboline-3-carboxylate; Cl 218,872, 3-methyl-6-[3-trifluoromethyl-phenyl]-1,2,4-triazolo[4,3-b]pyridazine; Ru 33203, 5-(6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)-3methyl-[1,2,4]-oxadiazole; PK 9084, 2-phenyl-4-(3-ethyl-piperidinyl)-quinoline; L-655,708, ethyl-7-methoxy-11,12,13,13a-tetrahydro-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c] [1,4]benzodiazepine-1-carboxylate; Ru 33356, 2-(6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)-4-methyl-thiazole; Ru 32698, 6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)phenylmethanone; PK 8165, 2-phenyl-4-(4-ethyl-piperidinyl)-quinoline; Ro 15-1788, ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate; Ru 31719, (7-ethyl-5-methoxyimidazo[1,2-a]quinolin2-yl)phenyl methanone; BZ, benzodiazepine; Ro 15-1788, flumazenil; Ro 15-4513, ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-1,4-benzodiazepine-3-carboxylate; -CCM, methyl-carboline-3-carboxylate; ANOVA, analysis of variance. 0026-895X/06/6902-640–649$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 2 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 17236/3079084 Mol Pharmacol 69:640–649, 2006 Printed in U.S.A. 640 http://molpharm.aspetjournals.org/content/suppl/2005/11/07/mol.105.017236.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A ril 0, 2017 m oharm .aspeurnals.org D ow nladed from The subunit composition determines the GABA sensitivity and the pharmacological properties of the GABAA receptor (Sieghart, 1995; Hevers and Luddens, 1998; Boileau et al., 2002). The subunit composition of the receptor also affects the time course of the GABA response (desensitization and deactivation of the chloride currents) (Bianchi et al., 2001; Boileau et al., 2003; Feng et al., 2004). Mutation of amino acid residues in and 2 subunits modulate the BZ sensitivity of the receptor, suggesting that the BZ binding pocket is located at the interface between and 2 (Sigel, 2002; Ernst et al., 2003). There is clear evidence that substitution of the 2 subunit by either 1 or 3 significantly alters the sensitivity for BZ (Hevers and Luddens, 1998). In contrast to the 2 subunit, which is ubiquitously expressed in the central nervous system, the 1 subunit is expressed in only a few areas of the brain such as the amygdala (central and medial nuclei), the pallidum, the septum, the substantia nigra, and the thalamus (centrolateral and medial nuclei) (Pirker et al., 2000; Korpi et al., 2002). Compounds selectively interacting with receptors containing 1 subunits thus might have a substantial clinical potential. Compared with receptors containing 2 subunits, little is known about the pharmacological profile of GABAA channels composed of 1, 2, and 1 subunits. Ymer et al. (1990) observed a loss in affinity for the benzodiazepine antagonist Ro 15-1788 and the inverse agonist methyl-6,7-dimethoxy-4ethyl-carboline-3-carboxylate (DMCM) when the 1 was substituted for 2 in 1 1 2 receptors. Negative modulatory effects of Ro 15-4513, -CCM, and DMCM for GABAA receptors composed of 1/2/3 1 2 subunits are changed to positive modulatory effects in 1/2/3 1 1 receptors (Puia et al., 1991; Wafford et al., 1993). Benke et al. (1996) observed a low affinity for clonazepam, zolpidem, and flunitrazepam and apparent insensitivity for flumazenil and Ro 15-4513 for 1-containing receptors. Wafford et al. (1993) demonstrated a reduced enhancement of chloride currents through 3 2 3 by diazepam, clonazepam, and bretazenil compared with 3 2 2 and a negative modulatory effect of zolpidem for 2 1 1 and alpidem for 3 1 1 receptors. Overall, in 4 different studies, a total of 14 compounds from 5 different compound classes have been investigated so far for their ability to modulate GABAA receptors containing 1 subunits. Unfortunately, most of these studies were carried out under different experimental conditions and with receptors containing different and subunits combined with 1. Thus, the relative efficacies of these compounds for 1 are not comparable (Hevers and Luddens, 1998). In the present study, we analyzed the modulation of 1 2 1 receptors expressed in Xenopus laevis oocytes by 21 compounds comprising distinct chemical structures. Triazolam, clotiazepam, midazolam, and CGS 20625 exhibited a significant potency and efficiency, whereas the other compounds were either inactive or displayed only a low potency on 1containing receptors. Materials and Methods Chemicals. Compounds were obtained from the following sources: flunitrazepam (7-nitro-1,3-dihydro-1-methyl-5-o-fluorophenyl-2H-1,4benzodiazepin-2-one), diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one), flurazepam (7-chloro-1,3-dihydro-1ethylaminodiethyl-5-o-fluorophenyl-2H-1,4-benzodiazepin-2-one), midazolam [8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5a][1,4]benzodiazepine], Ro 15-1788, and bretazenil [t-butyl(s)-8-bromo11,12,13,13a-tetrahydro-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c] [1,4]benodiazepine-1-carboxylate] were from Hoffmann La Roche (Basel, Switzerland); L-655,708 was purchased from Tocris Cookson Inc. (Bristol, UK); clotiazepam [5-(2-chlorophenyl)-7-ethyl-1,3-dihydro-1-methyl-2Hthieno[2,3-e][1,4]diazepin-2-one] was from Troponwerke (Köln, Germany); clobazam [7-chloro-1-methyl-5-phenyl-1H-1,5-benzodiazepine2,4(3H,5H)-dione] was from sanofi-aventis (Bridgewater, NJ); triazolam [8-chloro-6-(2-chlorophenyl)-1-methyl-4H-1,2,4-triazolo[4,3a][1,4]benzodiazepine] was from Sigma (Vienna, Austria); DMCM was from Ferrosan (Soeborg, Denmark); CGS 9896 and CGS 20625 were from Novartis (Basel, Switzerland); zolpidem [N,N,6-trimethyl-2-(4methylphenyl)imidazo[1,2-a]-pyridine-3-acetamide] was from Synthelabo Recherche (Bagneux, France); Cl 218,872 was from American Cyanamide Comp. (Wayne, NJ); Ru 31719, Ru 32698, Ru 33203, and Ru 33356 were from Roussel Uclaf (Romainville, France); PK 8165 and PK 9084 were from Pharmuka Laboratories (Gennevilliers, France). For chemical structures, see Ogris et al. (2004). Expression and Functional Characterization of GABAA Receptors. X. laevis oocytes were prepared and injected as described previously (Grabner et al., 1996). Female X. laevis (Nasco, Fort Atkinson, WI) were anesthetized by exposing them for 15 min to a 0.2% MS-222 (methane sulfonate salt of 3-aminobenzoic acid ethyl ester; Novartis) solution before surgically removing parts of the ovaries. Follicle membranes from isolated oocytes were enzymatically digested with 2 mg/ml collagenase (type 1A, Sigma). One day after isolation, the oocytes were injected with approximately 10 to 50 nl of a solution of diethyl pyrocarbonate water containing the different cRNAs at a concentration of approximately 300 to 3000 pg/nl/ subunit. The amount of cRNA was determined by means of a NanoDrop ND-1000 (Kisker-Biotech, Steinfurt, Germany). To ensure expression of the subunit in the case of 1 2 1 and 1 2 2S receptors, cRNAs were mixed in a ratio of 1:1:10, and for receptors comprising only 1 and 2 subunits, they were mixed in a ratio of 1:1 (Boileau et al., 2002). Oocytes were stored at 18°C in ND96 solution (Methfessel et al., 1986). Electrophysiological experiments were performed by the twoelectrode voltage-clamp method making use of a TURBO TEC 01C amplifier (NPI Electronic GmbH, Tamm, Germany) at a holding potential of 70 mV. The bath solution contained 90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 5 mM HEPES, pH 7.4. Perfusion System. GABA was applied by means of a modified version of a fast perfusion system according to Hering (1998). A schematic drawing of the perfusion chamber and drug application device is shown in Fig. 1A. As described previously, the voltageclamp experiments on X. laevis oocytes were performed in a small ( 15 l) bath that was covered by a glass plate. Two angular inlet channels in the glass cover (diameter, 1 mm) enabled access of the two microelectrodes to the oocyte. A funnel for drug application surrounded both access channels for the microelectrodes compared with a funnel surrounding a single access channel in Hering (1998). This modification increased the stability of oocyte perfusion. Drug or control solutions were applied to the funnel by means of a Miniprep 60 (Tecan, Durham, NC) that was controlled by a DigiData 1322A (Clampex version 9.2; Molecular Devices, Sunnyvale, CA), permitting automation of the experiments [see also Supplemental Videos S1 and S2 (files methods_1.avi and methods_2.avi) for animation of the solution exchange]. To estimate the rate of solution exchange independent of the ligand-receptor interaction, we expressed Kv1.1 channels in X. laevis oocytes and analyzed the time course of current decay during a rapid increase of the extracellular potassium concentration from 1 to 10 mM (sodium was reduced to 80 mM, respectively). Figure 1B illustrates a typical Kv1.1 current during a voltage-clamp step from 80 to 20 mV. The current decrease upon fast perfusion with 10 mM potassium at a speed of 1 ml/s is shown on the right in Fig. 1B. A GABAA Receptors Containing 1 Subunits 641 at A PE T Jornals on A ril 0, 2017 m oharm .aspeurnals.org D ow nladed from mean current decline time t10–90% of 140.8 17.5 ms (n 7) was estimated. To elicit GABA-induced chloride currents (IGABA), the chamber was perfused with 120 l of GABA-containing solution at the same volume rate (1 ml/s). The rise time of IGABA ranged usually between 100 and 250 ms (Fig. 1C), which is comparable with the rate of solution exchange estimated in Fig. 1B. After the initial fast perfusion step for rapid agonist application, the chamber was continuously perfused at a rate of 1 l/s for a total of 18 s. Before rapid washout of agonist and/or drug, the funnel was emptied by a suction pulse applied to the two funnel outlets (Fig. 1A). The time between this suction pulse and the application of new solution to the funnel was 1 s to avoid evaporation of the bath surrounding the oocyte. Duration of washout periods was extended from 3 to 30 min, with increasing concentrations of applied GABA to account for slow recovery from increasing levels of apparent desensitization. Oocytes with maximal current amplitudes 3 A were discarded to minimize voltage-clamp errors. Analyzing Concentration-Response Curves. Enhancement of chloride currents by modulators of the GABAA receptor was measured at a GABA concentration eliciting between 5 and 10% of the maximal current amplitude (EC5–10). The EC5–10 (usually ranging between 3 and 8 M) was determined at the beginning of each