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Pharmacological Properties of GABA_A Receptors Containing 1 Subunits

Department of Pharmacology and Toxicology, University of Vienna (S.K., I.B., E.N.T., A.H., S.H.), and Center of Brain Research, Medical University of Vienna, Division of Biochemistry and Molecular Biology (W.S.), Vienna, Austria

Received July 26, 2005; accepted November 4, 2005

	Abstract

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GABA_A 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 ₁₂₁-GABA_A 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 (I_GABA) 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[4,3-d]pyridin-3-one (CGS 20625), 2-(4-chlorophenyl)-pyrazolo[4,3-c]quinolin-3-one (CGS 9896), diazepam, zolpidem, and bretazenil at 1 µM concentrations were able to significantly (>20%) enhance I_GABA in ₁₂₁ 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[1,2-a]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[1,5-a]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)-4-methyl-thiazole (Ru 33356), and 6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)phenylmethanone (Ru 32698) (1 µM each) had no significant effect, and flunitrazepam and 2-phenyl-4-(4-ethyl-piperidinyl)-quinoline (PK 8165) inhibited I_GABA. The most potent compounds triazolam, clotiazepam, midazolam, and CGS 20625 were investigated in more detail on ₁₂₁ and _122S receptors. The potency and efficiency of these compounds for modulating I_GABA was smaller for ₁₂₁ than for _122S receptors, and their effects on ₁₂₁ could not be blocked by flumazenil. CGS 20625 displayed the highest efficiency by enhancing at 100 µM I_GABA (₁₂₂) by 775 ± 17% versus 526 ± 14% I_GABA (₁₂₁) and 157 ± 17% I_GABA (₁₂) (p < 0.05). These data provide new insight into the pharmacological properties of GABA_A receptors containing ₁ subunits and may aid in the design of specific ligands for this receptor subtype.

The subunit composition determines the GABA sensitivity and the pharmacological properties of the GABA_A 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 GABA_A 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-4-ethyl--carboline-3-carboxylate (DMCM) when the ₁ was substituted for ₂ in ₁₁₂ receptors. Negative modulatory effects of Ro 15-4513, -CCM, and DMCM for GABA_A receptors composed of _1/2/312 subunits are changed to positive modulatory effects in _1/2/311 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 ₃₂₃ by diazepam, clonazepam, and bretazenil compared with ₃₂₂ and a negative modulatory effect of zolpidem for ₂₁₁ and alpidem for ₃₁₁ 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 GABA_A 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 ₁₂₁ 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 ₁-containing receptors.

	Materials and Methods

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Chemicals. Compounds were obtained from the following sources: flunitrazepam (7-nitro-1,3-dihydro-1-methyl-5-o-fluorophenyl-2H-1,4-benzodiazepin-2-one), diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one), flurazepam (7-chloro-1,3-dihydro-1-ethylaminodiethyl-5-o-fluorophenyl-2H-1,4-benzodiazepin-2-one), midazolam [8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine], Ro 15-1788, and bretazenil [t-butyl(s)-8-bromo-11,12,13,13a-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-2H-thieno[2,3-e][1,4]diazepin-2-one] was from Troponwerke (Köln, Germany); clobazam [7-chloro-1-methyl-5-phenyl-1H-1,5-benzodiazepine-2,4(3H,5H)-dione] was from sanofi-aventis (Bridgewater, NJ); triazolam [8-chloro-6-(2-chlorophenyl)-1-methyl-4H-1,2,4-triazolo[4,3-a][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-(4-methylphenyl)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 GABA_A 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 Nano-Drop ND-1000 (Kisker-Biotech, Steinfurt, Germany). To ensure expression of the subunit in the case of ₁₂₁ and _122S 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 two-electrode 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 voltage-clamp experiments on X. laevis oocytes were performed in a small (15 µl) 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.

View larger version (18K): [in this window] [in a new window]   Fig. 1. A, cross-section view of the oocyte perfusion chamber. Two microelectrodes (M1 and M2) are inserted into the angular access inlets in a glass plate (the inlets serve simultaneously as perfusion holes) covering the small (15 µl) oocyte chamber. Drug was applied by a Tecan Miniprep 60 application tube (AT) to a quartz funnel surrounding the microelectrode access holes (MAH). Residual solution was removed from the funnel before drug application via the funnel outlets (see Hering, 1998 and the two Supplemental Video files S1 and S2). B, estimation of the rate of solution exchange on an oocyte expressing Kv 1.1 the extracellular potassium concentration was rapidly increased from 1 to 10 mM during a potassium outward current (voltage step from –80 to +20 mV). The time of current decrease (right) from 10 to 90% (t10–90%) was taken as a measure of the volume rate of oocyte perfusion. C, typical time courses of IGABA (122S receptors) activated by different GABA concentrations. Upon application of 300 µM GABA, IGABA increased from 10 to 90% within 100 ms. At 100, 30, and 10 µM GABA t10–90% was 140, 180, and 230 ms, respectively.

To estimate the rate of solution exchange independent of the ligand-receptor interaction, we expressed K_v1.1 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 K_v1.1 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 mean current decline time t_10–90% of 140.8 ± 17.5 ms (n = 7) was estimated.

To elicit GABA-induced chloride currents (I_GABA), the chamber was perfused with 120 µl of GABA-containing solution at the same volume rate (1 ml/s). The rise time of I_GABA 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 µl/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 µA were discarded to minimize voltage-clamp errors.

Analyzing Concentration-Response Curves. Enhancement of chloride currents by modulators of the GABA_A receptor was measured at a GABA concentration eliciting between 5 and 10% of the maximal current amplitude (EC_5–10). The EC_5–10 (usually ranging between 3 and 8 µM) was determined at the beginning of each experiment.

Enhancement of the chloride current (I_GABA) was defined as (I_{(GABA + Comp)}/I_GABA) – 1, where I_{(GABA + Comp)} is the current response in the presence of a given compound, and I_GABA is the control GABA current. To measure the sensitivity of the GABA_A receptor for a given compound, it was applied for an equilibration period of 1 min before concomitant application of GABA (EC_5–10) and increasing concentrations of the compound. None of the compounds investigated was able to induce chloride flux in the absence of GABA. Concentration-response curves were generated, and the data were fitted by nonlinear regression analysis using ORIGIN software (OriginLab Corp, Northampton, MA). Data were fitted to the equation 1/(1 + (EC₅₀/[Comp])^nH), where EC₅₀ is the concentration of the compound that increases the amplitude of the GABA-evoked current by 50%, and n_H is the Hill coefficient. Data are given as mean ± S.E. from at least four oocytes and 2 oocyte batches. Statistical significance was calculated using unpaired Student's t test with a confidence interval of p < 0.05.

View larger version (23K): [in this window] [in a new window]   Fig. 2. A, modulation of chloride currents through GABAA receptors composed of 121 subunits by 1 µM (), 10 µM (), and 100 µM () of the indicated compounds. Each value represents the mean ± S.E. from at least four oocytes and 2 oocyte batches. *, significantly different from 0 (p < 0.05, t test by ANOVA). B, typical traces for enhancement of chloride currents through 121 channels by triazolam, clotiazepam, and midazolam at EC5–10. Control currents (GABA, single bar) and corresponding currents elicited by coapplication of GABA and the indicated compound (double bar) are shown.

	Results

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In a first step, GABA_A receptors were activated by GABA concentrations corresponding to EC_5–10, and drug effects were screened at a single concentration of 1 µM. As illustrated in Fig. 2 (), only the benzodiazepines triazolam, midazolam, and diazepam, the thienodiazepine clotiazepam, the pyrazolopyridine CGS 20625, and the pyrazoloquinoline CGS 9896 induced an enhancement of >20% at this concentration. The other compounds induced either a very small but statistically significant enhancement (zolpidem and bretazenil), no statistically significant effect (DMCM, Cl 218,872, clobazam, flumazenil, Ru 33203, PK 9084, flurazepam, L-655,708, Ru 31719, Ru 33356, and Ru 32698), or even an inhibition of GABA-induced chloride currents (flunitrazepam and PK 8165), and details are given in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Modulation of the GABA-induced chloride currents by 1 µM concentration of the indicated compounds for receptors composed of 121 subunits (second column) and 12 subunits (third column) Each value represents the mean ± S.E. of at least four oocytes from at least two oocyte batches.

To establish compounds displaying low potency but high efficiency, all compounds were subsequently tested at higher concentrations (10 and 100 µM) (Fig. 2A). Modulation of GABA receptors at low GABA concentrations (EC_5–10 close to "tonic concentrations") can substantially differ from modulation at high concentrations (i.e., millimolar "synaptic concentrations"). We have, therefore, analyzed the effects of all 21 compounds at 1 mM GABA (Fig. 3A). None of the compounds, however, substantially enhanced or inhibited I_GABA. Representative chloride currents induced by 1 mM in the absence or presence of 1 µM triazolam or CGS 20625 are shown in Fig. 3B.

View larger version (23K): [in this window] [in a new window]   Fig. 3. A, modulation of chloride currents through GABAA receptors composed of 121 subunits at 1 mM GABA by 1 µM concentration of the indicated compounds. *, statistically significant differences from 0 (p < 0.05, t test by ANOVA). B, typical enhancement of IGABA through 121 channels induced by 1 mM GABA in the absence (control, left traces) and presence of 1 µM triazolam and 1 µM CGS 20625.

For the most potent stimulators of the ₁₂₁ receptors triazolam, clotiazepam, and midazolam, we also analyzed the inhibition of I_GABA in oocytes expressing only ₁₂ subunits at higher (10 µM) concentrations. Triazolam inhibited the GABA-induced chloride flux in ₁₂ receptors by –33 ± 4% (n = 12), clotiazepam by –30 ± 8% (n = 7), and midazolam by –31 ± 9% (n = 5) (experiments not shown).

Comparing the Effects of Benzodiazepine Site Ligands on ₁₂₁ and _122s Receptors. Triazolam, clotiazepam, midazolam, and CGS 20625 were subsequently analyzed in more detail by comparing their effects on ₁₂₁ and _122S receptors. Figure 4 illustrates the concentration-dependence of the enhancement of the currents (EC_5–10) by triazolam, clotiazepam, and midazolam. The EC₅₀ value was determined by fitting the concentration-effect data to the Hill equation. Triazolam enhanced the maximum chloride current of ₁₂₁ receptors by 85% while displaying the highest potency (EC₅₀ 90 nM) of all tested benzodiazepines. Clotiazepam elicited an enhancement of the GABA response of approximately 170% but had a 19-fold lower potency (EC₅₀ 1.7 µM) than triazolam. Midazolam was more potent than clotiazepam (EC₅₀ 1.2 µM) but was 13 times less potent than triazolam, with a maximum enhancement (92%) comparable with triazolam.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Concentration-effect curves for triazolam, clotiazepam, and midazolam on 121 () and 122S receptors () using an EC5–10 GABA concentration (EC50 values are given in Table 2). Data points represent means ± S.E. from at least four oocytes from 2 batches.

50/1

50/2S

View this table: [in this window] [in a new window]   TABLE 2 Potency and efficiency of triazolam, midazolam, clotiazepam, and CGS 20625 for GABAA receptors composed of 121 or 122S subunits The maximum enhancement (percentage of control) and EC50 values were calculated by fitting the data points to a standard concentration-response curve. Rightmost column represents ratios between EC50 values of 121 and 122S receptors.

Modulation of I_GABA by Triazolam, Clotiazepam, and Midazolam at Different GABA Concentrations. To gain insight into the mechanism of I_GABA enhancement, we studied the GABA dose-effect curves in the absence and presence of the modulators. The results are shown in Fig. 5, A to C. In control, the mean EC₅₀ value for GABA was 39 ± 3 µM in ₁₂₁ receptors and 50 ± 3 µM in _122S receptors. The three benzodiazepine receptor ligands shifted the dose-effect curves to the left without affecting the maximal response (Fig. 3A). It is noteworthy that the drug-induced shift was more pronounced for _122S than for ₁₂₁ receptors, reflecting the higher efficiency of these ligands on _122S.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Modulation of the GABA concentration-response curve of 121 (left) and 122S receptors (right) by 1 µM triazolam (A), 10 µM clotiazepam (B), and 10 µM midazolam (C). The corresponding mean EC50 values were 37 ± 2 µM (control), 26 ± 4 µM (triazolam) for 121 and 52 ± 7 µM (control), and 17 ± 3 µM (triazolam) for 122S (A); 40 ± 8 µM (control), 25 ± 4 µM (clotiazepam) for 121 and 50 ± 5 µM (control), and 19 ± 3 µM (clotiazepam) for 122S (B); and 41 ± 6 µM (control), 22 ± 2 µM (midazolam) for 121 and 47 ± 11 µM (control), and 13 ± 6 µM (midazolam) for 122S (C).

max/2

max/1

View larger version (23K): [in this window] [in a new window]   Fig. 6. Modulation of IGABA by CGS 20625. A, typical IGABA recordings illustrating concentration-dependent modulation of GABA-elicited chloride currents through 121-containing receptors. B, concentration-effect curves for CGS 20625 on 122S (), 121 (), and 12 () receptors. EC50 values and corresponding Hill coefficient were the following: 11.2 ± 0.7 µM, nH = 1.8 ± 0.1 (); 23.7 ± 6.8 µM, nH = 0.9 ± 0.1 (); and 4.3 ± 1.2 µM and nH = 1.4 ± 0.2 (), respectively. Each data point represents mean ± S.E. from at least four oocytes and 2 batches. IGABA at 300 µM (open symbols) were excluded from the fit. C, modulation of the GABA concentration response of 121 (left) and 122 receptors (right) by 100 µM CGS 20625. The corresponding EC50 values were 39 ± 16 µM (control) and 7 ± 2 µM (CGS 20625) in 121 receptors and 56 ± 14 µM (control) and 15 ± 3 µM (CGS 20625) in 122S receptors.

Figure 7, A and B (left), illustrates the inhibition by flumazenil of triazolam- or clotiazepam-induced I_GABA enhancement in _122S receptors. As shown on the right, the effects of triazolam or clotiazepam on ₁₂₁ receptors were not inhibited by 1 µM flumazenil. Moreover, we were unable to study possible antagonistic effects at higher concentrations, because flumazenil induced significant enhancement of I_GABA in oocytes expressing ₁₂₁ subunits at 10 (22 ± 2%, n = 4) and 100 µM (64 ± 8%, n = 4; Fig. 7C).

View larger version (20K): [in this window] [in a new window]   Fig. 7. A and B, effect of flumazenil on the enhancement of IGABA by triazolam and clotiazepam. The left graphs illustrates the enhancement of IGABA through 122s receptors by triazolam and clotiazepam in the absence and presence of flumazenil. The right graphs illustrate the lack of inhibition of IGABA through 121 receptors. C, typical IGABA through 121 receptors in the absence (GABA) and presence of the indicated concentrations of flumazenil.

	Discussion

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In the present study, we made use of a fast and automated perfusion technique (Fig. 1) to test a selection of 21 modulators on GABA_A receptors composed of ₁₂₁ subunits. Solution exchange occurred between 100 and 250 ms (see Materials and Methods and Fig. 1C), which reduced the effects of desensitization on peak current detection compared with conventional bath perfusion.

I_GABA of ₁₂₁ Subunit Receptors Is Enhanced by Some Benzodiazepines and the Pyrazolopyridine CGS 20625. The 21 compounds tested comprised benzodiazepines and representatives of other structural classes of ligands of the BZ binding site of GABA_A receptors. To determine the most potent modulators of ₁₂₁ subunit receptors, we first tested the compounds at a concentration of 1 µM. Of the 21 compounds, six induced an enhancement of the GABA response (EC_5–10) by more than 20% with the following order of potency: triazolam > clotiazepam > midazolam > CGS 20625 > CGS 9896 > diazepam. Zolpidem and bretazenil induced a small enhancement (13 and 10%, respectively). The effects of DMCM, Cl 218,872, clobazam, flumazenil, Ru 33203, PK 9084, flurazepam, L-655,708, Ru 31719, Ru 33356, and Ru 32698 were not significantly different from control. In contrast, flunitrazepam and PK 8165 significantly inhibited the GABA-induced chloride flux. Application of higher concentrations (10 and 100 µM) revealed a moderate enhancement at high concentrations (low-potency modulation) by bretazenil, DMCM, flumazenil, Ru 33203, Ru 32698, and flunitrazepam. All compounds were tested in the absence of GABA (data not shown). None of them induced measurable currents, even at high concentrations (up to 100 µM). Thus, the 1,4-benzodiazepines triazolam and midazolam, the 1,4-thienodiazepine clotiazepam, and the pyrazolopyridine CGS 20625 seemed to be the most promising candidates for further detailed analysis.

Contribution of the ₁ Subunit to the Enhancement of I_GABA. On injection of X. laevis oocytes with , , and subunits, not only are receptors containing all three subunits formed but possibly also receptors composed of and subunits only (Boileau et al., 2002). To investigate whether the observed drug effects were caused by effects on ₁₂₁ receptors or could also be explained by effects on ₁₂ receptors, the effects of drugs on the latter receptors were also investigated. A comparison of drug effects on ₁₂₁ and ₁₂ receptors revealed that the ₁ subunit was essential for enhancement of I_GABA by triazolam, clotiazepam, and midazolam (Table 1 and Fig. 2), because these compounds at 1 µM concentration induced a significant inhibition by –21 ± 4% (n = 12, p < 0.05), (–14 ± 2%, n = 6), and (–20 ± 5%, n = 6), respectively, of I_GABA through ₁₂ subunit receptors. In addition, the effects of several other drugs were clearly different in ₁₂₁ and ₁₂ receptors. Thus, CGS 20625, CGS 9896, diazepam, zolpidem, and bretazenil exhibited an enhancement of I_GABA that was higher in ₁₂₁ than in ₁₂ receptors, supporting the importance of the ₁ subunit for the effects observed. Finally, Cl 218,872 and flurazepam exhibited effects in opposite direction in ₁₂₁ and ₁₂ receptors, again supporting the conclusion that the drug effects observed on injection of oocytes with ₁, ₂, and ₁ subunits were predominantly caused by ₁₂₁ receptors (Table 1).

Our finding that triazolam, clotiazepam, and midazolam significantly inhibit I_GABA through ₁₂ receptors suggests that we might have underestimated the enhancement of I_GABA in ₁₂₁ receptors by these drugs because of simultaneously occurring inhibition of ₁₂ subunit receptors. The differential effects of various BZ binding site ligands on receptors composed of ₁₂ subunits are highly interesting by themselves and significantly extend previous evidence for the existence of a low-affinity benzodiazepine binding site at receptors (Wafford et al., 1993; Thomet et al., 1999; Walters et al., 2000).

To determine the potency and efficiency (maximum ability to enhance the GABA EC_5–10 response of the three most potent BZ; Table 2), we studied their concentration effect for receptors composed of ₁₂₁ subunits (Fig. 3). The 1,4-benzodiazepine triazolam displayed the highest potency (EC₅₀ 90 nM), followed by midazolam (EC₅₀ 1.2 µM) and the 1,4-thienodiazepine clotiazepam (EC₅₀ 1.7 µM, Table 2). A comparison of the concentration-effect curves for these benzodiazepine-type ligands with receptors composed of _122S receptors revealed a significantly lower efficiency and potency for ₁-containing receptors (Fig. 3 and Table 2).

The Pyrazolopyridine CGS 20625 Displays the Highest Efficiency in Enhancing GABA-Induced Chloride Flux of ₁₂₁ Subunit Receptors. CGS 20625 was identified as the most efficient compound in terms of maximum enhancement of the GABA-induced chloride currents through ₁₂₁ subunit receptors (Fig. 6). This compound induced a maximum enhancement of 645 ± 55% greater than control, which is approximately 3.75-fold the enhancement achieved with clotiazepam (172 ± 24% greater than control), and more than 7 times the enhancement induced by midazolam (92 ± 8% greater than control) or by triazolam (85 ± 7% above control), respectively (Fig. 3). CGS 20625, however, had a potency for ₁₂₁ receptors (EC₅₀ 20 µM) approximately 200 times lower than that of triazolam and 10 to 20 times lower than that of the other benzodiazepines (Table 2).

A closer inspection of the subunit composition specificity of CGS 20625 action revealed that this drug potentiates ₁₂₁ and _122S subunit receptors to an almost similar extent (Fig. 6B). However, a significantly lower efficiency on ₁₂ receptors revealed an essential role of a subunit.

CGS 20625 at 300 µM caused less enhancement than at 100 µM in ₁₂, ₁₂₁, and _122S subunit receptors (Fig. 6B), suggesting that this compound might inhibit chloride currents at high concentrations. Similar behavior was shown previously for the action of another pyrazolopyridine (tracazolate) on GABA_A channels (Thompson et al., 2002).

CGS 20625 thus represents a low-potency (EC₅₀ 20 µM) but high-efficiency modulator of ₁₂₁ and _122S subunit-containing receptors (Fig. 6B). This compound was almost not selective for either ₁ or ₂ subunits. ₁₂ Subunit receptors were, however, stimulated to a significantly lesser extent (160%) compared with _121/2S subunit receptors (640–730%) (Fig. 6B).

The competitive antagonist flumazenil (1 µM) inhibited I_GABA enhancement of _122S receptors but failed to affect the enhancement of I_GABA through ₁₂₁ receptors by triazolam and clotiazepam (Fig. 7, right). These data suggest that flumazenil exhibits either no or a very low affinity for the BZ binding site of ₁₂₁ receptors, or that flumazenil interacts with a binding site different from that for triazolam and clotiazepam at these receptors. The first conclusion is consistent with the observation that the affinity of flumazenil for its binding site was reduced approximately 1000-fold in GABA_A receptors in which phenylalanine 77 of the ₂ subunit was mutated to the corresponding residue (isoleucine) of the ₁ subunit (₂F77I; Buhr et al., 1997; Wingrove et al., 2002; Ogris et al., 2004). At higher concentrations, flumazenil displayed properties of a low-affinity agonist on ₁₂₁-receptors (10 µM potentiated I_GABA by 22 ± 2% and 100 µM by 64 ± 9%).

In addition to flumazenil, the affinities of bretazenil, L-655,708, DMCM, zolpidem, Cl 218,872, and PK 8165 were drastically reduced in receptors containing the ₂F77I point mutation (Ogris et al., 2004), as measured by [³H]flunitrazepam binding studies. A low affinity of these compounds for ₁₂₁ receptors could have contributed to their small effects on these receptors observed in the present study. In contrast, replacement of phenylalanine (₂F77) by the corresponding isoleucine of the ₁ subunit only weakly (2- to 7-fold) reduced the affinity of the classic 1,4-benzodiazepines, the 1,4-thienodiazepine clotiazepam, the 1,5-benzodiazepine clobazam, or the pyrazoloquinoline CGS 9896 (Ogris et al., 2004). The small (4- to 9-fold) reduction in potency of triazolam, midazolam, and clotiazepam for enhancing ₁₂₁ compared with ₁₂₂ receptors could be explained by a reduced apparent affinity of these compounds for ₁₂₁ receptors underlining the importance of Phe77 for high-affinity BZ binding (Buhr et al., 1997). For other compounds such as flunitrazepam (1 µM), we observed significant inhibition of I_GABA in ₁₂₁ receptors, indicating the importance of additional amino acids for drug binding and gating.

The different efficiency of triazolam, midazolam, clotiazepam, and CGS 20625 are explained by the different amounts of shifts of the GABA concentration-effect curves (Fig. 5, A–C). Larger shifts induced on _122S receptors reflect the higher apparent efficiency.

In summary we systematically investigated 21 ligands of the BZ binding site from chemically distinct classes to obtain insight in the pharmacological profile of GABA_A receptors comprising a ₁ subunit. Triazolam was identified as a high-potency and CGS 20625 as a high-efficiency modulator of this receptor subtype. Different potencies of triazolam, midazolam, clotiazepam, and CGS 20625 can be explained by different shifts of the GABA dose-effect curve, reflecting different apparent affinities of these compounds.

	Acknowledgements

 
We thank Drs. Roman Furtmüller and Stanislav Berjukow for helpful suggestions and Stanislav Beyl for preparing the video animation.

	Footnotes

 
This work was supported by the Austrian Science Fund (Fonds zur Förderung der wissenschaftlichen Forschung) grant P12649 [GenBank] -MED (to S.H.).

S.K. and I.B. contributed equally to this work.

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; I_GABA, 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)-3-methyl-[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]quinolin-2-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.

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Steffen Hering, Department of Pharmacology and Toxicology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. E-mail: steffen.hering{at}univie.ac.at

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