Estimating the efficiency of benzodiazepines on GABAA receptors comprising γ1 or γ2 subunits

Background and purpose: Heterologous expression of α1, β2 and γ2S(γ1) subunits produces a mixed population of GABAA receptors containing α1β2 or α1β2γ2S(γ1) subunits. GABA sensitivity (lower in receptors containing γ1 or γ2S subunits) and the potentiation of GABA-activated chloride currents (IGABA) by benzodiazepines (BZDs) are dependent on γ2S(γ1) incorporation. A variable γ subunit incorporation may affect the estimation of IGABA potentiation by BZDs. We propose an approach for estimation of BZD efficiency that accounts for mixed population of α1β2 and α1β2γ2S(γ1) receptors. Experimental approach: We investigated the relation between GABA sensitivity (EC50) and BZD modulation by analysing triazolam-, clotiazepam- and midazolam-induced potentiation of IGABA in Xenopus oocytes under two-microelectrode voltage clamp. Key results: Plotting EC50 versus BZD-induced shifts of GABA concentration-response curves (ΔEC50(BZD)) of oocytes injected with different amounts of α1, β2 and γ2S(γ1) cRNA (1:1:1–1:1:10) revealed a linear regression between γ2S(γ1)-mediated reduction of GABA sensitivity (EC50) and ΔEC50(BZD). The slope factors of the regression were always higher for oocytes expressing α1β2γ1 subunit receptors (1.8±0.1 (triazolam), 1.6±0.1 (clotiazepam), 2.3±0.2 (midazolam)) than for oocytes expressing α1β2γ2S receptors (1.4±0.1 (triazolam), 1.4±0.1 (clotiazepam), 1.3±0.1 (midazolam)). Mutant GABAA receptors (α1β2-R207Cγ2S) with lower GABA sensitivity showed higher drug efficiencies (slope factors=1.1±0.1 (triazolam), 1.1±0.1 (clotiazepam), 1.2±0.1 (midazolam)). Conclusions and implications: Regression analysis enabled the estimation of BZD efficiency when variable mixtures of α1β2 and α1β2γ2S(γ1) receptors are expressed and provided new insights into the γ2S(γ1) dependency of BZD action.

BZDs interact with amino-acid residues located at the interface between aand g-subunits (Macdonald and Barker, 1978;Sigel and Buhr, 1997). These drugs allosterically modulate activation of GABA A receptors either by increasing apparent affinity of at least one agonist-binding site (Gallager and Tallman, 1983;Serfozo and Cash, 1992;Lavoie and Twyman, 1996) or affecting the pore opening (Baur and Sigel, 2005). GABA A receptors carry two GABA-binding sites at the respective ab interfaces (see Twyman et al., 1990). By selective disruption of the one or the other of these sites in concatenated GABA A receptors, it recently has been demonstrated (Baur and Sigel, 2005) that chloride currents were potentiated by diazepam in both cases.
Heterologous expression systems are the basis for drug development and pharmacological characterization of different GABA A receptor subtypes. Transfection of mammalian cells or cRNA injection into Xenopus oocytes with a1, b2 and g2 subunits may, however, result in different mixtures of a1b2 and a1b2g2 receptors (Baumann et al., 2001;Boileau et al., 2002Boileau et al., , 2003, which complicates the unequivocal estimation of potencies and efficiencies of BZDs in heterologous expression systems. This problem can be partially overcome by injecting larger amounts of g2 subunit cRNA (Boileau et al., 2002) or, alternatively, by making use of concatenated GABA A receptor subunits (Baumann et al., 2002;Minier and Sigel, 2004;Boileau et al., 2005).
We have previously reported that different efficiencies of BZDs to enhance chloride currents through GABA A receptors comprising g1 or g2S subunits are related to their ability to shift the GABA concentration-response curves towards higher GABA sensitivities (Khom et al., 2006). Here we analyse the relation between g-induced inhibition of GABA sensitivity and GABA-induced chloride current (I GABA ) potentiation by three BZDs (triazolam, clotiazepam and midazolam) in oocytes expressing different populations of a1b2 and a1b2g2S(g1) subunit receptors. Correlation analysis yielded regression lines with slope factors reflecting higher drug efficiency in g2S than in g1 subunit-comprising receptors. GABA sensitivity and I GABA potentiation of concatenated subunits (g2-b2-a1 and b2-a1) fitted the regression lines of non-concatenated receptors supporting the hypothesis that their lower GABA sensitivity is due to complete g2S incorporation (rather than forced subunit arrangement). A simulation supports the hypothesis that even under conditions where a higher ratio of g2S cRNA relative to a1 and b2 cRNA is injected oocytes contain a significant population of a1b2 receptors.
Smaller slope factors of the regression lines were estimated for oocytes expressing receptors with lower GABA sensitivity (mutant b2-R207C; Wagner et al., 2004).

Materials and methods
Expression of GABA A receptors Stage V-VI oocytes from Xenopus laevis were prepared and cRNA was injected as previously described by Khom et al. (2006). Female X. laevis (NASCO, WI, USA) were anaesthetized by exposing them for 15 min to a 0.2% MS-222 (methanesulphonate salt of 3-aminobenzoic acid ethyl ester; Sandoz, Germany) solution before surgically removing parts of the ovaries. Follicle membranes from isolated oocytes were enzymically digested with 2 mg mL À1 collagenase (Type 1A; Sigma, Vienna, Germany). Synthesis of capped run-off poly(A þ ) cRNA transcripts was obtained from linearized cDNA templates (pCMV vector). At 1 day after enzymatic isolation, the oocytes were injected with 50 nL of water treated with diethylpyrocarbonate (Sigma) containing the different rat cRNAs at a concentration of approximately 300-3000 pg nL À1 per subunit. The amount of cRNA was determined by means of a NanoDrop ND-1000 (Kisker-Biotech, Steinfurt, Germany). To ensure expression of the g-subunit with different incorporation in the case of a1b2g1 and a1b2g2S receptors cRNAs were mixed in a ratios: 1:1:1, 1:1:3 and 1:1:10. The double b2-23-a1 (b2-a1) and triple g2-26-b2-23-a1 (g2-b2-a1) concatemers (kindly provided by E Sigel) and the b2-R207C mutant (kindly provided by C Czajkowski) have been described previously (Baumann et al., 2001(Baumann et al., , 2002Wagner et al., 2004). Oocytes were stored at 18 1C in ND96 solution (Methfessel et al., 1986). Voltage clamp measurements were performed between days 1 and 5 after cRNA injection.

Two-microelectrode voltage clamp studies
Electrophysiological experiments were performed by the two-microelectrode voltage clamp method making use of a TURBO TEC 01C amplifier (npi electronic GmbH, Tamm, Germany) at a holding potential of À70 mV. The bath solution contained 90 mM NaCl, 1 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 and 5 mM HEPES (pH 7.4).
Perfusion system GABA was applied by means of an automated fast perfusion system according to Baburin et al. (2006). To elicit I GABA the chamber was perfused with 120 mL of GABA-containing solution at volume rates of 300 mL s À1 . Duration of washout periods was extended from 3 to up to 20 min with increasing concentrations of applied GABA to account for slow recovery from increasing levels of desensitization (see Khom et al., 2007 for details).
Analysing concentration-response curves Concentration-response curves were generated and the data were fitted by non-linear regression analysis using Origin software (OriginLab Corporation, Northampton, MA, USA). Data were fitted to the equation: 1=ð1 þ ðEC 50 =GABAÞ nH Þ, where EC 50 is the concentration of the GABA that induces half-maximal GABA-evoked current and n H is the Hill coefficient. Concentration-response data for each oocyte were normalized to the maximum control GABA current for that oocyte. Statistical significance was calculated using unpaired Student's t-test with a confidence interval of Po0.05.
Calculation of EC 50 and DEC 50 of mixed population The EC 50 values and BZD-induced shifts in GABA sensitivity in oocytes expressing mixed receptor populations (different fractions of high GABA sensitive a1b2 versus low-sensitive a1b2g2S receptors) are described by the concentrationresponse curves for each receptor population by: and where R ab and R abg are peak I GABA currents (number of open channels in each population) at a given GABA concentration, The total normalized I GABA is a weighted sum of partial current responses: where F is the fraction (0oFo1) of current through g-containing GABA A receptors at saturating GABA concen- Under-application of a saturating concentration of BZD the receptor population consists of two subpopulations: (1) ab receptors with unchanged EC 50,ab and H ab and (2) modulated abg receptors with enhanced GABA sensitivity (midpoint, EC 50,mod and Hill coefficient, H mod ): The total current response is described now by: The apparent midpoints of the GABA concentrationresponse curves (EC 50 ) for oocytes expressing a given fractions (F) of a1b2g2S receptors and the shift of this curve (DEC 50 (BZD)) at saturating concentrations of triazolam, clotiazepam and midazolam can be obtained by varying the fractions (F) from 0 to 1 by maximum likelihood.

Results
Modulation by BZDs of chloride currents through GABA A receptors expressed in Xenopus oocytes is dependent on the incorporation of a g-subunit (Boileau et al., 2002). On the one hand injection of increasing amounts of g-encoding cRNA (that is, a1:b2:g2S ratios of 1:1:3 or 1:1:10) gradually reduces GABA sensitivity and, on the other hand, the increased fraction of a1b2g2S receptors results in an increased I GABA potentiation by BZDs. To quantify the relation between g-subunit-mediated inhibition of GABA sensitivity and BZD action we have analysed modulation of GABA-induced chloride currents (I GABA ) in a large population of oocytes injected with different mixtures (1:1:1, 1:1:3, 1:1:10) of wild-type or mutated a1, b2 and g2S or g1 GABA A receptor subunits, or g2-b2-a1/b2-a1 concatenated constructs.
Triazolam, clotiazepam and midazolam were chosen because these drugs modulate GABA A receptors incorporating g2S and g1 subunits that would allow us to compare their action at different efficiencies.
Modulation of I GABA by BZDs in oocytes expressing variable ratios of a1b2 versus a1b2g1 or a1b2g2S receptors Figure 1a illustrates the effect of triazolam on the concentration-response curves of I GABA through GABA A receptors in oocytes injected with different ratios of cRNAs encoding for a1, b2 and g2S subunits. A saturating concentration (1 mM) of triazolam shifted the GABA concentration-response curves leftwards without significant effects on the maximal response. Higher g2S expression and thus, presumably higher g2S incorporation resulted in reduced GABA sensitivity and larger triazolam-induced shifts of the GABA EC 50 values (DEC 50 (triazolam): 16 mM (cRNA ratio a1:b2:g2S 1:1:1), 24 mM (1:1:3), 50 mM (1:1:10; Figure 1a, see also legend to Figure 1 for individual EC 50 values).
The typical pattern of I GABA modulation of an oocyte (a1:b2:g2S cRNA ratio 1:1:3) is illustrated in Figure 1b. Triazolam enhanced I GABA predominantly at low GABA concentrations corresponding to EC 5À10 (concentrations of GABA that induce 5-10% of maximal GABA-evoked current) and had almost no effects at a saturating GABA concentration. Similar observations were made for a1b2g1 receptors ( Figure 1c).
Different efficiencies of I GABA potentiation in oocytes expressing a1b2g1 or a1b2g2S receptors Figure 1 illustrates that GABA potency (EC 50 ) and I GABA modulation by triazolam were both dependent on the amount of g2S (or g1) incorporation (see also Boileau et al., 2002). These experiments confirmed that apparent higher g-expression and correspondingly lower GABA sensitivity were always associated with larger BZD-induced shifts of the curves. In line with Boileau et al. (2002Boileau et al. ( , 2003 we observed a higher GABA sensitivity at cRNA ratios 1:1:1 and 1:1:3 compared to 1:1:10. The range of GABA EC 50 s for a given cRNA ratio reflected differences in g-incorporations. EC 50 s of individual oocytes were plotted versus the BZDinduced shifts of the GABA concentration-response curves (DEC 50 (BZD)s; Figure 2). For all three compounds applied at saturating concentrations, we obtained a clear correlation between EC 50 s and DEC 50 (BZD)s. The slope factors for oocytes expressing a1b2g1 subunit receptors (1.8 ± 0.1 (triazolam; Figure 2a), 1.6 ± 0.1 (clotiazepam; Figure 2b) and 2.3±0.2 (midazolam; Figure 2c)) were always higher than for oocytes expressing a1b2g2S receptors (1.4 ± 0.1 (triazolam; Figure 2d), 1.4 ± 0.1 (clotiazepam; Figure 2e) and 1.3±0.1 (midazolam; Figure 2f)). The regression line approached the ordinate in the range of the EC 50 of a1b2 receptors. The data suggest that the slope factors may reflect the efficiency of I GABA potentiation for these compounds (see Table 1). For clotiazepam, we estimated similar slopes for g1and g2S-incorporating receptors (difference statistically not significant; P40.05) whereas for triazolam and midazolam the slopes of the regression lines for the two receptor subtypes were significantly different (Po0.05).

Estimation of a1b2 and a1b2g2S receptor fractions
In an independent approach we theoretically calculated the behaviour of a mixed population of GABA A receptors with a  Figure 4 (solid lines). To calculate the putative fractions of a1b2 and a1b2g2S receptors for oocytes displaying different GABA sensitivities we made use of the concentration of half-maximal activation of the simulated curve of ab receptors (EC 50,a1b2 ¼ 8±2 mM, n ¼ 6; see also Sigel and Baur, 2000;Baumann et al., 2001) and EC 50,a1b2g2S (186 mM) of concatenated receptors and corresponding Hill coefficients (H a1b2 ¼ 1.0, H a1b2g2S ¼ 1.3) from our experiments (see Figure 3).
The maximum likelihood fit of the EC 50 versus DEC 50 relation predicts 100% g2S incorporation for the concatenated receptors (Figures 4d-f). Our calculations suggest, however, that even at a 1:1:10 cRNA ratio the fraction (F) of current through a1b2g2S receptors accounts for only between 50 and 70% of the total current. If we assume that the single channel currents through g incorporating receptors is n times larger than through a1b2 receptors,   then the fraction (f) of g incorporating receptors is given by f ¼ F/(nÀF(1Àn)). This formula accounts for differences in single channel conductance. The frequency of openings and mean open time also influences mean conductance of both subpopulations. The equation describing concentration-response curve used in our calculation (see Materials and methods) estimates the fraction of open channels that in turn reflects the mean open time of single channels at different GABA concentrations and thus accounts for differences in open time. Assuming a twofold greater single channel conductance for a1b2g2S receptors (that is, n ¼ 2, see Angelotti and Macdonald, 1993;Fisher and Macdonald, 1997) and FE70% would predict fE54%. In other words, our analysis suggests that even under conditions where a1:b2:g2S were injected at a ratio of 1:1:10 only about 50% of the expressed receptors contain a g2S subunit. This may explain the large difference in EC 50 s of concatenated and non-concatenated receptors (even at a ratio of 1:1:20; Figure 3).

Discussion
This study revealed a linear correlation between the g-subunit-mediated suppression and BZD-induced increase of GABA sensitivity in a1b2g2S and a1b2g1 receptors. We made use of a statistical approach because direct evaluation of this relationship is complicated by variable g-incorporation even under conditions where high ratios of g-subunit cRNA relative to a1 and b2 are injected. Injection of higher cRNA ratio of a1:b2:g2S than 1:1:10 induced only a slight further reduction in GABA sensitivity suggesting a non-linear relationship between cRNA ratios and g-subunit incorporation (see 1:1:20 ratio in Figure 3 and also Boileau et al., 2002). We have therefore induced different g-expression by injecting different cRNA ratios and analysed the relation between g-mediated inhibition and BZD-induced enhancement of GABA sensitivity.
Slopes of regression lines are inversely related to BZD efficiency An initially observed trend that larger BZD induced shifts of the concentration-response curves (DEC 50 (BZD)s) in oocytes expressing larger fractions of g-subunit-containing receptors (induced by injection of increasing amounts of cRNA encoding for g-subunits, Figure 1) was confirmed by correlation analysis (Figure 2). The linear relationship between EC 50 values and DEC 50 (BZD) suggests that BZDs reduce a g-subunit-mediated inhibition of GABA sensitivity. A slope of 1.0 would correspond to complete elimination of a g-subunit-induced inhibition of GABA sensitivity and BZDs would correspondingly shift the concentration-response curve of a1b2g1(2S) receptors to the position of a1b2 receptors. The observation that the tested BZDs never increased GABA sensitivity to that or above that of a1b2 receptors indicates that these drugs only partially can compensate for the g-subunit-induced inhibition.
The estimated slope factors ranged from 1.1 ± 0.1 in a1b2-R207Cg2S (triazolam, clotiazepam) to 2.3±0.2 (midazolam) in a1b2g1 subunit receptors. To evaluate the significance of the slopes we compared the slopes on oocytes expressing   (Khom et al., 2006) and indicate a reversed order of regression slopes (midazolam: 1.3 ± 0.1oclotiazepam: 1.4 ± 0.1 ¼ triazolam: 1.4 ± 0.1). Furthermore, steeper slopes of the regression lines for a1b2g1 compared to a1b2g2S receptors specify that midazolam and to a lesser extent triazolam and clotiazepam increase GABA sensitivity less efficiently in a1b2g1 than in a1b2g2S receptors (Figure 2), which confirms our previous studies (Khom et al., 2006). Taken together our data suggest that the slopes of the regression lines between DEC 50 (BZD)s and EC 50 s are inversely related to drug efficiency. The described approach is time consuming as it requires the injection of different cRNA ratios and the measurements of a large number of concentration-response curves. Our experiments under different conditions (including different BZDs, g-subunits and a mutation in the b2 subunit; Figures 2, 4 and 6) revealed, however, that the regression lines intercept the y-axis close to the EC 50 of a1b2 receptors. This finding prompted us to evaluate the possibility to use the y-intercept and three randomly selected data points (cRNA ratio 1:1:10; Figures 2d-f) for correlation analysis. The simplified procedure yielded regression lines with slopes that were statistically not significantly different from larger data sets (data not shown).
The relevance of the slope factor was further evaluated making use of the mutation b2-R207C that is known to induce a more than 60-fold reduction in GABA sensitivity (Wagner et al., 2004). Figure 5a illustrates that co-expression of g2S decreased the GABA sensitivity analogously to wild-type receptors (Figure 3). The estimated slope factors (1.1 ± 0.1 (triazolam), 1.1 ± 0.1 (clotiazepam) and 1.2 ± 0.1 (midazolam); Figure 6) were always smaller than in wild type (though only in the case of triazolam and clotiazepam that difference was significant, Po0.05), suggesting even higher BZD efficiencies compared to wild type.

Concatenated receptor subunits fit the regression lines
Forced g2 subunit incorporation in oocytes was previously shown to be associated with a significant loss in GABA sensitivity compared to oocytes injected with 1:1:10 cRNA ratios (Baumann et al., 2002; see also Figure 3; but see Baur et al., 2006). Here we show that the higher EC 50 s and corresponding DEC 50 (BZD)s of concatenated subunits fit the predictions of the regression lines of non-concatenated subunits (Figures 4d-f, closed triangles). The mean EC 50 of the concatenated g2-b2-a1/b2-a1 subunits (Figure 3) corresponds to the data of Baumann et al. (2002). Lower GABA sensitivity and higher BZD efficiencies of concatenated subunits expressed in Xenopus oocytes may accordingly result from more complete g-incorporation (rather than from forced subunit arrangement or interactions). Our data suggest that the studied GABA A receptor composed of two concatenated constructs may represent a model of completely assembled receptor with intact GABAand BZD-binding sites. This hypothesis (based on the extrapolation of the regression line; Figure 4) requires, however, further studies.
These experimental data could be reproduced by a mathematical model describing BZD modulation in oocytes expressing different current fractions of high GABA sensitive a1b2 versus low-sensitive a1b2g2S receptors. Maximum likelihood analysis predicted (in line with the experimental observations) a linear relationship that enables the calculation of a putative fraction of a1b2g2S receptors at a given EC 50 and DEC 50 (BZD). This calculation had, however, to account for the different single channel conductance of the two receptor subtypes (Angelotti and Macdonald, 1993;Fisher and Macdonald, 1997). Interestingly these independent calculations reproduced not only the linear correlation but predicted the expected 100% g2S incorporation for the concatemers and thus would support the experimental findings of the concatenated receptors and our conclusions.
Percentage of a1b2g2 receptors in oocytes expressing these subunits Boileau et al. (2002) have clearly demonstrated that injecting higher ratios of g2 subunit cRNA results in a purer population of a1b2g2 subunit receptors. In addition, they observed a remarkable run-down of BZD modulation, suggesting a decay of g2 subunit-incorporating receptors over time after injection into oocytes that makes it difficult to compare data from different labs. Furthermore, small changes in the applied effective GABA concentration (usually between EC 3 and EC 10 ) can substantially affect the apparent BZD efficiency, thus further confounding a calculation of the percentage of expressed a1b2g2S receptors. The method suggested above for determining the slope of the regression line, however, also allows the determination of the percentage of a1b2g2S receptors. After correction for single channel conductances (assuming a twofold difference in the single channel conductance of a1b2 and a1b2g2S receptors, Angelotti and Macdonald, 1993;Fisher and Macdonald, 1997), a 70% current ratio would correspond to about 50% of g2S subunit incorporation.
The regression slopes are independent from variations in expression of a1b2 versus a1b2g2S(g1) fractions or variations in experimental conditions (GABA concentration). The statistical analysis described here is based on the different shifts of the concentration-response curves and, therefore, independent of these possible experimental errors. The low percentage of a1b2g2S receptors formed under the conditions used indicate that at least in the Xenopus oocyte system (but not necessarily in other heterologous expression systems or under native conditions) the efficiency of assembly of a1b2 receptors might be comparable to or even higher than that of a1b2g2S receptors, resulting in comparable amounts of a1b2 or a1b2g2S receptors even when high concentrations of g-subunits are available.

Implications for the mechanism of BZD action
The different shifts in the GABA concentration-response curves for GABA A receptors with different subunit compositions or mutants can be interpreted in terms of a mechanism where BZDs increase the microscopic affinity of the GABAbinding site or, alternatively, by a mechanism where BZDs facilitate channel gating (for example, Jones-Davis et al., 2005). The second scenario is supported by previous findings suggesting that BZD-like modulators enhance the amplitude of the GABA response by stabilizing the open channel conformation (Downing et al., 2005;Rusch and Forman, 2005;Campo-Soria et al., 2006).
We are tempted to speculate that the BZD-induced I GABA increase reflects an increase in GABA efficacy (as defined by Colquhoun, 1998) by a direct transduction of the BZD effect to the channel region (Akabas, 2004;Ernst et al., 2005). An increase of efficacy from apparent low level (for example, in mutant b2-R207C or concatemers) could explain an increase of the maximal GABA response ('over-potentiation' of the BZDs on the a1b2-R207Cg2S isoform or concatenated receptors) and a simultaneous shift of the concentration-response curve (Figures 4a-c and 6a-c; Colquhoun, 1998;Downing et al., 2005;Rusch and Forman, 2005;Campo-Soria et al., 2006). These theoretical considerations need, however, more experimental validation including the investigation of further mutations (for example, in the putative GABA-binding site and/or pore region) and the use of BZDs with a broad range of efficiencies.