Baburin I et al. (2008), Estimating the efficiency of benzodiazepines on...

XB-ART-38073

Br J Pharmacol 2008 Oct 01;1553:424-33. doi: 10.1038/bjp.2008.271.

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Estimating the efficiency of benzodiazepines on GABA(A) receptors comprising gamma1 or gamma2 subunits.

Baburin I , Khom S , Timin E , Hohaus A , Sieghart W , Hering S .

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Heterologous expression of alpha1, beta2 and gamma2S(gamma1) subunits produces a mixed population of GABA(A) receptors containing alpha1beta2 or alpha1beta2gamma2S(gamma1) subunits. GABA sensitivity (lower in receptors containing gamma1 or gamma2S subunits) and the potentiation of GABA-activated chloride currents (I(GABA)) by benzodiazepines (BZDs) are dependent on gamma2S(gamma1) incorporation. A variable gamma subunit incorporation may affect the estimation of I(GABA) potentiation by BZDs. We propose an approach for estimation of BZD efficiency that accounts for mixed population of alpha1beta2 and alpha1beta2gamma2S(gamma1) receptors.We investigated the relation between GABA sensitivity (EC50) and BZD modulation by analysing triazolam-, clotiazepam- and midazolam-induced potentiation of I(GABA) in Xenopus oocytes under two-microelectrode voltage clamp.Plotting EC50 versus BZD-induced shifts of GABA concentration-response curves (DeltaEC50(BZD)) of oocytes injected with different amounts of alpha1, beta2 and gamma2S(gamma1) cRNA (1:1:1-1:1:10) revealed a linear regression between gamma2S(gamma1)-mediated reduction of GABA sensitivity (EC50) and DeltaEC50(BZD). The slope factors of the regression were always higher for oocytes expressing alpha1beta2gamma1 subunit receptors (1.8 +/- 0.1 (triazolam), 1.6 +/- 0.1 (clotiazepam), 2.3 +/- 0.2 (midazolam)) than for oocytes expressing alpha1beta2gamma2S receptors (1.4 +/- 0.1 (triazolam), 1.4 +/- 0.1 (clotiazepam), 1.3 +/- 0.1 (midazolam)). Mutant GABA(A) receptors (alpha1beta2-R207Cgamma2S) 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)).Regression analysis enabled the estimation of BZD efficiency when variable mixtures of alpha1beta2 and alpha1beta2gamma2S(gamma1) receptors are expressed and provided new insights into the gamma2S(gamma1) dependency of BZD action.

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	Figure 1. (a) Typical GABA concentration–response curves of oocytes injected with cRNAs of α1, β2 and γ2S subunits (cRNA stoichiometry: 1:1:1, left panel; 1:1:3, middle panel and 1:1:10, right panel) in the absence (control) and presence of 1 μM triazolam. The corresponding EC50 values for 1:1:1 were 24 μM (control), 8 μM (triazolam); for 1:1:3: 38 μM (control), 14 μM (triazolam) and for 1:1:10: 71 μM (control), 21 μM (triazolam). (b) Corresponding IGABA through α1β2γ2S (1:1:3) channels modulated by 1 μM triazolam at indicated GABA concentrations. (c) GABA concentration–response curves of oocytes injected with cRNAs of α1, β2 and γ1 subunits (1:1:1, left panel; 1:1:3, middle panel and 1:1:10, right panel) in the absence (control) and presence of 1 μM triazolam. The corresponding EC50 values for 1:1:1 were: 21 μM (control), 12 μM (triazolam); for 1:1:3: 31 μM (control), 16 μM (triazolam); for 1:1:10: 66 μM (control), 29 μM (triazolam). Each graph in (a) and (c) represents one experiment on one oocyte.
	Figure 2. Correlation between EC50 values of the GABA concentration–response curves (EC50s) of oocytes expressing α1β2γ1 receptors (a–c), α1β2γ2S receptors (d–f) and shifts of these EC50 values (ΔEC50(BZD)s) by modulation of the GABA concentration–response curve by 1 μM triazolam (a, d), 10 μM clotiazepam (b, e) and 10 μM midazolam (c, f); α-, β- and γ-subunit cRNA stoichiometries are 1:1:1, 1:1:3 and 1:1:10. Correlation coefficients were 0.94 (triazolam), 0.94 (clotiazepam), 0.94 (midazolam) (P<0.0001 in all cases, α1β2γ1) and 0.97 (triazolam), 0.94 (clotiazepam), 0.90 (midazolam) (P<0.0001 in all cases, α1β2γ2S). Each data point represents one experiment on one oocyte.
	Figure 3. GABA concentration–response curves for oocytes expressing α1β2 (cRNA injection 1:1), α1β2γ1 (1:1:10), α1β2γ1 (1:1:20), α1β2γ2S (1:1:10), α1β2γ2S (1:1:20) and γ2-β2-α1/β2-α1 (1:1) receptors. The corresponding mean EC50 values and Hill coefficients were α1β2 (1:1): 8±2 μM, nH=1.0±0.2 (n=6); α1β2γ1 (1:1:10): 48±3 μM, nH=1.3±0.1 (n=24); α1β2γ1 (1:1:20): 61±3 μM, nH=1.5±0.1 (n=4); α1β2γ2S (1:1:10): 51±3 μM, nH=1.4±0.1 (n=27); α1β2γ2S (1:1:20): 62±4 μM, nH=1.5±0.1 (n=4); γ2-β2-α1/β2-α1 (1:1): 186±13 μM, nH=1.3±0.1 (n=18).
	Figure 4. GABA concentration–response curves of oocytes injected with concatenated subunits (γ2-β2-α1/β2-α1) in the absence (control) and presence of 1 μM triazolam (a), 10 μM clotiazepam (b) and 10 μM midazolam (c). The corresponding EC50 values were: 158 μM (control), 34 μM (triazolam); 150 μM (control), 60 μM (clotiazepam); 132 μM (control), 43 μM (midazolam). EC50 values of the GABA concentration–response curves of oocytes expressing concatenated subunit constructs (γ2-β2-α1 and β2-α1 in 1:1 ratio) and ΔEC50(BZD)s induced by (d) 1 μM triazolam, (e) 10 μM clotiazepam and (f) 10 μM midazolam were added to the regression lines from Figures 2d–f. Corresponding regression lines (dashed) were taken from Figures 2d–f and extended towards the values of the concatenated subunits. Solid lines (d–f) represent the non-linear fit of Equations 1–5 (see Materials and methods) by the maximal likelihood. Calculated current fractions of γ-incorporating receptors (0, 50, 70 and 100%) are indicated. Each graph (a, b and c) and each data point (d, e and f) represent one experiment on one oocyte.
	Figure 5. (a) GABA concentration–response curves of oocytes expressing α1β2-R207C (1:1, EC50=486±81 μM, nH=1.2±0.2, n=6) and α1β2-R207Cγ2S (1:1:10, EC50=3217±378 μM, nH=0.9±0.1, n=18) receptors. (b) Representative traces for enhancement of IGABA through α1β2-R207Cγ2S. Control currents (GABA) in the absence of clotiazepam and corresponding currents elicited by co-application of GABA and clotiazepam are shown.
	Figure 6. GABA concentration–response curves of oocytes injected with cRNA ratios of α1, β2-R207C and γ2S subunits of 1:1:1 (a), 1:1:3 (b) and 1:1:10 (c) in the absence (control) and presence of 1 μM triazolam. The corresponding EC50 values for 1:1:1 were: 827 μM (control), 406 μM (triazolam); for 1:1:3: 1945 μM (control), 997 μM (triazolam); for 1:1:10: 3891 μM (control), 991 μM (triazolam). (d–f) Correlation between individual EC50 values of the GABA concentration–response curves (EC50s) of oocytes expressing α1β2-R207Cγ2S receptors and shifts of these EC50 values (ΔEC50(BZD)s) caused by modulation of the GABA concentration–response curve by 1 μM triazolam (d), 10 μM clotiazepam (e) and 10 μM midazolam (f). The slopes of regression lines were 1.1±0.1 (triazolam), 1.1±0.1 (clotiazepam) and 1.2±0.1 (midazolam). Correlation coefficients were 0.96 (triazolam), 0.98 (clotiazepam), 0.97 (midazolam) (P<0.0001 in all cases). Each graph (a, b and c) and each data point (d, e and f) represent one experiment on one oocyte.

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