Che Has AT , Absalom N , van Nieuwenhuijzen PS , Clarkson AN , Ahring PK , Chebib M .

	Figure 1. GABA-evoked responses at α1β3 and α1β3γ2 GABAA receptors.Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage-clamp electrophysiology as described in the methods. For experimentation, oocytes were clamped at −60 mV and full GABA concentration response relationships were obtained on each oocyte. (A) Representative GABA-evoked traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate application periods and GABA concentrations and “/” a wash period. Dotted lines indicate a 0 nA baseline and holding currents were −150 ± 93 nA, n = 6 for α1 + β3 (1:1) and −13 ± 10 nA, n = 9 for α1 + β3 (30:1). (B,C) Baseline subtracted peak current amplitudes for full GABA concentration-response curves at oocytes injected with the indicated cRNA mixtures using free subunits (B) or a concatenated β3-α1 construct (C) were fitted to the Hill equation using non-linear regression (fixed bottom of 0 and slope of 1) and normalized to the maximal fitted value (IGABA_max_fit). Averaged normalized data points are depicted as means ± S.E. as a function of the GABA concentration, fitted to the Hill equation and regression results are presented in Table 1. Each data point represents experiments from n = 5–9 oocytes from ≥2 batches. (D) α1β3 GABAA receptors can express in two stoichiometries of 2α1:3β3 (left) and 3α1:2β3 (right). The two binding sites for GABA at the β3(+)-α1(−) subunit interface are indicated by red arrowheads.
	Figure 2. Zn2+ inhibition of GABA-evoked currents from α1β3 and α1β3γ2 GABAA receptors.Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. Control currents (Icontrol) were evoked using a GABA concentration corresponding to ~EC50 and inhibition by Zn2+ was evaluated by co-applications with GABAcontrol. (A) Representative GABA-evoked current traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate GABA and Zn2+ application periods. Dotted lines indicate the peak current amplitude by GABAcontrol and “/” a wash period. (B,C) Concentration-response relationships of Zn2+ inhibition of GABAcontrol-evoked currents at α1β3 or α1β3γ2 receptors stemming from injecting the indicated cRNA mixtures using free subunits (B) or a concatenated β3-α1 construct (C). Averaged Zn2+ inhibition values were depicted as means ± S.E.M as a function of the Zn2+ concentration and fitted to the Hill equation by non-linear regression. Regression results for α1 + β3 (1:1) were IC50 = 0.84 (95% CI: 0.53–1.3), nH = −0.5 ± 0.32 and for β3-α1 + β3 (1:2) were IC50 = 1.6 (95% CI: 1.1–2.4), nH = −0.6 ± 0.06. For the remaining cRNA mixtures, Zn2+ inhibition at the maximal tested concentration was too low to allow for meaningful fitting. Each data point represent experiments from n = 5–8 oocytes of ≥2 batches. (D) The depiction of α1β3 GABAA receptor stoichiometries from Fig. 1D was modified to indicate Zn2+ binding in the β3(+)-β3(−) subunit interface (red/orange arrowhead).
	Figure 3. Zolpidem modulation of GABA-evoked currents from α1β3 and α1β3γ2 GABAA receptors.Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. Control currents (Icontrol) were evoked using a GABA concentration corresponding to ~EC5–10 and modulation by zolpidem was evaluated by co-applications with GABAcontrol. (A–C) Representative GABA-evoked current traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate GABA, zolpidem (Zolp) and flumazenil (Flu) concentrations and application periods. Dotted lines indicate the peak current amplitude by GABAcontrol and “/” a wash period. For the specific traces, zolpidem had no robust effects at receptors from α1 + β3 (1:1) injection (A), but showed 130% modulation at receptors from α1 + β3 (30:1) injection which was inhibited 95% by co-application of flumazenil (B). Zolpidem likewise modulated receptors from injection of α1 + β3 + γ2 (1:1:5) by 160% which could be inhibited 85% by flumazenil (C). (D,E) Concentration-response relationships of zolpidem modulation of GABAcontrol-evoked currents at α1β3 or α1β3γ2 receptors stemming from injecting the indicated cRNA mixtures using free subunits (D) or a concatenated β3-α1 construct (E). Average modulatory values were depicted as means ± S.E.M as a function of the zolpidem concentration and fitted to the Hill equation by non-linear regression. Each data point represents experiments from n = 5–7 oocytes from ≥2 batches and regression results are presented in Table 1.
	Figure 4. Mechanism of zolpidem modulatory actions at α1β3 and α1β3γ2 GABAA receptors.Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. (A–C) Full GABA concentration-response relationships were obtained in presence of the indicated concentrations of zolpidem (Zolp) at receptors stemming from injection of cRNA of free subunits (A,C) or a concatenated construct (B). Baseline subtracted GABA + zolpidem peak current amplitudes were normalized to a maximal GABA control response (3 mM) in the same oocytes. Averaged normalized data points are depicted as means ± S.E.M. as a function of the GABA concentration and fitted to the Hill equation with regression results are presented in Table 1. Each data point represents experiments from n = 5–7 oocytes from ≥2 batches. GABA concentration response relationships in absence of zolpidem (from Fig. 1) are included for comparison. (D) The depiction of α1β3 GABAA receptor stoichiometries from Fig. 2D was modified to indicate zolpidem binding in the α1(+)-α1(−) subunit interface (red/green arrowhead).
	Figure 5. Diazepam modulation of GABA-evoked currents from α1β3 GABAA receptors.Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. Control currents (Icontrol) were evoked using a GABA concentration corresponding to ~EC5–10 and modulation by diazepam was evaluated by co-applications with GABAcontrol. (A,B) Representative GABA-evoked current traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate GABA, diazepam (Diaz) and flumazenil (Flu) concentrations and application periods. Dotted lines indicate the peak current amplitude by GABAcontrol and “/” a wash period. (C) Concentration-response relationships of diazepam modulation of GABAcontrol-evoked currents at α1β3 receptors stemming from injecting the indicated cRNA mixtures using free subunits or a concatenated β3-α1 construct. Averaged modulatory values were depicted as means ± S.E as a function of the diazepam concentration and fitted to the Hill equation by non-linear regression. Each data point represents experiments from n = 6 oocytes from ≥2 batches. Regression results with 95% confidence intervals for α1 + β3 (30:1) were: EC50 value of 0.040 μM (0.010–0.12) and Emax value of 40% (33–48) whereas results for β3-α1 + α1 (1:2) were: EC50 value of 0.020 μM (0.010–0.04) and Emax value of 51% (46–56).

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