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	Fig 2. Subunit combinations with detectable function but were not activated by 2’MeO6MF.(A) Representative traces illustrating the robust response elicited by 3 mM GABA, and the lack of activity of 100 μM Zn2+ and 100 μM 2’MeO6MF at α2β3 (20:1) GABAARs (n = 6). (B) The injection of γ2L mRNA at high amounts (28–35 ng/oocyte) resulted in constitutively active channels which were inhibited by 100 μM Zn2+, but were not sensitive to 60 mM GABA, 100 μM etomidate and 100 μM 2’MeO6MF (n = 5). (C) The injection of β3 mRNA (5 ng/oocyte) resulted in the formation of functional receptors. Representative traces of β3 homomeric receptors responses to 1–60 mM GABA (top;), 3–300 μM of 2’MeO6MF and 100 μM Zn2+ (bottom). (D) 2’MeO6MF concentration-response curve for β3 homomeric receptors (n = 5). The efficacy of 2’MeO6MF as an inverse agonist is expressed as a fraction of the inhibited spontaneous current (I) normalised against the holding current (Iholding). Data are presented as mean ± SEM. Bars indicate durations of drug application. The holding current values are represented by the dotted lines.
	Fig 3. 2’MeO6MF activity at β3γ2L GABAARs.(A) 2’MeO6MF exhibits a complex spectrum of activity across β3γ2L GABAARs expressed at various injection ratios. Representative traces of 3 mM GABA (black) and 100 μM 2’MeO6MF (red) are shown for each ratio. Bars indicate durations of drug application. The holding current values are represented by the dotted lines. (1:1; n = 7), 2’MeO6MF inhibited the constitutive activity of receptors expressed. (1:5; n = 5) and (1:10; n = 10), 2’MeO6MF exhibited mixed agonist and inverse agonist activity. (1:15; n = 30), 2’MeO6MF directly activated receptors expressed efficaciously. (1:20; n = 49), 2’MeO6MF activated β3γ2L (1:20) receptors with variable efficacy. Sample traces of cell 1 and 2 were taken from a simultaneous experiment conducted on two different oocytes injected at the same time. (1:50; n = 15) and (1:100; n = 21), 2’MeO6MF showed activation with low efficacy at these receptors. (B) Mean efficacy of 100 μM 2’MeO6MF direct activation at β3γ2L (1:15), (1:20), (1:50) and (1:100) GABAARs. Data are normalised to the 3 mM GABA response. The mean efficacy of 100 μM 2’MeO6MF at various ratios was compared using Tukey’s test, and the significance levels are indicated with n.s. (not significant) and *** (p ≤ 0.001).
	Fig 4. Characterisation of β3γ2L GABAARs expressed at 1:15, 1:50 and 1:100 ratios.The level of constitutive activity is indicated by (A) holding current of injected oocytes and (B) the inhibition of baseline current by 100 μM Zn2+. (A) β3γ2L GABAARs expressed at 1:15 ratio showed significantly larger holding current (-260 ± 40 nA; n = 30) than at 1:50 (-28 ± 6.0 nA; n = 15) and 1:100 (-15 ± 5.0 nA; n = 21) ratios (p ≤ 0.001; Tukey’s test). (B) Representative traces demonstrating current responses of 3 mM GABA, 100 μM 2’MeO6MF and 100 μM Zn2+. At 1:15 ratio, receptors were sensitive to the inhibition of 100 μM Zn2+ (reduction in inward current; n = 10). Zn2+ did not have any effects at 1:50 (n = 7) and 1:100 (n = 8) ratios. (C) Representative traces demonstrating 2’MeO6MF’s direct activation from 1 to 300 μM in comparison to 3 mM GABA response. (D) 2’MeO6MF concentration-response curves of β3γ2L (1:15; n = 8), (1:50; n = 7) and (1:100; n = 6) GABAARs. Data are normalised to the 3 mM GABA response. (E) Representative traces of GABA current responses from 1 μM to 30 mM at β3γ2L (1:15), (1:50) and (1:100) GABAARs. (F) GABA concentration-response curves of β3γ2L (1:15; n = 6), (1:50; n = 8) and (1:100; n = 9) GABAARs. Data are presented as mean ± SEM. Bars indicate durations of drug application. The holding current values are represented by the dotted lines.
	Fig 5. Etomidate activates both β3γ2L and β3 GABAARs.(A) Representative traces of etomidate (10 μM) direct activation in comparison to 3 mM GABA at β3γ2L (1:15), (1:50) and (1:100) GABAARs. (B) Mean efficacy of 10 μM etomidate activation at β3γ2L (1:15; n = 7), (1:50; n = 6) and (1:100; n = 7) receptors. Data are normalised to the 3 mM GABA response. The mean efficacy of 10 μM etomidate at various ratios was compared using Tukey’s test, and the significance levels are indicated with n.s. (not significant) and *** (p ≤ 0.001). (C) Concentration-response curves of etomidate activation at β3γ2L receptors expressed at 1:15 (n = 5), 1:50 (n = 6) and 1:100 (n = 7) ratios. Recording was conducted at -60 mV. Data are normalised to the 3 mM GABA response. (D) Concentration-response curves of etomidate activation at β3γ2L receptors expressed at 1:15 (n = 4) and 1:100 (n = 5) ratios. Recording was conducted at -30 mV. Data are normalised to the 3 mM GABA response. (E) Representative traces of β3 homomeric receptors responses to 0.1–300 μM etomidate in comparison to 60 mM GABA response. (F) Etomidate concentration-response curve for β3 homomeric receptors (n = 5). Data are normalised against 60 mM GABA responses. Data are presented as mean ± SEM. Bars indicate durations of drug application. The holding current values are represented by the dotted lines.
	Fig 6. Propofol activates β3γ2L (1:15) and (1:100) receptors with different relative efficacies.(A) Representative traces of propofol (1–300 μM) direct activation in comparison to 3 mM GABA at β3γ2L (1:15) and (1:100) GABAARs. (B) Concentration-response curves of propofol activation at β3γ2L receptors expressed at 1:15 (n = 4) and 1:100 (n = 4) ratios. Data are normalised to the 3 mM GABA response.
	Fig 1. Characterisation of α2β3γ2L GABAARs expressed at a 3:1:3 injection ratio.(A) Left panel, Representative traces of α2β3γ2L (3:1:3) GABAARs responses to 3 mM GABA and 100 μM Zn2+ alone. Right panel, Mean holding current of oocytes expressing α2β3γ2L (3:1:3) GABAARs (-93 ± 19 nA; n = 8). (B) Left panel, Continuous traces demonstrating two consecutive applications of control (100 μM GABA) followed by the co-application of 10 μM Zn2+ with control. Right panel, Modulation of 100 μM GABA responses by 10 μM Zn2+ (n = 9). (C) Left panel, Continuous traces demonstrating two consecutive applications of control (1 μM GABA) followed by the co-application of 1 μM diazepam with control; 1 μM diazepam and 10 μM flumazenil with control; and control. Right panel, Potentiation by 1 μM diazepam of 1 μM GABA responses (n = 6). Representative traces demonstrating (D) GABA current responses from 1 μM to 10 mM and (E) 2’MeO6MF’s direct activation from 1 to 300 μM (red) in comparison to 3 mM GABA response. Concentration-response curves of GABA (black; n = 6) and 2’MeO6MF (red; n = 5) are shown in (F). Data are presented as mean ± SEM. Bars indicate durations of drug application. The holding current values are represented by the dotted lines.

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