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	Figure 1. Chemical structure of compounds 31, 132, 4-O-methylhonokiol, and the high-affinity benzodiazepine antagonist Ro 15-1788.
	Figure 2. Compounds 31 and 132 stimulate GABA currents mediated by α1β2γ2 receptors in a concentration-dependent manner.α1β2γ2 receptors were expressed in Xenopus oocytes and electrophysiological experiments were performed. Original current traces of an experiment with compound 31 (a) and with compound 132 (b). Numbers indicate applied concentrations of the respective compounds. (c) Concentration dependence of the positive allosteric modulation by compound 31 (closed circles) and compound 132 (closed squares) in oocytes expressing α1β2γ2 receptors. Mean data ± SD for both compounds is shown, n = 3.
	Figure 3. Compounds 31 and 132 do not act at the classical high affinity site for benzodiazepines.GABA at a concentration eliciting 0.5% of the maximal current amplitude (EC0.5, single bars) was applied until a stable response was obtained. Subsequently, the same concentration of GABA was co-applied with 3 μM of compounds 31 (a) or 132 (b), which resulted both in a large increase of current amplitude. Co-application of Ro 15-1788 with compound and GABA did not reduce the degree of modulation in both cases. Experiments were repeated 4 times, with three different batches of oocytes, with a similar outcome.
	Figure 4. (a) Model structure of the GABAA receptor transmembrane domain. The major isoform of the GABAA receptor is composed of two α1, two β2, and one γ2 subunits. The model structure depicts the crystalized homomeric β3 GABAA receptor (PDB structure 4COF)38. In this figure, some of the β3 subunits were renamed α1 (yellow), β2 (blue) and γ2 (red); structures are shown in ribbon representation. The mutated residues α1S269, β2N265, and γ2S280 are located at the interfaces between subunits. (b) Potentiation of the GABA response by compound 31 (3 μM), compound 132 (3 μM), and 4-O-methylhonokiol (1 μM, abbreviated Mh) in wild-type α1β2γ2, single mutant (α1M, β2M, γ2M), and triple mutant receptors expressed in Xenopus oocytes. The bars indicate mean ± SD, n = 3.
	Figure 5. Individual roles of the two β+/α− interfaces in channel modulation by compounds 31, 132 and 4-O-methylhonokiol.(a) Scheme showing the four concatenated wild-type and mutant receptors. 1 and 2 refer to the two different β+/α− subunit interfaces, interface 1 and interface 2. The location of the β2N265I mutations is indicated in red color. Concatenated receptors were prepared containing no mutation (α1-β2-α1/γ2-β2, non M), a mutation at interface 1 (α1-β2-α1/γ2-β2M, interface 1 M), a mutation at interface 2 (α1-β2M-α1/γ2-β2, interface 2 M), or mutations in both sites (α1-β2M-α1/γ2-β2M, double M). Interface 2 harbors a binding site for GABA with higher apparent affinity for channel gating than the one positioned at the interface 154. (b) Potentiation by compound 31 (3 μM), compound 132 (3 μM), and 4-O-methylhonokiol (1 μM), using an EC0.5–1.5 concentration of GABA for each concatenated receptor subtype. Bars indicate mean ± SD, n = 3.
	Figure 6. Effect of the β2N265S mutation on modulation by compounds 31 and 132.Wild-type α1β2γ2 and mutated α1β2N265Sγ2 receptors were expressed in Xenopus oocytes and studied. Potentiation of GABA currents was determined using 3 μM of compound 31 or 132. Bars indicate mean ± SD, n = 4–11.
	Figure 7. Subunit specificity of current potentiation in different GABAA receptors.Different subunit combinations were expressed in Xenopus oocytes. Potentiation of GABA currents at a GABA concentration eliciting 0.5–1.5% of the maximal current amplitude was determined using 3 μM of compound 31 (a) or 132 (b). Bars indicate mean ± SD, n = 4–11.
	Figure 8. Alignment of the rat amino acid residue sequences of different α (a) and β (b) subunit isoforms.(a) Sequences preceding M1 and the first part of M1 in α subunits are shown. (b) Sequences preceding M3 and the first part of M3 in β subunits are shown.
	Figure 9. Concentration-response curves for loss of righting reflexes (LORR) in tadpoles for compounds 31 (closed circle), 132 (closed square), and 4-O-methylhonokiol (closed triangle).The percent of animals anesthetized is plotted against aqueous anesthetic concentration, overlaid with logistic fits. Each point represents data from ten animals. Data were fitted to a Hill equation.

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