Simeone X , Siebert DCB , Bampali K , Varagic Z , Treven M , Rehman S , Pyszkowski J , Holzinger R , Steudle F , Scholze P , Mihovilovic MD , Schnürch M , Ernst M .

W 1232 Austrian Science Fund FWF

	Figure 1. Chemical structures of the ligands (1–7) employed in this study. The letters “A, B, C and D” refer to the different rings in the scaffold. The position and numbering of the residues (R6, R8 and R′4) are depicted in the general structure (bottom right corner). The nomenclature “chloro-methoxy” or “methoxy-methoxy” respectively describes first the residue in position R8 and then the residue in position R′4. For compound 7 residues in position R8-R6-R′4 are indicated.
	Figure 2. Compound 1 modulates GABA-evoked currents from α1β3, α1β3γ2 and α1β3δ similarly. Concentration-dependent modulation of GABA EC3–5 current at α1β3, α1β3γ2 and α1β3δ. Data represent means ± SEM (n = 3–8). α1β3 and α1β3γ2 data are identical with those published previously22.
	Figure 3. Compounds 1–6 show potency selectivity for β1-containing receptors. Dose-response data of compounds 1–6 at α1β1, α1β2 and α1β3 subunit combinations; (a–c) Left, aggregate dose-response curves of R8 = chloro compounds 1–3 co-applied with GABA EC3–5. Right, EC50 values obtained by fitting data of each cell individually; (d–f) Left, aggregate dose-response curves of R8 = methoxy compounds 4–6 co-applied with GABA EC3–5. Right, EC50 values obtained by fitting data of each cell individually. Highest potency was consistently observed at α1β1 receptors. Compound 6 (f) lacked efficacy at α1β2 and α1β3, therefore EC50 values could not be obtained. In those instances where high compound concentrations elicited substantial desensitization (see panels a, c, d, f and sample traces in (g,h)), the highest compound concentration was excluded from the fit. Statistically significant differences were assessed by one-way ANOVA with Tukey’s multiple comparison test; *p < 0.05, ***p < 0.01, ****p < 0.001, *****p < 0.0001, n.s. = not significant, n.d. = not determined. n = 3–8. (g–i) Sample traces obtained with compound 1. Note the desensitization in α1β1 (g) at 10 µM and 30 µM, increasingly limiting maximum current amplitudes. Tabulated data corresponding to panels a–f are provided in Supplementary Tables S1–S6. Additional sample traces are provided in Supplementary Fig. S7.
	Figure 4. Comparison of EC50 and maximum efficacy among α1β1, α1β3 and α1β3N41R. (a,b) The plots show the mean EC50 on the x-axis (note that the axis is broken to accommodate the range) and the mean maximum efficacy at 10 μM (% of control current at EC3–5) on the y-axis (note the different scales on the two panels) of compounds 1–6. The difference between wild type α1β3 and α1β3N41R is indicated with a black arrow, statistically significant EC50 differences are indicated. The potency differences between α1β3 and α1β3N41R for compounds 1, 3 and 4 are statistically significant (**,**,****, respectively). Arrows pointing to the left show a decrease of the EC50 value between wild type and mutated receptors, which corresponds to an increase in potency. Simultaneously, changes in efficacy can be seen (arrows with upward or downward component indicating increase or decrease in maximum efficacy, respectively). The values obtained with wild type α1β3 and α1β1 receptors are connected with a blue dotted line. The dotted purple line visualizes the difference between α1β1 and α1β3N41R. The EC50 values for the mutated receptors are 0.98 µM, 3.44 µM, 0.2 µM, 1.2 µM, 2.47 µM and 1.87 µM for compounds 1–6, respectively. EC50 values were calculated for each individual experiment and are presented as mean ± SEM. Statistically significant differences were assessed by one-way ANOVA with Tukey’s multiple comparison test. Note that the EC50 value of compound 6 in α1β3 receptors is not depicted, since this compound has nearly no efficacy in this receptor subtype. Bars indicate mean ± SEM, n = 3–8. The dose-response curves of compounds 1–6 in α1β3N41R receptors are depicted in Supplementary Fig. S12.
	Figure 5. Compound 1 modulates GABA-evoked currents in α1β1, α1β1γ1 and α1β1δ receptors with similar potencies. (a) Concentration-dependent modulation of GABA EC3–5 current at α1β1, α1β1γ1 and α1β1δ. Data represent means ± SEM (n = 3–10). (b) EC50 values were calculated for each individual experiment and are presented as mean ± SEM. One-Way ANOVA was used for multiple comparisons followed by a Tukey post hoc test and showed no significant differences between the mean EC50 values for each subtype.
	Figure 6. Compound 7 modulates GABA-evoked currents in α1β1, α1β1γ1 and α1β1δ receptors. (a,b) Concentration-dependent modulation of GABA EC3–5 current at α1β1, α1β1γ1 and α1β1δ receptors. Data represent means ± SEM (n = 4–17) (see Supplementary Table S19).

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