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	Figure 1. Symmetrical BPCs with a C3 linker inhibit α7 and muscle-type nAChR currents with micromolar potency and in a noncompetitive way. Human α7 and (α1)2β1εδ nAChRs were expressed in Xenopus laevis oocytes. Oocytes were clamped at −70 mV, and BPCs were preincubated (20 s) and coapplied with ACh. (A) Representative current traces in response to 100 μM ACh (α7) or 30 μM ACh (muscle-type) before and after application of 300 nM PTM0022 (red) or 3 μM MB327 (magenta). (B) Full dose inhibition curves of BPCs with at least a low micromolar potency at α7 and muscle nAChR subtypes. Error bars represent the SD of the mean from n = 3–6 individual oocytes. (C) ACh dose response curves without black filled circles and in the presence of the following concentrations of BPCs: 3 μM MB327, 3 μM PTM0002, 10 μM PTM0007, 3 μM PTM0015, and 300 nM PTM0022. Individual BPCs are indicated in the legend. MB327 and the most potent derivate PTM0022 are colored. Error bars represent the SD from the mean of 5–7 individual oocytes. Best-fit values from parts B and C are shown in Tables 1 and 2.
	Figure 2. Influence of the α7M253L allosteric binding site mutation on BPC potency and interactions between BPC and PNU120596 binding. (A) Human α7 and α7M253L nAChRs were expressed in Xenopus laevis oocytes and clamped at −70 mV. Representative current traces in response to 100 μM ACh (black bar) are shown before (black) and after preincubation (20 s) and coapplication of the indicated compounds (colored lines and bars). Note the more than 10-fold increase of ACh-elicited current amplitude by PNU120596. (B) Dose-inhibition curves for the indicated BPCs at the human α7 and α7 M253L mutant (n = 3–6, the mean and SD are shown). Dotted/solid lines represent data from Figure 1B, for comparison. (C) Co-application of BPCs or QX-314 upon sequential preapplication of 10 μM PNU120596 and 100 μM ACh. Averaged current traces from at least five different oocytes are shown. SD is shown in light gray. Current traces were normalized to the ACh-elicited signal before the co-application. PNU125096-amplified signals were between 20 and 30 μA. Note that, due to the normalization, absolute current values are not shown.
	Figure 3. Voltage-dependency of α7 and (α1)2β1εδ nAChR inhibition by selected BPCs. Representative current traces and statistical analysis of ACh (100 μM)-induced current responses from α7 (A) and (α1)2β1εδ nAChRs (B) before and after inhibition by the indicated compounds. Gray current traces represent equilibrated control currents at −50 mV (solid) and −100 mV (dotted). Colored lines represent the respective responses following 20 s of preincubation and co-application of the indicated compounds with ACh. Note that current traces are larger at −100 mV. Paired analysis comparing normalized responses upon antagonist application at a holding potential of −50 mV (filled symbols) and −100 mV (empty symbols) from 4–6 individual oocytes is shown. Responses were normalized to the currents in the absence of antagonists. Single values are shown, with the mean displayed as a black line. Statistical significance was determined with a paired t-test with p < 0.05 *, p < 0.01 **, and p < 0.001 ***. MLA (gray), PTM0022 (red), MB327 (magenta), and QX-314 (gold).
	Figure 4. Inhibition of α7 chimeras and mutants by BPCs. Normalized responses of 1 μM PTM0022 (red triangle), 10 μM MB327 (magenta dot), and 30 μM QX-314 (golden dot) at the indicated chimeras and mutants (shown as pictograms, see Table S1 for more details). Single values are shown with the mean displayed as black line. Error bars represent 95% CIs (n = 4–6). Statistical analysis was done using Brown–Forsythe and Welch ANOVA with posthoc Dunnett T3 test compared to α7 with p < 0.05 *, p < 0.01 **, and p < 0.001 ***, to −α7 with p < 0.001 #, and to 5HT3A with p < 0.05°, p < 0.01°°. EC80 concentration of agonist was applied, and currents were normalized to currents evoked before incubation and co-application of the indicated compound.
	Figure 5. Molecular docking of PTM0022, MB327, and QX-314 in open, closed, and desensitized states of the α7 nAChR. Results with the highest binding energy are shown for PTM0022, MB327, and QX-314 in the resting (PDB ID: 7KOO, A), open (PDB ID: 7KOX, B), and desensitized (PDB ID: 7KOQ, C) states of the α7 nAChR. Cartoon structures of each receptor state with all three compounds are shown on the left. Detailed surface representation and interacting amino acid residues for PTM0022 (red), MB327 (magenta), and OX-314 (yellow) are shown on the right. Color coding follows YRB script (Hagemans et al., 2015) with hydrophobic C atoms in yellow and polar interaction partners in blue (positive) and red (negative). Black rings indicate the position of the labeled pore lining residues. Carbon, nitrogen, and oxygen atoms of amino acid residues are colored in gray, blue, and red, respectively. ECD—extracellular domain, TMD—transmembrane domain, and ICD—intracellular domain.
	Figure 6. Analysis of α7 nAChR mutants to identify residues involved in the inhibition by PTM0022. (A) Representative current traces (Scale bars represent 0.5 μA and 2 s) and statistical analysis showing inhibition of the indicated α7 mutants by 1 μM PTM0022 (red triangle). For details of the mutants, refer to Table S1. Mean of the individual values is displayed as black line. Error bars represent the 95% CIs from n = 4–6 experiments. Statistical analysis was done using Brown–Forsythe and Welch ANOVA with posthoc Dunnett T3 test compared to α7 with p < 0.05 *, p < 0.01 **, and p < 0.001 ***, to 5HT3A with p < 0.01°. Current responses (EC80 agonist) were normalized to responses before—preincubation and co-application of PTM0022. (B) ACh dose response curves (EC50 values are in Table 5) for the indicated α7 mutants and chimeras with the mean and SD (left panel) and detailed analysis of responses to 10 mM ACh showing single values with means and 95% CIs (right panel).
	Figure 7. Correlation analysis of the potency of the BPCs at the human α7 nAChR and their chemical properties. (A) Pearson correlation of the determined pIC50 values and relevant QSAR chemical properties (see Table S5) obtained from http://chemicalize.com/ (ChemAxon) with significance threshold of p < 0.05 (Holm corrected) with (+, n = 8) or without (−, n = 7) QX-314. Negative and positive correlations are indicated with red and blue, respectively. (B) Scatter plots of the five significant chemical properties from A without (−) QX-314 with a smooth fit (x–y, dashed line) and 95% confidence interval (light gray) of the fit. QX-314 is shown as red point, independent from the fit. Note that pIC50 values could not be determined for PTM0008 and 09 and they are not included in this analysis (C) chemical structures of the symmetrical BPCs and QX-314. Oxygen and nitrogen atoms are colored red and blue, respectively. Access, accessible; mol, molar; refract, refractivity; sol, solvent; top, topological; VdW, van der Waals.
	Figure 8. BPC do not resensitize oocyte-expressed α7 nAChR and show weak inhibition of some other cation-selective ion channels. (A) Oocytes were clamped at −70 mV, and 5 s-pulses of 100 μM ACh (gray lines) were applied in 1 min intervals until stable responses were obtained. Receptors were then desensitized by a 1 min application of 1 mM ACh (black lines) with or without 10 μM PNU120596 (blue line) or 3 μM PTM0022 (red line). After a 5 s wash-out, 1 mM ACh was reapplied. Representative current traces from three different oocytes per experiment are shown. Note that PNU120596 and PTM0022 were added 7 s after the beginning of 1 mM ACh exposure to allow complete receptor desensitization. (B) Superposition of representative current traces before (black) and after preincubation and co-application (red) of the same PTM0022 concentrations as indicated in (C) for respective ligand-gated ion channels clamped at −100 mV. Scale bars represent 0.5 μA and 2 s. (C) Voltage-dependency of the inhibition shown in (B). Single values with the mean as black line for −50 and −100 mV are shown. Paired t-test with p < 0.05 *, p < 0.01 **, and p < 0.001 ***. (D) Left panel, typical current traces of NaV1.4 before (black) and 12 min after (red) application of 10 μM PTM0022. Currents were elicited by 50 ms pulses to −10 mV from a holding potential of −120 mV every 20 s. Right panel, sodium peak currents normalized to the control pulse before application of PTM0022. Means and SD values of four different oocytes are shown.

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