Sure F , Einsiedel J , Gmeiner P , Duchstein P , Zahn D , Korbmacher C , Ilyaskin AV .

	Figure 1. S3969 activates human but not mouse ENaC, and the ECL of human β-ENaC is essential for this stimulatory effect. A–D, first panels: Pictograms symbolize α-, β-, and γ-ENaC subunits, which were co-expressed in the corresponding group of oocytes. Murine or human isoforms and parts of the chimeric β-ENaC are shown in white with a red outline or in blue with a black outline, respectively. Transmembrane domains are represented as black bars. Second panels: A representative whole-cell current trace is shown for an oocyte expressing ENaC with the subunit composition as indicated in the corresponding first panel. Amiloride (ami, 2 μM) and S3969 (at the indicated concentration in μM) were present in the bath solution as indicated by black and gray shaded bars, respectively. Dotted lines correspond to the zero current level. Third panels: ENaC-mediated amiloride-sensitive whole-cell current values (ΔIami) were determined from similar experiments as shown in the second panel. Gray lines connect data points obtained in an individual oocyte. Mean ± SD are shown in black (A: N = 7, n = 29; B: N = 4, n = 19; C: N = 2, n = 7; D: N = 2, n = 19; N indicates the number of different batches of oocytes, n indicates the number of individual oocytes studied per experimental group). Fourth panels: Concentration-response relationship of the S3969 effect on ENaC currents. In each individual recording shown in the third panel, ΔIami was normalized to ΔIami obtained with 0.03 μM of S3969. Mean ± SD are shown and fitted to Equation 1 (in A, C, and D) to estimate EC50, the half maximal stimulatory concentration. In (B), data were fitted to a spline function. Dotted lines indicate normalized ΔIami values of one (no effect). One-way ANOVA with Bonferroni’s post hoc test was used to calculate p-values for comparisons with the baseline current values: in third panels (A: 1/1/0.009/<0.0001/<0.0001/<0.0001; B: 1/1/1/1/1/1; C: 1/0.8/0.004/<0.0001/<0.0001/<0.0001; D: 1/1/0.106/<0.0001/<0.0001/<0.0001); in fourth panels (A: 1/<0.0001/<0.0001/<0.0001/<0.0001/<0.0001; B: 1/1/1/0.633/0.010/<0.0001; C: 1/0.030/<0.0001/<0.0001/<0.0001/<0.0001; D: 1/0.400/<0.0001/<0.0001/<0.0001/<0.0001). ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 indicate significant stimulation by S3969. ENaC, epithelial sodium channel.
	Figure 2. Mouse-human chimeric β-ENaCs used to identify a portion of the ECL critically involved in the stimulatory effect of S3969. The pictograms highlight in color the parts of the ECL in mouse β-ENaC that were replaced by the corresponding human ECL sequence. The different colors represent the domain organization with palm in yellow, β-ball in red, finger in brown, GRIP in violet, thumb in green, and knuckle in blue. Unmodified portions of mouse β-ENaC are shown in white with a red outline. Transmembrane domains are represented as black bars. Similarity between mouse and human sequences, as well as the number of nonidentical residues in the replaced channel portion, were calculated from the sequence alignment shown in Fig. S3 . Ø: no stimulatory effect of S3969; ↔, marginal stimulatory effect with 10 μM of S3969; ↑, ↑↑, ↑↑↑, ↑↑↑↑: significant stimulatory effect of S3969 ranked according to the estimated EC50 values and/or maximal normalized ΔIami achieved with 10 μM of S3969. ENaC, epithelial sodium channel.
	Figure 3. Identification of a β-ECL portion critically involved in the stimulatory effect of S3969. A–C, left panels: representative whole-cell current traces obtained in individual oocytes expressing mouse αm- and γm-subunits together with a chimeric mouse-human β subunit (βm,h) as indicated. Pictograms illustrate which portion of mouse β-ENaC were replaced by the corresponding region of human β-ENaC. The explanation for the color code is given in Figure 2 . The experimental protocol was similar to that described in Figure 1 . Middle and right panels: Concentration-response relationship of the stimulatory effect of S3969 on absolute (middle panel) or normalized ΔIami (right panel). Summary data obtained from similar experiments as shown in left panels and analyzed as described in Figure 1 . Mean ± SD are shown. In right panels, mean values are fitted to a spline function (A: N = 5, n = 31; N indicates the number of different batches of oocytes, n indicates the number of individual oocytes studied per experimental group) or to Equation 1 (B: N = 2, n = 12; C: N = 2, n = 11). One-way ANOVA with Bonferroni’s post hoc test was used to calculate p-values for comparisons with the baseline current values: in middle panels (A: 1/1/1/1/0.0534/0.005; B: 1/1/1/0.021/0.002/0.0003; C: 1/1/1/0.265/0.040/0.015); in right panels (A: 1/1/1/<0.0001/<0.0001/<0.0001; B: 1/0.041/<0.0001/<0.0001/<0.0001/<0.0001; C: 1/0.002/<0.0001/<0.0001/<0.0001/<0.0001). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate significant stimulation by S3969. ENaC, epithelial sodium channel.
	Figure 4. An arginine residue (Arg388) in β-ENaC is functionally important for channel stimulation by S3969. A, ribbon diagram of the ECL of human αβγ-ENaC generated using atom coordinates from PDB entry 6WTH ( 8 , 71 ). α-subunit is shown in dark gray, γ-subunit in white, and β-subunit is colored according to its domain organization as indicated. The putative location of unresolved transmembrane domains is indicated with a gray box placed within a schematically depicted lipid bilayer. The inset shows the location of a salt bridge interaction (visualized by blue dotted lines and electric charge symbols) between an arginine residue Arg388 in the α4 helix of the thumb domain and an aspartate residue Asp331 in the β-ball domain. Both residues are shown in stick representation with carbon atoms in green for Arg388 or pink for Asp331, nitrogen in blue, oxygen in red, and hydrogen in white. Nonpolar hydrogen atoms are omitted for clarity. B and C, representative whole-cell current trace (left panel) obtained in an oocyte expressing the mutant human ENaC (αhβh R388Hγh, B) or WT human ENaC (αhβhγh, C) and summary data obtained from similar experiments showing the concentration-response relationship of the stimulatory effect of S3969 on absolute (middle panel) or normalized ΔIami (right panel). Analysis was performed as described in Figure 1 . Mean ± SD are shown. In right panels, mean values are fitted to a spline function (B: N = 2, n = 15; N indicates the number of different batches of oocytes, n indicates the number of individual oocytes studied per experimental group) or to Equation 1 (C: N = 2, n = 15). One-way ANOVA with Bonferroni’s post hoc test was used to calculate p-values for comparisons with the baseline current values: in middle panels (B: 1/1/1/1/1/0.042; C: 1/1/0.0581/0.003/<0.0001/<0.0001); in right panels (B: 1/1/1/0.0096/<0.0001/<0.0001; C: 1/1/<0.0001/<0.0001/<0.0001/<0.0001). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate significant stimulation by S3969. ENaC, epithelial sodium channel.
	Figure 5. Atomistic molecular dynamics simulations reveal stable interactions between S3969 and Arg388, Phe391 and Tyr406 residues in β-ENaC. A, putative binding site of S3969 in the lower part of the cavity localized in the ECL of human β-ENaC. Snapshot is taken from the same MD simulation as in (B) at t = 405 ns. β-subunit is shown in surface representation and colored according to its domain organization using the same color code as in Figure 2 . α- and γ-subunits are in ribbon representation in dark gray and white, respectively. S3969 is shown in stick representation with carbon atoms in tan, nitrogen in blue, oxygen in red, sulfur in yellow, and hydrogen in white. Insets show the S3969-binding site on an expanded scale in surface (left inset) or ribbon representation (right inset). In the right inset, Arg388, Phe391, and Tyr406 residues forming the most stable interactions with S3969 are shown in stick representation with carbon atoms in green, oxygen in red, nitrogen in blue, and polar hydrogen in white. Apolar hydrogen atoms of β-ENaC are omitted for clarity. Blue and pink dotted lines visualize hydrogen bond and hydrophobic interactions, respectively. B, diagram shows interactions formed between β-ENaC residues, which contribute to the putative S3969-binding site and labeled in the left inset in (A), and a S3969 molecule during 500 ns MD simulations. Interaction types are represented by different colors as indicated. Each line represents the formation of at least one interaction of the specified type per trajectory frame. A darker color intensity indicates multiple interactions of the same type per trajectory frame. ENaC, epithelial sodium channel.
	Figure 6. Mutations F391G or Y406A in human β-ENaC nearly abolish the stimulatory effect of S3969. A–C, representative whole-cell current traces (left panels) obtained in individual oocytes expressing mutant human ENaC (αhβh F391Gγh in A or αhβh Y406Aγh in B) or WT human ENaC (αhβhγh, C) and summary data obtained from similar experiments showing concentration-response relationships of the stimulatory effect of S3969 on absolute (middle panel) or normalized ΔIami (right panel). Analysis was performed as described in Figure 1 . Mean ± SD are shown. In right panels, mean values are fitted to a spline function (A: N = 3, n = 18; B: N = 3, n = 18; N indicates the number of different batches of oocytes, n indicates the number of individual oocytes studied per experimental group) or to Equation 1 (C: N = 3, n = 7). One-way ANOVA with Bonferroni’s post hoc test was used to calculate p-values for comparisons with the baseline current values: in middle panels (A: 1/1/1/1/1/1; B: 1/1/1/1/1/1; C: 1/1/1/1/0.364/0.212); in right panels (A: 1/1/1/1/1/1; B: 1/1/0.725/1/1/<0.0001; C: 1/0.006/<0.0001/<0.0001/<0.0001/<0.0001). ∗∗p < 0.01 and ∗∗∗p < 0.001 indicate significant stimulation by S3969. ENaC, epithelial sodium channel.
	Figure 7. Binding of S3969 increases the distance between the α5 helix of the thumb domain in β-ENaC and the palm domain in γ-ENaC. A and B, left panels: Human αβγ-ENaC was simulated with (A) or without (B) S3969. Snapshots are taken from corresponding MD simulations at t = 405 ns. ENaC is shown in ribbon representation and S3969 in spheres representation using the same color code as in Figure 2 . The insets show a part of the β-γ-subunit interface at a larger scale to illustrate the conformational change of the α5 helix of the thumb domain of β-ENaC (in green), elicited by S3969 binding. Side chains of selected residues are shown in stick representation to illustrate the increase in the intersubunit distance caused by S3969. Distances were calculated between the selected atoms as indicated by dotted brown lines. Carbon atoms of β-ENaC are in green, of γ-ENaC in white, nitrogen atoms in blue, oxygen in red, and hydrogen in white. Apolar hydrogen atoms are omitted for clarity. Right panels: For each β-ENaC residue from 431 to 441, the distance between its center of mass and the center of mass of the closest residue in γ-ENaC was calculated in each MD trajectory frame. The distance values are represented by different colors using the scientific color map “lajolla” ( 76 ) shown on the right. ENaC, epithelial sodium channel.
	Figure 8. Replacing Arg437 in β- and Ser298 in γ-ENaC with cysteines to form a disulfide-bond prevents ENaC stimulation by S3969 which can be rescued by DTT. A and B, left panel: Representative whole-cell current traces are shown for an oocyte expressing mutant human ENaC (αhβh R437Cγh S298C) without (A) or with (B) 15 min pre-incubation in 30 mM DTT. Amiloride (ami, 2 μM) and S3969 (10 μM) were present in the bath solution as indicated by black and white bars, respectively. Dotted lines indicate zero current level. Right panel: Summary of ΔIami values measured before (−) and after (+) application of S3969 from similar experiments as shown in the corresponding left panel. Gray lines connect data points obtained in an individual oocyte. Mean ± SD are shown in black (N = 2, n = 15; N indicates the number of different batches of oocytes, n indicates the number of individual oocytes studied per experimental group). Paired t test was used to calculate p-values (A: <0.0001, B: <0.0001). ∗∗∗p < 0.001 indicates significant stimulation by S3969. ENaC, epithelial sodium channel.
	Figure 9. S3969 stimulates ENaC in H441 cells. A and B, left panel: Representative equivalent short circuit current (ISC) recordings are shown. S3969 (10 μM) and amiloride (ami, 10 μM) were present in the apical bath solution as indicated by gray and black bars, respectively. Initial parts of recordings (∼30 min) corresponding to the equilibration phase after transferring the cells into Ussing chambers and applying Ringer's solution to the apical compartment are omitted for clarity. The dotted lines indicate zero current level. Right panel: Summary data obtained in similar experiments as shown in the left panel demonstrate the effect of S3969 on ISC in the absence (A) or the presence (B) of apical amiloride. ISC values were measured in each individual recording before the application of S3969 (A) or amiloride (B), before the subsequent application of amiloride in the presence of S3969 (A) or of S3969 in the presence of amiloride (B), and at the end of the experiment. Gray lines connect data points obtained from an individual H441 cell monolayer. Mean ± SD are shown in black (A: n = 14; B: n = 12). One-way ANOVA with Bonferroni’s post hoc test was used to calculate p-values. ENaC, epithelial sodium channel.
	Figure 10. Putative molecular mechanism underlying ENaC activation by S3969. Binding of S3969 to β-ENaC moves the α5 helix of the thumb domain of β-ENaC away from the palm domain of γ-ENaC probably causing a conformational change resulting in channel activation. ENaC, epithelial sodium channel.
	Figure 1. S3969 activates human but not mouse ENaC, and the ECL of human β-ENaC is essential for this stimulatory effect.A–D, first panels: Pictograms symbolize α-, β-, and γ-ENaC subunits, which were co-expressed in the corresponding group of oocytes. Murine or human isoforms and parts of the chimeric β-ENaC are shown in white with a red outline or in blue with a black outline, respectively. Transmembrane domains are represented as black bars. Second panels: A representative whole-cell current trace is shown for an oocyte expressing ENaC with the subunit composition as indicated in the corresponding first panel. Amiloride (ami, 2 μM) and S3969 (at the indicated concentration in μM) were present in the bath solution as indicated by black and gray shaded bars, respectively. Dotted lines correspond to the zero current level. Third panels: ENaC-mediated amiloride-sensitive whole-cell current values (ΔIami) were determined from similar experiments as shown in the second panel. Gray lines connect data points obtained in an individual oocyte. Mean ± SD are shown in black (A: N = 7, n = 29; B: N = 4, n = 19; C: N = 2, n = 7; D: N = 2, n = 19; N indicates the number of different batches of oocytes, n indicates the number of individual oocytes studied per experimental group). Fourth panels: Concentration-response relationship of the S3969 effect on ENaC currents. In each individual recording shown in the third panel, ΔIami was normalized to ΔIami obtained with 0.03 μM of S3969. Mean ± SD are shown and fitted to Equation 1 (in A, C, and D) to estimate EC50, the half maximal stimulatory concentration. In (B), data were fitted to a spline function. Dotted lines indicate normalized ΔIami values of one (no effect). One-way ANOVA with Bonferroni’s post hoc test was used to calculate p-values for comparisons with the baseline current values: in third panels (A: 1/1/0.009/<0.0001/<0.0001/<0.0001; B: 1/1/1/1/1/1; C: 1/0.8/0.004/<0.0001/<0.0001/<0.0001; D: 1/1/0.106/<0.0001/<0.0001/<0.0001); in fourth panels (A: 1/<0.0001/<0.0001/<0.0001/<0.0001/<0.0001; B: 1/1/1/0.633/0.010/<0.0001; C: 1/0.030/<0.0001/<0.0001/<0.0001/<0.0001; D: 1/0.400/<0.0001/<0.0001/<0.0001/<0.0001). ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 indicate significant stimulation by S3969. ENaC, epithelial sodium channel.
	Figure 2. Mouse-human chimeric β-ENaCs used to identify a portion of the ECL critically involved in the stimulatory effect of S3969. The pictograms highlight in color the parts of the ECL in mouse β-ENaC that were replaced by the corresponding human ECL sequence. The different colors represent the domain organization with palm in yellow, β-ball in red, finger in brown, GRIP in violet, thumb in green, and knuckle in blue. Unmodified portions of mouse β-ENaC are shown in white with a red outline. Transmembrane domains are represented as black bars. Similarity between mouse and human sequences, as well as the number of nonidentical residues in the replaced channel portion, were calculated from the sequence alignment shown in Fig. S3. Ø: no stimulatory effect of S3969; ↔, marginal stimulatory effect with 10 μM of S3969; ↑, ↑↑, ↑↑↑, ↑↑↑↑: significant stimulatory effect of S3969 ranked according to the estimated EC50 values and/or maximal normalized ΔIami achieved with 10 μM of S3969. ENaC, epithelial sodium channel.
	Figure 3. Identification of a β-ECL portion critically involved in the stimulatory effect of S3969.A–C, left panels: representative whole-cell current traces obtained in individual oocytes expressing mouse αm- and γm-subunits together with a chimeric mouse-human β subunit (βm,h) as indicated. Pictograms illustrate which portion of mouse β-ENaC were replaced by the corresponding region of human β-ENaC. The explanation for the color code is given in Figure 2. The experimental protocol was similar to that described in Figure 1. Middle and right panels: Concentration-response relationship of the stimulatory effect of S3969 on absolute (middle panel) or normalized ΔIami (right panel). Summary data obtained from similar experiments as shown in left panels and analyzed as described in Figure 1. Mean ± SD are shown. In right panels, mean values are fitted to a spline function (A: N = 5, n = 31; N indicates the number of different batches of oocytes, n indicates the number of individual oocytes studied per experimental group) or to Equation 1 (B: N = 2, n = 12; C: N = 2, n = 11). One-way ANOVA with Bonferroni’s post hoc test was used to calculate p-values for comparisons with the baseline current values: in middle panels (A: 1/1/1/1/0.0534/0.005; B: 1/1/1/0.021/0.002/0.0003; C: 1/1/1/0.265/0.040/0.015); in right panels (A: 1/1/1/<0.0001/<0.0001/<0.0001; B: 1/0.041/<0.0001/<0.0001/<0.0001/<0.0001; C: 1/0.002/<0.0001/<0.0001/<0.0001/<0.0001). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate significant stimulation by S3969. ENaC, epithelial sodium channel.
	Figure 4. An arginine residue (Arg388) in β-ENaC is functionally important for channel stimulation by S3969.A, ribbon diagram of the ECL of human αβγ-ENaC generated using atom coordinates from PDB entry 6WTH (8, 71). α-subunit is shown in dark gray, γ-subunit in white, and β-subunit is colored according to its domain organization as indicated. The putative location of unresolved transmembrane domains is indicated with a gray box placed within a schematically depicted lipid bilayer. The inset shows the location of a salt bridge interaction (visualized by blue dotted lines and electric charge symbols) between an arginine residue Arg388 in the α4 helix of the thumb domain and an aspartate residue Asp331 in the β-ball domain. Both residues are shown in stick representation with carbon atoms in green for Arg388 or pink for Asp331, nitrogen in blue, oxygen in red, and hydrogen in white. Nonpolar hydrogen atoms are omitted for clarity. B and C, representative whole-cell current trace (left panel) obtained in an oocyte expressing the mutant human ENaC (αhβhR388Hγh, B) or WT human ENaC (αhβhγh, C) and summary data obtained from similar experiments showing the concentration-response relationship of the stimulatory effect of S3969 on absolute (middle panel) or normalized ΔIami (right panel). Analysis was performed as described in Figure 1. Mean ± SD are shown. In right panels, mean values are fitted to a spline function (B: N = 2, n = 15; N indicates the number of different batches of oocytes, n indicates the number of individual oocytes studied per experimental group) or to Equation 1 (C: N = 2, n = 15). One-way ANOVA with Bonferroni’s post hoc test was used to calculate p-values for comparisons with the baseline current values: in middle panels (B: 1/1/1/1/1/0.042; C: 1/1/0.0581/0.003/<0.0001/<0.0001); in right panels (B: 1/1/1/0.0096/<0.0001/<0.0001; C: 1/1/<0.0001/<0.0001/<0.0001/<0.0001). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate significant stimulation by S3969. ENaC, epithelial sodium channel.
	Figure 5. Atomistic molecular dynamics simulations reveal stable interactions between S3969 and Arg388, Phe391 and Tyr406 residues in β-ENaC.A, putative binding site of S3969 in the lower part of the cavity localized in the ECL of human β-ENaC. Snapshot is taken from the same MD simulation as in (B) at t = 405 ns. β-subunit is shown in surface representation and colored according to its domain organization using the same color code as in Figure 2. α- and γ-subunits are in ribbon representation in dark gray and white, respectively. S3969 is shown in stick representation with carbon atoms in tan, nitrogen in blue, oxygen in red, sulfur in yellow, and hydrogen in white. Insets show the S3969-binding site on an expanded scale in surface (left inset) or ribbon representation (right inset). In the right inset, Arg388, Phe391, and Tyr406 residues forming the most stable interactions with S3969 are shown in stick representation with carbon atoms in green, oxygen in red, nitrogen in blue, and polar hydrogen in white. Apolar hydrogen atoms of β-ENaC are omitted for clarity. Blue and pink dotted lines visualize hydrogen bond and hydrophobic interactions, respectively. B, diagram shows interactions formed between β-ENaC residues, which contribute to the putative S3969-binding site and labeled in the left inset in (A), and a S3969 molecule during 500 ns MD simulations. Interaction types are represented by different colors as indicated. Each line represents the formation of at least one interaction of the specified type per trajectory frame. A darker color intensity indicates multiple interactions of the same type per trajectory frame. ENaC, epithelial sodium channel.
	Figure 6. Mutations F391G or Y406A in human β-ENaC nearly abolish the stimulatory effect of S3969.A–C, representative whole-cell current traces (left panels) obtained in individual oocytes expressing mutant human ENaC (αhβhF391Gγh in A or αhβhY406Aγh in B) or WT human ENaC (αhβhγh, C) and summary data obtained from similar experiments showing concentration-response relationships of the stimulatory effect of S3969 on absolute (middle panel) or normalized ΔIami (right panel). Analysis was performed as described in Figure 1. Mean ± SD are shown. In right panels, mean values are fitted to a spline function (A: N = 3, n = 18; B: N = 3, n = 18; N indicates the number of different batches of oocytes, n indicates the number of individual oocytes studied per experimental group) or to Equation 1 (C: N = 3, n = 7). One-way ANOVA with Bonferroni’s post hoc test was used to calculate p-values for comparisons with the baseline current values: in middle panels (A: 1/1/1/1/1/1; B: 1/1/1/1/1/1; C: 1/1/1/1/0.364/0.212); in right panels (A: 1/1/1/1/1/1; B: 1/1/0.725/1/1/<0.0001; C: 1/0.006/<0.0001/<0.0001/<0.0001/<0.0001). ∗∗p < 0.01 and ∗∗∗p < 0.001 indicate significant stimulation by S3969. ENaC, epithelial sodium channel.
	Figure 7. Binding of S3969 increases the distance between the α5 helix of the thumb domain in β-ENaC and the palm domain in γ-ENaC.A and B, left panels: Human αβγ-ENaC was simulated with (A) or without (B) S3969. Snapshots are taken from corresponding MD simulations at t = 405 ns. ENaC is shown in ribbon representation and S3969 in spheres representation using the same color code as in Figure 2. The insets show a part of the β-γ-subunit interface at a larger scale to illustrate the conformational change of the α5 helix of the thumb domain of β-ENaC (in green), elicited by S3969 binding. Side chains of selected residues are shown in stick representation to illustrate the increase in the intersubunit distance caused by S3969. Distances were calculated between the selected atoms as indicated by dotted brown lines. Carbon atoms of β-ENaC are in green, of γ-ENaC in white, nitrogen atoms in blue, oxygen in red, and hydrogen in white. Apolar hydrogen atoms are omitted for clarity. Right panels: For each β-ENaC residue from 431 to 441, the distance between its center of mass and the center of mass of the closest residue in γ-ENaC was calculated in each MD trajectory frame. The distance values are represented by different colors using the scientific color map “lajolla” (76) shown on the right. ENaC, epithelial sodium channel.
	Figure 8. Replacing Arg437 in β- and Ser298 in γ-ENaC with cysteines to form a disulfide-bond prevents ENaC stimulation by S3969 which can be rescued by DTT.A and B, left panel: Representative whole-cell current traces are shown for an oocyte expressing mutant human ENaC (αhβhR437Cγh S298C) without (A) or with (B) 15 min pre-incubation in 30 mM DTT. Amiloride (ami, 2 μM) and S3969 (10 μM) were present in the bath solution as indicated by black and white bars, respectively. Dotted lines indicate zero current level. Right panel: Summary of ΔIami values measured before (−) and after (+) application of S3969 from similar experiments as shown in the corresponding left panel. Gray lines connect data points obtained in an individual oocyte. Mean ± SD are shown in black (N = 2, n = 15; N indicates the number of different batches of oocytes, n indicates the number of individual oocytes studied per experimental group). Paired t test was used to calculate p-values (A: <0.0001, B: <0.0001). ∗∗∗p < 0.001 indicates significant stimulation by S3969. ENaC, epithelial sodium channel.
	Figure 9. S3969 stimulates ENaC in H441 cells.A and B, left panel: Representative equivalent short circuit current (ISC) recordings are shown. S3969 (10 μM) and amiloride (ami, 10 μM) were present in the apical bath solution as indicated by gray and black bars, respectively. Initial parts of recordings (∼30 min) corresponding to the equilibration phase after transferring the cells into Ussing chambers and applying Ringer's solution to the apical compartment are omitted for clarity. The dotted lines indicate zero current level. Right panel: Summary data obtained in similar experiments as shown in the left panel demonstrate the effect of S3969 on ISC in the absence (A) or the presence (B) of apical amiloride. ISC values were measured in each individual recording before the application of S3969 (A) or amiloride (B), before the subsequent application of amiloride in the presence of S3969 (A) or of S3969 in the presence of amiloride (B), and at the end of the experiment. Gray lines connect data points obtained from an individual H441 cell monolayer. Mean ± SD are shown in black (A: n = 14; B: n = 12). One-way ANOVA with Bonferroni’s post hoc test was used to calculate p-values. ENaC, epithelial sodium channel.
	Figure 10. Putative molecular mechanism underlying ENaC activation by S3969. Binding of S3969 to β-ENaC moves the α5 helix of the thumb domain of β-ENaC away from the palm domain of γ-ENaC probably causing a conformational change resulting in channel activation. ENaC, epithelial sodium channel.

Akçay, Pseudohypoaldosteronism type 1 and respiratory distress syndrome. 2002, Pubmed

Akçay, Pseudohypoaldosteronism type 1 and respiratory distress syndrome. 2002, Pubmed
Askwith, Neuropeptide FF and FMRFamide potentiate acid-evoked currents from sensory neurons and proton-gated DEG/ENaC channels. 2000, Pubmed , Xenbase
Baconguis, Structural plasticity and dynamic selectivity of acid-sensing ion channel-spider toxin complexes. 2012, Pubmed
Baconguis, X-ray structure of acid-sensing ion channel 1-snake toxin complex reveals open state of a Na(+)-selective channel. 2014, Pubmed
Balchak, The epithelial Na+ channel γ subunit autoinhibitory tract suppresses channel activity by binding the γ subunit's finger-thumb domain interface. 2018, Pubmed , Xenbase
Bohnert, Experimental nephrotic syndrome leads to proteolytic activation of the epithelial Na+ channel in the mouse kidney. 2021, Pubmed
Bohnert, Aprotinin prevents proteolytic epithelial sodium channel (ENaC) activation and volume retention in nephrotic syndrome. 2018, Pubmed , Xenbase
Boiko, Pseudohypoaldosteronism type 1 and Liddle's syndrome mutations that affect the single-channel properties of the epithelial Na+ channel. 2015, Pubmed
Boucher, Airway surface dehydration in cystic fibrosis: pathogenesis and therapy. 2007, Pubmed
Boucher, Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation. 1986, Pubmed
Canessa, Membrane topology of the epithelial sodium channel in intact cells. 1994, Pubmed , Xenbase
Chang, Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. 1996, Pubmed
Collier, Extracellular chloride regulates the epithelial sodium channel. 2009, Pubmed , Xenbase
Collier, Intersubunit conformational changes mediate epithelial sodium channel gating. 2014, Pubmed , Xenbase
Czikora, A novel tumor necrosis factor-mediated mechanism of direct epithelial sodium channel activation. 2014, Pubmed
Dawson, Structure of the acid-sensing ion channel 1 in complex with the gating modifier Psalmotoxin 1. 2012, Pubmed
Eaton, The contribution of epithelial sodium channels to alveolar function in health and disease. 2009, Pubmed
Essigke, Zymogen-locked mutant prostasin (Prss8) leads to incomplete proteolytic activation of the epithelial sodium channel (ENaC) and severely compromises triamterene tolerance in mice. 2021, Pubmed , Xenbase
Fronius, Treatment of pulmonary edema by ENaC activators/stimulators. 2013, Pubmed
Garty, Epithelial sodium channels: function, structure, and regulation. 1997, Pubmed
Gründer, A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel. 1997, Pubmed , Xenbase
Haerteis, The delta-subunit of the epithelial sodium channel (ENaC) enhances channel activity and alters proteolytic ENaC activation. 2009, Pubmed , Xenbase
Haerteis, Proteolytic activation of the human epithelial sodium channel by trypsin IV and trypsin I involves distinct cleavage sites. 2014, Pubmed , Xenbase
Hartmann, TIP peptide inhalation in experimental acute lung injury: effect of repetitive dosage and different synthetic variants. 2014, Pubmed
Hartmann, An inhaled tumor necrosis factor-alpha-derived TIP peptide improves the pulmonary function in experimental lung injury. 2013, Pubmed
Hinrichs, Mechanisms of sodium retention in nephrotic syndrome. 2020, Pubmed
Huber, Functional characterization of a partial loss-of-function mutation of the epithelial sodium channel (ENaC) associated with atypical cystic fibrosis. 2010, Pubmed , Xenbase
Hummler, Role of the epithelial sodium channel in lung liquid clearance. 1997, Pubmed
Ilyaskin, Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis. 2021, Pubmed , Xenbase
Irwin, ZINC20-A Free Ultralarge-Scale Chemical Database for Ligand Discovery. 2020, Pubmed
Jasti, Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. 2007, Pubmed
Kasahara, Novel indole and benzothiophene ring derivatives showing differential modulatory activity against human epithelial sodium channel subunits, ENaC β and γ. 2019, Pubmed
Kellenberger, International Union of Basic and Clinical Pharmacology. XCI. structure, function, and pharmacology of acid-sensing ion channels and the epithelial Na+ channel. 2015, Pubmed
Keszler, Pseudohypoaldosteronism. 1983, Pubmed
Kleyman, Epithelial Na+ Channel Regulation by Extracellular and Intracellular Factors. 2018, Pubmed
Kleyman, Regulating ENaC's gate. 2020, Pubmed
Lemmens-Gruber, The Epithelial Sodium Channel-An Underestimated Drug Target. 2023, Pubmed
Li, Interaction of the aromatics Tyr-72/Trp-288 in the interface of the extracellular and transmembrane domains is essential for proton gating of acid-sensing ion channels. 2009, Pubmed , Xenbase
Lingueglia, Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel. 1995, Pubmed , Xenbase
Liu, Structure and mechanism of a neuropeptide-activated channel in the ENaC/DEG superfamily. 2023, Pubmed
Liu, Molecular mechanism and structural basis of small-molecule modulation of the gating of acid-sensing ion channel 1. 2021, Pubmed
Lorenz, Heteromultimeric CLC chloride channels with novel properties. 1996, Pubmed , Xenbase
Lu, Small molecule activator of the human epithelial sodium channel. 2008, Pubmed , Xenbase
Lucas, The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit. 2016, Pubmed
Lucas, Mapping the lectin-like activity of tumor necrosis factor. 1994, Pubmed
Malagon-Rogers, A patient with pseudohypoaldosteronism type 1 and respiratory distress syndrome. 1999, Pubmed
Mall, Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. 2004, Pubmed
Mansley, Prostaglandin E2 stimulates the epithelial sodium channel (ENaC) in cultured mouse cortical collecting duct cells in an autocrine manner. 2020, Pubmed
Mutchler, Epithelial Sodium Channel and Salt-Sensitive Hypertension. 2021, Pubmed
Noreng, Molecular principles of assembly, activation, and inhibition in epithelial sodium channel. 2020, Pubmed
Noreng, Structure of the human epithelial sodium channel by cryo-electron microscopy. 2018, Pubmed
Patel, Feedback inhibition of ENaC: acute and chronic mechanisms. 2014, Pubmed , Xenbase
Pearce, Regulation of distal tubule sodium transport: mechanisms and roles in homeostasis and pathophysiology. 2022, Pubmed
Pettersen, UCSF Chimera--a visualization system for exploratory research and analysis. 2004, Pubmed
Rauh, A mutation in the β-subunit of ENaC identified in a patient with cystic fibrosis-like symptoms has a gain-of-function effect. 2013, Pubmed , Xenbase
Rossier, Epithelial sodium channel (ENaC) and the control of blood pressure. 2014, Pubmed
Rossier, Activation of the epithelial sodium channel (ENaC) by serine proteases. 2009, Pubmed
Rotin, Function and Regulation of the Epithelial Na+ Channel ENaC. 2021, Pubmed
Sanner, Reduced surface: an efficient way to compute molecular surfaces. 1996, Pubmed
Shabbir, Mechanism of action of novel lung edema therapeutic AP301 by activation of the epithelial sodium channel. 2013, Pubmed
Shabbir, Glycosylation-dependent activation of epithelial sodium channel by solnatide. 2015, Pubmed
Shi, Role of the wrist domain in the response of the epithelial sodium channel to external stimuli. 2012, Pubmed , Xenbase
Shi, Base of the thumb domain modulates epithelial sodium channel gating. 2011, Pubmed , Xenbase
Snyder, Membrane topology of the amiloride-sensitive epithelial sodium channel. 1994, Pubmed , Xenbase
Strautnieks, Localisation of pseudohypoaldosteronism genes to chromosome 16p12.2-13.11 and 12p13.1-pter by homozygosity mapping. 1996, Pubmed
Sun, Structural insights into human acid-sensing ion channel 1a inhibition by snake toxin mambalgin1. 2020, Pubmed
Sure, Transmembrane serine protease 2 (TMPRSS2) proteolytically activates the epithelial sodium channel (ENaC) by cleaving the channel's γ-subunit. 2022, Pubmed , Xenbase
Tarran, Regulation of normal and cystic fibrosis airway surface liquid volume by phasic shear stress. 2006, Pubmed
Vullo, Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating. 2017, Pubmed
Wichmann, Incorporation of the δ-subunit into the epithelial sodium channel (ENaC) generates protease-resistant ENaCs in Xenopus laevis. 2018, Pubmed , Xenbase
Wichmann, Evolution of epithelial sodium channels: current concepts and hypotheses. 2020, Pubmed
Willam, TNF Lectin-Like Domain Restores Epithelial Sodium Channel Function in Frameshift Mutants Associated with Pseudohypoaldosteronism Type 1B. 2017, Pubmed
Yoder, Gating mechanisms of acid-sensing ion channels. 2018, Pubmed
Zhou, Solnatide Demonstrates Profound Therapeutic Activity in a Rat Model of Pulmonary Edema Induced by Acute Hypobaric Hypoxia and Exercise. 2017, Pubmed