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Figure 1. Location of variants in the human ENaC structure. A, location of ï¡R350 in a trimeric human. ENaC model (PDB 6BQN, (21)). The ï¡, ï¢ and ï§ subunits are shown as red, blue and green ribbons, respectively, using PyMol 2.0 (60). Side chain of ï¡R350 is shown as yellow spheres. Left, side view and right, top view. Both views show that ï¡R350 is near palm domain of ï¢ENaC. B, locations of ï¡R350,
ï¡V351, ï¡H354 and ï¡G355 in the extracellular ï¢-ball domain of ï¡ENaC. Helical domains are displayed in red and ï¢ strands in yellow. All four residues are shown as sticks with carbons in cyan, oxygen in red and nitrogen in blue. C, sequence alignments of ENaC/degenerin members. Alignments were performed using
Vector NTI 11 (ThermoFisher Scientific). Only sequences of the ï¢7 strand and its following residues are shown. Amino acid numbers of the first residue in all sequences are shown in parentheses. Four residues where variants of this study reside are shown in red letters.
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Figure 2. Three variants of human ï¡ENaC changed channel activity in Xenopus oocytes. Oocytes expressing WT and mutant human ENaCs were clamped at -100 mV (membrane potential) and whole-cell currents were recorded in a bath solution (NaCl-110, containing 110 mM NaCl) in the absence and presence
of 10 ïM amiloride. A, C, E and G, representative recordings of WT and mutant ENaCs. Traces were superimposed with the same time and current scales. Negative values reflect inward Na+ currents. B, D, F and H, normalized currents, representing amiloride-sensitive currents in all cells that were divided by the
mean of the WT group in the same batch of oocytes. Data were pooled from 3 to 5 batches of oocytes. Dot plots were overlaid with mean ï± SD. The p values were from Studentâs t-test.
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Figure 3. Functional variants do not alter ENaC surface expression in oocytes. A, surface expression levels in oocytes injected with cRNAs for WT ï¡Î²F
γ (ï¢F for ï¢FLAG), ï¡R350Wï¢F ï§ or WT ï¡ï¢ï§ (no FLAG control) ENaCs. B, surface expression levels in oocytes injected with WT ï¡ï¢F ï§, ï¡V351AβFγ, ï¡G355RβF
γ or WT ï¡ï¢ï§ ENaC. Levels of surface expression were assessed using a chemiluminescence assay 48 h after cRNA injection. Relative light units measured from individual oocytes were normalized to the mean relative light units of the same batch of oocytes expressing WT (ï¡Î²Fγ). Data were combined from three batches of oocytes. Similar relative surface expression levels of WT (ï¡Î²F
γ) and mutants (ï¡R350Wï¢Fï§ in A,V351AβFγ, and ï¡G355RβFγ in B) were observed (NS, not significant). However, levels of FLAG-tagged WT (ï¡Î²F
γ, positive control) were significantly greater than non-tagged WT (ï¡ï¢ï§, negative control, p <0.0001, one-way ANOVA with Dunnettâs post hoc test).
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Figure 4. Function variants do not alter ENaC surface expression in FRT cells. Cells were transfected with a WT or mutant (ï¡R350W, ï¡V351A or ï¡G355R) ï¡ subunit with an N-terminal HA and C-terminal V5 epitope tag, together with non-tagged WT ï¢ and ï§ subunits. Surface proteins were labeled with NHSSS-biotin and following cell lysis isolated with avidin beads. Following SDS-PAGE proteins were immunoblotted with a horseradish peroxidase-conjugated anti-HA antibody. Chemiluminescence was quantified with Bio-Rad ChemiDocTM System, and were normalized to GAPDH expression. Resultant values were normalized to WT to obtain the relative expression levels for each experiment. Relative ï¡ENaC
surafce expression levels are shown in A (full-length, 90 kDa), C (cleaved, 22 kDa) and E (full length +cleaved, 90 kDa + 22 kDa). Relative total ï¡ENaC expression levels (5% of the cell lysate) are shown in B (full-length, 90 kDa), D (cleaved, 22 kDa) and F (full length + cleaved, 90 kDa + 22 kDa). G, representative blots for surface and total (5% of the cell lysate) expression of ï¡ENaC. NC (negative control) represents
results with cells transfected with non-tagged human ï¡, ï¢ and ï§ cDNAs. WT, 350, 351 and 355 represent the cells transfected with cDNAs for HA-ï¡-V5, HA-ï¡R350W-V5, HA-ï¡V351A-V5 or HA-ï¡G355R-V5, respectively, accompanied with non-tagged ï¢ and ï§ ENaCs. Data were from four independent experiments.
Bars are mean  S.D. All values in A-F were not significant different (p > 0.05, n=3-4, one-way ANOVA).
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Figure 5. ï¡R350W, ï¡H354R and ï¡G355R reduce the Na+ self-inhibition response. A, C, E and G, representative recordings in oocytes expressing WT or mutant ENaCs to show the Na+ self-inhibition response. Oocytes were clamped at -100 mV while bath Na+ concentration was increased from 1 mM (NaCl1, grey bar) to 110 mM (NaCl-110, black bar). Traces were superimposed with the same time and current scales. B, D, F and H, Iss/Ipeak represents the magnitude of Na+ self-inhibition. Values were obtained from amiloride-sensitive Iss and Ipeak. Horizontal bars are mean ï± S.D. Data were collected from three batches of
oocytes. The p values were from Studentâs t tests, NS, not significant.
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Figure 6. ï¡R350W has an increased channel open probability. A, representative traces of single channel recordings from oocytes expressing WT ï¡ï¢ï§, ï¡V351Aï¢ï§ and ï¡R350Wï¢ï§. Cell-attached patches were performed at -80 mV (opposite to pipette potential) with a NaCl-110 bath solution and a pipette solution containing 110 mM LiCl. Letters âCâ and âOâ represent closed and open states. Recordings were further filtered at 20 Hz with a low-pass Gaussian following 100 Hz of low-pass Gaussian algorithm by ClampFit 10 (Molecular Devices) for display. The filtering did not eliminate transitions, judged by visual inspection. B, single channel conductances of WT and the two variants, determined by linear regression of unitary currents and clamping voltages in the range of -20 to -100 mV. Values were not significantly different (p > 0.05, one-way ANOVA). C, NPo as the product of N (channel number in patches) and Po (open
probability) of WT and the mutant channels. D, N (channel numbers) of WT and mutant channels. E, Po of WT and mutant channels. In B-E, data are shown as dot plots with mean and S.D. bars. Significantly different values in C-E are noted (p < 0.05, one-way ANOVA followed with a Dunnettâs post hoc test). NS,
not significant.
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Figure 7. Other variants at ï¡R350 increase ENaC activity. Normalized currents obtained in the same batches of oocytes expressing WT and mutant channels are shown in A (ï¡R350G and WT), B (ï¡R350Q and WT) and C (ï¡R350L and WT). Normalized currents (measured at -100mV) were obtained as described in Fig 2 legend. Data were pooled from three batches of oocytes for each WT and mutant pair. Bars are mean ï± S.D. The p values were from Studentâs t tests.
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Figure 8. Comparisons of the effects of ï¡R350W and other known gain-of-function human ENaC variants. ENaC activities and Na+ self-inhibition responses were examined in the same batches of oocytes expressing human ENaC ï¡ï¢ï§ (WT), ï¡R350Wï¢ï§, ï¡C479Rï¢ï§ or ï¡W493Rï¢ï§. A, normalized currents of WT,
ï¡C479R, ï¡R350W and ï¡W493R mutants, obtained in three batches of oocytes with numbers of cells in parentheses. B, Iss/Ipeak of WT and the three variants, obtained in three batches of oocytes. Significantly different values in both A and B are noted (p < 0.0001, one-way ANOVA followed by Tukeyâs post hoc
test). NS, not significant. C, locations of ï¡R350, ï¡W493 and ï¡C479 in a trimeric model of human ENaC. Three subunits (ï¡, ï¢ and ï§) are shown as three colored ribbons, from PDB 6BQN (21), using PyMol 2.0 (60). Three residues where the gain-of-function variants reside are shown in yellow spheres. D, zoomed-in
view of the same model as C. For clarity, only ï¡R350 in ï¢7 of ï¡ subunit and a partial palm domain of ï¢ subunit are shown. Eight ï¢ENaC residues within 8 Ã
of the side chain of ï¡R350, predicted by PyMol are shown. The dash line identifies ï¡/ï¢ subunit interface at the level.
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