Wang XP , Balchak DM , Gentilcore C , Clark NL , Kashlan OB .

P30 DK079307 NIDDK NIH HHS , P30 DK120531 NIDDK NIH HHS , R01 DK125439 NIDDK NIH HHS , R01 EY030546 NEI NIH HHS

	Figure 1. Sequence conservation in epithelial Na+ channel (ENaC) subunits. (A) Space filling model of ENaC (pdb code: 6BQN) with plane indicating position of outer membrane border. The α and γ subunits are white and gray, respectively. The β subunit is colored by domain as indicated in panel B. Intracellular structures are absent in this structural model. (B) Linear model of human ENaC subunits showing domain organization and highlighting position of polybasic cleavage sites and PY motifs. (C) Sequences (Supplementary file 1) were aligned using MUSCLE (Edgar, 2004; Figure 1—source data 1). Residue symbol sizes are proportional to frequency at a given position. Key features in the sequence are indicated, as are the approximate position of α helices (rounded rectangles) and β-strands (arrows). Colors correspond to protein domains, as indicated in panel B. The GRIP (gating release of inhibition by proteolysis) domain is unique to ENaC subunits in the ENaC/Deg family. The P1 and P2 β-strands in the GRIP domain are not indicated, but are likely near the inhibitory tract and distal site, respectively, if present.
	Figure 2. Phylogenetic tree of epithelial Na+ channel (ENaC) subunits. Maximum likelihood tree calculated from ENaC subunit sequences of marine species and select terrestrial vertebrates, and ENaC-related proteins. Branch support bootstrap values are shown. Scale bar indicates the number of substitutions per site. Key ancestral nodes are indicated by circled numbers. A. Lungfish = Australian Lungfish, E. Shark = Elephant Shark, E. Lamprey = European River Lamprey, J. Lamprey = Japanese Lamprey, J. Medaka = Japanese Medaka, S. Lamprey = Sea Lamprey, W. Lungfish = West African Lungfish.
	Figure 3. Predicted cleavage sites in the epithelial Na+ channel (ENaC) γ subunit from Australian lungfish are functional. (A) Schematic of Aγ topology. Aγ has two predicted furin cleavage sites in its extracellular GRIP (gating release of inhibition by proteolysis) domain. All γ subunits were labeled with C-terminal epitope tags to facilitate detection of full-length subunits and the larger of the cleaved fragments. (B) Xenopus oocytes were injected with cRNAs encoding mα∆, mβT, and hemagglutinin (HA)-tagged γ subunits, as indicated. One day after injection, whole cell lysates and cell surface isolates were blotted and probed for HA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Full-length and cleaved bands are indicated and band densities were quantified. An overexposed blot is shown to highlight cell surface bands. Over exposed areas are red. (C) Cleavage %, calculated as cleaved/(cleaved + full length) × 100 is shown. Data were analyzed by repeated measures two-way analysis of variance (ANOVA) with Šidák’s multiple comparison test. p values are shown for indicated comparisons. Cleavage was also greater for Aγ than for mγ (p = 0.05). (D) Normalized total expression was calculated by normalizing the sum of full-length and cleaved bands to the mean of mγ after normalizing each sample for loading based on GAPDH from the same blot. (E) Surface expression % was calculated using band densities adjusted for the fraction of the respective sample loaded. (F) Normalized surface expression was calculated by multiplying values from the same sample in D and E, and then normalizing to the mean of mγ. Data in D–F were analyzed by one-way ANOVA with Tukey’s multiple comparison test. No significant differences between groups were found. Note that due to the lack of GAPDH data for one blot, the number of replicates for D and F (n = 6) is one fewer than for C and E (n = 7). (G) Whole cell currents were measured in injected oocytes by two-electrode voltage clamp, with voltage clamped at −100 mV. Representative traces of indicated subunit combinations are shown. Currents were continuously recorded in a bath solution containing 110 mM Na+. The ENaC-blocking drug amiloride (100 µM) was added at the end of each experiment to determine the ENaC-mediated current. (H) Log-transformed amiloride-sensitive inward currents are plotted, and were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. p values for comparisons where p < 0.05 are shown. Bars indicate mean values; errors shown are standard deviation (SD).
	Figure 4. Tissue distribution of (A) Erpetoichthys calabaricus (ropefish) and (B) Xenopus laevis epithelial Na+ channel (ENaC) subunit transcripts by reverse transcription-PCR (RT-PCR). cDNA libraries were generated from tissue homogenates. PCR reactions were performed using primers indicated in Supplementary file 3. Figure 4—source data 1 Uncropped gels are shown with bands at the target size for a given primer pair indicated. https://cdn.elifesciences.org/articles/75796/elife-75796-fig4-data1-v2.zip Download elife-75796-fig4-data1-v2.zip

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