Gettings SM et al. (2021), Two Functional Epithelial Sodium Channel Isofor...

XB-ART-58728

Mol Biol Evol 2021 Dec 09;3812:5704-5725. doi: 10.1093/molbev/msab271.

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Two Functional Epithelial Sodium Channel Isoforms Are Present in Rodents despite Pronounced Evolutionary Pseudogenization and Exon Fusion.

Gettings SM , Maxeiner S , Tzika M , Cobain MRD , Ruf I , Benseler F , Brose N , Krasteva-Christ G , Vande Velde G , Schönberger M , Althaus M .

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The epithelial sodium channel (ENaC) plays a key role in salt and water homeostasis in tetrapod vertebrates. There are four ENaC subunits (α, β, γ, δ), forming heterotrimeric αβγ- or δβγ-ENaCs. Although the physiology of αβγ-ENaC is well understood, for decades the field has stalled with respect to δβγ-ENaC due to the lack of mammalian model organisms. The SCNN1D gene coding for δ-ENaC was previously believed to be absent in rodents, hindering studies using standard laboratory animals. We analyzed all currently available rodent genomes and discovered that SCNN1D is present in rodents but was independently lost in five rodent lineages, including the Muridae (mice and rats). The independent loss of SCNN1D in rodent lineages may be constrained by phylogeny and taxon-specific adaptation to dry habitats, however habitat aridity does not provide a selection pressure for maintenance of SCNN1D across Rodentia. A fusion of two exons coding for a structurally flexible region in the extracellular domain of δ-ENaC appeared in the Hystricognathi (a group that includes guinea pigs). This conserved pattern evolved at least 41 Ma and represents a new autapomorphic feature for this clade. Exon fusion does not impair functionality of guinea pig (Cavia porcellus) δβγ-ENaC expressed in Xenopus oocytes. Electrophysiological characterization at the whole-cell and single-channel level revealed conserved biophysical features and mechanisms controlling guinea pig αβγ- and δβγ-ENaC function as compared with human orthologs. Guinea pigs therefore represent commercially available mammalian model animals that will help shed light on the physiological function of δ-ENaC.

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Species referenced: Xenopus laevis
Genes referenced: scnn1d

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	Fig. 1. Proposed nomenclature of SCNN1 genes and structural features of the human αβγ-ENaC. (A) The SCNN1 genes share a canonical organization in which the coding DNA is distributed over at least 12 exons (exons 2–13). Due to the high variability of exon 2, different predicted start codons located on alternative exons preceding exon 2 as well as the absence of a likely start codon on exon 2 in certain species, make an additional exon(-s) necessary and are therefore depicted in a dashed box. Structural features obtained from the cryo-EM-derived structure of human αβγ-ENaC were imposed to the respective encoding exons. All structural features are highlighted with colored boxes. Only exon sizes and not intron sizes are drawn to scale. (B) SCNN1 proteins share an overall hand-like structure, including regions representing “finger,” “thumb,” “palm,” “wrist,” and “knuckle,” holding a “ball of β-sheets” (shown in magenta). Transmembrane regions are termed TM1 and TM2. The image shows the human ENaC α-subunit (Noreng et al. 2018). (C) Surface model of the cryo-EM-derived structure of human αβγ-ENaC (Noreng et al. 2018). Gating Relief of Inhibition by Proteolysis (GRIP) domains are highlighted in darker colors.
	Fig. 2. Presence of SCNN1D in rodent families. Families marked in magenta include species which lost a functional SCNN1D, that is, families with pseudogene versions of SCNN1D. SCNN1D is completely absent in the Heteromyidae marked in red. All families marked in blue maintained intact SCNN1D genes. Families highlighted in dark blue contain species in which exons 11 and 12 of SCNN1D are fused to a “super-exon,” whereas light blue families do not include species with SCNN1D exon fusion (w/o=without exon fusion). There is currently no available genomic information of species representing the families highlighted in gray. Note that the Caviidae family contains species with and without intact SCNN1D. A list of all species that were analyzed is provided in table 1. The classification of rodents into 35 families is based on D’Elía et al. (2019), the taxon Eumuroida was suggested by Steppan et al. (2004).
	Fig. 3. Genomic organization and peptide sequences of guinea pig SCNN1D. (A) All SCNN1 family members are depicted relative to scale with the exception of an intron following SCNN1B exon 1, which is located roughly 41 kb upstream of SCNN1B exon 2. Blue boxes represent coding regions, gray boxes represent 3′-UTR, that is, sequences immediately downstream of the respective stop codons to the first poly-adenylation sequence motif. 5′RACE analyses encompass exon/exon junctions and resulted in 5′-UTR sequence information depicted in magenta. This is reflected in either alternative exons upstream of SCNN1G (exons 1a and 1b) or alternative transcriptional start sites of SCNN1D formally lacking any upstream exon. The fusion of exon 11 and exon 12 to a “super-exon” 11* is depicted in black. (B) Alignment of the amino acid sequences of the extracellular GRIP domains, based on Noreng et al. (2020), of human and guinea pig α- and δ-ENaC. Consensus sites for the protease furin are highlighted in bold letters. P1 to P4 strands of the Gating Relief of Inhibition by Proteolysis (GRIP) domains are highlighted in blue. (C) Alignment of amino acid sequences that are part of the extracellular acidic cleft with the putative sodium coordination sites (Asp-338 in human α-ENaC) highlighted in magenta. (D) Alignment of amino acid sequences close to the beginning of the second transmembrane domain (TM2) with the “super-exon” 11* of guinea pig δ-ENaC highlighted in yellow. A full sequence alignment is provided in supplementary data 2, Supplementary Material online. (E) The region coded by exons 11 (blue) and 12 (red) are highlighted in the structure of the extracellular domain of the human ENaC α-subunit (Noreng et al. 2020). The magnification shows the region between Gln-509 and Ala-522 of the knuckle domain in yellow. This corresponds to the region of incorporated amino acids in guinea pig δ-ENaC due to exon fusion. (F) Surface representation of the extracellular domain of human αβγ-ENaC, highlighting the presence of the region between Q509 and A522 in yellow at the protein surface.
	Fig. 4. Guinea pig δβγ-ENaC forms a functional channel when expressed in Xenopus oocytes. (A) Transmembrane current (IM) traces of oocytes expressing guinea pig αβγ- and δβγ-ENaC as well as water-injected control oocytes at −60 mV holding potential. Application of 100 µM amiloride is represented by black bars (a). (B) Amiloride-sensitive current fractions (ΔIami) for guinea pig αβγ- and δβγ-ENaCs (Student’s unpaired t-test with Welch’s correction). (C) Amiloride IC50 values were determined from concentration-response experiments for guinea pig αβγ- (black) and δβγ-ENaC (blue). (D) Representative IM traces of oocytes expressing human αβγ- and δβγ-ENaC at −60 mV holding potential. (E) ΔIami for human αβγ- and δβγ-ENaC (Student’s unpaired t-test). (F) Amiloride IC50 values for human αβγ- (black) and δβγ-ENaC (gray) as determined from concentration-response experiments. (G) Representative current traces of guinea pig αβγ- and δβγ-ENaC expressing oocytes from cell-attached patch-clamp recordings at a holding potential of −100 mV (c=closed; 1–2, number of open channels). (H) Slope conductance (Gslope) of guinea pig αβγ- and δβγ-ENaC, derived from linear regression of unitary channel conductance at holding potentials between −100 to −20 mV. Numbers in parentheses indicate (n).
	Fig. 5. Isoform-specific control of ENaC activity by proteases and sodium. (A) Representative transmembrane current (IM) traces of human αβγ- and δβγ-ENaC expressing oocytes, showing the determination of amiloride-sensitive fractions of IM (ΔIami) before and after application of chymotrypsin (2 µg/ml, CT, gray bar) with amiloride (100 µM, a, black bar). (B) ΔIami were calculated as the difference between IM at 3 min after wash-out of amiloride and the IM under subsequent presence of amiloride. The fold change (ΔIami after chymotrypsin/ΔIami before chymotrypsin) is shown for human αβγ- and δβγ-ENaC expressing oocytes that were exposed to chymotrypsin as shown in panel (A), in comparison with identical control experiments without chymotrypsin (Student’s paired t-test). (C, D) Data from experiments with guinea pig ENaC expressing oocytes that were identical to those shown in panels (A) and (B). Statistical analysis shown in panel (D) was performed with a Mann–Whitney U test. (E) Representative IM trace showing sodium self-inhibition of guinea pig αβγ- and δβγ-ENaC expressing oocytes. Application of amiloride is represented by black bars (a) and [Na+] is represented by white (90 mM) and gray (1 mM) bars. The perfusion was at a fast speed of 12 ml/min. (F) The percentage of SSI is shown for guinea pig αβγ- and δβγ-ENaC (Student’s unpaired t-test). SSI was calculated as (ΔIM peak−ΔIM 3 min)/ΔIM peak×100, where ΔIM peak= IM under 90 mM [Na+] peak − IM under 1 mM [Na+], and ΔIM 3 min=IM after 3 min under 90 mM [Na+]−IM under 1 mM [Na+]. (G, H) Data obtained from experiments using human αβγ- and δβγ-ENaC expressing oocytes. Experiments were identical to those shown in panels (E) and (F). Statistical analysis of data shown in panel (F) was performed using a Student’s unpaired t-test with Welch’s correction. Numbers in parentheses indicate (n).
	Fig. 6. Both human and guinea pig δβγ-ENaC have increased activity compared with αβγ-ENaC at high extracellular Na+ concentrations. (A) Representative transmembrane current (IM) traces for guinea pig αβγ- and δβγ-ENaC expressing oocytes as well as water-injected control oocytes. Boxes shaded in gray represent the different extracellular [Na+] in mM. (B) The IM values of guinea pig ENaC expressing oocytes were plotted against the extracellular [Na+] and fitted to the Michaelis–Menten equation allowing the estimation of Vmax and the KM. (C) The Vmax values and KM values of guinea pig αβγ- and δβγ-ENaC expressing oocytes (Mann–Whitney U test). (D–F) Similar to A–C except for oocytes expressing human ENaC orthologs and separate water-injected control oocytes. Data shown in panel (F) were statistically analyzed with Student’s unpaired t-test with Welch’s correction. Numbers in parentheses indicate (n).
	Fig. 7. Reduced sodium self-inhibition is pivotal to ENaC activity at high extracellular Na+ concentrations. (A) Representative transmembrane current (IM) traces showing sodium self-inhibition (SSI), determined with guinea pig αβγ-ENaC expressing oocytes, with and without prior incubation with chymotrypsin (2 µg/ml in NMDG-ORS for 5 min). Application of amiloride (100 µM) is represented by black bars (a) and [Na+] is represented by white (90 mM) and gray (1 mM) bars. The perfusion speed was 12 ml/min. (B) The percentage of SSI was plotted for guinea pig αβγ-ENaC with and without prior incubation with chymotrypsin (Student’s unpaired t-test). SSI was calculated as (ΔIM peak−ΔIM 3 min)/ΔIM peak×100, where ΔIM peak=IM under 90 mM [Na+] peak −IM under 1 mM [Na+], and ΔIM 3 min=IM after 3 min under 90 mM [Na+]−IM under 1 mM [Na+]. (C) Representative IM traces for guinea pig αβγ-ENaC expressing oocytes with and without prior incubation with chymotrypsin (2 µg/ml for 5 min) across a range of extracellular Na+ concentrations ([Na+]), gray-shaded boxes). (D) The IM from experiments shown in panel (C) were plotted against the extracellular [Na+] and fitted to the Michaelis–Menten equation allowing the estimation of the maximum IM (Vmax) and the [Na+] at which half of Vmax is reached (KM). (E) The Vmax values of guinea pig αβγ-ENaC with and without prior incubation with chymotrypsin (Student’s unpaired t-test with Welch’s correction). (F) The KM values of guinea pig αβγ-ENaC with and without prior incubation with chymotrypsin (Mann–Whitney U test). Numbers in parentheses indicate (n).
	Fig. 8. The reduced sodium self-inhibition of human and guinea pig δβγ-ENaC generates increased activity at high extracellular Na+ concentrations. (A) Representative transmembrane current (IM) traces of guinea pig αβγ- and δβγ-ENaC at different extracellular Na+ concentrations ([Na+]). Recordings of a given extracellular [Na+] were performed in individual oocytes. The perfusion speed was 16 ml/min. (B, C) The percentage of sodium self-inhibition for guinea pig and human ENaC expressing oocytes plotted against logarithmic transformations of concentrations of extracellular [Na+]. SSI was calculated as (ΔIM peak−ΔIM 3 min)/ΔIM peak×100, where ΔIM peak=IM under X mM [Na+] peak −initial IM under 1 mM [Na+], and ΔIM 3 min=IM after 3 min under X mM [Na+]−initial IM under 1 mM [Na+]. Slopes (sl.) were derived from linear regressions and P values, derived from ANCOVA, demonstrate the difference between slopes. Numbers in parentheses indicate (n).
	Fig. 9. The loss of SCNN1D does not generally correlate with habitat aridity. Geolocation data of the rodent species listed in table 1 were extracted from the Global Biodiversity Information Facility (GBIF) and were used to plot the global distribution of noninvasive rodent species of the clades Sciuromorpha, Supramyomorpha, and Hystricomorpha. Global distribution of individual species is provided in supplementary figures 2–5, Supplementary Material online. Observations for the indicated species were also plotted with corresponding habitat aridity. Mixed effects models suggest that the absence or presence of functional SCNN1D in the investigated species does not explain any significant proportion variation in habitat aridity. The colors indicate absence or presence of functional SCNN1D: Light blue=SCNN1D without exon fusion; Dark blue=SCNN1D with exon fusion; Magenta=species without functional SCNN1D (pseudogene and complete gene loss). Background colors differentiate habitats following the generalized climate classification scheme for aridity index values (Middleton and Thomas 1997). Species are ordered based on ascending median aridity within each clade. The numbers to the right of the plots indicate the number of GBIF observations that were extracted for each species.

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