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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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The limited sodium availability of freshwater and terrestrial environments was a major physiological challenge during vertebrate evolution. The epithelial sodium channel (ENaC) is present in the apical membrane of sodium-absorbing vertebrate epithelia and evolved as part of a machinery for efficient sodium conservation. ENaC belongs to the degenerin/ENaC protein family and is the only member that opens without an external stimulus. We hypothesized that ENaC evolved from a proton-activated sodium channel present in ionocytes of freshwater vertebrates and therefore investigated whether such ancestral traits are present in ENaC isoforms of the aquatic pipid frog Xenopus laevis Using whole-cell and single-channel electrophysiology of Xenopus oocytes expressing ENaC isoforms assembled from αβγ- or δβγ-subunit combinations, we demonstrate that Xenopus δβγ-ENaC is profoundly activated by extracellular acidification within biologically relevant ranges (pH 8.0-6.0). This effect was not observed in Xenopus αβγ-ENaC or human ENaC orthologs. We show that protons interfere with allosteric ENaC inhibition by extracellular sodium ions, thereby increasing the probability of channel opening. Using homology modeling of ENaC structure and site-directed mutagenesis, we identified a cleft region within the extracellular loop of the δ-subunit that contains several acidic amino acid residues that confer proton-sensitivity and enable allosteric inhibition by extracellular sodium ions. We propose that Xenopus δβγ-ENaC can serve as a model for investigating ENaC transformation from a proton-activated toward a constitutively-active ion channel. Such transformation might have occurred during the evolution of tetrapod vertebrates to enable bulk sodium absorption during the water-to-land transition., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Wichmann et al.)

The epithelial sodium channel (ENaC) is a critical regulator of vertebrate electrolyte homeostasis. ENaC is the only constitutively open ion channel in the degenerin/ENaC protein family, and its expression, membrane abundance, and open probability therefore are tightly controlled. The canonical ENaC is composed of three subunits (α, β, and γ), but a fourth δ-subunit may replace α and form atypical δβγ-ENaCs. Using Xenopus laevis as a model, here we found that mRNAs of the α- and δ-subunits are differentially expressed in different tissues and that δ-ENaC predominantly is present in the urogenital tract. Using whole-cell and single-channel electrophysiology of oocytes expressing Xenopus αβγ- or δβγ-ENaC, we demonstrate that the presence of the δ-subunit enhances the amount of current generated by ENaC due to an increased open probability, but also changes current into a transient form. Activity of canonical ENaCs is critically dependent on proteolytic processing of the α- and γ-subunits, and immunoblotting with epitope-tagged ENaC subunits indicated that, unlike α-ENaC, the δ-subunit does not undergo proteolytic maturation by the endogenous protease furin. Furthermore, currents generated by δβγ-ENaC were insensitive to activation by extracellular chymotrypsin, and presence of the δ-subunit prevented cleavage of γ-ENaC at the cell surface. Our findings suggest that subunit composition constitutes an additional level of ENaC regulation, and we propose that the Xenopus δ-ENaC subunit represents a functional example that demonstrates the importance of proteolytic maturation during ENaC evolution., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2018 Wichmann et al.)