Epithelial sodium channel (ENaC) family: Phylogeny, structure-function, tissue distribution, and associated inherited diseases

Abstract

The epithelial sodium channel (ENaC) is composed of three homologous subunits and allows the flow of Na(+) ions across high resistance epithelia, maintaining body salt and water homeostasis. ENaC dependent reabsorption of Na(+) in the kidney tubules regulates extracellular fluid (ECF) volume and blood pressure by modulating osmolarity. In multi-ciliated cells, ENaC is located in cilia and plays an essential role in the regulation of epithelial surface liquid volume necessary for cilial transport of mucus and gametes in the respiratory and reproductive tracts respectively. The subunits that form ENaC (named as alpha, beta, gamma and delta, encoded by genes SCNN1A, SCNN1B, SCNN1G, and SCNN1D) are members of the ENaC/Degenerin superfamily. The earliest appearance of ENaC orthologs is in the genomes of the most ancient vertebrate taxon, Cyclostomata (jawless vertebrates) including lampreys, followed by earliest representatives of Gnathostomata (jawed vertebrates) including cartilaginous sharks. Among Euteleostomi (bony vertebrates), Actinopterygii (ray finned-fishes) branch has lost ENaC genes. Yet, most animals in the Sarcopterygii (lobe-finned fish) branch including Tetrapoda, amphibians and amniotes (lizards, crocodiles, birds, and mammals), have four ENaC paralogs. We compared the sequences of ENaC orthologs from 20 species and established criteria for the identification of ENaC orthologs and paralogs, and their distinction from other members of the ENaC/Degenerin superfamily, especially ASIC family. Differences between ENaCs and ASICs are summarized in view of their physiological functions and tissue distributions. Structural motifs that are conserved throughout vertebrate ENaCs are highlighted. We also present a comparative overview of the genotype-phenotype relationships in inherited diseases associated with ENaC mutations, including multisystem pseudohypoaldosteronism (PHA1B), Liddle syndrome, cystic fibrosis-like disease and essential hypertension.

Keywords: Epithelia; Evolution; Ion channels; Kidney; Renin–angiotensin–aldosterone system; Transmembrane proteins.

Fig. 1

Schematic illustration of the location and function of ENaC in epithelia.

Fig. 2

Intron-exon organization of the human ENaC genes, SCNN1A, SCNN1B, SCNN1G and SCNN1D and their primary transcripts based on the NCBI Homo sapiens Annotation Release 107 (2015-03-13). The name of each gene and its chromosomal location are noted at the left-edge of the diagrams. Under each exon-intron map, there are two coordinates: the upper one specifies the chromosomal coordinates, and the lower one specifies the position of the nucleotide (in kb) starting at the 5'-end of the RNA transcript (marked as 0). The codes above the diagrams represent the ID numbers of the RNA transcript (starting with NM_) and the encoded protein (starting with NP_) in the NCBI Gene database. For SCNN1A, two transcripts are shown as examples of alternative splicing products. Notes: 1) SCNN1A coordinates are given in a scale that descends from left-to-right because the gene is located in the reverse strand of the chromosome. 2) The x-axis for SCNN1B intron #1 includes a break between 5 kb and 45 kb marks. Display of the full sequence (i.e., without a break) would lead to the visible merger of exons 9 and 10 and hence disappearance of the intron 9 because of the short size of intron 9. Additional information about the genes and their products is provided in Table 1 and Table 2.

Fig. 3

Schematic illustration of the transmembrane localization of an ENaC subunit. The sequence shown is of human α subunit (see Table 1). All homologous ENaC subunits have two transmembrane segments. The TM segments for this figure was predicted by the Phobius program (see Table 3) and drawn using Protter (Omasits et al., 2014). The extracellular domain includes about 70% of the sequence of amino acids of an ENaC subunit.

Fig. 4

Aligned sequences of human α, β, γ and δ-ENaC subunits and conserved positions of introns in all four subunits. Residues that are identical in all four subunits are shaded. The numbers (2 to 12) below the sequences mark the position and number of the intron located in or at the end of the codon of the specific residue above the number. In the 5' portion of the gene encoding δ-ENaC subunit there are additional introns that are not shown here. The sequences were aligned using the ClustalW2 program, and the alignment of some residues in the amino and carboxy termini were manually edited to eliminate some gaps without affecting percent identity score. TM1 and TM2 mark the predicted transmembrane segments of the proteins.

Fig 5

A. Ribbon structure model of subunit A of chicken ASIC1 (PDB ID: 2QTS). Segments in helical conformation are red colored and segments in sheet conformation are blue colored. B. The surface structure of subunits A and B of ASIC1. The four hydrophobic helices of the A and B subunits are embedded in the lipid bilayer marked by gray shading. The third subunit (C) surface is not shown to allow visibility of the central pore predicted by the Porewalker software. Red colored small spheres represent water molecules placed at the center of the predicted pore and extracellular vestibule in each 3 Angstrom slice of 2QTS calculated by Porewalker.

Fig. 6

Topology diagram of chicken ASIC1 structure. The cylinders represent helical segments, and the arrows represent β-strands. The transmembrane (TM), and secondary structural domains (palm, β-ball, finger, thumb and knuckle) were colored distinctly and named as in (Jasti et al., 2007). Certain features of the diagram were adopted from previous diagrams (Eastwood and Goodman, 2012; Kashlan and Kleyman, 2011).

Fig. 7

Secondary structures in the sequence of chicken ASIC1. The positions of the structures were taken from the PDB file of 2QTS. The numbering of the structures is based on (Jasti et al., 2007). Note that some short stretches of helix and β-strand are not numbered. For comparison of sequence conservation, human β-ENaC is globally aligned with the ASIC1 sequence and identical residues were gray color shadowed. Note that most but not all secondary structures are associated with conserved sequences.

Fig. 8

Correlation between the sequence identities among α, β and γ subunits of ENaC for 20 species relative to human ENaC. A) Correlation of the extent of identity of α and β subunits with their human counterparts. B) Correlation of the extent of identity of β and γ subunits with their human counterparts. The x, y coordinates of each point are percent identities between human sequence and the sequence of another species for the subunit indicated in the x and y axes. The sequences were from human, chimpanzee, gorilla, rhesus, elephant, bovine, dog, mouse, rat, rabbit, orca, Tasmanian devil, platypus, chicken, flycatcher, alligator, turtle, Xenopus, lungfish, and coelacanth.

Fig. 9

A hypothetical phylogenetic tree for paralogs of ENaC. "Anc." is used as an abbreviation for "Ancestor". A "duplication node" represents a gene duplication event that yields two genes within one genome. A "speciation node" represents the formation of a new species that carries the gene of interest. By the convention of Ensembl Gene Tree, collapsed trees for paralogs are shown in blue color. The figure is based on a Gene Tree constructed for 540 ENaC homologs in the Ensembl genome database (release 79) of vertebrate and eukaryotic species using EnsemblCompara GeneTrees paralogy prediction method. The figure includes several modifications from the Gene Tree: The nodes for C. elegans degenerins and one homolog from a fish were omitted from the figure, and the positions of the nodes were modified to show branches in parallel. The number of homologs in each collapsed branch is written on the right side of the collapsed tree marking.

Fig. 10

Comparison of α, β, and γ sequences in the N-terminal, pre-TM1, and TM1 segments from twenty species. For each subunit, residues that are identical in at least 19 out of 20 species (95% identity) are shaded. The location of the predicted TM1 is shown above the sequences. The α subunits have N-termini of highly variable lengths (the numbers at the beginning of each sequence marks the number of additional residues that did not fit into the page), with little or no sequence conservation in this variable region. In contrast, the β and γ subunit N-termini are mostly of similar length and show a high degree of conservation within a ~40 residues-long segment prior to the TM1. The row of red letters, in between the β and γ sequence groups, mark the residues that are identical in both β and γ subunits. In four sequences (β: chicken; γ: gorilla, chicken and coelacanth) 2–5 residues prior to the first methionine were deleted to be consistent with other Uniprot sequences that start with Met as the first translated codon. There may be also sequencing errors in the unusually short platypus α sequence, and flycatcher γ sequence.

Fig. 11

Serine protease cleavage sites in the extracellular domain of α, β and γ ENaC subunits from 20 species. Key basic amino acids (Arg (R) and Lys (K)) in the putative cleavage site are marked with yellow shading. The sequences of the respective subunits from 20 species were aligned by CLUSTALW. The conserved sequences of the inhibitory tracts located in between the two SP sites are marked light blue background. The residues of the substrate protein that are recognized by proteases are numbered based on their position relative to the cleaved peptide bond. P₁ marks the putative residue after which the peptide bond is cleaved by the SP (Antalis et al., 2010).

Fig. 12

Comparison of α, β, and γ sequences in the pre-TM2 and TM2 segment from twenty species. For each subunit, residues that are identical in at least 19 out 20 species (95% identity) are shaded. The location of the TM2 based on homology to ASIC1 is shown above the sequences. In the preTM2 region, only three charged residues are conserved in all three subunits. The positions of these charged residues are marked at the top of the alignments by the corresponding cASIC1 homologs, Ala413, Glu417 and Gln421. Column headers: Deg: degenerin or "Deg" residue. Ami: amiloride binding residues. Sel.: selectivity filter.

Fig. 13

Location of the cASIC1 E417 and Q421 in ASIC1 structure (PDB ID: 2QTS). The three ribbon structures shown represent the β12-strand region (from L414 to K423) of all three subunits of chicken ASIC1, termed in order A, B, and C (PDB 2QTS). For each subunit, only two residues, E417, and Q421, are shown in CPK style. In 20 species examined, the residue homologous to E417 is an arginine or lysine (K534 in α, R505 in β and R514 in γ subunit of human ENaC). The space in the center of the figure is part of the vestibule along the three-fold axis of symmetry that is thought to be part of the ion pathway.

Fig. 14

Conservation of the PY motif in the C-termini of α, β and γ ENaC subunits from 20 species.