Keywords: Ion channels, Epithelia, Evolution, Transmembrane proteins, Kidney, Renin-angiotensin-aldosterone system

2.1. Definitions: Homolog, paralog, ortholog

In studies of protein evolution, the word "homologous" isused to describe proteins that share significant sequence similarity that isassumed to derive from a common ancestral origin. This concept of homologycovers both proteins that are homologous across species as well as proteins thatare present in multiple copies in the genome of a single species. To distinguishbetween these two types of homologous proteins, two separate terms were coinedby Walter Fitch (Fitch, 1970):orthologous and paralogous. Within the genome of a single species, there aremany genes that represent duplicate copies encoding isoforms of proteins withsimilar functions. The most common example is the globin family that includesα-globin, β-globin, and myoglobin. Homologous proteins thatexist "in parallel" within one species are called"paralogs", a hybrid word combining "parallel"with "homolog". The word "ortholog" is used forhomologous proteins that originate from a single ancestral gene in the lastcommon ancestor of the compared species. Continuing the globin example, theortholog of human α-globin is any of the α-globins in relatedprimates. Further examples of these terms are provided by (Koonin, 2005).

2.2. ENaC paralogs

In the human genome there are nine genes that encode for ENaC paralogs.These paralogs are grouped into two families based on their homology: 1.Non-voltage gated sodium channel family that is composed of four genes encodingENaC homologs and 2. acid-sensing (proton-gated) ion channels (ASIC) family thatis composed of five homologous genes. The four ENaC genes have been assignedabbreviations as SCNN1A, SCNN1B, SCNN1G, and SCNN1D by the Human GenomeOrganization (HUGO) Gene Nomenclature Committee (http://www.genenames.org/)following the Greek letters assigned to the four ENaC subunits α,β, γ, and δ (Table1 andTable 2). The second"N" in "SCNN1" was added to distinguish betweenthe NON-voltage gated ENaC and the SCN1 symbol assigned to the "sodiumchannel, voltage-gated, type I" that is expressed in neurons and muscle.The UniProt protein database (UniProt,2014) uses an abbreviated code for ENaC subunits (SCNNA, SCNNB, SCNNGand SCNND) to which the abbreviated species name is appended (Table 2). For the mouse genome, theconvention for gene nomenclature starts with an uppercase letter, followed byall lowercase letters as shown inTable1. For mouse, the gene for SCNN1D is not listed as it was not found inthe mouse genome (Giraldez et al.,2012). Another common name for ENaC subunits is"amiloride-sensitive sodium channel" as ENaC is inhibited byamiloride (Garty and Palmer, 1997;Kashlan and Kleyman, 2011).

As detailed below, the HUGO nomenclature appears to be sufficient fornaming ENaC orthologs in other vertebrate species whose genomes have beensequenced.

2.3. ASICs and other homologs

The five genes that code for the five Acid-Sensing Ion Channel (ASIC)subunits in the human genome have been numbered as ASIC1, ASIC2, ASIC3, ASIC4and ASIC5 by the HUGO Gene Nomenclature. The same abbreviation is used by theUniProt database (e.g. ASIC1_HUMAN). These channels were previously called asACCN and BNaC (García-Añoveroset al., 1997). One example of the proliferation of names is ASIC5.The product of this gene was initially named "brain, liver, intestineNa+ channel" (BLINaC) in mouse and rat. The homologous protein in humanswas found to be expressed in the intestine. Therefore, it was named"intestine Na+ channel (INaC)" in humans (Schaefer et al., 2000). A more recent study renamed thesame protein as "bile acid-sensitive ion channel" (BASIC) (Lefèvre et al., 2014). Althoughreferred to as ASIC5, it is not an acid-activated ion channel. The multiplicityof names for one protein emphasizes the need to adhere to names standardized byinternational nomenclature.

Many of the ENaC homologs were named based on the proteincharacteristics such as, sites of expression (e.g. "INaC","BLINaC"), physiologic consequences of activating mutations(e.g. "degenerin"), ligand interactions (e.g."FMRFamide-activated", "amiloride-sensitive","acid-sensing"), organism (e.g. HyNaC for channels in Hydra) andoriginal gene name (e.g. pickpocket in Drosophila). As noted with ASIC5, the useof different terms to name homologous proteins results in unrelated names forproteins that are highly homologous or orthologous. Moreover, homologousproteins may be expressed in different cell types and fulfill multiple functionsin different species, as observed with ENaC/Degenerin superfamily members. Thus,assignment of one name for a protein may not be relevant for an orthologousprotein in a different species.

As an alternative to naming proteins based on functionalcharacteristics, HUGO has taken the approach of a serial numbering system basedon homologous groupings (e.g. ASIC1… ASIC5, SCNN1A…SCNN1D). Inour view, this is a better approach for the nomenclature of ENaC/Degenerinsuperfamily, as it provides identical names to orthologs across species. In thecurrent genomic era, protein sequences are predicted based on genomic sequenceanalysis that includes comparisons between predicted and known proteinsequences. This approach of naming proteins based on sequence homology avoidsthe problems of names associated with protein characteristics.

In numerous invertebrate Metazoan species there is a multitude of highlydivergent proteins that show sequences homologous to ENaC/Degenerin superfamilymembers, but clearly represent different families based on low sequencesimilarity. As there is no standardized nomenclature for these proteins, in thisreview we used the names as in the original database records.

Table 3.

4.1. Trimeric structure and channel pore

In the trimeric structure of ASIC1, one of the issues that have beenintensively studied is the location of the channel pore through which ions flowacross the membrane. ASIC1 has six transmembrane segments - three of each of TM1and TM2. The structure of ASIC1 revealed that the TM1 and TM2 helices areorganized in two separate concentric triads. The central pore is formed by thetriad of TM2s. TM1s form a triad around the TM2 triad (Baconguis et al., 2014;Gonzales et al., 2009;Li et al.,2011). Most studies on ENaC suggest a similar organization of the TMsegments in ENaC as well (Tolino et al.,2011). Section 11 on conserved motifs presents the properties ofthese segments in detail.

One of the major unresolved questions in ENaC function is the path(s) ofions into the channel pore in the membrane as described above. On top of thechannel pore, the extracellular regions of the three subunits form a tripartitefunnel with rotational symmetry (Fig. 5)(Baconguis et al., 2014;Jasti et al., 2007). However, the threesubunits are not completely tightly juxtaposed along their entire lengths andthere are fenestrations between the subunits above the pore around the regioncalled "extracellular vestibule" (Baconguis et al., 2014). The hollow space along the centralaxis of rotational symmetry of this channel has been called a"vestibule". This vestibule leads from the top opening in thelumen to the channel pore embedded in the membrane. Under differentcrystallization conditions, segments of this vestibule may be constricted orexpanded (Baconguis et al., 2014). Thesedifferent states suggest that dynamic vestibule constriction and expansion mayregulate ion flow into the channel pore.

The extracellular and central segments of the vestibule are surroundedby beta-strands of the palm domain two of which are connected to the TM helices(β1 to TM1 and β12 to TM2) (Fig.5). Thus, changes in the angles of TM helices may effect constrictionof the vestibule. Conversely, movement of the β1 and β12 strandsmay effect opening or closing of the channel gate by modulating the position ofthe TM helices. For ASIC1, there is evidence that the movement of the coiledlinker region immediately prior to β1 and β12 strands may effectchannel opening and closing (Li et al.,2010;Springauf et al.,2011). The dynamics of these parts are also affected by the interactionsbetween the thumb and finger domains (Gwiazdaet al., 2015;Yang et al.,2009). There is a variety of intracellular and extracellular factorsthat can affect the dynamics of these segments, e.g. cytoplasmic Ca²⁺(Gu, 2008), binding to actin andother cytoskeletal proteins (Ilatovskaya etal., 2012;Sasaki et al.,2014), phosphoinositides that serve as second messengers inintracellular signaling cascades (Hille et al.,2015;Pochynyuk et al.,2008), extracellular ions, including Na⁺ andCl⁻, pH and cleavage by extracellular proteases (Kashlan and Kleyman, 2012;Kellenberger and Schild, 2015).

Table 4.

Table 5.

Table 6.

5.1. Sites of divergence among ENaC and ASIC paralogs

The divergence of N- and C- termini of ENaC/Degenerin superfamilymembers (noted above) represents a general trend in protein families. Previousstudies on other proteins have shown that changes in protein domain architectureare most common in the N- and C-termini of proteins (Björklund et al., 2005;Forslund and Sonnhammer, 2012). In contrast to α-and δ-ENaC, the N- and C-termini of human β- and γ-ENaCare highly conserved. The structures of these terminal segments are currentlynot known, but there are studies indicating that these cytoplasmic domainsinteract, either directly or indirectly, with other cytoplasmic and cytoskeletalproteins such as syntaxin (Berdiev et al.,2004;Condliffe et al., 2003),actin (Copeland et al., 2001), ubiquitinligase Nedd4 and protein kinases (Asher et al.,2001;Bobby et al., 2013;Shi et al., 2002).

Since the structure of the extracellular region of ASIC1 has beenresolved and in this region there is a significant homology between ASIC1 andENaC subunits, we shall present the sites of divergence in this region in termsof the secondary structural segments of ASIC1. The original study on the crystalstructure of ASIC1 noted that ASIC1 structure resembles a hand holding a ball(Jasti et al., 2007). Hence, domainswithin the extracellular regions are referred to as palm, thumb, knuckle, fingerand β-ball (Jasti et al., 2007).The palm and β-ball domains are formed by non-contiguousβ-strands and loops, and are in close proximity to the membrane (Fig. 6). More peripheral domains (thumb,knuckle and finger) are formed by contiguous α-helices and loops (Fig. 6).

To facilitate location of divergent regions in ENaC relative to thestructural domains of ASIC1 inFig. 7 weprovide an alignment of the β-subunit with ASIC1 sequence includingmarking of the positions of the secondary structural elements according to thePDB ID 2QTS (Fig. 7).

In the extracellular region of ENaC subunits, there are several highlydivergent segments where insertions/deletions are found (Fig. 4 andFig. 7). Onedivergent area is in the finger domain in between helix #1 andβ-strand #3 (Fig. 4,Fig. 6 andFig.7). This segment is divergent in four ENaC paralogs and ischaracterized by poorly aligned sequences including large insertions anddeletions (Fig. 4 andFig. 7). This "finger" domain shows thehighest variability among ENaC/Degenerin superfamily members indicating thatthis region may have an important role in conferring functional specificity(Eastwood and Goodman, 2012;Kashlan and Kleyman, 2011). For example,the α and γ-subunit finger domains have inhibitory tracts thatare released following proteolytic processing (Bruns et al., 2007;Carattino etal., 2008a,2006;Kashlan et al., 2011;Passero et al., 2010).

Another divergent segment in ENaC starts at about residue 376 of thehuman β-ENaC and includes an insertion of three residues (Fig. 4). In alignment with ASIC1 this regionis located in the region between β-9 and α-4 (Fig. 7). This is the region that connects thepalm domain of ASIC1 to the thumb domain (Jastiet al., 2007). This region has been proposed to transmitconformational changes in the periphery of the extracellular region to thechannel pore and gate (Jasti et al.,2007;Li et al., 2011;Shi et al., 2011). Other divergent areasinclude the knuckle domain and the loop connecting the β-6 andβ-7 strands. Residues in the β-6 - β-7 loop of theα subunit have been proposed to function as an extracellularNa⁺ binding site that is involved in Na⁺self-inhibition (Kashlan et al.,2015).

In conclusion, it appears that areas of divergence that are seen in ENaCand ASIC1 comparisons are located in the connecting segments within the fingerand thumb domains. In additions to these, there are a few other sequencedifferences but the sequence homology predominates especially in theβ-strand segments in the palm and β ball domains (Fig. 6, andFig. 7).

It is interesting that the most divergent areas within members of theENaC/Degenerin family are in the periphery of the extracellular region. There isgrowing evidence that these divergent areas have sites of direct interactionwith extracellular regulatory factors that modulate channel activity, such asproteases (Bruns et al., 2007;Vallet et al., 1997), inhibitory peptidereleased by proteases (Carattino et al.,2006;Kashlan et al., 2010),extracellular chloride (Cl⁻) ions (Collier and Snyder, 2011), extracellular Na⁺(Chraibi and Horisberger, 2002;Edelheit et al., 2014;Winarski et al., 2010), protons (Collier et al., 2012;Krauson et al., 2013), and laminar shear stress induced byfluid flow (Shi et al., 2012). As thedifferent ENaC/Degenerin family members are regulated by distinct factors,evolutionary divergence within the peripheral domains may have been a key factorin allowing this family to evolve with different functional properties.

6.1. Cyclostomata and Chondrichthyes (cartilaginous fishes)

In the phylogeny of vertebrates, the most ancient taxon is Cyclostomata,i.e. jawless vertebrates. Lampreys and hagfishes are common extant species thatrepresent this taxon. These fishes have only cartilaginous elements as aprimitive skeleton that supports their body parts (Shimeld and Donoghue, 2012). The genome of sea lampreyincludes three genes that code for the orthologs of α, β andγ-ENaC, but apparently does not include a gene for the delta subunit(Table 7) (Smith et al., 2013). The sequence of lamprey αsubunit is not complete (S4RTA3_PETMA).

Next steps in the evolution of vertebrates include the development ofjaw and skeleton leading to the formation of Gnathostomata (jawed vertebrates)(Donoghue et al., 2006;Kawasaki and Weiss, 2006;Kuratani, 2012). The earliestrepresentatives of this branch include cartilaginous fish species, includingrays and sharks. The genome of the cartilaginous elephant shark has beendetermined and it includes three orthologous ENaC genes (Venkatesh et al., 2007) (Table 7).

6.2. Euteleostomi (bony vertebrates)

In evolution, the development of jaw is followed by the development ofbony fishes. The clade of Euteleostomi (bony vertebrates) includes two branches:

1. Actinopterygii (ray-finned fishes): The"ray-finned" description is based on spinyprojections in the fins of these fishes.

   Comparison of shark, human and teleost ray-finned fishgenomes has revealed that 154 genes (including ENaC paralogs) thathave orthologs in the shark genome are not present in ray-finnedfish genomes (Venkatesh et al.,2007). Thus, the whole clade of Actinopterygii (rayfinned-fishes), which includes Zebrafish, do not have ENaC genes.However, they have ASIC genes. During the course of evolution, ENaCgenes may have been lost at the onset of the branch of ray-finnedfishes for lack of a functional need or were replaced functionallyby alternative genes and proteins (Uchiyama et al., 2014;Venkatesh et al., 2007).

   The EnsemblCompara GeneTree shows one"SCNN1A" gene for Lepisosteus oculatus (spotted gar)that is a freshwater ray-finned fish. Our comparison of this proteinwith human ASIC and ENaC paralogs showed that it is more homologousto ASIC than ENaC paralogs. UniProt database includes 9 proteinfragments from the spotted-gar genome that show homology to"amiloride-sensitive sodium channel family". Ourcomparison of 4 partial sequences (with lengths >400residues) from the UniProt database with human ASIC and ENaCparalogs showed that all four sequences share 49–61%sequence identity with human ASIC1, while they share13–15% with human ENaC paralogs. Therefore, thenaming of the single ray-finned fish spotted gar protein(ENSLOCP00000013400) as "SCNN1A" appears to be inerror. Thus with the elimination of this case, so far ray-finnedfish genomes do not appear to have ENaC orthologs as notedabove.

2. Sarcopterygii (lobe-finned fish): The"lobe-finned" description was given because of theirfleshy paired fins which are considered an early form of limbdevelopment in tetrapod vertebrates with four limbs. Therefore, thisclade also includes all Tetrapoda species.

   Sarcopterygii includes two ancient taxa with extant fishes:Coelacanthiformes (lobe finned fishes, coelacanth) and Dipnoi(lungfishes) (Table 7). Thethree ENaC genes are present in the genomes of these fish (Amemiya et al., 2013;Uchiyama et al., 2014,2012). Tetrapoda is considereda branch that emerged in parallel to Dipnoi.

6.3. Amphibia

In the evolutionary ladder, the development of bony vertebrates wasfollowed by the emergence of tetrapods with four limbs. Amphibians (frogs, toadsand salamanders) represent the first class of tetrapods. Xenopus tropicalis(frog) genome includes genes encoding the four ENaC paralogs (Hellsten et al., 2010) (Table 7).

6.4. Sauropsida

The second group of tetrapods is Amniota (amniotes) characterized byhaving an egg or embryo covered with an amniotic membrane. Amniotes include twoclades: Sauropsida that includes birds and reptiles, and Mammalia (mammals).

The genome sequences of three crocodilians have been recently reported(Green et al., 2014). Currently,NCBI Genome database Genome Assembly and Annotation report (including a list ofpredicted proteins) is available only for Alligator mississippiensis (Americanalligator). Search of this database for amiloride-sensitive sodium channelyielded four sequences (XP_006258424.1,XP_006268483.1,XP_006268484.1, andXP_006277862.1). In this report, the first and the fourth sequences were namedas "amiloride-sensitive sodium channel subunit alpha-like",while the second and the third sequences were named as "…subunit beta" and "… subunit gamma". Percentidentities of these four sequences are shown onTable 6. These results show that the second sequence(XP_006277862.1) that was labeled as "alpha-like", matches otherdelta-ENaC sequences in terms of its percent identity with the other alligatorENaC subunits (Table 6) and human ENaCsubunits (results not shown). Thus, we conclude that this alligator has fourENaC paralogs including one gene coding for the delta subunit.

The Ensembl (release 79) Gene Tree view includes two reptiles:soft-shell turtle and green anole lizard (Table7). Both of these genome sequences also include four genes coding forthe four ENaC paralogs (Table 7).

Bird genomes that are listed in Ensembl (release 79) Gene Tree view,include four genes coding for ENaC subunits. The recently determined genome ofsunbittern (Eurypyga helias) (Zhang et al.,2014) is not yet included in the Ensembl database. Similar to thecase of alligator genome noted above, NCBI Genome database Genome Assembly andAnnotation report includes four amiloride-sensitive sodium channel entries oneof which was listed as "…alpha-like". Our sequenceidentity analysis unequivocally classifies this "alpha-like" asthe δ subunit. Therefore, this genome also includes four ENaC heterologs(Table 7). Since birds andcrocodilians are considered evolutionary descendants of dinosaurs (Green et al., 2014), it is likely thatdinosaurs also had four genes coding for ENaC subunits.

6.5. Mammalia

The class of Mammalia includes three taxa: egg-laying mammals(Monotremata), marsupials (Metatheria) and placental mammals (Eutheria). Innearly all mammals in these three clades, there are four ENaC genes (Table 8). Ensembl genome database (release79) includes 38 mammalian species, including 34 placental mammals, 3 marsupials(opossum, Tasmanian devil, wallaby) and egg-laying platypus. All of thesespecies have four paralogs of ENaC with the exception of the mouse genome thatappears to have lost the gene for the delta subunit (Ensembl Gene Tree for ENaChomologs). The rat genome, that is a very close phylogenetic relative of themouse, includes four ENaC paralogs, but the δ subunit sequence ispresently available only as a fragment (NCBI Accession:NC_005104.4).

In the Ensembl (release 79) Gene Tree view, there are only one to threeENaC paralogs for some mammalian species. Our examination of the genome in eachof these cases showed that in most cases the genome sequence does include themissing paralog(s); in other cases the genome sequence is incomplete.

6.6. Summary for Tetrapoda

For the genomes of tetrapods where sequence information is available,including amphibians and amniotes (lizards, crocodiles, birds, and mammals)there are four paralogs of ENaC with the exception of mouse that has lost thegene for the delta subunit (Table 7 andTable 8) (Giraldez et al., 2012).

Table 9.

Table 10.

Table 11.

Table 12.

8.1. Insertions and deletions in orthologs

In phylogenetic comparisons above, we noted that in some ENaC/Degenerinhomologs, in addition to sequence divergence, there are majorinsertions/deletions (extending for tens to hundreds of residues) relative toENaC. Thus, we concluded that such proteins belong to different families withinthe ENaC/Degenerin superfamily. The major differences in the functions of thesefamilies of proteins are associated with specific structural features built uponthe major common scaffold of these channels. Whereas ENaC is constitutivelyactive and functions in transport of Na⁺ across epithelia andconsequently regulates extracellular fluid volume, ASIC and degenerin typechannels fulfill mainly sensory functions (Ben-Shahar, 2011) (see Section 14). The large insertions in thefinger domain (Fig. 6) of DEG family ofproteins are apparently part of the complex of mechano-sensitivity of thesechannels (Eastwood and Goodman,2012).

In the alignment of α subunit sequences from tetrapod species,it can be seen that the N- and C-termini show divergence (see Section 11).However, the extracellular regions do not have major insertions and deletions.Several sequences have deletion/insertion of 2–6 residues relative tothe human ortholog. Nearly all of these are located at sites of sequencedivergence when compared with ASIC1 (see Section 5.1).

Alignments of β-ENaC sequences also show no majorinsertions/deletions for 20 species. The anole lizard β-ENaC has a16-residue insert starting at residue 406. The status of this protein iscurrently "uncharacterized protein" implying it may haveerrors.

Alignment of γ subunit sequences from 20 species shows highhomology in the extracellular region, with the exception of the chimpanzeesequence that has a ~65 residue deletion. Such a deletion is not foundin other mammalian species, and could reflect an error.

Overall, ENaC family orthologs are highly conserved throughout thespectrum of vertebrate species. The degree of their sequence identity is relatedto their phylogenetic/taxonomic distance. ENaC orthologs do not have majorinsertions/deletions and can be readily distinguished from members of otherfamilies within the ENaC/Degenerin superfamily by their high percent of sequenceidentity.

9.1. Threshold for orthologs

The sequences of ENaC orthologs across species show a high degree ofconservation with the lowest sequence identity of 39% between tetrapodspecies and lobe-finned fish coelacanth in global alignment (Tables 10 and11). The termini of α subunit orthologs are moredivergent, while the sequences of the extracellular region have about10% higher sequence identity. Thus, in a case where the classificationof a sequence is unclear, extracellular regions should be compared. Secondly,insertion/deletion of a large segment (>10 residues) should raiseconcerns regarding subfamily classification (see Section 8.1).

Protein structure database SCOP employed a minimal criteria of30% sequence identity for assignment of proteins into the same proteinfamily (Murzin et al., 1995). CATHdatabase uses >35% sequence similarity as the criteria forclassification as members of a family (Sillitoeet al., 2015). The observation that among ENaC orthologs sequenceidentity is >38%, matches the requirements of these twodatabases for the classification of these proteins as members of the same familyof ENaC. As sequence identity with ASIC homologs (seeTable 4) and other Degenerin type proteins are generallyless than 20%, these proteins represent members of families differentfrom ENaC.

9.2. Threshold for paralogs

Multisequence comparisons presented here show a consistent picture.Global alignments within species show that ENaC paralogs generally share>20% sequence identity with one another (Table 4 andTable 6).In contrast, all four ENaC subunits share less than 20% sequenceidentity with ASIC. This also extends to other homologs, such as Degenerins.Thus within species, 20% sequence identity appears as the cut-off pointfor the ENaC family as opposed to membership in the ASIC family amongvertebrates.

11.1. Cytoplasmic amino terminus

As can be seen in the alignments of the human ENaC sequences, theN-termini of α and δ subunits show heterogeneity in both theirsequence and length (Fig. 4). A similarpattern of heterogeneity is observed in the alignment of the N-terminalsequences from 20 species (Fig. 10). Incontrast to the α and δ subunits, the β and γsubunits from 20 species are highly conserved and most are of similar length(Fig. 10).

Chalfant et al. examined activities of rat ENaC subunits withN-terminal deletions. They found that deletion of residues 2–67 in theN-terminus of the α subunit reduced endocytosis of ENaC and increasedthe half-life of the channel in the membrane, suggesting that the N-terminuscontains an endocytotic motif (Chalfant et al.,1999). Deletion of longer segments of α, β, andγ N-terminus (94, 50, and 94 residues respectively), drastically reduceENaC activity (Bachhuber et al.,2005).

Yue et al. noted that the N-terminal segments of β andγ subunits contain a stretch of basic residues that is characteristic ofPhosphatidylinositol 4,5-bisphosphate (PIP2) binding sites in other proteins(Yue et al., 2002). Mutation of 4 ofthese basic residues to nonpolar residues in the β but not in theγ subunit drastically reduced ENaC currents in the Xenopus oocyteexpression system (Kunzelmann et al.,2005).

In a stretch of about 30 to 40 residues prior to the start of TM1, allfour human ENaC paralogs have some strictly conserved residues (Fig. 4 andFig. 10). These residues are conserved in the four human ENaCparalogs (Fig. 4) as well as in all 20species examined (Fig. 10). Theconservation of these sequences across species suggests that this region has animportant functional role. A Gly37Ser missense mutation in this region of theβ subunit causes multi-system PHA and this mutation reduces the openprobability (P_o) of ENaC (Gründer et al., 1997). Mutations of the correspondingresidue in the α and γ subunits also reduced channel activity,suggesting that this site has an important role in regulating channel gating(Gründer et al., 1997).Gründer et al. also examined the roles of the residues flanking the keyGly residue by systematic alanine mutagenesis of 28 residues (H77 to H104 in ratα subunit) (Gründer et al.,1999). The expression of ENaC with these mutant subunits in Xenopusoocytes showed that most mutants decreased channel activity, likely due to areduction in channel P_o (Gründer et al., 1999). The stretch of ten residues from T92to C101 showed the highest sensitivity to alanine mutagenesis, with G95, H94 andR98 mutants showing the strongest reduction (Gründer et al., 1999). These residues are conserved in all20 species with the exception of the platypus, which has an exceptionally shortN-terminus (Uniprot ID: F7F7U2_ORNAN) (Fig.10). Since the status of this sequence is marked as an"uncharacterized protein" it may have an error.

11.2. TM1

The resolved structure of ASIC1, and sequence similarities betweenASICs and ENaCs provide important clues about the stretch of ENaC residues thatform TM1. We have also used algorithms to predict the location of the TM1 (Fig. 4 andTable 3). Relative to the alignment with ASIC1 sequence, predictedTM1 segment starts three residues (KKK in human β subunit) after thestart of the ASIC1 TM1 (cf.Fig. 4,Fig. 7, andFig. 10). According to this prediction, TM1 is preceded by2–3 Arg/Lys residues that are conserved in all ENaC orthologs (Fig. 3, andFig. 10). Studies on the distributions of charged residues inα-helical TM segments indicated that positively charged residues Arg andLys are present at much higher proportions on the cytoplasmic side of the TMsegment of proteins. This trend was named the "positive-inside"rule (von Heijne, 1992). A recent studyhas shown that in 191 transmembrane α-helical segments, the residues Argand Lys are present at highest proportion just before the start of the lipidbilayer (Pogozheva et al., 2014). Atthis location, the positively charged residues interact with the polar headgroups of membrane lipids and contribute to the strength of membrane anchoringof ENaC. The conserved appearance of Arg and Lys just before the predicted TM1provide support for the predicted location of the TM1 (Table 3,Fig. 3, andFig. 10).

In the predicted TM1 location, a tryptophan (W) appears as one of thefirst three residues conserved in all 20 ENaC orthologs (Fig. 10). In ASIC1, a Trp appears as the third residue fromthe beginning of the first helix (Fig. 7).Analysis of 191 α-helical TM proteins showed that aromatic residues Trpand Tyr are predominantly located at the membrane-water interface (Pogozheva et al., 2014). These aromaticamino acids are known to partition into the interface region of membranes.Hence, it has been suggested that they contribute strongly to the anchoring andprecise positioning of TM segments in the lipid bilayer (Hong et al., 2007). The appearance of Trp at the beginningof the TM1 in all ENaC orthologs in 20 species provides further support for thepredicted location of the TM1 (Fig. 3,Fig. 4 andFig. 10). Analyses of TM1 of the α subunit of ENaCby tryptophan-scanning mutagenesis suggested two functionally different regions.N-terminal tryptophan residues altered both channel activity and cationselectivity, with a periodicity consistent with a helical structure. WhileC-terminal tryptophan residues also affected activity and selectivity, there wasno apparent periodicity (Kashlan et al.,2006).

11.3. Extracellular region

The extracellular region, as revealed in the resolved structures ofASIC1, has a complex structure that resembles an outstretched hand holding aball (Figs. 3 and4). Hence, domains within the extracellular regions arereferred to as palm, thumb, knuckle, finger and β ball (Jasti et al., 2007) (Fig. 7). The palm and β ball domains are formed bynon-contiguous β strands and loops, and are in close proximity to themembrane (Figs. 3 and4). More peripheral domains (thumb, knuckle and finger) areformed by contiguous α helices and loops, and are poorly conserved amongENaC/Degenerin family members, when compared with other parts of theextracellular region. Based on sequence homology and predicted secondarystructure, it is likely that the structural features of the extracellular regionof ASIC1 is shared among all members of the ENaC/Degenerin superfamily. This isone of the key defining features of this ion channel family. Below we presentsome of these conserved segments the functions of which have been examined.

11.4. TM2

Prior to the report on ASIC1 structure, the location of the TM2 in ENaCsubunits was predicted by various software based on hydrophobicity of thisregion (Canessa et al., 1994a;Saxena et al., 1998). After thepublication of the ASIC1 structure in 2007, the helical TM2 segment of ASIC1 hasbeen generally adopted as the location of TM2 in ENaC subunits as well, based onthe strong sequence homology in this region and subsequent empirical studies(Kashlan and Kleyman, 2011). Whilethere is overlap between earlier predictions and ASIC1 structure based segment,a segment identified as "pre-M2" (i.e. before the TM2) inearlier work (Kellenberger et al., 1999;Schild et al., 1997), resides withinTM2. The terms used in earlier studies should be examined to avoid confusionabout regions studied.

Fig. 12 presents alignments ofα, β and γ subunits in the TM2 segments in 20 specieswhere the TM2 was marked based on homology with ASIC1. These alignments showthat TM2 is highly conserved in all subunits.

A number of key functional sites are within the TM2 region of ENaCsubunits, including the channel gate, amiloride binding site and selectivityfilter.Fig. 10 illustrates how thesesites are conserved through evolution. Functional studies as well as resolvedASIC1 structures suggest that the channel gate is within the outer part of thesecond membrane spanning domain, within the region encompassing an LLSN motifthat is conserved among ENaC subunits across species (Fig. 12). The Ser in this motif is a site where theintroduction of large residues has been found to dramatically increase channelopen probability (Kellenberger et al.,2002;Sheng et al., 2001a;Snyder et al., 1999). This site hasbeen referred to as the degenerin or "Deg" site, as theintroduction of large residues in mechanosensitive ENaC/Degenerin family memberin C. elegans results in neurodegeneration, in association with an increase inchannel open probability (Goodman et al.,2002;Sherwood et al.,2012).

One of the characteristics of αβγ ENaC is itsinhibition by relatively low concentrations of amiloride (Kleyman and Cragoe, 1988). Other family members, includingδβγ and ASICs, are inhibited by amiloride or itsderivatives at higher concentrations of these drugs (Table 13) (Diochot et al.,2007;Ji et al., 2012). Anamiloride binding site has been described at a site in the secondmembrane-spanning domain, consisting of a Ser in the α subunit, and aGly in the beta and gamma subunits (Fig.12). The introduction of specific mutations at these sites led to aprofound loss of the efficacy of amiloride (Kashlan et al., 2005;Schild etal., 1997).

Another defining characteristic of ENaC is its cation selectivity. Withregard to its ability to discriminate Na⁺ and K⁺, ENaC isthe most Na selective mammalian ion channel (Table 13). A three-residue selectivity filter, consisting of aG/S-X-S motif, is present in the TM2 of ENaC subunits (Fig. 12). The introduction of specific mutations in thefirst or third residue of this motif resulted in channels that allow for modestK⁺ permeation (Kellenberger etal., 1999;Sheng et al.,2000;Snyder et al., 1999).

At the distal end of TM2 there are three charged residues (within thestretch of EMAELVFD in human α subunit) that are conserved in all 20species examined (Fig. 12). Mutation ofthese acidic residues reduced channel conductance (Langloh et al., 2000;Sheridan et al., 2005) and also affect ion selectivity (Sheng et al., 2001b).

11.5. Carboxy terminus

In general, the cytoplasmic C-terminus contains sites of interactionwith other proteins, signal transduction molecules, and ions that regulate ENaCfunction.

Alpha, beta and gamma subunits of ENaC

Most of the studies on tissue localization of ENaC subunits have beencarried out by immunohistochemical studies using antibodies generated againstsmall segments of expressed proteins or synthetic peptides that represent shortsegments of ENaC subunits (Brouard et al.,1999;Coric et al., 2004,2003;Duc et al., 1994;Hager et al.,2001;Masilamani et al.,1999;Tousson et al., 1989;S. Wang et al., 2013). These studiesprovided evidence for the localization of ENaC in kidney, lung, salivary glands,skin, placenta and the colon. Expression of ENaC subunits has been also examinedby in situ hybridization of tissue sections using cDNA probes (Greig et al., 2003). But, this approachdoes not provide an image of the intracellular localization of the subunitsthemselves.

To enhance the immunofluorescence signal, we generated polyclonalantibodies against the complete extracellular region of ENaC subunits (Enuka et al., 2012). These antibodiesallowed us to visualize ENaC expression in the bronchial epithelia of human lungand the female reproductive tract extending from the uterus to the fallopiantube at a high resolution by immunofluorescence and 3D confocal microscopy(Enuka et al., 2012).

The expression and sites of localization of ENaC in the kidney nephrontubules have been recently extensively reviewed (Rossier, 2014) and the complexities of this subject arebeyond the scope of the present review.

Tissue specificity of expression of ENaC subunits has been alsoinvestigated by large-scale microarray and high-throughput RNA sequencingexperiments. The results of these studies can be accessed via the EMBL-EBIExpression Atlas database of gene expression (Petryszak et al., 2014). Experiments included in the ExpressionAtlas (https://www.ebi.ac.uk/gxa/home) report many tissues wherein ENaCsubunits are expressed at varying levels. Consistent with theimmunohistochemical studies, these studies report highest levels of expressionfor the α, β and γ subunits in the kidney, lung, andcolon. Other tissues reported include the fallopian tube, esophagus, placenta,prostate, skin, stomach, thyroid, tongue and vagina (Expression Atlas).

Some recent studies have reported immunolocalization of ENaC subunitsin astrocytes in the brain (Miller and Loewy,2013), human eye (Krueger et al.,2012), nasal mucosa (Jiang et al.,2015), mouse ear (Morris et al.,2012), rat muscle (Simon et al.,2010), vascular endothelium (Kusche-Vihrog et al., 2014), vascular smooth muscle cells (Drummond et al., 2008), lymphocytes (Ottaviani et al., 2002) and platelets(Cerecedo et al., 2014).

There is also evidence for the expression of ENaC in mammary epithelia(Wang and Schultz, 2014). Thefunctional significance of ENaC in organs, such as kidney, lung and therespiratory tract, sweat and salivary glands and the reproductive tract whereENaC is expressed at relatively high levels, has been established, based on thefacts that mutations in ENaC genes result in major dysfunction in these tissuesin multi-system PHA (Chang et al., 1996;Enuka et al., 2012;Hanukoglu, 1991;Hanukoglu et al., 2008). However, even in most severecases of multi-system PHA there does not appear to be an abnormal function inthe eye, ear, muscle or neural tissue function. Thus, the physiologicalsignificance of the low-level expression of ENaC in other tissues remains to beestablished.

Expression of ENaC has also been detected in the lingual epithelium andin taste receptor cells (TRCs) (Chandrashekar etal., 2010;Kretz et al.,1999). Most mammals can sense five basic tastes, sweet, sour, bitter,umami and salt by TRCs specific for each taste. In fungiform taste buds of micethere are amiloride-sensitive TRCs that are responsive specifically to NaCl(Shigemura et al., 2008). Ingenetically engineered mice lacking the α subunit specifically in TRCs,the neural response to low NaCl (<120 mM) in a subpopulation of TRCs wasabolished while response to high NaCl remained intact (Chandrashekar et al., 2010). These studies suggest thatENaC in TRCs plays an essential role in the salt-taste receptor system (Chandrashekar et al., 2010;Oka et al., 2013).

In the mammalian order Cetacea that includes whales and dolphins, tastereceptor buds have atrophied and appear in degenerate form (Tinker, 1988). Sequencing of the variouscetacean genes for the receptors of the five tastes revealed that the receptorgenes for four tastes are non-functional pseudogenes because of accumulation ofmutations (Zhu et al., 2014). However,in contrast to these, the three ENaC subunits, α, β, andγ, that also serve as salt taste receptor have remained intact andfunctional. As the authors note, the conservation of ENaC genes in Cetacea isbecause of the significance of ENaC in osmoregulation and other physiologicalfunctions and it is still not known whether Cetacea are capable of sensing saltytaste (Zhu et al., 2014).

Delta subunit of ENaC

The first report on the cloning of the human δ subunit alsoshowed that in northern blots the highest levels of its expression are observedin brain, pancreas, testis and ovary with only low levels in the kidney and lung(Waldmann et al., 1995). Consistentwith these initial results, the Expression Atlas database results show that theexpression of the delta subunit gene (SCNN1D) is relatively much lower in thekidney and lung but highest in neural tissues, (including cerebral cortex,cerebellum, hippocampus, hypothalamus and pituitary gland) and testis(Expression Atlas, 53 GTEx). Expression of the δ subunit has been alsodetected in the human nasal epithelium (Bangel-Ruland et al., 2010) and eye (Krueger et al., 2012). Overall, the tissue distributionpattern of the δ subunit is distinctly different from that of α,β and γ subunits.

ASICs

ASIC subunits are expressed mainly in the central and peripheralnervous systems and the gastrointestinal tract. ASIC tissue distribution hasbeen recently extensively reviewed (Holzer,2015;Lin et al., 2015;Zha, 2013).

ENaC on cilia

A cilium is a finger like protrusion on the cell surface that has amicrotubular skeleton called axoneme. The ciliary membrane that covers theaxoneme is continuous with the cell membrane (Satir and Christensen, 2007). Most cells in mammals contain a singlecilium (primary cilium) that is built upon the microtubular structure of acentriole. In three major organs, the lung, the reproductive tract, and thecentral nervous system, the epithelial surface of many cells have multiple cilia(200–300 per cell). These cilia are motile and beat in concert at apredetermined direction generating waves that move the fluid, particles andcells in the lumen of the epithelium (Brooks andWallingford, 2014).

Using antibodies we generated, we showed that in multi-ciliated cellsin the human bronchus and the female reproductive tract (extending from theuterus to the fimbria of fallopian tube), ENaC is specifically located in cilia(Enuka et al., 2012). This was alsoconfirmed by co-localization of ENaC immunofluorescence with that ofcilia-specific β-tubulin IV (Enuka etal., 2012).

The discovery that ENaCs are highly expressed in multi-ciliated cellsin the lung and the reproductive tract and that they are specifically locatedover the entire length of cilia has increased our understanding of the functionof ENaC in epithelia with motile cilia (Enuka etal., 2012). The depth of the fluid that bathes the cilia in the lumenhas to be precisely regulated for normal cilial function (Choi et al., 2015;Tilleyet al., 2015). Since Na⁺ is the major solute in the ECF,regulation of ENaC activity directly affects osmolarity of the periciliary fluidand consequently the flow and volume of the fluid in the lumen. Cilial locationallows ENaC to serve as a sensor and regulator of osmolarity of the periciliaryfluid along the entire length of the cilia. Thus, ENaC mediated changes inosmolarity would then modulate the fluid volume on the epithelial surface (Enuka et al., 2012). Since all airwayepithelia are Na⁺ absorptive, ENaC plays a key role in pulmonaryepithelia, but it should be noted that in the airway epithelium there areadditional ion channels and transporters that contribute to the regulation ofthe composition and volume of the periciliary liquid (Hollenhorst et al., 2011).

The functional importance of ENaC in the multi-ciliated cells of therespiratory airway is illustrated by the contrasting phenotypes of two diseases:1) In multi-system pseudohypoaldosteronism (see section 15.1), theloss-of-function of ENaC leads to an increase in the volume of airway surfaceliquid (ASL) (Kerem et al., 1999). 2) Incystic fibrosis (see section 15.3), dysfunction of the chloride transporter CFTR(Cystic Fibrosis Transmembrane Conductance Regulator) leads to reducedinhibition of ENaC by chloride ions. Enhanced activity of ENaC contributes tothe drastic reduction of the airway surface liquid (ASL) volume observed incystic fibrosis. Normally, the undulating movement of the cilia rapidly movesthe mucus gel on top of the cilial layer, and together with the mucus, it pushesinhaled particles and microbes in the respiratory airways for clearance via themouth (Rogers, 2007;Tilley et al., 2015). When ASL volume isreduced, clearance of foreign particles, dust, microbes and viruses is impairedcontributing to the chronic infections characteristic of cystic fibrosis.

In epithelia with motile-cilia, the interaction between ENaC and CFTRin the respiratory airways has been extensively studied because of theinvolvement of these channels in cystic fibrosis (Althaus, 2013). There are conflicting views regarding the mechanismof CFTR and ENaC interactions (Nagel et al.,2005). Some studies suggested that CFTR interacts directly with ENaC(Berdiev et al., 2009). But, whileENaC is located on the cilial surface, CFTR is located on the apical membraneoutside of cilial borders (Enuka et al.,2012). Thus, the mechanism of CFTR action in multi-ciliated cellscannot be via direct interaction with ENaC. There is evidence that chloride ionsinhibit ENaC (Bachhuber et al., 2005).Thus, CFTR may regulate ENaC activity via its modulation ofCl⁻ levels.

Table 14.

15.1. Multi-system pseudohypoaldosteronism type 1 (PHA1B)

Type I pseudohypoaldosteronism (PHA) is a syndrome of unresponsiveness(also called resistance) to mineralocorticoid hormone aldosterone. This diseasewas first described by Cheek and Perry as an aldosterone unresponsivenesssyndrome resulting in salt wasting in a child (Cheek and Perry, 1958). Subsequently over 50 studies reported manycases of PHA with characteristics of aldosterone resistance with varying degreesof salt wasting. In 1991, Hanukoglu established that PHA includes twoindependent syndromes (called renal and multi-system forms of PHA) that differin their pathogenesis, mode of inheritance, the involvement of aldosteronetarget organs and the severity of salt wasting (Hanukoglu, 1991). Later studies confirmed this distinction and thesetwo forms have been assigned two separate entries in the OMIM database(http://omim.org/): 1) Renal form (PHA1A): OMIM #177735,inherited as an autosomal-dominant disease; and 2) multi-system form (PHA1B):OMIM #264350, inherited as an autosomal-recessive disease.

As commonly observed for other steroid hormone resistance diseases,initially the cause of PHA was suspected to be a mutation(s) in themineralocorticoid receptor (MR) gene (Armaniniet al., 1985). However, analysis of the linkage between PHA andpolymorphisms adjacent to the mineralocorticoid receptor gene on chromosome 4 in10 consanguineous families excluded mutations in the MR gene in multi-system PHApatients (Chung et al., 1995). By furtherhomozygosity mapping, multi-system PHA locus was mapped to two regions codingfor SCNN1A and SCNN1B and SCNN1G in chromosomes 12p and 16p respectively (Strautnieks et al., 1996). Indeed,sequencing of the genes coding for the α, β and γsubunits revealed that the multi-system form results from mutations in thesethree genes (Chang et al., 1996). Twoyears later Geller et al. reported that the milder renal form (PHA1A) is causedby mutations in the mineralocorticoid receptor gene (Geller et al., 1998).

15.2. Liddle syndrome

Liddle syndrome (OMIM #177200) is an autosomal dominantdisorder characterized by early-onset hypertension associated with hypokalemia,metabolic alkalosis, and low levels of plasma renin activity (PRA) andaldosterone (Bogdanović et al.,2012;Hansson et al., 1995;Liddle et al., 1963) (seeTable 14). The degree of phenotypicexpression may vary even within the same family (Bogdanović et al., 2012;Findling et al., 1997).

Liddle syndrome is caused by missense mutations in the PY motif ofβ or γ subunit of ENaC (Fig.14), and nonsense or frameshift mutations that result in truncationof the C-terminus leading to the loss of the PY motif in these subunits (Furuhashi et al., 2005;Hansson et al., 1995;Ma et al., 2008;Schild et al., 1996;Shimkets et al., 1994). Mutation or elimination of the PY motifdisrupts ubiquitin ligase Nedd4-2 binding to the PY motif (Rotin and Staub, 2011). This leads to accumulation ofactive channels at the cell surface and increased Na⁺ reabsorption inthe kidney, resulting in elevated blood volume and blood pressure. Although inthe α subunit there is also a PY-motif (Fig. 14), so far no case of Liddle syndrome has been reported with amutation in the α subunit.

In addition to mutations in the PY motif region, two mutations in theC-terminus (β subunit, R563Q) and the TM2 segment (γ subunit,N530S) have been reported to be associated with Liddle syndrome phenotype. Inthe β subunit, an R563Q mutation is associated with low plasma reninactivity (PRA), low aldosterone and hypertension in a minority of theindividuals carrying the variant (Rayner etal., 2003). In the γ subunit, Hiltunen et al. found an N530Smutation in a patient who developed Liddle syndrome like symptoms at the age of25 years (Hiltunen et al., 2002). Yet,in the same study the N530S mutation was also found in a healthy person withnormal blood pressure (Hiltunen et al.,2002). Thus, the authors raise the possibility that the γN530S mutation may not be the sole cause of hypertension and that there may beadditional factors (such as other genes or environmental factors) responsiblefor the phenotype observed (Hiltunen et al.,2002). Functional expression of ENaC carrying the N530S mutationshowed that the mutation increases ENaC open probability two fold. The increasedENaC activity was observed without a change in the surface expression of ENaCrelative to the wild type (Hiltunen et al.,2002). It is probable that more mutations will be found that increaseENaC activity and that are associated with Liddle syndrome like symptoms,similar to the two cases noted above.

Generally Liddle syndrome is a rare disease. However, an extensivestudy that included a sample of 330 Chinese young hypertensive patients revealedthat 1.5% of the patients had mutations associated with a loss of the PYmotif (Wang et al., 2015). In aretrospective study in a cohort of 149 hypertensive US veterans, 6% werefound to have biochemical abnormalities compatible with Liddle syndrome (Tapolyai et al., 2010). Thus, Liddlesyndrome may be a common cause of monogenic hypertension in some populations(Padmanabhan et al., 2015).

Undiagnosed and untreated individuals with Liddle syndrome are at highrisk of premature cardiovascular morbidity and mortality. Diuretics that blockENaC activity (amiloride or triamterene) and low salt diet, are usually adequateto control hypertension.

15.3. Cystic fibrosis-like disease

Cystic fibrosis (CF) is an autosomal recessive disease caused bymutations in the cystic fibrosis transmembrane conductance regulator (CFTR)gene. CFTR dysfunction affects epithelial chloride transport in multiple organsincluding the digestive system, sweat glands, pancreas, and the reproductivetract, but, progressive lung disease continues to be the major cause ofmorbidity and mortality.

Respiratory tract infections result from dehydration of airway surfacesthat reduces mucociliary clearance and creates an environment conducive tobacterial infections leading to progressive respiratory insufficiency andeventually respiratory failure.

In the lumen of the respiratory tract, ENaC function is essential fornormal mucociliary clearance (Althaus,2013;Hobbs et al., 2013). Asnoted in section 13, ENaC is expressed in the respiratory tract, located on thesurface of cilia and regulates the volume of luminal fluid (Enuka et al., 2012). PHA1B patients withENaC mutations suffer frequently from lower respiratory tract infections (Hanukoglu et al., 1994;Kerem et al., 1999;Schaedel et al., 1999). Increased airway-specificexpression of the β subunit of ENaC results in CF-like symptoms in mice(Mall et al., 2004). This is thoughtto result in over-expression of channels composed of only α andβ subunits, which have a high intrinsic open probability (Mall et al., 2010). Therefore, it has beensuggested that mutations in ENaC genes may be involved in some forms of cysticfibrosis. To examine the hypothesis that ENaC mutations may be associated withthe degree of severity of CF, a French group screened genomic DNA of 56 CFpatients for the presence of variants in SCNN1B and SCNN1G genes (Viel et al., 2008). By using denaturinghigh-performance liquid chromatography (DHPLC), they found 4 missense mutationsin three patients out of 56 (T313M and G589S in β, and L481Q and V546Iin γ subunit). However, nasal potential difference measurements did notindicate a functional effect of these variants on Na⁺ transport, atleast in the nasal epithelium of these patients. Thus, the authors concludedthat variants in SCNN1B and SCNN1G genes are not associated with CF severity inthe cohort examined (Viel et al.,2008).

Cases that present with classical features of cystic fibrosis (such aschronic lung infections with elevated sweat chloride concentration), but withoutCFTR mutation or a single allele CFTR mutation, have been referred to as cysticfibrosis-like (CF-like) disease (Table14). These cases lack a genetic diagnosis, and a commonly suspectedcause is mutations in ENaC genes (Collawn etal., 2012).

Screening 185 patients with non-classic CF, Sheridan at al. identified20 patients who had elevated sweat chloride concentrations, and pulmonarydisease but without CFTR mutations (Sheridan etal., 2005). Sequencing of the ENaC genes (SCNN1A, B or G) revealedthat two of the patients carry compound heterozygous mutations in the SCNN1Bgene: a missense mutation (P267L) with a splice site mutation in one patient andtwo missense mutations (G294S and E539K) in the other. Neither patient hadabnormal renin or aldosterone levels. In functional expression studies, P267Land E539K mutants showed decreased activity and G294S mutant showed increasedactivity (Sheridan et al., 2005). Theauthors concluded that the compound heterozygous mutations identified in theβ ENaC genes that have a mild effect on ENaC activity are associatedwith CF-like disease without causing severe renal salt loss (Sheridan et al., 2005).

In a multi-center European study, 30 ENaC variants were found in 76patients with CF-like disease (Azad et al.,2009). Only two (hypoactive F61L and hyperactive V114I in SCNN1A) ofthe 30 variants were found in patients but not in the control populations. ENaCsubunit variants had a significantly higher frequency in the patients ascompared to controls. The variant W493R in the α subunit showed the mostsignificant difference, and in functional expression in Xenopus oocytes showedover four-fold higher ENaC activity. Thus, the authors concluded that thesevariants may be involved in CF-like disease by a polygenetic mechanism (Azad et al., 2009). In a study including 99Italian patients with CFTR-related diseases, 12 ENaC variants were found, butthe allele frequency of these variants was not significantly different fromcontrols (Amato et al., 2012).

Fajac et al. screened a group of 55 patients with diffuse idiopathicbronchiectasis (permanent dilation of the airways as a result of chronicbronchial infection) by sequencing SCNN1B and SCNN1G exons and identified fiveheterozygous missense variants (S82C, P369T, N288S in β, and G183S,E197K in γ subunit) in eight patients (Fajac et al., 2008). The S82C mutation was found in three unrelatedpatients who were also heterozygous for a CFTR mutation. The authors thusconcluded that trans-heterozygous mutations in ENaC and CFTR may be responsiblefor the CF-like symptoms.

In a study including 60 Rwandan children with CF-like symptoms, fivepatients were found to have a heterozygous CFTR mutation. Two of these patientshad a missense ENaC variant (V573I in α, V348M and G442V in βsubunit); of these only V348M was not found in the control group (Mutesa et al., 2009). Functionalexpression of the V348M mutant showed that the mutation enhances ENaC activity(Rauh et al., 2013). Since the fullENaC subunit gene sequences were not determined in 55 of the patients, therelationship between ENaC variants and CF-like disease cannot be determined forthe whole group.

A recent study on CF-like phenotypes examined the sequences of fivegenes (CFTR, SCNN1A, SCNN1B, SCNN1G and SERPINA1) in six patients by whole exomesequencing (Ramos et al., 2014). Theauthors detected three missense variants in SCNN1A (R204W, A357T, C641F) and onemissense (R563Q) in SCNN1B, and four additional nucleotide variants. Two ofthese mutants (C641F and R563Q) appeared also in two CF controls but not inhealthy controls. The authors suggest that the variants that appear at a higherfrequency in patients with CF-like phenotype than in controls may be responsiblefor this phenotype. By their family analysis they also stress the importance ofgenetic /environmental factors in the development of CF-like disease (Ramos et al., 2014).

Brenan et al. examined 33 nonwhite, non-Hispanic patients with aCF-like disease whose CFTR gene analysis was non-diagnostic (19 with nomutations in CFTR gene and 14 with a heterozygous change in the CFTR gene)(Brennan et al., 2015). Sequencing ofthe exons and introns of SCNN1A, SCNN1B, and SCNN1G in all patients revealed 21variants. Since the variants found in conjunction with a CFTR mutation werecommon polymorphisms, the authors concluded that there is no conclusiveassociation of ENaC genetic variants with CF in their cohort.

InTable 15 we listed all themissense mutations identified in the studies reviewed above. Among the threeENaC genes, most of the variants have been observed in SCNN1A and SCNN1B.Proportionately, the highest number of mutants is in SCNN1A. Two of the majorstudies examined only two genes: SCNN1B and SCNN1G, thus the number of variantsin SCNN1A inTable 15 isunderrepresented. In our analysis of PHA1B mutants, we observed a similar trendthat most of the PHA1B causing mutants appear in SCNN1A and only a few in SCNN1G(Edelheit et al., 2005).

As detailed in the studies cited, many of the variants are also presentin control groups. Yet, some of the mutants that were shown to affect adverselyENaC activity have been reported in independent studies (Table 15). The total number of cases is still too small toreach definitive conclusions about the role of these variants/mutants in causingCF-like disease. In any case, the number of variants that may be associated withCF-like disease represents a small percentage of the total patients in eachcohort and differs between ethnic groups.

15.4. Hypertension

In modern industrial societies, hypertension has emerged as one of mostwidespread health problems (Toka et al.,2013). A minority of the cases of hypertension can be ascribed tomonogenic conditions such as Liddle's syndrome or Gordon'ssyndrome (Padmanabhan et al., 2015). Theremainder of the cases is generally grouped as "essentialhypertension" with multifactorial etiology, including multiple genetic,humoral, environmental and dietary factors (Suand Menon, 2001). The major systems that are responsible for theregulation of blood pressure include the renin-angiotensin-aldosterone systemand Na⁺ transporters in the kidney (Padmanabhan et al., 2015;Rossier,2014;Soundararajan et al.,2010;Su and Menon, 2001).Moreover, the Liddle syndrome described above firmly established the importanceof ENaC in blood pressure regulation. Therefore, several large-scale studieshave examined the association of ENaC variants with essential hypertension. Liuet al. examined 2880 Chinese subjects (GenSalt study) and found an associationof blood pressure with SCNN1B and SCNN1G SNPs and variants (Liu et al., 2015). Rayner et al. screened139 South African black hypertensives for the R563Q variant of β subunitand found that the variant was significantly associated with hypertension (Rayner et al., 2003). In a study in aFinland, the authors sequenced only exon 13 (that codes for TM2 and theC-terminal segment) of β and γ subunits (seeFig. 7) in 27 hypertensive patients. Thethree identified variants were then screened in 347 hypertensives. The frequencyof all variants in the hypertensives was significantly higher (~3 fold)than that in two control groups. Functional expression of one variant (βG589S) showed slightly enhanced activity in Xenopus oocytes (Hannila-Handelberg et al., 2005).

Ambrosius et al. reported a small but significant association between acommon αT663A variant and normal blood pressure (Ambrosius et al., 1999). The αA663 variant reducesENaC activity and functional expression in Xenopus oocytes (Samaha et al., 2004;Tong et al., 2006), but the change in activity wasopposite of that predicted by the results of Ambrosius et al. Furthermore, noassociation was found between αT663A variant and blood pressure in asample of 247 Japanese hypertensives (Sugiyamaet al., 2001). Nonetheless, homozygous A663 allele appears to have adifferential effect on lung function (Foxx-Lupoet al., 2011).

Another polymorphism with conflicting reports is β-T594M. In asample of black hypertensives from London, 8.3% (17 out of 206) wereheterozygous for the β T594M variant, but in the control group only2.1% had the same variant (Baker et al.,1998). However, in a much larger sample, including 1666 Jamaicanblacks, no association was found between the β T594M allele andhypertension (Hollier et al., 2006).This variant did not alter ENaC activity in a heterologous expression system(Persu et al., 1998)

Persu et al. identified seven variants in the β subunits of,mostly white, 525 probands of hypertensive families, but could not identify anassociation between a variant and essential hypertension (Persu et al., 1998).

In summary, currently there does not appear to be a clear associationbetween ENaC variants and essential hypertension. Yet, this area of research isjust at its beginnings and requires examination of larger sets of SNPs andvariants in selected populations. Some of the past studies have examined onlyspecific segments (such as exon 13) of subunits. This approach skews theresults. The SNPs that are accumulating in whole genome sequencing will presentlarger and more comprehensive databases for future examination. It is likelythat such studies will reveal new variants associated with hypertension similarto that found in the Chinese GenSalt study. Since the majority of the variantsso far screened do not seem to be associated with hypertension, the proportionof ENaC variants associated with hypertension would be expected to be small.

Highlights.

• A comprehensive review of the structure and function of four ENaCsubunits from an evolutionary perspective.

• Comparison of the sequences of ENaC homologs and identification ofstructural motifs conserved throughout vertebrates.

• Establishing criteria for distinguishing ENaC family members fromother families within the ENaC/Degenerin superfamily including ASIC, deg,mec, unc, ppk type gene products.

• Review of tissue-specific expression and functions of ENaC paralogsand inherited diseases associated with mutations in ENaC genes.

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