*2.2. Pathophysiology and Genetics*

ENaC is a amiloride-sensitive epithelial sodium channel, localized in the apical portion of epithelial cells of distal nephron, distal colon, lung and ducts of exocrine glands [8]. Under physiological conditions, its expression and activity in the distal nephron are positively regulated by aldosterone and antidiuretic hormone and they are influenced by numerous extracellular factors, such as sodium, chloride, protons and proteases [9,10]. This channel is crucial, together with ROMK (renal outer medullary K+) channels and Na+/K+ ATPase, for Na+ reabsorption and, thus, for electrolytes homeostasis [9] (Figure 1A). The channel is a heteromeric complex constituted of three homologous subunits, α, β and γ [8,11,12], encoded by the *SCNN1A*, *SCNN1B* and *SCNN1G* genes, respectively. *SCNN1A* is located on chromosome 12p13.31, while *SCNN1B* and *SCNN1G* are located on chromosome 16p12.2 [9]. Although the α subunit alone is sufficient to induce a Na<sup>+</sup> current, the expression of the three subunits induces a maximal amiloride-sensitive Na+ current [8]. The amino acid sequences of the three homologous subunits share 30–40% identity [8,9] and the protein structures are very similar, composed of two short intracellular N-terminus and C-terminus, two transmembrane domains (identified as TM1 and TM2) and a big extracellular loop [9,13]. Within the C-terminus of all three ENaC subunits, there is a highly conserved sequence, named the PY (Proline Tyrosine) motif [14]. This proline-rich sequence, PPxY, is a binding site for a member of the ubiquitin ligase family, Nedd4 (Neural precursor cell expressed, developmentally down-regulated 4), that mediates the internalization and the proteasomal degradation of the channel [9,14–16].

(**B**) **Figure 1.** *Cont.*

**Figure 1.** (**A**) Physiopathology of ENaC. Under physiological conditions, the epithelial Na+ channel (ENaC) is expressed on the luminal side of distal nephron epithelium. ENaC is positively regulated by aldosterone and antidiuretic hormone and allows the passage of Na+ ions from lumen toward cytoplasm. The proline-rich sequence (indicated as PY), located at the C-terminus of each subunit, regulates channel internalization and degradation, through Nedd4 binding and ubiquitination. ENaC function is combined with K+ channel ROMK (green triangles) and Na+/K+ ATPase (blue ovals) and it is crucial for hydroelectrolytic homeostasis, consisting in sodium renal reabsorption and potassium excretion; (**B**) β and γ subunits mutations. The germline mutations of the *SCNN1B* and *SCNN1G* genes causes the loss or disruption of proline-rich sequence that has a pivotal role in negative regulation of the channel. These mutations are gain-of-function and determine an increased membrane density of ENaC and a consequent increase in renal Na+ reabsorption; (**C**) α subunit mutation. The germline mutation of the *SCNN1A* gene affects the extracellular domain, causing the disruption of a disulphide bridge. It is a gain-of-function mutation that leads to an increase of the open probability of the channel and a consequent increase in Na+ current, without affecting the PY motif.

Liddle syndrome results from germline mutations in *SCNN1A*, *SCNN1B* or *SCNN1G* genes. The first mutation to be identified was the nonsense p.Arg566\* substitution of the β subunit, in the large kindred described by Liddle et al. and subsequently by Botero-Velez et al. [4,6,7,17]. This mutation causes a truncation of the C-terminus of the β subunit with loss of the PY motif.

The first germinal mutation in the *SCNN1G* gene, resulting in the nonsense substitution p.Trp573\*, was identified by Hansson et al. in 1995 [18]. Again, this mutation erases the γ subunit's C-terminus, causing the loss of the PY motif. In the following years, 24 different mutations of the β subunit and six different mutations of the γ subunit were identified in 72 families from different countries (Table 1). The vast majority of the reported cases are determined by missense (ten different in 30 families), nonsense (eight in 21 families) or frameshift mutations (12 in 20 families) in *SCNN1B* or *SCNN1G* genes, that cause loss or disruption of the PY motif [9,19]. The loss of the proline-rich sequence prevents the internalization and degradation of the channel via the ubiquitination-proteasomal pathway and allows the accumulation of ENaC in the distal nephron apical membrane leading to an increase in sodium reabsorption [9,20,21]. The mutations are in fact responsible for an augmented apical membrane channel density and a subsequent increase in amiloride-sensitive inward sodium current, as demonstrated by in vitro studies in *Xenopus laevis* oocytes (Figure 1B) [21]. In 1996, Firsov et al. developed a quantitative method, based on the binding of a monoclonal antibody against a FLAG epitope inserted in the extracellular domain of α, β and γ subunits, and demonstrated a significant correlation between the entity of Na+ inward current and the number of ENaC on the cellular membrane [22].

Interestingly, additional mechanisms have been implicated in the augmented Na+ reabsorption, including an increase in channel open probability [23], an increase in the fraction of proteolitically cleaved channel (active) [24], together with a reduced feedback inhibition of ENaC by intracellular Na+ [25].

**Table 1.** Clinical and biochemical phenotype of patients affected by Liddle's syndrome. \*—in the original manuscript the mutation is reported according to the homologous rat sequence, HT—hypertension, SD—sudden death, LVH—left ventricular hypertrophy, TOD—target organ damage, CV—cardio vascular, n.a.—not available. Hypokalemia defined as serum K+ <3.5 mmol/L, hypoaldosteronemia defined as serum aldosterone <5 ng/dL or urinary aldosterone <5 μg/24 h.


**Table 1.** *Cont.*


**Table 1.** *Cont.*


As an example, the reported mutation p.Asn530Ser in the γ subunit [44] which is located in the TM2 segment and does not affect the PY motif, causes a two-fold increase in amiloride-sensitive Na<sup>+</sup> current, that was not associated to an increase in cell surface expression of the channel [44].

Recently, a germline mutation in the α subunit (p.Cys479Arg) was identified in a Caucasian family affected by Liddle syndrome (Table 1) [26]. This missense mutation is localized in the highly conserved extracellular domain of the subunit and leads to the disruption of a disulphide bridge. The p.Cys479Arg substitution increases the open conformation of the channel, resulting in a two-fold increase in Na<sup>+</sup> current, without affecting channel density at the plasma membrane [26].

In vivo studies conducted on mice homozygous for the *SCNN1B* p.Arg566\* mutation, indicate that the transition zone between the late distal convoluted tubule and the connecting tubule, is the main nephron site of ENaC hyperactivity in LS [69], where its activity is largely aldosterone independent [70]. However, ENaC is also expressed in several brain structures, including the supraoptic nucleus, magnocellular paraventricular nucleus, hippocampus, choroid plexus, ependyma, and brain blood vessels [71]. Mice lacking Nedd4-2 (Nedd4−/−) develop a phenotype of LS and display an increased ENaC expression in the central nervous system together with an increased blood pressure response after the infusion of Na+-rich cerebrospinal fluid compared to wild-type animals [72]. Similarly, Nedd4-2−/<sup>−</sup> mice display a marked increase in cerebrospinal fluid Na+ concentration, following a high sodium diet. Both effects were largely prevented by the intra-cerebro-ventricular infusion of the ENaC blocker benzamil, raising the question as to whether a similar mechanism could be implicated in the pathogenesis of arterial hypertension in patients affected by LS as well [72].

Interestingly, specific β ENaC single nucleotide polymorphisms (SNPs) have been associated with arterial hypertension. In particular, the SNP rs3743966 in intron 12 (c.1543-112A>T) was significantly associated with essential hypertension in Chinese hypertensive families [73] and the intronic variants rs7205273 (c.-9+11091C>T) and rs8044970 (c.311+1599T>G) were associated with blood pressure in a large Korean population [74]. The missense SNPs (rs1799979, rs149868979 and rs1799980 leading to the substitutions p.Thr594Met, p.Arg563Gln and p.Gly442Val), have been found to be associated with arterial hypertension and with increased markers of Na+ channel activity [75–78]. In particular, the p.Thr594Met substitution was highly prevalent in a large population of black African origin, its frequency increased with the severity of hypertension [77] and was significantly associated with low plasma renin activity [76].The association of α ENaC polymorphisms (rs2228576, rs11542844, rs3741913) (resulting in the substitutions p.Thr663Ala, p.Ala334Thr and p.Cys618Phe) have been associated with high blood pressure in some studies, but not in others [79]. Functional studies in *Xenopus laevis* oocytes showed that the p.Cys618Phe and p.Ala663Thr polymorphisms (but not the p.Thr633Ala) increased channel activity by 3.3 and 1.6-fold, respectively [79]. Similarly, after different studies showed an association between *SCNN1G* locus and blood pressure variation [80,81], four *SCNN1G* intronic SNPs, rs13331086 (c.914-468T>G), rs11074553 (c.1077+2571G>A), rs4299163 (c.1077+3271C>G) and rs5740 (c.1176+14A>G) resulted to be associated to systolic blood pressure in the general Australian white population, after adjustment for age, sex and body mass index [82]. In particular, the association of rs13331086 was confirmed in a much larger cohort including more than 8000 individuals and the minor allele of this SNP was associated with a 1 mmHg increase in systolic blood pressure and 0.52 mmHg increase in diastolic blood pressure [83].
