Open access

New Insights into the Epithelial Sodium Channel Using Directed Mutagenesis

Written By

Ahmed Chraibi and Stéphane Renauld

Submitted: 05 October 2011 Published: 05 February 2013

DOI: 10.5772/50309

From the Edited Volume

Genetic Manipulation of DNA and Protein - Examples from Current Research

Edited by David Figurski

Chapter metrics overview

2,086 Chapter Downloads

View Full Metrics

1. Introduction

Directed mutagenesis is a fundamentally important DNA technology that seeks to change the base sequence of DNA and test the effect of the change on gene or DNA function. It can be accomplished using the polymerase chain reaction (PCR). For more than 20 years, many applications in both basic and clinical research have been revolutionized by PCR. The development of this technique allowed the substitution, addition or deletion of single or multiple nucleotides in DNA (Mullis and Faloona, 1987). Because of redundancy in the genetic code, such mutations do not always alter the primary structure of proteins. In this chapter, we will review the contribution of PCR-directed mutagenesis in the determination of the structure-function relationship of the epithelial sodium channel (ENaC), particularly with respect to the domains involved in proteolytic activation and ligand-induced stimulation of the channel.

Advertisement

2. Physiological role and structure of ENaC

ENaC is a key component of the transepithelial sodium transport. It is expressed at the apical membrane of a variety of tissues, such as the distal nephron of the kidney, lungs, exocrine glands (e.g., sweat and salivary glands) (Brouard et al., 1999; Duc et al., 1994; Perucca et al., 2008; Roudier-Pujol et al., 1996) and distal colon (Kunzelmann and Mall, 2002). In aldosterone-sensitive distal nephron (ASDN) and distal colon, this channel plays a major role in the control of sodium balance and blood pressure (Frindt and Palmer 2003; Garty and Palmer 1997). In lungs, ENaC regulates mucus secretion and aids in the protection of the airway surface (Randell and Boucher, 2006). Its role was clearly demonstrated in mice in which the ENaC gene was inactivated by homologous recombination (Hummler et al., 1996). ENaC belongs to a gene family with members found throughout the animal kingdom, the so-called ENaC/degenerin family, including the acid sensing ion channel (ACIC) and the Phe-Arg-Met-Phe amide-gated ion channel (FaNaCh), (Kellenberger and Schild. L, 2002). Using the Xenopus oocyte expression system and a distal colon cDNA library, the primary structure of ENaC was identified; and electrophysiologic characteristics of ENaC channel were determined (Canessa et al., 1993; Canessa et al., 1994; Lingueglia et al., 1993). ENaC is a heteromeric channel made of three subunits (α, β and γ) encoded by 3 different genes SCNN1a, SCNN1b and SCNN1g, respectively. Each subunit exhibits ~30% identity at the amino acid level and shares highly conserved domains. The membrane topology of each subunit predicts the presence of two transmembrane domains (M1 and M2), a large extracellular loop (~70% of the size of the channel) and relatively short amino and carboxyl termini. The stoichiometry of ENaC was much discussed: several examples of biochemical and functional evidence are consistent with a heterotetrameric structure (2α, 1β, 1γ) (Anantharam A, 2007; Dijkink et al., 2002; Firsov et al., 1998), but octameric or nonameric structures have also been suggested (Eskandari et al., 1999; Snyder et al., 1998). Recent crystallographic data obtained on the related ASIC1 channel suggest ENaC most likely exists functionally as an αβγ heterotrimer complex (Jasti et al., 2007; Stockand et al., 2008). ENaC is characterized by high sodium selectivity (PNa+/PK+ > 100), a low single-channel conductance (4-5 pS), gating kinetics characterized by long opening and closing times, and a specific block by amiloride (Ki: 100 -200 nM).

Sodium homeostasis requires that the entry of sodium through the apical membrane of epithelial cells is tightly controlled. This control may be realized by regulation of ENaC activity and expression. The role of different domains involved in this regulation has been determined by directed mutagenesis.

Advertisement

3. Mutations in ENaC subunits cause hereditary human disease

The role of ENaC in the regulation of blood pressure and regulation of extracellular fluid volume has been highlighted by the discovery of two severe human diseases. The diseases are due to loss or gain of function of ENaC. Homozygous inactivating mutations in the α, β or γ ENaC subunits cause pseudohypoaldosteronism type 1 (PHA-1), characterized by hypotension and severe hyperkalemic acidosis (Chang et al., 1996). Activating mutations in the genes for the β or γ ENaC subunits lead to Liddle’s syndrome, characterized by autosomal-dominant hypertension accompanied by hypokalemic Alkalosis and volume expansion (Shimkets et al., 1994).

The mutations causing PHA-1 have been identified, and the mechanisms by which they led to a hypofunction of ENaC have been addressed. See (Kellenberger and Schild. L, 2002) for review.

In particular Chang et al. (1996) showed that a single point mutation (G37S) in the coding region for a highly conserved motif in the amino-terminal domain of the β subunit induces PHA-1. Grunder and co-authors (1997) showed that this domain is involved in the gating of ENaC. They identified that the mutation G37S in the gene for the β subunit and homologous mutations in the other subunit genes reduce channel function by changing the open probability.

Liddle syndrome has been linked genetically to mutations that delete or alter a conserved PY (proline-tyrosine) motif located in the carboxy-terminal domain of either β or γENaC (Hansson et al., 1995; Hansson et al., 1995; Shimkets et al., 1994; Tamura et al., 1996). Such deletions or point mutations lead to elevated channel function after expression in Xenopus oocytes, suggesting that the PY motif is involved in the regulation of activity and the density of ENaC channels at the cell surface (Firsov et al., 1996; Kellenberger et al., 1998; Schild et al., 1995; Schild et al., 1996; Shimkets et al., 1997). Mutations within the coding region for the PY motif were generated in vitro by directed mutagenesis. They have been widely studied to investigate the role of Nedd4-2 in the regulation of the number of ENaCs at the cell surface (Abriel and Horisberger, 1999; Debonneville et al., 2001; Kamynina and Staub, 2002; Renauld et al., 2010; Staub et al., 1997). Thus, this has allowed the identification of tyrosine- carrying ubiquitin residues involved in Nedd4-2 dependent-internalization of the channel.

Intracellular C termini also harbor multiple phosphorylation sites and participate in the activity of the channel, suggesting that aldosterone, insulin, SGK1, PKA and PKC modulate the activity of ENaC by phosphorylation (Renauld et al., 2010; Shimkets et al., 1997).

Advertisement

4. Directed mutagenesis and regulation of ENaC by extracellular factors

Several members of the ENaC/degenerin family are clearly extracellular-ligand-gated channels. Numerous studies suggest that ENaC may also be a ligand-gated channel (Horisberger and Chraibi, 2004). A number of extracellular factors of various types have been shown to activate or inhibit ENaC. Amongst these factors, there are serine proteases, sodium itself, other inorganic cations, organic cations, and small molecules.

4.1. Activation by Serine proteases

In 1997 we cloned a serine protease that acts as a channel-activating protease, called CAP1 (Vallet et al., 1997); and we explored the mechanism by which it stimulates ENaC (Chraibi et al., 1998). We showed that the effect of CAP1 is done on the extracellular part of the channel, and it can be mimicked by trypsin or chymotrypsin. Ion selectivity, single channel conductance and channel density are not modified, which suggests that the serine proteases increase the open probability. During the last ten years, many studies showed that ENaC can be activated by other proteases, such as prostasin or furin (Hughey et al., 2004; Vuagniaux et al., 2000). Further progress in the understanding of the mechanism by which serine proteases activate ENaC has been made by functional investigation in heterologous expression systems combined with directed mutagenesis. Mutation of the CAP1 GPI-anchored consensus motif completely abolishes ENaC activation. However, catalytic mutants of CAP1 do not fully stimulate ENaC, suggesting that a noncatalytic mechanism is partly involved in this regulation pathway (Vallet et al., 2002). Thus, a putative site for CAP1 and trypsin action has been identified. However, there is no clear evidence of their role in the proteolytic activation of ENaC. Masilamani and co-authors (1999) first provided evidence for a possible cleavage of the γENaC subunit. These authors were able to show that the aldosterone infusion, or salt restriction, induced a shift in molecular weight of the gamma subunit from 85 to 70 KDa. Subsequently, it was shown that the serine proteases, including prostasin, plasmin, elastase and furin, cleave the extracellular domain of the α and γ subunits (Bruns et al., 2007; Caldwell et al., 2005; Hughey et al., 2004; Passero et al., 2008; Rossier, 2004; Vuagniaux et al., 2002). A basic motif (RKRK186) has been identified as a cleavage site for CAP1/Prostasin in the extracellular loop of γENaC (Bruns et al., 2007; Diakov et al., 2008). Additional cleavage sites within extracellular loop of α and γ subunits have been described (Garcia-Caballero et al., 2008; Myerburg et al., 2006). However, no site for furin was described in βENaC (see Figure 1).

4.2. Effects of extracellular sodium and other small molecules

4.2.1. Self-inhibition

We have shown that the external sodium exerts a fast inhibitory effect on ENaC activity, a phenomenon called sodium self-inhibition (Chraibi and Horisberger, 2002). We observed that the apparent affinity constant for the site responsible for self-inhibition was significantly lower, with a K½ of 100-200 mM. The kinetics of this phenomenon strongly depended on temperature and the extent of proteolytic processing of the ENaC subunits. We demonstrated that the effect of temperature was due to a large decrease in the probability of channel opening at high temperatures, while the unitary current increased with temperature (Chraibi and Horisberger, 2003). Later Sheng et al. (2004, 2002) showed that the mutation of His282 in the α subunit or His239 in the γ subunit (these amino acids reside in close proximity to the defined sites for furin cleavage) enhanced and eliminated the sodium self-inhibition response, respectively.

Figure 1.

Schematic representation of the rat ENaC subunits and their identified and putative sites for furin, CAP1, trypsin and prostasin. M1, M2: transmembrane domains; N, C: intracellular amino- and carboxy-termini, respectively.

4.2.2. Effects of cpt-cAMP and cpt-cGMP

cpt-cAMP, a membrane permeant cAMP analogue, has been described to be a species- dependent extracellular activator of ENaC. Rat and Xenopus laevis ENaC expressed in Xenopus oocytes are not sensitive to cpt-cAMP (Awayda et al., 1996). However, guinea pig (gp) channels could be activated by cpt-cAMP perfusion in the oocyte expression system (Liebold et al., 1996). The gp αENaC has been shown to be essential for this stimulation (Schnizler et al., 2000). However, the mechanism leading to ENaC stimulation did not exclude the possibility of an intracellular pathway involving protein kinase A (PKA). Further experiments demonstrated that PKA inhibitor PKI 6-22 did not prevent cpt-cAMP stimulation of gpENaC expressed in Xenopus oocytes. Furthermore, the α subunit containing the gp extracellular loop with rat intracellular C and/or N termini expressed in Xenopus oocytes together with rat βγ ENaC were sensitive to cpt-cAMP (Chraibi et al., 2001). This chimeric channel demonstrated that the extracellular domain of the gp α subunit was the determinant for ENaC stimulation by cpt-cAMP. Thus, the molecule can be considered to be a ligand for the channel. Moreover, the outside-out configuration of the patch clamp showed an increase of the open probability and the number of open channels (N.Po) exposed to cpt-cAMP, confirming a direct interaction with the extracellular domain of the gpα, ratβγ chimera expressed in Xenopus oocytes. To determine which part of the extracellular domain of αENaC is involved in this regulation, we made four chimeric constructions of that subunit (Figure 2).

Figure 2.

Schematic representation of the ENaC subunits. Chimeric constructions of α subunits by fusion of the coding region for the αgp part (bold line) with the αrat part (thin line). Numbers indicate residues at corresponding positions on the αgp sequence.

To do so, two restriction sites were generated in guinea pig and rat αENaC cDNAs at homologous positions using a PCR technique. Then the appropriate fragment of gp cDNA was inserted into the rat cDNA between the restriction sites. Amiloride-sensitive current was measured in the presence and absence of 10 µM cpt-cAMP. We generated eleven swapping mutants of rat and gp αENaC using PCR-directed mutagenesis and expressed each of these mutants with the rat β and γ subunits in Xenopus oocytes. Among the eleven substitutions, Ile481 in the gp αENaC extracellular domain plays a major role in cpt-cAMP-induced ENaC activation. The I481N mutation in the gene for the αgp subunit completely abolished stimulation of ENaC. The N510I mutation in the gene for the αrat subunit caused intermediate sensitivity to cpt-cAMP. All other mutations or combination of mutations, including N510I in the αrat gene, did not increase the cpt-cAMP effect (Renauld et al., 2008).

Similarly to what we described with cpt-cAMP, Hong-Guan and coworkers (Nie et al., 2009) suggested that cpt-cGMP stimulates human, rat and mouse ENaC through direct interaction and not through the intracellular pathway. Indeed directed mutagenesis of the coding regions for potential phosphorylation sites for the cGMP-dependent kinases on ENaC did not affect cpt-cGMP-induced activation in Xenopus oocytes. Furthermore, knockdown of PKG isoforms did not prevent cpt-cGMP-dependent activation. Han and colleagues (2011) confirmed that cpt-cGMP-induced ENaC activation was mediated through direct interaction and an increases of N.Po. By directed mutagenesis, these authors were able to show that the mutations abolishing self-inhibition (βΔV348 and γH233R) lost their responses to cpt-cGMP. The mutations augmenting this phenomenon (αY458A and γM432G) facilitated the stimulatory effects of this compound. Thus, these data suggest that the elimination of self-inhibition may be a novel mechanism for cpt-cGMP to stimulate ENaC.

α subunitIcpt/IctlSEMunpaired t-Test VS α rat wt
α gp wt2.280.05P<0.001
α rat wt1.130.01
construction 11.140.01NS
construction 21.280.03P<0.001
construction 31.340.03P<0.001
construction 41.850.06P<0.001
αr L493S1.060.02NS
αr S500N1.080.03NS
αr S507N1.060.03NS
αr I509T1.050.02NS
αr N510I1.450.03NS
αr K524T1.020.03NS
αr E531Q1.120.02NS
αr N542S1.050.04NS
αr K550N1.120.02NS
αr F554Y1.160.02NS
αr K561R1.120.02NS
αgp I481N1.580.07P<0.001

Table 1.

Effect of cpt-cAMP on different constructions and mutants of the αENaC subunit expressed in Xenopus oocytes together with the β and γrat subunits. Results are presented as a ratio of amiloride-sensitive current measured after and before cpt-cAMP perfusion (Icpt/Ictl). gp, guinea pig; r, rat; wt, wild type; NS, not significant relative to αrat wt

4.2.3. Effects of glibenclamide

The same experimental approach was used to study the stimulation of ENaC by glibenclamide (Renauld and Chraibi, 2009). Glibenclamide, a high affinity-blocker of the KATP channel, has been shown to stimulate Xenopus ENaC (but not rat ENaC) expressed in Xenopus oocytes. The α subunit has been shown to be critical for this activation (Chraibi and Horisberger, 1999). As described with cpt-cAMP, patch clamp recordings in the outside-out configuration showed an increase of N.Po when Xenopus ENaC was exposed to glibenclamide. Another study has demonstrated that the αgp subunit, but not the αrat subunit, conferred sensitivity of ENaC to glibenclamide (Schnizler et al., 2003). Using mutagenesis, these authors were able to produce other chimeric rat/gp α subunits; and they suggested that the extracellular loop or the transmembrane domain of the αgp subunit is involved in the activation of the ENaC channel by glibenclamide. Thus, similarly to cpt-cAMP activation, channels composed of the αgp subunit and the β and γ subunits from rat are sensitive to glibenclamide, while channels composed of the α, β, and γ subunits from rat are resistant. We used the chimeras of the α subunit previously generated and found that construction 4 was also important for glibenclamide stimulation of the channel. Unlike cpt-cAMP, glibenclamide had no effect on the other constructions expressed with the β and γ subunits from rat. Moreover, directed mutagenesis did not reveal particular residues involved in this regulation.

α subunitIglib/IctrlSEMunpaired t-Test VS α rat wt
α gp wt1.630.02P<0.001
α rat wt0.960.01
construction 11.030.04NS
construction 21.040.05NS
construction 31.030.01NS
construction 41.270.04P<0.001
αr L493S0.920.03NS
αr S500N1.010.03NS
αr S507N0.980.02NS
αr I509T0.990.02NS
αr N510I1.000.01NS
αr K524T0.970.02NS
αr E531Q0.990.05NS
αr N542S0.980.02NS
αr K550N0.910.01NS
αr F554Y0.990.02NS
αr K561R0.960.02NS

Table 2.

Effect of glibenclamide on different constructions and mutants of αENaC expressed in Xenopus oocytes together with the β and γ subunits from rat. Results are presented as a ratio of amiloride-sensitive current measured after and before glibenclamide perfusion (Iglib/Ictl). gp, guinea pig; r, rat; wt, wild type; NS, not significant relative to αrat wt

4.2.4. Effects of other molecules

Capsazepine has been described as the first selective active activator for the δENaC subunit (Yamamura et al., 2004). Indeed capsazepine specifically stimulates human-made δ subunit, but not the α subunit expressed in Xenopus oocytes. Moreover, this molecule can stimulate the δENaC monomer, whereas no other vanilloid compound can produce changes in sodium amiloride-sensitive current. However, the authors did not determine any amino acids involved in this activation. Directed mutagenesis could be a powerful tool to understand differences between the α and δ subunits and resolve the structure-function relationships of both proteins.

S3969 is a small molecule described as a reversible activator of human, but not mouse, αβγ ENaC through direct interaction with the extracellular side of the channel by increasing N.Po (Lu et al., 2008). Interestingly, S3969 stimulates amiloride-sensitive current in oocytes expressing the δ subunit instead of α. The authors showed that βENaC was critical for this activation. Mouse-human chimeras of the β subunit confirmed the implication of the extracellular domain. More specifically, deletion of Val348 in βENaC completely abolished S3969 activation of ENaC. Maturation and optimal transport of ENaC to the plasma membrane requires furin cleavage of the β and γ subunits at a specific Arg. Mutations of the furin cleavage site in which Arg was replaced by Ala did not prevent ENaC activation by S3969, suggesting that proteolytic activation prior to S3969 stimulation is not necessary. Mutations producing pseudohypoaldosteronism type 1 (PHA1), resulting in salt-wasting, a genetic disease, have been generated in the α (R508STOP) and β (G37S) subunits. These mutants decreased amiloride-sensitive current, but the S3969 compound was still able to stimulate ENaC activity.

Advertisement

5. Conclusion

The epithelial sodium channel (ENaC) has been used for decades as a therapeutic target against type 1 hypertension and Liddle syndrome. More recently, several studies pointed to ENaC as a potential target for cystic fibrosis (Zhou et al., 2011), a pathology characterized by an impaired Cl- secretion through the cystic fibrosis transmembrane conductance regulator (CFTR) and an increase of Na+ reabsorption through ENaC. The studies of mutations involved in these diseases have been extremely helpful in determining the molecular mechanisms by which they lead to a dysfunction of ENaC. Furthermore, the experiments carried out on this topic have shown the contribution of the PCR-directed mutagenesis technique in the determination of the structure-function relationships of ENaC. These studies have led to a better understanding of the domains involved in ion selectivity, gating and expression of the channel at the cell membrane. Additional studies are needed to define other key domains of ENaC. They may provide a new strategy for the treatment of pathologies linked to dysfunction of this channel.

References

  1. 1. AbrielHHorisbergerJ. D1999Feedback inhibition of rat amiloride-sensitive epithelial sodium channels expressed in Xenopus laevis oocytes.J Physiol 5163143
  2. 2. AnantharamAP.L. 2007Determination of epithelial na+ channel subunit stoichiometry from single-channel conductances.J Gen Physiol. 1305570
  3. 3. AwaydaM. SIsmailovI. IBerdievB. KFullerC. MBenosD. J1996Protein kinase regulation of a cloned epithelial Na+ channel.J Gen Physiol 1084965
  4. 4. BrouardMCasadoMDjelidiSBarrandonYFarmanN1999Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation.J Cell Sci 112 (Pt 19):3343 EOF52 EOF
  5. 5. BrunsJ. BCarattinoM. DShengSMaaroufA. BWeiszO. APilewskiJ. MHugheyR. PKleymanT. R2007Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit.J Biol Chem 282615360
  6. 6. CaldwellR. ABoucherR. CStuttsM. J2005Neutrophil elastase activates near-silent epithelial Na+ channels and increases airway epithelial Na+ transport.Am J Physiol Lung Cell Mol Physiol 288:L8139
  7. 7. CanessaC. MHorisbergerJ. DRossierB. C1993Epithelial sodium channel related to proteins involved in neurodegeneration.Nature36146770
  8. 8. CanessaC. MSchildLBuellGThorensBGautschiIHorisbergerJ. DRossierB. C1994Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.Nature3674637
  9. 9. ChangS. SGrunderSHanukogluARoslerAMathewP. MHanukogluISchildLLuYShimketsR. ANelson-williamsCRossierB. CLiftonR. P1996Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1.Nat Genet 1224853
  10. 10. ChraibiAHorisbergerJ. D1999Stimulation of epithelial sodium channel activity by the sulfonylurea glibenclamideJ Pharmacol Exp Ther 2903417
  11. 11. ChraibiAHorisbergerJ. D2002Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases.J Gen Physiol 12013345
  12. 12. ChraibiAHorisbergerJ. D2003Dual effect of temperature on the human epithelial Na+ channel.Pflugers Arch 447316320
  13. 13. ChraibiASchnizlerMClaussWHorisbergerJ. D2001Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes.J Membr Biol 1831523
  14. 14. DebonnevilleCFloresS. YKamyninaEPlantP. JTauxeCThomasM. AMunsterCChraibiAPrattJ. HHorisbergerJ. DPearceDLoffingJStaubO2001Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression.Embo J 2070529
  15. 15. DiakovABeraKMokrushinaMKruegerBKorbmacherC2008Cleavage in the {gamma}-subunit of the epithelial sodium channel (ENaC) plays an important role in the proteolytic activation of near-silent channelsJ Physiol 5864587608
  16. 16. DijkinkLHartogAVan OsC. HBindelsR. J2002The epithelial sodium channel (ENaC) is intracellularly located as a tetramer.Pflugers Arch 44454955
  17. 17. DucCFarmanNCanessaC. MBonvaletJ. PRossierB. C1994Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.J Cell Biol 127190721
  18. 18. EskandariSSnyderP. MKremanMZampighiG. AWelshM. JWrightE. M1999Number of subunits comprising the epithelial sodium channel.J Biol Chem 274272816
  19. 19. FirsovDGautschiIMerillatA. MRossierB. CSchildL1998The heterotetrameric architecture of the epithelial sodium channel (ENaC).Embo J 1734452
  20. 20. FirsovDSchildLGautschiIMerillatA. MSchneebergerERossierB. C1996Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approachProc Natl Acad Sci U S A 93153705
  21. 21. Garcia-caballeroADangYHeHStuttsM. J2008ENaC proteolytic regulation by channel-activating protease 2.J Gen Physiol 13252135
  22. 22. GrunderSFirsovDChangS. SJaegerN. FGautschiISchildLLiftonR. PRossierB. C1997A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.Embo J 16899907
  23. 23. HanD. YNieH. GSuX. FShiX. MBhattaraiDZhaoMZhaoR. ZLandersKTangHZhangLJiH. L2011CPT-cGMP Stimulates Human Alveolar Fluid Clearance by Releasing External Na+ Self-Inhibition of ENaC. Am J Respir Cell Mol Biol
  24. 24. HanssonJ. HNelson-williamsCSuzukiHSchildLShimketsRLuYCanessaCIwasakiTRossierBLiftonR. P1995Hypertension caused by a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle syndrome.Nat Genet 117682
  25. 25. HanssonJ. HSchildLLuYWilsonT. AGautschiIShimketsRNelson-williamsCRossierB. CLiftonR. P1995A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity.Proc Natl Acad Sci U S A 92114959
  26. 26. HorisbergerJ. DChraibiA2004Epithelial Sodium Channel: A Ligand-Gated Channel? Nephron Physiology 963741
  27. 27. HugheyRBrunsJKinloughCHarkleroadKTongQCarattinoMJohnsonJStockandJKleymanT2004Epithelial sodium channels are activated by furin- dependent proteolysisJ. Biol. Chem. 30181114
  28. 28. HummlerEBarkerPGatzyJBeermannFVerdumoCSchmidtABoucherRRossierB. C1996Early death due to defective neonatal lung liquid clearance in alpha- ENaC-deficient miceNat Genet 123258
  29. 29. JastiJFurukawaHGonzalesE. BGouauxE2007Structure of acid-sensing ion channel 1 at 1.9A° resolution and low pH. nature449316324
  30. 30. KamyninaEStaubO2002Concerted action of ENaC, Nedd42and Sgk1 in transepithelial Na(+) transport.Am J Physiol Renal Physiol 283:F377-87
  31. 31. KellenbergerSGautschiIRossierB. CSchildL1998Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system.J Clin Invest 101274150
  32. 32. KellenbergerSSchildL2002Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure.Physiol Rev. 8273567
  33. 33. KunzelmannKMallM2002Electrolyte transport in the mammalian colon: mechanisms and implications for disease.Physiol Rev 8224589
  34. 34. LieboldK. MReifarthF. WClaussWWeberW1996cAMP-activation of amiloride- sensitive Na+ channels from guinea-pig colon expressed in Xenopus oocytes. Pflugers Arch 43191322
  35. 35. LinguegliaEVoilleyNWaldmannRLazdunskiMBarbryP1993Expression cloning of an epithelial amiloride-sensitive Na+ channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett 318959
  36. 36. LuMEcheverriFKalabatDLaitaBDahanD. SSmithR. DXuHStaszewskiLYamamotoJLingJHwangNKimmichRLiPPatronEKeungWPatronAMoyerB. D2008Small molecule activator of the human epithelial sodium channel. J Biol Chem 2831198111994
  37. 37. MasilamaniSKimG. HMitchellCWadeJ. BKnepperM. A1999Aldosterone- mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney. J Clin Invest 104:R1923
  38. 38. MullisK. BFaloonaF. A1987Specific synthesis of DNA in vitro via a polymerase- catalyzed chain reaction. Methods Enzymol 15533550
  39. 39. MyerburgM. MButterworthM. BMckennaE. EPetersK. WFrizzellR. AKleymanT. RPilewskiJ. M2006Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance: a mechanism for sodium hyperabsorption in cystic fibrosis. J Biol Chem 281279429
  40. 40. NieH. GChenLHanD. YLiJSongW. FWeiS. PFangX. HGuXMatalonSJiH. L2009Regulation of epithelial sodium channels by cGMP/PKGII. J Physiol 587266376
  41. 41. PasseroC. JMuellerG. MRondon-berriosHTofovicS. PHugheyR. PKleymanT. R2008Plasmin activates epithelial Na+ channels by cleaving the gamma subunit. J Biol Chem 2833658691
  42. 42. PeruccaJBichetD. GBardouxPBoubyNBankirL2008Sodium excretion in response to vasopressin and selective vasopressin receptor antagonists. J Am Soc Nephrol 19172131
  43. 43. RandellS. HBoucherR. C2006Effective mucus clearance is essential for respiratory health. Am J Respir Cell Mol Biol 35208
  44. 44. RenauldSAllacheRChraibiA2008Ile481 from the guinea pig alpha-subunit plays a major role in the activation of ENaC by cpt-cAMP. Cell Physiol Biochem. 22(1-4):101-8
  45. 45. RenauldSChraibiA2009Role of the C-Terminal Part of the Extracellular Domain of the alpha-ENaC in Activation by Sulfonylurea Glibenclamide. J Membr Biol
  46. 46. RenauldSTremblayKAit-benichouSSimoneau-royMGarneauHStaubOChraibiA2010Stimulation of ENaC activity by rosiglitazone is PPARgamma- dependent and correlates with SGK1 expression increase. J Membr Biol 23625970
  47. 47. RossierB2004The epithelial sodium channel: activation by membrane-bound serine proteases. Proc Am Thorac Soc: 149
  48. 48. Roudier-pujolCRochatAEscoubetBEugeneEBarrandonYBonvaletJ. PFarmanN1996Differential expression of epithelial sodium channel subunit mRNAs in rat skin. J Cell Sci 109 (Pt 2):379-85
  49. 49. SchildLCanessaC. MShimketsR. AGautschiILiftonR. PRossierB. C1995A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci U S A 925699703
  50. 50. SchildLLuYGautschiISchneebergerELiftonR. PRossierB. C1996Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. Embo J 1523817
  51. 51. SchnizlerMBerkAClaussW2003Sensitivity of oocyte-expressed epithelial Na+ channel to glibenclamide. Biochim Biophys Acta 16091706
  52. 52. SchnizlerMMastroberardinoLReifarthFWeberW. MVerreyFClaussW2000cAMP sensitivity conferred to the epithelial Na+ channel by alpha- subunit cloned from guinea-pig colon. Pflugers Arch 43957987
  53. 53. ShengSBrunsJKleymanT2004Extracellular histidine residues crucial for Na+ self- inhibition of epithelial Na+ channels. JBC 27997439
  54. 54. ShengSPerryCKleymanT2002External nickel inhibits epithelial sodium channel by binding to histidine residues within the extracellular domains of alpha and gamma subunits and reducing channel open probability. J Biol. Chem. 27750098111
  55. 55. ShimketsR. ALiftonR. PCanessaC. M1997The activity of the epithelial sodium channel is regulated by clathrin- mediated endocytosis. J Biol Chem 2722553741
  56. 56. ShimketsR. AWarnockD. GBositisC. MNelson-williamsCHanssonJ. HSchambelanMGillJ. RJr., Ulick, S., Milora, R.V., Findling, J.W., et al. 1994Liddle’s syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial sodium channel. Cell 7940714
  57. 57. SnyderP. MChengCPrinceL. SRogersJ. CWelshM. J1998Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits. J Biol Chem 2736814
  58. 58. StaubOGautschiIIshikawaTBreitschopfKCiechanoverASchildLRotinD1997Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. EMBO J 1663256336
  59. 59. StockandJ. DStaruschenkoAPochynyukOBoothR. ESilverthornD. U2008Insight Toward Epithelial Na1 Channel Mechanism Revealed by the Acid-sensing Ion Channel 1 Structure. IUBMB Life 60620628
  60. 60. TamuraHSchildLEnomotoNMatsuiNMarumoFRossierB. C1996Liddle disease caused by a missense mutation of beta subunit of the epithelial sodium channel gene. J Clin Invest 9717804
  61. 61. ValletVChraibiAGaeggelerH. PHorisbergerJ. DRossierB. C1997An epithelial serine protease activates the amiloride-sensitive sodium channel. Nature 38960710
  62. 62. ValletVPfisterCLoffingJRossierB. C2002Cell-surface expression of the channel activating protease xCAP-1 is required for activation of ENaC in the Xenopus oocyte. J Am Soc Nephrol 1358894
  63. 63. VuagniauxGValletVJaegerN. FHummlerERossierB. C2002Synergistic Activation of ENaC by Three Membrane-bound Channel- activating Serine Proteases (mCAP1, mCAP2, and mCAP3) and Serum- and Glucocorticoid- regulated Kinase (Sgk1) in Xenopus Oocytes. J Gen Physiol 120191201
  64. 64. VuagniauxGValletVJaegerN. FPfisterCBensMFarmanNCourtois-coutryNVandewalleARossierB. CHummlerE2000Activation of the amiloride- sensitive epithelial sodium channel by the serine protease mCAP1 expressed in a mouse cortical collecting duct cell line. J Am Soc Nephrol 1182834
  65. 65. YamamuraHUgawaSUedaTNagaoMShimadaS2004Protons activate the delta-subunit of epithelial Na+ channel in humans. JBC in preess (published on january 15, manuscript M400274200)
  66. 66. ZhouZDuerrJJohannessonBSchubertS. CTreisDHarmMGraeberS. YDalpkeASchultzCMallM. A2011The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease. J Cyst Fibros 10 Suppl 2:S17282

Written By

Ahmed Chraibi and Stéphane Renauld

Submitted: 05 October 2011 Published: 05 February 2013