Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Curr Opin Nephrol Hypertens. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2895494

Regulation of sodium transport by ENaC in the kidney


Purpose of review

The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in the regulation of sodium transport in the collecting duct and hence sodium balance. This review describes recent findings in the regulation of ENaC function by serine proteases in particular and other regulatory aspects.

Recent findings

Regulation of ENaC occurs at many levels (biophysical, transcriptional, post-translational modifications, assembly, membrane insertion, retrieval, recycling, degradation, etc.). Recent studies have recognized and delineated proteolytic cleavage, particularly of the α and γ subunits, as major mechanisms of activation. Release of peptide fragments from these two subunits appears to be an important aspect of activation. These proteolytic mechanisms of ENaC activation have also been demonstrated in vivo and strongly suggested in clinical circumstances, particularly the nephrotic syndrome. In the nephrotic syndrome, filtered plasminogen may be cleaved by tubular urokinase to yield plasmin which can activate ENaC. In addition to these mechanisms, regulation by ubiquitination and deubiquitination represents a pivotal process. Several important deubiquitinating enzymes have been identified as important in ENaC retention in, or recycling to, the apical membrane. New aspects of the genomic control of ENaC transcription have also been found including histone methylation.


The mechanisms of regulation of ENaC are increasingly understood to be a complex interplay of many different levels and systems. Proteolytic cleavage of α and γ subunits plays a major role in ENaC activation. This may be particularly clinically relevant in nephrotic syndrome in which plasmin may activate ENaC activity.

Keywords: Epithelial sodium channel, aldosterone, prostasin, SGK-1, plasmin


The collecting duct is the site of final regulation of urinary sodium (Na) excretion. The importance of this regulation has been dramatically illustrated by the fact that several forms of monogenic hypertension have been associated with genetic defects targeting this site. Sodium transport in the collecting ducts occurs via rate limiting amiloride-sensitive epithelial sodium channels (ENaC) in series with basolateral Na-K-ATPase. ENaC is composed of a complex of three homologous subunits, α, β, and γ with the α subunit as the transporting subunit with regulatory roles for the β and γ subunits. Each subunit has a large extracellular domain in the luminal space, two transmembrane domains, and relatively small cytoplasmic domains. Aldosterone is the pivotal hormone regulating sodium transport in the collecting duct via its genomic effects on the mineralocorticoid receptor. Aldosterone induces a number of proteins, including ENaC and Na-K-ATPase, but it also has early effects on apical Na transport via stimulation of SGK1 [1]. SGK1 phosphorylates Nedd4-2, thus reducing its role in ubiquitination, retrieval, and degradation of ENaC [2]. Aldosterone may also have a number of other mechanisms to stimulate ENaC (including synthesis of subunits over a longer time scale) including increasing expression of glucocorticoid-induced leucine zipper protein (GILZ) which acts by inhibition of extracellular signal–regulated kinase (ERK) [3]. In addition to classic systemic hormones, more recent studies implicate paracrine and intracellular factors in modulating Na transport.

This review will focus on regulation of ENaC by serine proteases, and also selectively provide some update on recent studies addressing other regulatory factors involved in Na transport. ENaC has become one of the most widely investigated renal transport proteins and many aspects have been unraveled, from biophysical properties to intracellular trafficking [4]. Proteolytic processing of ENaC has been one of several areas of concentrated investigation recently [5;6]. The scope of this review does not allow recent findings related to a variety of other hormones affecting ENaC, certain other signaling pathways, and many new biophysical findings.

Protease activation of ENaC

Intense interest in serine protease effects on ENaC began when Vallet et al identified a novel serine protease called CAP1, for channel-activating protease 1, which activates ENaC [7]. CAP1 is an apical GPI anchored protein which can also be secreted in some tissues. CAP1 is the ortholog to the protein prostasin previously cloned from prostate [8;9] and is highly expressed in kidney and other epithelial tissues that express ENaC. (Subsequent reference to CAP1 herein will refer to prostasin.) Two other homologous proteins CAP2 (TMPRSS4) and CAP3 (matriptase) have been identified [10;11] and a variety of other proteases such as elastase [12;13] have been found to potentially activate ENaC. Although initial studies did not suggest actual cleavage of ENaC subunits, subsequent studies indicated critical roles for cleavage of subunits by the intracellular protease furin, and extracellular activation by prostasin (or by exogenous trypsin) [14-16]. Recently significant progress has been made in several directions regarding proteolytic activation of ENaC.

Recent studies suggest that proteolytic activation of ENaC results from proteolytic release of inhibitory peptides from within the α and γ subunits (figure1). Carattino et al first reported that cleavage of the α subunit by furin at two sites released a 26-amino acid tract, leading to the activation of ENaC [17]. Bruns et al [18], following the work of Hughey et al [16] on the cleavage sites for the γ subunit by furin, found that the γ subunit was further cleaved by prostasin at a site distal to the furin cleavage site. Cleavage of the γ subunit by furin and prostasin released a 43-residue tract leading to an increase of ENaC activity. A synthetic peptide, representing the cleaved tract from γ subunit, inhibited ENaC activity [18]. Most recent studies suggest that the γ subunit cleavage has the dominant role in activation of ENaC. Carattino et al [19] reported that in Xenopus oocytes expressing ENaC subunits, prostasin fully activated ENaC even with mutation of furin cleavage sites in the α subunit, whereas the activation of ENaC by prostasin was blocked by the mutation of furin and prostasin cleavage sites in the γ subunit. Deletion of the γ subunit inhibitory tract fully activated ENaC despite the mutation of furin cleavage sites in the α subunit or the presence of α subunit inhibitory tract. When the γ subunit inhibitory tract was removed, the cleavage of α subunit was required to fully active sodium channels. Removal of the γ subunit inhibitory tract reduced the response of ENaC activity to α subunit 26-residue inhibitory tract. These results suggested that proteolytic cleavage of the γ subunit has a major role in the regulation of the ENaC activity. Smaller peptide sequences from both α and γ inhibitory tracts are sufficient for channel inhibition. Diakov et al [20] also found that the γ subunit was critical for regulation by proteases, but in a manner that involves both gating of active channels and more importantly recruitment of “near silent channels”. The γ subunit is also critical for elastase and CAP2 activation of ENaC [21][22]. This seeming importance of the γ subunit does contrast with other studies which demonstrated the importance of α subunit cleavage by furin [15][13][23].

Figure 1
Model of proteolytic cleavage sites in α and γ ENaC subunits by furin, prostasin, neutrophil and pancreatic elastase, plasmin, CAP2 and trypsin. Adapted from refs. 5 and 6.. The dashed line is shown to illustrate possible inhibitory fragments ...

In terms of regulation of these processes, aldosterone and volume status alter prostasin and the abundance of cleaved subunits as discussed below. SGK1, discussed below, may also regulate γ ENaC cleavage [24]. Also Synder’s group has found that the rate of proteolytic cleavage and activation of ENaC are reduced by increased levels of intracellular Na (“Na feedback inhibition”) and altered also by apical membrane resident time and by Nedd4, the E3 ubiquitin ligase that targets ENaC for degradation [25;26][27]. Deubiquitylation also alters proteolytic cleavage of ENaC [28]. Other aspects of regulation are discussed below.

In addition to the proteases mentioned above, two other proteases have recently been found to have intriguing possible pathophysiologic relevance: tissue kallikrein and plasmin (discussed below). Kallikrein is known to be secreted into the urinary space and to have significant relevance to sodium balance and blood pressure control. Picard et al [29] recently demonstrated that luminal tissue kallikrein (TK) activates Na current in cortical collecting duct principal cells microperfused in vitro. The 70 KD cleaved fragment of γ ENaC subunit was absent in TK knockout mouse, but it reappeared when incubated with TK. These results strongly suggest that TK might activate sodium transport via the cleavage of γ ENaC subunit. Plasmin and its possible role in nephrotic syndrome is discussed below.

In contrast to the above studies that implicate direct channel cleavage by serine proteases, some studies have shown activation of ENaC can occur without active proteolysis [30][31]. Also, indirect activation of ENaC by proteases, rather than by proteolytic cleavage, may also contribute to channel activation [32]; protease activated receptors present in the kidney could be important in this regard. Several investigators have proposed the possibility of proteolytic cascades with multiple components [6]. Another aspect of serine protease action on distal nephron transport is that protease inhibitors increase paracellular permeability and proteases decrease permeability [11][33].

Protease inhibitors and TGF-β1

Pharmacologic inhibitors of serine proteases reduce Na transport, but importantly an endogenous inhibitor of prostasin, protease nexin 1 (PN-1) has been identified [34]. PN-1 is a member of the serpin family (serine protease inhibitor) and is an endogenous inhibitor for α-thrombin, plasmin, and plasminogen activators. PN-1 decreases sodium current by ENaC in vitro. And knockdown of PN-1 gene expression increases baseline sodium current in M-1 cells. Furthermore prostasin and PN-1 appear to be reciprocally regulated by aldosterone (increased prostasin and decreased PN-1) and TGF-β (decreased prostasin and increased PN-1) [34]. However, as with many hormones, the actions of TGF-β1 are also more complicated than a single mechanism: TGF-β1 significantly increases the amount of IkBα, and may inhibit prostasin promoter activity through the induction of IkBα and subsequent inhibition of c-Rel activity in M-1 cells [35]. But activation of the NFkB pathway inhibits SGK1 expression [36;37], a key modulator of ENaC as discussed below. Edinger et al [37] also showed that Nedd-4 function and phosphorylation by IKKB are required for IKKB regulation of ENaC activity. In another study, TGF-β1 inhibited ENaC activity through smad-4 and α ENaC mRNA expression reduction in mpkCCDcl4 mouse collecting duct cells. Removal of N-terminus of Smad4 not only abolished the inhibitory effects of Smad4 on sodium current and αENaC mRNA expression, but also prevented the inhibition of sodium current by TGF-β1[38]. These studies suggest that TGF-β1 regulates sodium transport by a variety of mechanisms whose relative importance is yet to be determined.

The synthetic serine protease inhibitor nafamostat mesilate was shown to reduce renal sodium reabsorption and prostasin excretion in vivo [39]. More recently, Maekawa et al [40] demonstrated that an orally active synthetic serine protease inhibitor, camostat mesilate, decreased Na transport in vitro and blood pressure in Dahl salt-sensitive rats fed with high-salt diet. Proteinuria and renal function were also improved. This study raises the possibility that protease inhibitors could represent a potential new class of antihypertensive agent with renoprotective effects.

In vivo evidence of proteolytic cleavage of ENaC subunits

Although much of the above data is in vitro, substantial evidence supports these proteolytic mechanisms in vivo. Masilamani et al. [41] first demonstrated a shift in the molecular weight of γ ENaC from 85 KD to 70 KD with elevated circulating aldosterone. Subsequent studies by the Frindt and Palmer group, among others, have significantly expanded our understanding of the in vivo events [42-45]; these studies have combined biochemical information (western blots etc.) with physiologic data (whole cell patch clamp Na currents) from intact tubules from rats. The studies have demonstrated that salt deprivation and/or aldosterone increase the cleaved form of the α and γ subunits, that these changes can occur rapidly (hours) and correlate with Na conductance, and that these mechanisms are present in medullary collecting ducts as well as cortical collecting ducts [43-45]. The most recent of these studies have been able to demonstrate that apical surface membrane subunits increase with aldosterone or salt depletion, and decrease with salt repletion [42]; also the Na currents were not able to be further activated by addition of trypsin in tubules from salt depleted rats in contrast to those from salt replete rats [42]. Aldosterone and/or salt depletion both increase expression of the subunits at the membrane and increase their activity via cleavage. Increased glycosylation of the β subunit was also seen with salt depletion [42]. In addition to these and other studies of intact tubules [29;46], Nesterov et al [47] also showed by whole cell patch-clamp data that trypsin increases amiloride-sensitive sodium current in microdissected distal tubules of mice on low and normal salt diets. The stimulatory effect of trypsin on sodium current was blocked by pretreatment with a protease inhibitor.

Initial clinical studies indicated that urinary prostasin is elevated in patients with hyperaldosteronism [48]. Recent clinical studies have suggested more generally that urinary prostasin may serve as an in vivo marker of activation of ENaC [49], correlate with urinary aldosterone [50], and increase with pressure natriuresis [51]. Another study suggested that genetic polymorphisms in prostasin may be correlated with hypertension [52]. All of these clinical studies are intriguing but will need confirmation.

Role of plasmin in nephrotic syndrome

Two recent studies have demonstrated that plasmin activation of ENaC may contribute to Na retention in nephrotic syndrome. Passero et al [53] showed that plasmin activated Na current in oocyte expressing ENaC by cleaving lysine 194 in the γ subunit. Also plasminogen and plasmin were found in the urine of obese ZSF1 rats, but not in control rats. Svenningsen et al [54] found that urine of nephrotic rats showed a 10 fold increase of serine protease activity compared with that of control rats. Moreover, urine of nephrotic rats activated Na current which was blocked by aprotinin. Plasmin was identified as the primary serine protease in the nephrotic urine. In oocytes expressing ENaC subunits, sodium current was activated by combination of plasminogen and urokinase-type plasminogen activator (uPA), but not by plasminogen or uPA alone, suggesting that plasmin is important for ENaC activation. Furthermore, the combination of plasminogen and uPA also generated a 67 KD cleaved fragment of the γ ENaC subunit. In addition, urine of nephrotic patients was also shown to stimulate Na current which was blocked by amiloride or plasmin inhibitors. These two important studies suggest that plasminogen filtered in the nephrotic urine may be cleaved by tubule urokinase to generate plasmin which can then activate ENaC by cleavage of the γ subunit. This provides a possible mechanism of sodium retention in diseases with significant proteinuria.

Regulation of ENaC activity by membrane trafficking

The importance of ENaC insertion into the apical membrane, retrieval, and recycling have been recognized since shortly after the cloning of ENaC subunits and their identification as the genes mutated in Liddle’s syndrome. Significant progress continues in this area [4;55-57], but the scope here will be limited to selected recent findings (figure 2). The mechanisms that govern subunit synthesis, assembly, and insertion in the membrane (although most is degraded prior to insertion) have been investigated but still not completely understood [55]; but more attention has focused on retrieval and subsequent events. Basically, Nedd4-2 ubiquitinates surface subunits by binding to PPxY motifs in ENaC subunits [27;58], initiating their retrieval from the apical membrane to endosomes, to be either degraded in lysosomes or proteosomes, or recycled to the apical membrane. After ubiquitination, ENaC is probably retrieved from the membrane by clathrin mediated pathways predominantly. Epsin and PIP2 play significant roles in this process [59;60].The importance of Nedd4-2 in regulating ENaC and hence blood pressure was recently confirmed with a Nedd4-2 knockout mouse model that exhibited salt sensitive hypertension and increased expression levels of all three ENaC subunits [61].

Figure 2
Model of the regulation of Na transport in collecting duct principal cells. Ubiquitin ligase Nedd4-2 and deubiquitinase Usp2-45 reciprocally regulate ubiquitination and recycling of ENaC. Aldosterone regulates gene expression of ENaC subunits via Sgk1-induced ...

Aldosterone induces the PI3K-dependent kinase SGK1, which is now identified as a central modulator of the ubiquitination process by Nedd4-2. SGK1 phosphorylates Nedd4-2, inducing its interaction with 14-3-3 proteins and decreasing its interaction with ENaC. In a negative feedback manner, phosphorylated Nedd4-2 ubiquitinates and leads to degradation of SGK1. SGK1 also directly activates αENaC [62]. SGK1 also has a variety of other actions as discussed elsewhere. (A 5′ variant alternate transcript of SGK1 that encodes a Sgk1 isoform, Sgk1_i2 may be most efficient in regulating ENaC [63].) Despite the central role in regulation of ENaC, SGK1 knockout animals have a surprisingly mild phenotype and aldosterone is still able to activate ENaC [24;64]. Other signaling pathways also interact with Nedd4 binding. A Raf-1–MAPK/ERK kinase pathway phosphorylates ENaC, increasing its association with Nedd4, and decreasing activity, e.g. [65-67]. GILZ1, another aldosterone induced protein, disrupts the Raf-1/ ERK pathway, stimulating ENaC. Recently Pearce’s group have not only shown synergistic actions, but also physical interaction, between the GILZ1 and SGK1 pathways, specifically a complex of ENaC, Nedd4-2, and Raf-1 [68;69]. The G protein coupled kinase GRK2 also impinges at the intersection of Nedd4-2 interaction with ENaC [70]. The phosphatidylinositide 3′-kinase (PI3K) pathway (recently reviewed [71]) not only stimulates SGK1, but both PIP3 and PIP2 directly affect ENaC gating. A variety of other kinases including Akt, WNK1, IκB kinase-β, and AMP-activated kinase also affect ENaC activity indirectly [37;72-74].

Once ubiquitinated, ENaC is retrieved into endosomal compartments and much undergoes degradation in lysosomes. However recent studies indicate that deubiquitination occurs constitutively, maintaining ENaC at the surface and playing a role in recycling subunits from sorting endosomes to the surface. Recently, three deubiquitinating enzymes (DUBs) have been identified to influence ENaC. Butterworth et al [75] found a ubiquitin C-terminal hydrolase (UCH) isoform L3 was the predominant deubiquitinating enzyme in CCD cells. Fakitsas et al [76] showed that aldosterone increased expression of ubiquitin-specific protein 2-45 (Usp2-45) in mouse CCD. Usp2-45 increased ENaC activity and surface expression and has also been found to increase cleavage of α and γ ENaC subunits [28]. ENaC surface expression may be reciprocally regulated by deubiquitylation by DUBs and ubiquitylation by Nedd4-2. Another DUB, USP10, is induced by vasopressin and enhances ENaC activity by deubiquitinating sorting nexin 3 instead of ENaC subunits directly [77].

After deubiquitination, ENaC subunits may be recycled from certain endosomes back to the apical surface; a variety of Rab GTPases may be involved [4;78]. Recently, Martel et al. [79] identified melanophilin as an aldosterone induced protein that may be involved in ENaC trafficking. Fusion of ENaC containing vesicles with the plasma membrane likely involves lipid rafts and various SNARE components [4;80].

Recent findings related to genomic regulation of ENaC

Although most of the past focus has been on up-regulation of Na transport by aldosterone’s genomic actions, several recent findings have highlighted negative genomic effects by various factors. Zhang et al [81] found that Dot1a (a histone methyltransferase) represses αENaC transcription by histone methylation but that aldosterone, via SGK-1 phosphorylation of its binding partner AF9, down-regulates Dot1a [82][83]. Naray-Fejes-Toth [84] found another negative regulator of β and γ ENaC expression, promyelocytic leukemia zinc finger (PLZF) protein which is a transcriptional repressor. Overexpression of PLZF inhibited sodium current and mRNA expression of β and γ ENaC subunits in collecting duct cells.

Structural features of ENaC

ENaC is a member of a large gene family which also contains the acid sensing ion channels (ASCI) of the vertebrate nervous system. Recently the crystal structure of chicken ASIC1 was reported [85], leading to intense study for insights into the structural/functional correlates for ENaC [86][87]. Although there are significant differences between the two ion channels, this breakthrough will likely lead to significant insight into the structure of ENaC.


ENaC activity is regulated at many different levels. Two systems have recently received particular attention: activation by proteolytic cleavage of the α and γ subunits, and retrieval and recycling of ENaC by ubiquitination and deubiquitination.


Supported by a grant from the Institutional Award program of the National Center for Research Resources (P20RR017659) and a Merit Review grant from the Department of Veterans Affairs.


This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reference List

1. varez de la RD, Canessa CM, Fyfe GK, Zhang P. Structure and regulation of amiloride-sensitive sodium channels. Annu.Rev.Physiol. 2000;62:573–594. [PubMed]
2. Snyder PM, Olson DR, Thomas BC. Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel. J.Biol.Chem. 2002;277:5–8. [PubMed]
3. Soundararajan R, Zhang TT, Wang J, Vandewalle A, Pearce D. A novel role for glucocorticoid-induced leucine zipper protein in epithelial sodium channel-mediated sodium transport. J Biol.Chem. 2005;280:39970–39981. [PubMed]
4. Butterworth MB, Edinger RS, Frizzell RA, Johnson JP. Regulation of the epithelial sodium channel by membrane trafficking. Am J Physiol Renal Physiol. 2009;296:F10–F24. [PubMed]
5. Kleyman TR, Carattino MD, Hughey RP. ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases. J Biol.Chem. 2009;284:20447–20451. [PMC free article] [PubMed]
6. Rossier BC, Stutts MJ. Activation of the epithelial sodium channel (ENaC) by serine proteases. Annu.Rev.Physiol. 2009;71:361–379. [PubMed]
7. Vallet V, Chraibi A, Gaeggeler HP, Horisberger JD, Rossier BC. An epithelial serine protease activates the amiloride-sensitive sodium channel. Nature. 1997;389:607–610. [PubMed]
8. Yu JX, Chao L, Chao J. Molecular cloning, tissue-specific expression, and cellular localization of human prostasin mRNA. J.Biol.Chem. 1995;270:13483–13489. [PubMed]
9. Yu JX, Chao L, Chao J. Prostasin is a novel human serine proteinase from seminal fluid. Purification, tissue distribution, and localization in prostate gland. J.Biol.Chem. 1994;269:18843–18848. [PubMed]
10. Vuagniaux G, Vallet V, Jaeger NF, Hummler E, Rossier BC. Synergistic 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. 2002;120:191–201. [PMC free article] [PubMed]
11. Liu L, Hering-Smith KS, Schiro FR, Hamm LL. Serine protease activity in m-1 cortical collecting duct cells. Hypertension. 2002;39:860–864. [PubMed]
12. Caldwell RA, Boucher RC, Stutts MJ. Neutrophil elastase activates near-silent epithelial Na+ channels and increases airway epithelial Na+ transport. Am.J.Physiol Lung Cell Mol.Physiol. 2005;288:L813–L819. [PubMed]
13. Harris M, Firsov D, Vuagniaux G, Stutts MJ, Rossier BC. A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes. J.Biol.Chem. 2007;282:58–64. [PubMed]
14. Caldwell RA, Boucher RC, Stutts MJ. Serine protease activation of near-silent epithelial Na+ channels. Am.J.Physiol Cell Physiol. 2004;286:C190–C194. [PubMed]
15. Hughey RP, Mueller GM, Bruns JB, Kinlough CL, Poland PA, Harkleroad KL, Carattino MD, Kleyman TR. Maturation of the epithelial Na+ channel involves proteolytic processing of the alpha- and gamma-subunits. J.Biol.Chem. 2003;278:37073–37082. [PubMed]
16. Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, Kleyman TR. Epithelial sodium channels are activated by furin-dependent proteolysis. J.Biol.Chem. 2004;279:18111–18114. [PubMed]
17. Carattino MD, Sheng S, Bruns JB, Pilewski JM, Hughey RP, Kleyman TR. The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its alpha subunit. J.Biol.Chem. 2006;281:18901–18907. [PubMed]
18. Bruns JB, Carattino MD, Sheng S, Maarouf AB, Weisz OA, Pilewski JM, Hughey RP, Kleyman TR. Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit. J.Biol.Chem. 2007;282:6153–6160. [PubMed]
19. Carattino MD, Hughey RP, Kleyman TR. Proteolytic processing of the epithelial sodium channel gamma subunit has a dominant role in channel activation. J.Biol.Chem. 2008;283:25290–25295. [PubMed] •• Most recent in a series describing the release of inhibitory peptides from ENaC subunits, and demonstrating a predominant role of γ subunit proteolysis.
20. Diakov A, Bera K, Mokrushina M, Krueger B, Korbmacher C. Cleavage in the {gamma}-subunit of the epithelial sodium channel (ENaC) plays an important role in the proteolytic activation of near-silent channels. J.Physiol. 2008;586:4587–4608. [PubMed]
21. Adebamiro A, Cheng Y, Rao US, Danahay H, Bridges RJ. A segment of gamma ENaC mediates elastase activation of Na+ transport. J.Gen.Physiol. 2007;130:611–629. [PMC free article] [PubMed]
22. Garcia-Caballero A, Dang Y, He H, Stutts MJ. ENaC proteolytic regulation by channel-activating protease 2. J Gen.Physiol. 2008;132:521–535. [PMC free article] [PubMed]
23. Harris M, Garcia-Caballero A, Stutts MJ, Firsov D, Rossier BC. Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis. J.Biol.Chem. 2008;283:7455–7463. [PMC free article] [PubMed]
24. Fejes-Toth G, Frindt G, Naray-Fejes-Toth A, Palmer LG. Epithelial Na+ channel activation and processing in mice lacking SGK1. Am.J.Physiol Renal Physiol. 2008;294:F1298–F1305. [PubMed]
25. Knight KK, Olson DR, Zhou R, Snyder PM. Liddle’s syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage. Proc.Natl.Acad.Sci.U.S.A. 2006;103:2805–2808. [PubMed]
26. Knight KK, Wentzlaff DM, Snyder PM. Intracellular sodium regulates proteolytic activation of the epithelial sodium channel. J Biol.Chem. 2008;283:27477–27482. [PMC free article] [PubMed]
27. Kabra R, Knight KK, Zhou R, Snyder PM. Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels. J Biol.Chem. 2008;283:6033–6039. [PubMed]
28. Ruffieux-Daidie D, Poirot O, Boulkroun S, Verrey F, Kellenberger S, Staub O. Deubiquitylation regulates activation and proteolytic cleavage of ENaC. J.Am.Soc.Nephrol. 2008;19:2170–2180. [PubMed]
29. Picard N, Eladari D, El MS, Planes C, Bourgeois S, Houillier P, Wang Q, Burnier M, Deschenes G, Knepper MA, Meneton P, Chambrey R. Defective ENaC processing and function in tissue kallikreindeficient mice. J.Biol.Chem. 2008;283:4602–4611. [PubMed] •• Paper comprehensively demonstrating the likely involvement of kallikrein in ENaC proteolysis.
30. Andreasen D, Vuagniaux G, Fowler-Jaeger N, Hummler E, Rossier BC. Activation of epithelial sodium channels by mouse channel activating proteases (mCAP) expressed in Xenopus oocytes requires catalytic activity of mCAP3 and mCAP2 but not mCAP1. J Am Soc Nephrol. 2006;17:968–976. [PubMed]
31. Vallet V, Pfister C, Loffing J, Rossier BC. Cell-Surface Expression of the Channel Activating Protease xCAP-1 Is Required for Activation of ENaC in the Xenopus Oocyte. J Am Soc Nephrol. 2002;13:588–594. [PubMed]
32. Bengrine A, Li J, Hamm LL, Awayda MS. Indirect activation of the epithelial Na+ channel by trypsin. J.Biol.Chem. 2007;282:26884–26896. [PubMed]
33. Verghese GM, Gutknecht MF, Caughey GH. Prostasin regulates epithelial monolayer function: cellspecific Gpld1-mediated secretion and functional role for GPI anchor. Am.J.Physiol Cell Physiol. 2006;291:C1258–C1270. [PMC free article] [PubMed]
34. Wakida N, Kitamura K, Tuyen DG, Maekawa A, Miyoshi T, Adachi M, Shiraishi N, Ko T, Ha V, Nonoguchi H, Tomita K. Inhibition of prostasin-induced ENaC activities by PN-1 and regulation of PN-1 expression by TGF-beta1 and aldosterone. Kidney Int. 2006;70:1432–1438. [PubMed]
35. Tuyen DG, Kitamura K, Adachi M, Miyoshi T, Wakida N, Nagano J, Nonoguchi H, Tomita K. Inhibition of prostasin expression by TGF-beta1 in renal epithelial cells. Kidney Int. 2005;67:193–200. [PubMed]
36. de SS, Leroy V, Ghzili H, Rousselot M, Nielsen S, Rossier BC, Martin PY, Feraille E. NF-kappaB inhibits sodium transport via down-regulation of SGK1 in renal collecting duct principal cells. J.Biol.Chem. 2008;283:25671–25681. [PubMed]
37. Edinger RS, Lebowitz J, Li H, Alzamora R, Wang H, Johnson JP, Hallows KR. Functional regulation of the epithelial Na+ channel by IkappaB kinase-beta occurs via phosphorylation of the ubiquitin ligase Nedd4-2. J.Biol.Chem. 2009;284:150–157. [PMC free article] [PubMed]
38. Chang CT, Hung CC, Chen YC, Yen TH, Wu MS, Yang CW, Phillips A, Tian YC. Transforming growth factor-beta1 decreases epithelial sodium channel functionality in renal collecting duct cells via a Smad4-dependent pathway. Nephrol.Dial.Transplant. 2008;23:1126–1134. [PubMed]
39. Iwashita K, Kitamura K, Narikiyo T, Adachi M, Shiraishi N, Miyoshi T, Nagano J, Tuyen DG, Nonoguchi H, Tomita K. Inhibition of prostasin secretion by serine protease inhibitors in the kidney. J.Am.Soc.Nephrol. 2003;14:11–16. [PubMed]
40. Maekawa A, Kakizoe Y, Miyoshi T, Wakida N, Ko T, Shiraishi N, Adachi M, Tomita K, Kitamura K. Camostat mesilate inhibits prostasin activity and reduces blood pressure and renal injury in salt-sensitive hypertension. J.Hypertens. 2009;27:181–189. [PubMed]
41. Masilamani S, Kim GH, Mitchell C, Wade JB, Knepper MA. Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney. J.Clin.Invest. 1999;104:R19–R23. [PMC free article] [PubMed]
42. Frindt G, Ergonul Z, Palmer LG. Surface expression of epithelial Na channel protein in rat kidney. J.Gen.Physiol. 2008;131:617–627. [PubMed] •• Paper using novel new in vivo techniques to demonstrate the in vivo importance and regulation of ENaC subunit processing.
43. Ergonul Z, Frindt G, Palmer LG. Regulation of maturation and processing of ENaC subunits in the rat kidney. Am J Physiol Renal Physiol. 2006;291:F683–F693. [PubMed]
44. Frindt G, Ergonul Z, Palmer LG. Na channel expression and activity in the medullary collecting duct of rat kidney. Am J Physiol Renal Physiol. 2007;292:F1190–F1196. [PubMed]
45. Frindt G, Masilamani S, Knepper MA, Palmer LG. Activation of epithelial Na channels during short term Na deprivation. Am J Physiol Renal Physiol. 2001;280:F112–F118. [PubMed]
46. Morimoto T, Liu W, Woda C, Carattino MD, Wei Y, Hughey RP, Apodaca G, Satlin LM, Kleyman TR. Mechanism underlying flow stimulation of sodium absorption in the mammalian collecting duct. Am J Physiol Renal Physiol. 2006;291:F663–F669. [PubMed]
47. Nesterov V, Dahlmann A, Bertog M, Korbmacher C. Trypsin can activate the epithelial sodium channel (ENaC) in microdissected mouse distal nephron. Am.J.Physiol Renal Physiol. 2008;295:F1052–F1062. [PubMed]
48. Narikiyo T, Kitamura K, Adachi M, Miyoshi T, Iwashita K, Shiraishi N, Nonoguchi H, Chen LM, Chai KX, Chao J, Tomita K. Regulation of prostasin by aldosterone in the kidney. J.Clin.Invest. 2002;109:401–408. [PMC free article] [PubMed]
49. Olivieri O, Castagna A, Guarini P, Chiecchi L, Sabaini G, Pizzolo F, Corrocher R, Righetti PG. Urinary prostasin: a candidate marker of epithelial sodium channel activation in humans. Hypertension. 2005;46:683–688. [PubMed]
50. Koda A, Wakida N, Toriyama K, Yamamoto K, Iijima H, Tomita K, Kitamura K. Urinary prostasin in humans: relationships among prostasin, aldosterone and epithelial sodium channel activity. Hypertens.Res. 2009;32:276–281. [PubMed]
51. Zhu H, Chao J, Guo D, Li K, Huang Y, Hawkins K, Wright N, Stallmann-Jorgensen I, Yan W, Harshfield GA, Dong Y. Urinary prostasin: a possible biomarker for renal pressure natriuresis in black adolescents. Pediatr.Res. 2009;65:443–446. [PMC free article] [PubMed]
52. Zhu H, Guo D, Li K, Yan W, Tan Y, Wang X, Treiber FA, Chao J, Snieder H, Dong Y. Prostasin: a possible candidate gene for human hypertension. Am.J.Hypertens. 2008;21:1028–1033. [PMC free article] [PubMed]
53. Passero CJ, Mueller GM, Rondon-Berrios H, Tofovic SP, Hughey RP, Kleyman TR. Plasmin activates epithelial Na+ channels by cleaving the gamma subunit. J.Biol.Chem. 2008;283:36586–36591. [PMC free article] [PubMed]
54. Svenningsen P, Bistrup C, Friis UG, Bertog M, Haerteis S, Krueger B, Stubbe J, Jensen ON, Thiesson HC, Uhrenholt TR, Jespersen B, Jensen BL, Korbmacher C, Skott O. Plasmin in nephrotic urine activates the epithelial sodium channel. J.Am.Soc.Nephrol. 2009;20:299–310. [PubMed] †† Paper demonstrating the likely activation of ENaC by plasmin proteolysis of ENaC in nephrotic syndrome..
55. Butterworth MB, Weisz OA, Johnson JP. Some assembly required: putting the epithelial sodium channel together. J Biol.Chem. 2008;283:35305–35309. %19. [PMC free article] [PubMed]
56. Malik B, Price SR, Mitch WE, Yue Q, Eaton DC. Regulation of epithelial sodium channels by the ubiquitin-proteasome proteolytic pathway. Am J Physiol Renal Physiol. 2006;290:F1285–F1294. [PubMed]
57. Bhalla V, Hallows KR. Mechanisms of ENaC regulation and clinical implications. J Am Soc Nephrol. 2008;19:1845–1854. [PubMed]
58. Zhou R, Patel SV, Snyder PM. Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC. J Biol.Chem. 2007;282:20207–20212. [PubMed]
59. Wang H, Traub LM, Weixel KM, Hawryluk MJ, Shah N, Edinger RS, Perry CJ, Kester L, Butterworth MB, Peters KW, Kleyman TR, Frizzell RA, Johnson JP. Clathrin-mediated endocytosis of the epithelial sodium channel. Role of epsin. J Biol.Chem. 2006;281:14129–14135. %19. [PubMed]
60. Weixel KM, Edinger RS, Kester L, Guerriero CJ, Wang H, Fang L, Kleyman TR, Welling PA, Weisz OA, Johnson JP. Phosphatidylinositol 4-phosphate 5-kinase reduces cell surface expression of the epithelial sodium channel (ENaC) in cultured collecting duct cells. J Biol.Chem. 2007;282:36534–36542. [PubMed]
61. Shi PP, Cao XR, Sweezer EM, Kinney TS, Williams NR, Husted RF, Nair R, Weiss RM, Williamson RA, Sigmund CD, Snyder PM, Staub O, Stokes JB, Yang B. Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2. Am.J.Physiol Renal Physiol. 2008;295:F462–F470. [PubMed]
62. Diakov A, Korbmacher C. A novel pathway of epithelial sodium channel activation involves a serum- and glucocorticoid-inducible kinase consensus motif in the C terminus of the channel’s alpha-subunit. J Biol.Chem. 2004;279:38134–38142. [PubMed]
63. Raikwar NS, Snyder PM, Thomas CP. An evolutionarily conserved N-terminal Sgk1 variant with enhanced stability and improved function. Am.J.Physiol Renal Physiol. 2008;295:F1440–F1448. [PubMed]
64. Wulff P, Vallon V, Huang DY, Volkl H, Yu F, Richter K, Jansen M, Schlunz M, Klingel K, Loffing J, Kauselmann G, Bosl MR, Lang F, Kuhl D. Impaired renal Na(+) retention in the sgk1-knockout mouse. J.Clin.Invest. 2002;110:1263–1268. [PMC free article] [PubMed]
65. Falin RA, Cotton CU. Acute downregulation of ENaC by EGF involves the PY motif and putative ERK phosphorylation site. J Gen.Physiol. 2007;130:313–328. [PMC free article] [PubMed]
66. Liu L, Duke BJ, Malik B, Yue Q, Eaton DC. Biphasic regulation of ENaC by TGF-{alpha} and EGF in renal epithelial cells. Am J Physiol Renal Physiol. 2009;296:F1417–F1427. [PubMed]
67. Bugaj V, Pochynyuk O, Mironova E, Vandewalle A, Medina JL, Stockand JD. Regulation of the epithelial Na+ channel by endothelin-1 in rat collecting duct. Am J Physiol Renal Physiol. 2008;295:F1063–F1070. [PubMed]
68. Soundararajan R, Melters D, Shih IC, Wang J, Pearce D. Epithelial sodium channel regulated by differential composition of a signaling complex. Proc.Natl.Acad.Sci.U.S.A. 2009;106:7804–7809. [PubMed] • Demonstration of a physical signaling complex of regulatory molecules with ENaC.
69. Bhalla V, Soundararajan R, Pao AC, Li H, Pearce D. Disinhibitory pathways for control of sodium transport: regulation of ENaC by SGK1 and GILZ. Am J Physiol Renal Physiol. 2006;291:F714–F721. [PubMed]
70. Sanchez-Perez A, Kumar S, Cook DI. GRK2 interacts with and phosphorylates Nedd4 and Nedd4-2. Biochem.Biophys.Res.Commun. 2007;359:611–615. [PubMed]
71. Pochynyuk O, Bugaj V, Stockand JD. Physiologic regulation of the epithelial sodium channel by phosphatidylinositides. Curr.Opin.Nephrol Hypertens. 2008;17:533–540. [PMC free article] [PubMed]
72. Lee IH, Dinudom A, Sanchez-Perez A, Kumar S, Cook DI. Akt mediates the effect of insulin on epithelial sodium channels by inhibiting Nedd4-2. J Biol.Chem. 2007;282:29866–29873. [PubMed]
73. Bhalla V, Oyster NM, Fitch AC, Wijngaarden MA, Neumann D, Schlattner U, Pearce D, Hallows KR. AMP-activated kinase inhibits the epithelial Na+ channel through functional regulation of the ubiquitin ligase Nedd4-2. J Biol.Chem. 2006;281:26159–26169. [PubMed]
74. Naray-Fejes-Toth A, Snyder PM, Fejes-Toth G. The kidney-specific WNK1 isoform is induced by aldosterone and stimulates epithelial sodium channel-mediated Na+ transport. Proc.Natl.Acad.Sci.U.S.A. 2004;101:17434–17439. [PubMed]
75. Butterworth MB, Edinger RS, Ovaa H, Burg D, Johnson JP, Frizzell RA. The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel. J.Biol.Chem. 2007;282:37885–37893. [PubMed]
76. Fakitsas P, Adam G, Daidie D, van Bemmelen MX, Fouladkou F, Patrignani A, Wagner U, Warth R, Camargo SM, Staub O, Verrey F. Early aldosterone-induced gene product regulates the epithelial sodium channel by deubiquitylation. J.Am.Soc.Nephrol. 2007;18:1084–1092. [PubMed]
77. Boulkroun S, Ruffieux-Daidie D, Vitagliano JJ, Poirot O, Charles RP, Lagnaz D, Firsov D, Kellenberger S, Staub O. Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3. Am.J.Physiol Renal Physiol. 2008;295:F889–F900. [PubMed]
78. Saxena SK, Kaur S. Regulation of epithelial ion channels by Rab GTPases. Biochem.Biophys.Res.Commun. 2006;351:582–587. [PubMed]
79. Martel JA, Michael D, Fejes-Toth G, Naray-Fejes-Toth A. Melanophilin, a novel aldosterone-induced gene in mouse cortical collecting duct cells. Am J Physiol Renal Physiol. 2007;293:F904–F913. [PubMed]
80. Hill WG, Butterworth MB, Wang H, Edinger RS, Lebowitz J, Peters KW, Frizzell RA, Johnson JP. The epithelial sodium channel (ENaC) traffics to apical membrane in lipid rafts in mouse cortical collecting duct cells. J Biol.Chem. 2007;282:37402–37411. [PubMed]
81. Zhang W, Xia X, Reisenauer MR, Hemenway CS, Kone BC. Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner. J.Biol.Chem. 2006;281:18059–18068. [PMC free article] [PubMed]
82. Zhang W, Xia X, Reisenauer MR, Rieg T, Lang F, Kuhl D, Vallon V, Kone BC. Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha. J.Clin.Invest. 2007;117:773–783. [PMC free article] [PubMed]
83. Zhang D, Yu ZY, Cruz P, Kong Q, Li S, Kone BC. Epigenetics and the control of epithelial sodium channel expression in collecting duct. Kidney Int. 2009;75:260–267. [PMC free article] [PubMed]
84. Naray-Fejes-Toth A, Boyd C, Fejes-Toth G. Regulation of epithelial sodium transport by promyelocytic leukemia zinc finger protein. Am.J.Physiol Renal Physiol. 2008;295:F18–F26. [PubMed]
85. Jasti J, Furukawa H, Gonzales EB, Gouaux E. Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. Nature. 2007;449:316–323. %20. [PubMed]
86. Stockand JD, Staruschenko A, Pochynyuk O, Booth RE, Silverthorn DU. Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure. IUBMB.Life. 2008;60:620–628. [PubMed]
87. Maarouf AB, Sheng N, Chen J, Winarski KL, Okumura S, Carattino MD, Boyd CR, Kleyman TR, Sheng S. Novel determinants of epithelial sodium channel gating within extracellular thumb domains. J Biol.Chem. 2009;284:7756–7765. %20. [PMC free article] [PubMed]