Mechanisms of Enac Regulation and Clinical Implications

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Mechanisms of ENaC Regulation and Clinical Implications

Vivek Bhalla* and Kenneth R. Hallows†

*Division of Nephrology, Department of Medicine, Stanford University School of Medicine, Stanford, California; and †Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

ABSTRACT ϩ ϩ The epithelial Na channel (ENaC) transports Na across tight epithelia, including the others and has two presumed mem- the distal nephron. Different paradigms of ENaC regulation include extrinsic and brane-spanning domains, a large extra- intrinsic factors that affect the expression, single-channel properties, and intracel- cellular loop, and intracellular N- and C- lular trafficking of the channel. In particular, recent discoveries highlight new termini (Figure 1).4–6 ENaC is a member findings regarding proteolytic processing, ubiquitination, and recycling of the of a gene superfamily that includes genes channel. Understanding the regulation of this channel is critical to the understand- identified in Caenorhabditis elegans ing of various clinical phenomena, including normal physiology and several dis- based on mutations that result in mech- eases of kidney and lung epithelia, such as blood pressure (BP) control, edema, and anosensation defects (mecs) or neurode- airway fluid clearance. Significant progress has been achieved in this active field of generation (degs),3,7,8 acid-sensing ion research. Although ENaC is classically thought to be a mediator of BP and volume channels (ASIC),9,10 and mechanosensi- ϩ status through Na reabsorption in the distal nephron, several studies in animal tive cation channels present in skin and models highlight important roles for ENaC in lung pathophysiology, including in on cochlear hair cells.11–15 Recent crystal- cystic fibrosis. The purpose of this review is to highlight the various modes and lographic data obtained on the related mechanisms of ENaC regulation, with a focus on more recent studies and their ASIC1 channel suggest ENaC most likely clinical implications. exists functionally as an ␣, ␤, ␥ heterotrimer at the plasma membrane (Figure J Am Soc Nephrol 19: 1845–1854, 2008. doi: 10.1681/ASN.2008020225 1)16; however, it is conceivable that ENaC subunit composition inside cells and at the plasma membrane is not nec- ϩ The epithelial Na channel (ENaC) is illustrated by naturally occurring ENaC essarily fixed.17–19 expressed at the apical plasma mem- mutations and conditions that cause in- brane in many epithelial tissues through- creased or decreased ENaC activity, such out the body, including principal cells in as Liddle syndrome, type I pseudohy- ENaC REGULATION the distal nephron of the kidney and ep- poaldosteronism (PHA-I), cystic fibrosis ithelial cells in the urinary bladder, lung (CF), and high-altitude pulmonary Given the need for rapid, dynamic airway, distal colon, and ducts of salivary edema. changes in salt and water reabsorption and sweat glands.1 In the kidney, filtered Since the cloning of ENaC,3,4 major and secretion, it is not surprising that Naϩ exits the urinary space of the collect- advances have been made in our under- ENaC is regulated by a variety of extrin- ing duct by crossing the apical plasma standing of the structure and molecular sic and intrinsic factors. The diverse membrane through ENaC, which is the characteristics of the channel. ENaC is pathways that are important in control- rate-limiting step for salt reabsorption in composed of three structurally related ling ENaC activity ultimately impinge on this segment. Working in conjunction subunits, termed ␣-, ␤-, and ␥-ENaC, ϩ ϩ with a basolateral Na /K -ATPase, this which are each 85 to 95 kD in size in their Published online ahead of print. Publication date channel regulates salt reabsorption and unmodified state; however, glycosyla- available at www.jasn.org. plays a major role in the control of total- tion, proteolytic cleavage, and other Correspondence: Dr. Kenneth R. Hallows, Renal- body salt and water homeostasis and posttranslational modifications play very Electrolyte Division, University of Pittsburgh School 1,2 of Medicine, S976.1 Scaife Hall, 3550 Terrace BP. In airway epithelial cells, ENaC ac- important roles in the regulation of Street, Pittsburgh, PA 15261. Phone: 412-648-9580; tivity is an important modulator of air- ENaC activity and expression (see next Fax: 412-383-8956; E-mail: [email protected] way surface liquid clearance. The physi- section). Each subunit shares approxi- Copyright ᮊ 2008 by the American Society of ologic importance of this channel is mately 30 to 40% sequence identity with Nephrology

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changes in [Ca2ϩ] or membrane trafficking.44 It has been proposed that large extracellular loops of ENaC function as an- tennae that are deflected by shear stress, transducing flow stimuli to the channel α gating region.39 Finally, the cytoskeleton regulates ENaC, as the COOH-terminus βγ of ␣-ENaC interacts directly with the actin cytoskeleton,45 and ENaC activity is enhanced by actin-disrupting agents or M2 M2 M1 M1 by addition of short actin filaments in vitro.46,47 C Cytosol N N PY PY Proteolytic Cleavage. There has been a recent flurry of inves- C C tigations into the regulation of ENaC activity through cleavage by intra- and Figure 1. ENaC architecture: Structural features of the epithelial sodium channel. As extracellular proteases. The first observa- predicted by Jasti et al.,16 ENaC may exist as a heterotrimer with a single ␣, ␤, and ␥ tion that ENaC is regulated by endoge- subunit. Each subunit has two membrane-spanning domains (M1 and M2) with intracel- nous serine proteases was made using lular N- and C-termini. The ␤ and ␥ subunits each contain a canonical “PY” motif in their Xenopus A6 collecting duct cells, in COOH-termini. See text for details. which it was found that the general protease inhibitor aprotinin partially blocks ϩ one or more of several functional modes lated kinase (ERK) in the case of aldoste- amiloride-sensitive Na uptake.48 Ex- of channel regulation, which include the rone,21,31,32 protein kinase A for AVP and pression cloning was used to identify regulation of channel expression/synthe- ANP regulation,30,33,34 phosphatidylino- channel-activating peptidase 1, also sis, intracellular channel trafficking, and sitol 3-kinase–dependent signaling for called prostasin, as a factor that aug- single-channel properties such as open insulin,35 and SRC family kinases in the ments ENaC currents when coexpressed 1,20 27 probability (Po). endothelin regulatory pathway. in oocytes. Similar effects are observed through addition of trypsin in mouse Extrinsic Factors Mechanical and Cytoskeletal Activity. collecting duct M-1 cells.49 Masilamani Hormonal. There is growing evidence that ENaC is a et al.17 found direct biochemical evi- Epithelial Naϩ transport is regulated by mechanosensitive channel. Mechano- dence for cleavage, showing the apparent the action of the volume-regulatory hor- sensitivity of ENaC is not unexpected molecular weight of the ␥ subunit shifts mones aldosterone,21,22 arginine vaso- given its close phylogenetic relationship from approximately 85 to approximately pressin (AVP),23,24 and atrial natriuretic to other, more recognized mechanosen- 70 kD in rats treated with aldosterone or peptide (ANP),25 as well as other hor- sitive channels such as Mecs and a low-salt diet. mones such as insulin and endothe- Degs.11,36 Indeed, ENaC subunits are ENaC activation by proteolytic cleav- lin.26,27 Increased apical targeting of found in nonepithelial tissues and medi- age was elegantly shown through patch- ENaC subunits, although induced pri- ate mechanosensation. For example, ␤- clamp studies to confer dramatic in- ␥ marily by aldosterone-induced miner- and -ENaC play an important role in creases in single-channel Po of previously alocorticoid receptor (MR) action, also mechanotransduction involving both “near-silent” channels at the plasma occur through MR-independent mecha- neurons innervating the aortic arch and membrane.50 At approximately that nisms.28 In addition, chronic AVP treat- vascular smooth muscle.37,38 time, it was reported that furin, a mem- ment upregulates distinct ENaC sub- Whether ENaC is activated by member of the pro-protein convertase family units29 while not affecting subcellular brane stretch caused by hydrostatic pres- thought to reside in the trans-Golgi net- localization of ENaC, as seen acutely sure or cell swelling remains controver- work, was important in cleaving the ␣ with second messengers of the AVP re- sial39; however, laminar shear stress subunit at two sites and the ␥ subunit at sponse.30 Hormonal regulation of ENaC induced by fluid flow under physiologi- one site in the extracellular loops occurs through receptor-mediated mod- cally relevant conditions elegantly acti- (ECL).51,52 Furin-mediated cleavage of ulation of intracellular signaling path- vates ENaC in isolated rabbit collecting ␣-ENaC activates ENaC by releasing an ways involving various kinase cascades, ducts in vivo and in ENaC-expressing inhibitory peptide from the ECL.53 such as stimulation of serum and glu- Xenopus oocytes.40 Shear force–induced Furin-dependent proteolysis of ␣-ENaC cocorticoid-regulated kinase (SGK1) or ENaC activation is mediated by an in- activates the channel by relieving Naϩ 41–43 inhibition of extracellular signal–regu- crease in Po and is not affected by self-inhibition (see Intrinsic Factors be-

1846 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1845–1854, 2008 www.jasn.org BRIEF REVIEW low), thereby increasing Naϩ transport made of multiple ENaC subunits are syn- eral studies using heterologous expres- at higher extracellular Naϩ concentra- thesized and modified along the biosyn- sion suggested that ENaC is sorted to ly- tions.54 Recently, it was also shown that thetic pathway en route to the apical sosomes for degradation, but other prostasin, likely present in the extracellu- plasma membrane.51,52,61,62 ENaC is re- studies of endogenous ENaC have dem- lar apical compartment, also cleaves trieved from the plasma membrane in onstrated only proteasomal degrada- ␥-ENaC at a site in the ECL near furin clathrin-coated vesicles63,64 and may tion.80–82 cleavage sites, which as with the dual then be shuttled to the lysosome for deg- furin cleavage sites in ␣-ENaC, releases radation or recycled back to the plasma Nedd4-2 and ENaC Ubiquitination/De- an inhibitory peptide that further acti- membrane.65 ubiquitination. vates ENaC.55 Proteolysis of the channel Only a minority of ENaC channels re- Liddle syndrome is caused by one of sev- per se is not strictly necessary for activa- side in the apical plasma membrane,66–68 eral mutations in the COOH-terminal tion, because deletion of these inhibitory but ENaC surface expression may be rap- tails of ␤-or␥-ENaC83 and is responsible tracts in ␣ and ␥ subunits is sufficient to idly increased. Stimulation of apical for gain-of-function of these channels enhance channel activity without any ENaC by cAMP is demonstrable in (see the Clinical Correlations section). cleavage. Moreover, addition of the in- cells,66 but the origin of these channels is These mutations uniformly decrease inhibitory peptides to wild-type ENaC still controversial. Lu et al.,67 using live- teraction with the ubiquitin-protein li- confers inhibition of the channel.53,56 cell imaging and immunocytochemistry gase Nedd4-2.84 Several recent discover- Both processed (complex glycosylated with overexpressed ENaC in polarized ies regarding Nedd4-2 and ENaC are with cleaved subunits; active) and non- MDCK cells, posited that a large pool of noteworthy: Nedd4-2 ubiquitinates sur- processed (noncleaved; inactive) forms of synthesized, intact channels rest in a sub- face ENaC,85 this ubiquitination is neces- ENaC subunits exist in cells and tissues apical compartment available for rapid sary but not sufficient for endocytosis of expressing endogenous ENaC.17,51,57 In- transit to the apical membrane. Con- the channel,85,86 disruption of this inter- active, near-silent channels at the cell versely, an elegant study by Butterworth action primarily affects internalization of surface are probably uncleaved and may et al.,66 examining endogenous channels the channel,67 and ubiquitination of the serve as a channel pool that can be acti- in mouse polarized cortical collecting channel marks it for degradation.80,85 As vated by extracellular proteases. Recent duct (CCD) cells, argued that a recycling a potent inhibitor of sodium transport, evidence suggests the extent of ENaC pool of channels is responsible for rapid the cellular pathways that affect ENaC ϩ proteolysis is dependent on channel res- replenishment of Na transport. Several through regulation of Nedd4-2 are also idency time in the plasma membrane, other studies also addressed apical inser- well documented. Three primary mech- because channels with mutations that tion of ENaC. A small portion of ENaC anisms include phosphorylation, feed- ϩ block ubiquitin-dependent endocytosis channels in CCD cells are resident within back inhibition by intracellular Na ,87 remain on the surface longer, have an in- lipid rafts, and this pool may be part of and dietary sodium intake (likely through creased proportion of mature (cleaved) the biosynthetic pathway.69 In addition, aldosterone).88 subunits, and have a correspondingly heat-shock proteins, known to promote ENaC is mono- or polyubiquitinated Ϫ higher activity.58 At least another level of apical expression of the CFTR Cl chan- (linked chains of ubiquitin) at multiple regulation exists as a result of the pres- nel, stimulate plasma membrane insertion residues.82,85 Interpretation of various ence of endogenous protease inhibitors, of ENaC.70,71 Similarly, other components studies is complicated because some are such as protease nexin 1.59 Indeed, recent of the biosynthetic pathway that alter bio- performed using cultured cells that en- studies suggest the proteolytic state of genesis of ENaC have now been identi- dogenously express ENaC or are modi- ENaC subunits reflect a net balance be- fied.72,73 fied to overexpress the channel. A recent tween the expression of activating pro- Surface ENaC is internalized, but its study by Weimuth et al.82 suggested teases and protease inhibitors.60 ENaC paths and modes of degradation are still ENaC, similar to other membrane pro- regulation by proteases and protease in- not clear. In CCD cells, ENaC is endocy- teins, is monoubiquitinated at the cell hibitors is likely to be physiologically sig- tosed through clathrin-coated pits into surface, which targets ENaC for lysoso- nificant in several organs, including lung early endosomes within minutes.64 This mal degradation.65 Conversely, polyu- and kidney. Of note, this balance may process is dependent on ENaC binding biquitinated ENaC may be derived from play an important role in the pathogene- to epsin through phosphoinositides at the large proportion of synthesized sis of CF lung disease (see the Clinical the plasma membrane.74 Overexpressed channels that never reach the apical Correlations section). ENaC has also been localized separately membrane and are targeted for proteo- to recycling endosomes and late endo- somal degradation. Biochemical studies Intrinsic Factors somes.67 Saxena et al.75–79 contended that to date have not been able to discern ENaC Trafficking. ENaC is present in multiple endocytic whether either of these modes of regula- Several new communications shed light compartments, which are coordinated tion can distinguish between mature or on how ENaC traffics to and from the by sorting machinery, including syntaxin immature ENaC (i.e., proteolytically plasma membrane. Composite channels 1A as well as several Rab GTPases. Sev- cleaved or uncleaved channels) and

J Am Soc Nephrol 19: 1845–1854, 2008 Mechanisms of ENaC Regulation 1847 BRIEF REVIEW www.jasn.org whether ubiquitin ligases other than ing ENaC activity.87,96 Nedd4-mediated rectly phosphorylates and stimulates Nedd4-2 are involved. ubiquitination, altered in the Liddle phe- ␣-ENaC.109 The G-protein–coupled re- Further evidence for the importance notype, mediates this effect.97 Thus, ceptor kinase GRK2, which is implicated in of ENaC ubiquitination comes from acute changes in [Naϩ] affect open prob- cases of human hypertension, disrupts two studies that highlighted reversal of ability, whereas chronic changes in Nedd4-2–ENaC interaction by direct the ubiquitination process (de-ubiq- [Naϩ] affect trafficking. phosphorylation of the COOH-terminal ϩ uitination) as another mode of ENaC Both self-inhibition and Na feed- tail of ␤-ENaC.87 regulation. In CCD cells, Usp-45, a de- back are likely important physiologically Kinases that indirectly regulate ENaC ubiquitinating enzyme, is potently up- in limiting Naϩ absorption under condi- trafficking are also noteworthy. I␬B regulated by aldosterone and reverses the tions of high salt delivery, thereby miti- kinase-␤ interacts with ENaC to upregu- inhibitory effects of Nedd4-2 on ENaC gating large changes in intracellular late cell surface expression and Naϩ cur- ϩ retrieval.89 UCH-L3 is also identified in [Na ] and epithelial cell volume.98,99 In rent in CCD cells.110 With No lysines [K] CCD cells to de-ubiquitinate ENaC and addition, Kellenberger et al.96 described (WNK1) was recently demonstrated to enhance rapid recycling of ENaC to the that the effects of Liddle mutants on alter SGK1 activity and hence stimulate apical plasma membrane.90 UCH-L3 is ENaC trafficking occur only in the pres- ENaC. Akt (similar to SGK1) also dis- localized to early endosomes, arguing ence of high Naϩ influx; that is, wild- rupts ENaC–Nedd4-2 binding and that it plays a direct role in modifying type and Liddle mutant channels had hence stimulates ENaC-mediated Naϩ Nedd4-2–dependent, ubiquitinated equivalent ENaC activity when incu- current in response to insulin.111 Protein ENaC upon retrieval from the plasma bated in a low-Naϩ bath. This finding kinase A was shown previously to inhibit membrane. could explain why patients with Liddle Nedd4-2112 (similar to SGK1) but has syndrome lack respiratory manifesta- also now been suggested to modulate ty- Naϩ Self-Inhibition and Feedback Inhibi- tions, because broad ranges in luminal rosine kinase activity to increase apical ϩ tion. airway [Na ] are not as evident as in the expression of ENaC.113 Protein kinase D ENaC activity is regulated by changes in distal nephron. also rapidly promotes insertion of ENaC both extracellular and intracellular into the plasma membrane.114 The met- [Naϩ]. A fast change (over seconds) in Phosphorylation. abolic sensor AMP-activated kinase channel activity as a result of changes in Phosphorylation of ENaC was first de- (AMPK), which is sensitive to a host of ϩ ϩ extracellular [Na ] is known as Na self- scribed by Shimkets et al.100 as a possible external stimuli and intracellular energy inhibition.91 This process is steeply tem- mode of its hormone-mediated regula- stores, indirectly inhibits ENaC surface perature dependent, and treatment with tion. Aldosterone- and insulin-mediated expression by promoting an interaction external trypsin blunts the self-inhibi- regulation of ENaC occurs through between Nedd4-2 and ENaC.115,116 Inter- tion effect, suggesting intrinsic channel SGK1-mediated inhibition of Nedd4- estingly, phosphatidylinositol 3-kinase in- ϩ cleavage relieves Na self-inhibition.92 2.31,101 Since these initial discoveries, a fluences both ENaC trafficking (through 93 117 Additional work by Sheng et al. identi- complex array of kinases has been shown SGK1 and/or Akt) and Po. fied key ECL histidine residues that mod- to regulate ENaC directly and indirectly. These myriad signaling pathways ulate Naϩ self-inhibition, which are in- For the purposes of this review, only re- demonstrate a complex regulatory net- terestingly located near furin cleavage cent discoveries are discussed. work that directly and indirectly influ- ␣ ␥ sites in the and subunits of ENaC (see ERK2 and casein kinase 2 (CK2) di- ences ENaC surface expression and Po. Intrinsic Factors above). Noncleaved rectly phosphorylate the COOH-termini Such mechanisms are likely operative in ␤ ␥ channels have a low intrinsic Po that re- of - and -ENaC and promote Nedd4-2– acute, subacute, and chronic changes in sults from enhanced channel inhibition mediated ENaC internalization.102,103 Pro- ENaC regulation. Further study is war- ϩ by external Na .54 tein kinase C (PKC), which may be stimu- ranted to determine how many of these Feedback inhibition, a slower change lated through extracellular purinergic pathways alter Naϩ transport in vivo.

(over hours) in channel activity caused receptor activation, inhibits both ENaC Po by increased intracellular [Naϩ], was first and surface expression by signaling Metabolic Depletion and pH. described in 1961 by MacRobbie and through the mitogen-activated protein ki- It has long been recognized that inhibi- Ussing.94 Epithelial cells exposed to inhib- nase/ERK pathway.104,105 A recently dis- tion of cellular metabolism through de- ϩ ϩ itors of the Na /K -ATPase did not swell covered functional polymorphism in the pletion of metabolic substrates118–121 ϩ ϩ from continued Na influx. Recent inves- COOH-terminal tail of human ␣-ENaC and hypoxia122,123 inhibits apical Na tigations demonstrated that increases in also prompted investigation of its regula- channel activity in epithelia, but the un- Naϩ influx leading to elevated intracellular tion by kinase-mediated changes in ENaC derlying mechanisms involved in this in- ϩ [Na ] indirectly downregulate ENaC trafficking.106 The COOH-terminal tail of hibition are unclear. Several ion trans- activity.95 Gain-of-function Liddle mu- ␣-ENaC is regulated by CK1 or PKC-␦ to port proteins are downregulated by tations in ENaC may operate by decreas- increase insertion of channels in the depletion of energy substrates, particu- ing feedback inhibition, thereby enhanc- plasma membrane.107,108 SGK1 also di- larly ATP. The metabolic sensor AMPK is

1848 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1845–1854, 2008 www.jasn.org BRIEF REVIEW implicated in several of these transport Kidney. with a lack of hypertension among Afri- proteins, reviewed elsewhere.124 We ENaC gain-of-function mutations in pa- can-American individuals.106 The amino propose that AMPK plays an important tients with Liddle syndrome lead to vol- acid substitution may be a site for threo- role in this ENaC-metabolism coupling ume expansion, hypertension, hypo- nine phosphorylation by either PKC-␦ or process and represents another mode kalemia, low aldosterone levels, and CK1107,108 to regulate the intracellular traf- of intrinsic regulation of the channel. In- metabolic alkalosis.83,129–131 ENaC loss- ficking of the channel. Despite these at- tracellular acidification also inhibits of-function mutations are found in pa- tempts to correlate the molecular mecha- ␣-ENaC, and intracellular alkalinization tients with PHA-I, a disorder character- nisms of a given SNP with BP, different stimulates ENaC activity through an in- ized by volume depletion, hypotension, investigators have reached differing con- 125,126 132–134 crease in Po. Hypoxia also has direct and hyperkalemia. Recent molecular clusions about the significance of this mu- effects on expression of ENaC subunits and studies also implicated ENaC in PHA-II, tation, demonstrating the difficulty in activity in rodent airway epithelial but this has not been confirmed in vivo.135 proving a cause-and-effect relationship in cells.127,128 Further clinical correlations of The importance of ENaC in the genesis ENaC polymorphisms for essential hyper- these phenomena are warranted to ascer- of essential hypertension has also been ex- tension. tain their physiologic or pathophysiologic plored in subsets of the population. Several Pathophysiologic implications of ENaC importance. A schematic working model studies showed single-nucleotide poly- trafficking in diabetes and fluid overload of the regulation of ENaC by various sig- morphisms (SNP) and sensitivity to amilo- states were examined recently in animal naling pathways is shown in Figure 2. ride associated with hypertension among models. Insulin increases apical targeting African-American individuals136–139 (re- of distinct ENaC subunits after acute Clinical Correlations viewed elsewhere140). The most detailed treatment. Chronic hyperinsulinemia is The importance of ENaC is underscored correlation between SNP and ENaC func- associated with antinatriuresis, but in- by continued discoveries of the relevance tion is the Thr663Ala polymorphism in creased ENaC activity in models of type of ENaC activity in renal, gastrointestinal, ␣-ENaC. This variant is more common II diabetes has not yet been demon- and pulmonary physiology (Figure 3). among white individuals and segregates strated.141 Two models of hyperinsulinemia and hypertension in mice reveal a role for insulin-dependent SGK1 activation and ENaC-mediated sodium re- Proteolytic EXTRINSIC FACTORS 142,143 cleavage absorption. Increased apical target- Mechanosensation ing of ENaC subunits is seen in sodium avid states such as certain models of ne- α phrotic syndrome or cirrhosis.144–146 β γ Hormonal

Ub N C N Ub Ub Ub Ub C C P-Ser PULMONARY Ubiquitination/ – Neonatal Respiratory De-Ub P-Thr Distress Syndrome – Cystic Fibrosis ARDS Phosphorylation High-Altitude Pulmonary Edema + GASTROINTESTINAL Na Ulcerative Colitis Inhibition by AMP Diarrhea sodium RENAL Metabolic Hypo/Hypertension depletion Trafficking Edema Nephrotic Syndrome Cirrhosis INTRINSIC FACTORS

Figure 3. Clinical correlations of ENaC regulation. ENaC has been well characterized as a regulator of volume status and BP, but Figure 2. Modes of ENaC regulation. The various mechanisms of ENaC regulation can more recent studies have demonstrated its be divided into extrinsic and intrinsic mechanisms. Extrinsic regulation of the channel may role in edema formation and gastrointestinal be due to hormone activation, mechanical stretch, and/or proteolytic cleavage. Intrinsic and respiratory disorders, including CF, neo- regulation may be due to intracellular trafficking, ubiquitination, various kinases, sodium, natal respiratory distress syndrome, and and metabolic substrates. Several examples of each are delineated in the text. high-altitude pulmonary edema.

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Colon. its regulation is complex. Several extrin- gated ion channel. Proc Natl Acad Sci Inhibition of sodium and chloride ab- sic and intrinsic motifs are now known in USA95: 10240–10245, 1998 11. Benson CJ, Xie J, Wemmie JA, Price MP, sorption in the colon is important for this regulatory network, and, most re- Henss JM, Welsh MJ, Snyder PM: Hetero- diarrheal disorders such as CF and ul- cently, exciting discoveries in cellular multimers of DEG/ENaC subunits form cerative colitis. Decreased expression and animal models have been made in Hϩ-gated channels in mouse sensory neu- of ENaC subunits in human biopsy the proteolytic processing and intracel- rons. Proc Natl Acad Sci U S A 99: 2338– samples from patients with colitis is a lular trafficking of the channel. Although 2343, 2002 12. 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1854 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1845–1854, 2008