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The Proceedings of the American Thoracic Society 1:4-9 (2004)
© 2004 The American Thoracic Society

The Epithelial Sodium Channel

Activation by Membrane-Bound Serine Proteases

Bernard C. Rossier

Departments of Pharmacology and of Toxicology, University of Lausanne, Lausanne, Switzerland

Correspondence and requests for reprints should be addressed to Bernard C. Rossier, M.D., Bugnon 27, CH-1005-Lausanne, Switzerland. E-mail: Bernard.Rossier{at}ipharm.unil.ch


   ABSTRACT
TOP
 ABSTRACT
ENAC IS A LIMITING...
 ENaC AND ITS ACTIVATION...
 CONCLUSIONS AND PERSPECTIVES
 REFERENCES
 
The epithelial sodium channel (ENaC) was cloned just 10 years ago. Since that time, the study of human monogenic diseases (pseudohypoaldosteronism type 1 [PHA-1] and Liddle syndrome), as well as mouse models mimicking salt-losing syndromes (PHA-1) or salt-sensitive hypertension (Liddle syndrome), have greatly contributed to our understanding of the function of ENaC in vivo. In this brief review, I will first discuss ENaC as a limiting factor in the control of ionic composition of the extracellular fluid and then, more specifically, the activation of ENaC by membrane-bound serine proteases. Recent in vitro and in vivo experiments indicate that membrane-bound serine proteases (channel activating proteases [CAP-1, -2, or-3]) may be of critical importance in the activation of ENaC in different organs, such as the kidney, the lung or the cochlea.


   ENAC IS A LIMITING FACTOR IN THE CONTROL OF IONIC COMPOSITION OF THE EXTRACELLULAR FLUID
TOP
 ABSTRACT
 ENAC IS A LIMITING...
ENaC AND ITS ACTIVATION...
 CONCLUSIONS AND PERSPECTIVES
 REFERENCES
 
In aldosterone-responsive epithelial cells (kidney, colon), the epithelial sodium channel (ENaC) plays a critical role in the control of sodium balance, blood volume, and blood pressure (1, 2). In glucocorticoid-responsive epithelia, as in distal lung airways, ENaC has a distinct role in controlling fluid reabsorption at the air–liquid interface, thereby determining the rate of mucociliary transport (2).

In 1994, we reported that ENaC was made of three homologous subunits ({alpha}, ß, {gamma}), sharing 30% homology at the protein level (3). The membrane topology of each subunit predicts the presence of two transmembrane domains with short cytoplasmic amino- and carboxytermini and a large extracellular loop. ENaC has been proposed to be a heterotetrameric protein ({alpha}ß{alpha}{gamma}), but octameric or nonameric structures have also been suggested (4, 5).

ENaC is characterized by a high cation selectivity (permeability to sodium/permeability to potassium over 20), a low unitary conductance (4–5 picosiemens in the presence of sodium), gating kinetics characterized by long closing and opening times and a high sensitivity to amiloride (inhibition constant: 0.1 µM). Many recent reviews address the question of the structure–function relationship and ENaC regulation (6–8).

Tissue-specific expression of ENaC is observed in sodium transporting epithelia sensitive to aldosterone. ENaC subunits are found in the aldosterone-sensitive distal nephron, in the surface epithelia of the colon, and in the duct cells of exocrine glands. ENaC is expressed in epithelial cells of nontransporting epithelia (keratinocytes and air follicles), which are mineralocorticoid-responsive but not involved in overall sodium balance. Aldosterone-dependent ENaC expression in taste buds suggests an important role in salt tasting. ENaC is expressed in distal and proximal airways, as well as in nasal mucosa. In these tissues, ENaC participates in the control of the extracellular fluid at the air–cell interface by controlling the ionic composition of the airway surface liquid and mucociliary clearance (9). The process is not under mineralocorticoid control but, rather, glucocorticoid control (10). Lung epithelial fluid transport may be important in the resolution of pulmonary edema (11)

The molecular mechanisms by which hormones regulate ENaC activity to increase the net sodium reabsorption in various epithelia can be classified in the following way: (1) change of the number of channel molecules expressed at the plasma membrane, (2) change of the open probability (Po) of the channel, and (3) change of the unitary conductance of the channel. The gating properties of ENaC are unique. ENaC is not voltage-gated. The gating properties are weakly voltage-dependent (i.e., Po tends to increase at negative membrane potentials). The Po is subject to great variability from channel to channel, even within the same patch. In A6 kidney cells (12), frog skin, (13) and rat cortical collecting ducts (14) from salt-depleted rats, the Po ranged from 0.05 to 0.9 (mean = 0.5), suggesting distinct gating modes. In the Xenopus oocyte system, it has been possible to quantitate precisely the number of channel molecules expressed at the cell surface and to measure in the same oocyte the amiloride-sensitive current generated by the expression of ENaC at the membrane (15, 16). This assay allows the measurent of an average Po in an intact cell that we have termed whole cell Po (15–17), the rapid assessment of whether a mutation in ENaC causes a change in cell surface expression and/or its Po,and the demonstration of the preferential assembly of {alpha}2ß,{gamma} channels. Maximal ENaC activity measured by the amiloride-sensitive inward current is observed only when {alpha}, ß, {gamma} subunits are co-injected in the Xenopus oocyte expression system. There is an excellent correlation between amiloride-sensitive inward current and the number of channel molecules expressed at the cell surface. When {alpha} subunit is injected alone (presumably {alpha}4 tetramers), only 1 or 2% of maximal activity is observed. When {alpha}ß or {alpha}{gamma} subunits are coinjected ({alpha}2ß2 or {alpha}2{gamma}2, respectively), 5 to 15% of maximal activity is recorded. ß or {gamma} subunit alone do not lead to any significant channel activity, whereas ß2{gamma}2 channels lead to a very low level of activity (2%), and only after a long incubation (6 to 7 days after injection) (18). The data indicate that the {alpha} subunit has a specific chaperone role to bring ß and {gamma} subunits to the plasma membrane, but ß and {gamma} subunits are also required to bring {alpha} subunits to the cell surface. To test whether ENaC is limiting in vivo, gene inactivation experiments have been performed and each subunit has been independently knocked out (19–21). Of these experiments, one can deduce that each subunit is limiting for survival, because a lethal phenotype is observed soon after birth in each case. There were, however, interesting, distinct phenotypes in each gene inactivation experiment. When the {alpha} subunit was inactivated, the newborn mice died early from failure of lung clearance and the inability to reabsorb fluid from the distal airspaces of the lung. According to the data obtained in vitro in the Xenopus oocyte system, the {alpha} knock-out should leave only the possibility to make ß{gamma} channels (presumably ß2{gamma}2) with very low activity (1 or 2% of maximal activity). When the {gamma} subunit was inactivated, the animals died from a severe salt-losing syndrome and lethal hyperkaliemia. Presumably, the remaining {alpha}ß channels ({alpha}2ß2 with 5 or 10% of maximal activity) were enough to prevent a severe lung clearance failure but not a lethal salt-losing syndrome. A similar situation was observed when the ß subunit was deleted ({alpha}2{gamma}2 with 5 to 10% of maximal activity). One can conclude that 5 or 10% of EnaC activity is enough to establish a normal air–liquid interface at birth but is not enough to prevent a salt-losing syndrome in the kidney. Classically, PHA-1 is characterized by: (1) early and severe manifestation of salt-wasting and dehydration; (2) life threatening hyperkaliemia and metabolic acidosis; and (3) activation of the renin angiotensin aldosterone system, associated with normal adrenal function. Plasma aldosterone concentrations are elevated, as is plasma renin activity, demonstrating the peripheral resistance of target tissues to these hormones. Treatment by the potent mineralocorticoid fludrocortisone is without effect. Salt supplementation alone is sufficient to compensate for sodium wasting. Potassium chelators are needed to treat life-threatening hyperkaliemia. The very severe lung phenotype observed in the mouse model is usually not observed in newborns affected by PHA-1. This prompted investigators to study mucociliary clearance in the lung of adult patients with PHA-1 (22). The authors were able to demonstrate a highly significantly increased mucociliary clearance in the lung of these patients, consistent with an important inhibition of sodium and chloride reabsorption in distal and proximal airways. Interestingly, a case of recessive neonatal systemic PHA-1 was reported, in which pulmonary manifestations established the diagnosis. In this case, the salt-losing syndrome was accompanied by severe and recurrent episodes of respiratory problems at birth (23). The differences between the human and the mouse lung phenotype appear therefore to be more quantitative than qualitative. The gene inactivation in the mouse demonstrates a lethal phenotype within 4 days after birth, which is independent of genetic backgrounds, suggesting no redundancy between ENaC subunits mimicking human pathophysiology in lung and kidney. One of the major limitations of these experiments is the fact that the functional consequences of ENaC inactivation in adults obviously cannot be studied. There is therefore a need for conditional tissue-specific gene targeting. Conditional gene targeting of the {alpha} ENaC gene locus was recently achieved (24). Mice carrying the {alpha} ENaC floxed allele were bred to Hoxb-7 Cre strains. Using this promoter, it was possible to inactivate {alpha} ENaC only in the collecting duct but not in the more proximal part of the aldosterone-sensitive distal nephron (25). As predicted from the Xenopus expression system, the lack of {alpha} subunit in collecting duct lead to the lack of translocation of ß and {gamma} subunits to the plasma membrane (apical membrane of cortical collecting duct [CCD] principal cells). We conclude that in vivo {alpha} subunit also has a chaperone-specific role in bringing ß and {gamma} subunits to the apical membrane. In conclusion, ENaC appears to be a major limiting factor in the control of ionic composition of the extracellular fluid, either in the kidney or in airways.


   ENaC AND ITS ACTIVATION BY MEMBRANE-BOUND SERINE PROTEASES: A NOVEL EXTRACELLULAR SIGNALING PATHWAY
TOP
 ABSTRACT
 ENAC IS A LIMITING...
 ENaC AND ITS ACTIVATION...
CONCLUSIONS AND PERSPECTIVES
 REFERENCES
 
Despite large changes in water and salt intake, the kidney is able to maintain the extracellular osmolarity and volume within narrow margins (1). Such fine control requires specific factors or hormones: among them, aldosterone and vasopressin play the key role. As discussed in the previous section, hormones or various regulatory factors could change ENaC activity by modifying the number of channels expressed at the cell surface and/or its Po and/or its unitary ionic conductance. As far as the latter effect is concerned, to date, no experimental evidence has been provided to support the idea that regulation of ENaC activity could be achieved through changes in the single channel conductance. This is probably related to the unique ionic selectivity of ENaC, not allowing any significant change without being lethal. We are therefore left with the possibility of changing the number of channel molecules and/or Po. As discussed recently (26), the experimental evidence for the specific control of the number of channel molecules by aldosterone (14, 27–30) and vasopressin is now quite strong (31). A number of cytoplasmic (sodium, pH, calcium, protein kinase A, actin filament) or external factors (self-inhibition by sodium) have been shown to affect Po in patch-clamp or in bilayer experiments (see review in [7]). The relevance of these effects to the regulation of ENaC in intact native cells remains, however, to be established. Structure–function relationship studies in the Xenopus oocyte system indicate that at least two protein domains are involved in ENaC gating. Mutation (G to S) in the aminoterminus of the ß subunit causes a severe pseudohypoaldosteronism (32). The mutation does not change the number of channels expressed at the plasma membrane, but decreases the whole-cell Po by roughly 50% (33). Another domain has been recently identified in the large putative extracellular loop, preceding the second transmembrane domain M2. Mutations identified in Caenorhabditis elegans (degenerin site) lead to a permanent opening of this mechanosensitive channel, leading to cell swelling and neuronal death (34, 35). A corresponding mutation in ENaC causes a three-fold increase in the Po, an effect that is due to a shortening of channel-closed times and an increase in the number of long openings (see review in [6]). We have recently identified a membrane-bound serine protease that acts as a channel activating protease (CAP), namely CAP-1 (36–38). This regulation defines a novel extracellular signaling pathway, which appears to be highly conserved throughout evolution from Xenopus (xCAP-1) (36) to mice (38), rats (39), and humans (40). CAP-1 activates ENaC through its extracellular serine protease activity, as evidenced by patch-clamp experiments (41). The activation of ENaC by CAP-1 can be mimicked by external addition of trypsin, showing that both CAP-1 and trypsin act via the same pathway (Figure 1) . Aprotinin, an inhibitor of serine proteases, can block this effect (36, 38). In epithelial cell lines, like the Xenopus kidney cell line A6, ENaC appears to be constitutively activated through the presence of endogenously expressed serine proteases, because basal transepithelial sodium transport cannot be further activated by addition of trypsin. However, the baseline sodium transport can be inhibited by the addition of aprotinin on the apical side of the cell, and this inhibition can be reversed by the addition of trypsin (Figure 1). In Xenopus kidney cells, up to 90% of the amiloride-sensitive electrogenic sodium transport can be blocked by aprotinin, whereas the mouse mpk-CCDC14 cell line, derived from the cortical collecting duct (42), appears to be only 50% sensitive to aprotinin (38). In human bronchial epithelial cells, it was found that aprotinin and the humanized version of aprotinin (BAY-39–9437) inhibited basal current by approximately 30%. These findings suggested that ENaC activation is achieved by either a constitutive serine protease–independent mechanism or, alternatively, that the activation depends on more than one serine protease with different sensitivities to aprotinin that acts in combination within the same cells. This latter hypothesis was recently supported by the identification of two additional CAPs, termed mCAP-2 and mCAP-3 (17). mCAP-2 is the mouse ortholog of the transmembrane serine protease (TMPRSS4) (17) and a mouse homolog of TMPRSS3 found in humans (43), whereas mCAP-1 or its human ortholog, prostasine, is a glycosylphosphatidylinositol (GPI)-anchored protein (44, 45). mCAP-2 is a type 2 oriented membrane-bound serine protease with a predicted transmembrane segment, a short aminoterminus and an extracellular carboxyterminus containing the catalytic domain with a classical catalytic triad (HDS) and with a potential proteolytic activation cleavage site (17). Interestingly, the catalytic domain is preceded by a low-density lipoprotein receptor class A domain and a group A scavenger receptor domain (17). mCAP-3 is a third-channel activating protease, identified in the same kidney cell line. It is also a predicted type 2 membrane-bound protein. The catalytic domain is preceded by two complement factor of sea-urchin embryonic bone morphogenetic protein domains and four low-density lipoprotein receptor domains. Each of these membrane-bound serine proteases is able to activate ENaC by four to seven-fold, depending on the experimental conditions; the effect is always an increase of Po (up to six-fold), without any significant change in cell surface expression (17). The mechanism of activation remains obscure. In the Xenopus oocyte expression system, cell surface expression of CAP is required for activation of ENaC (44). When the GPI-anchored consensus motif is mutated, CAP-1 is secreted and ENaC is no longer activated. Interestingly, catalytic mutants of CAP-1 significantly decreased ENaC activation but did not fully abolish the effect of xCAP-1. The data indicated the critical role of the GPI anchor in ENaC activation and suggested that catalytic and noncatalytic mechanisms were involved (44). Other Type II transmembrane serine proteases belonging to the human airway trypsin-like protease/differently expressed in squamous cell carcinoma (HAT/DESC) subfamily, the Matriptase subfamily or the Corin subfamily could also play a role, especially in some pathological states (see a recent review in [45]).



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  		Figure 1. Model amiloride-sensitive sodium transport across an epithelial cell.

 

   CONCLUSIONS AND PERSPECTIVES
TOP
 ABSTRACT
 ENAC IS A LIMITING...
 ENaC AND ITS ACTIVATION...
 CONCLUSIONS AND PERSPECTIVES
REFERENCES
 
Our present working hypothesis is shown in Figure 2 . We postulate the existence at the apical membrane of CCD cells of three membrane-bound CAPs, termed 1, 2, and 3. In the inactive state (upper panel), endogenous serine protease inhibitors are bound to one of the CAPs. CAPs 1, 2, and 3 are proenzyme catalytically inactive. ENaC is closed and sodium transport is shut down. Under the effect of CAP cascade activator(s) (termed "X") (lower panel), serine protease inhibitor is released from its binding site to CAP (1). CAP (1) would activate autocatalytically and then activate CAP (2), whereupon CAP (2) would activate CAP (3) that in turn would trigger the opening of ENaC, leading to the establishment of transepithelial sodium transport.



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  		Figure 2. Working model of ENaC activation by epithelial membrane–bound serine proteases. Upper panel: State. Lower panel: Open state. See text for explanations.

 
At present, we do not know where to place mCAP-1, mCAP-2, and mCAP-3 in this signaling cascade, and our working hypothesis is not supported by any biochemical or cell biology data. A number of intriguing questions remain therefore to be solved experimentally. Among those questions are the following:
  1. What is the molecular mechanism of ENaC activation? In other words, what is the specific substrate for each of the CAP? Is ENaC a substrate for any of the three CAPs? Recent evidence tends to support the concept that {alpha} and {gamma} ENaC are direct substrate for membrane-bound serine proteases (47)
  2. What is the nature of the endogenous serine protease inhibitors? Prostasin has been shown to be associated with serine protease inhibitors in the seminal fluid of the prostate, but the molecular identity of this protein is not yet known. Interestingly, the analysis of the transcriptome of CCD cells indicates that a number of serine protease inhibitors are expressed at a relatively high level (48).
  3. Are the three CAPs expressed biochemically in the same membrane and in close association with ENaC in kidney, colon, or lung epithelia? Immunofluorescent labeling and colocalization with ENaC have not yet been performed. Recently, studies of primary nasal epithelial cultures in Ussing chambers have revealed that inhibition of endogenous serine protease activity with aprotinin markedly decreased EnaC-mediated current and sensitized the epithelia to subsequent channel activation by exogenous trypsin. The data suggests that protease-mediated regulation of sodium absorption is a function of human airway epithelia, and prostasin is a likely candidate for this activity (49). Similar results were obtained in human bronchial epithelial cell cultures. Bikunin is a plasma proteinase inhibitor (50). BAY-39–9437 is a recombinant kunitz-type serine protease inhibitor of the human placental bikunin. This compound decreased the amiloride-sensitive short circuit current of normal or cystic fibrosis HBE cells (51). The data indicate that sodium transport in HBE cells is activated by an endogenously expressed serine protease. Of importance, the authors showed that both aprotinin and BAY-39 equally inhibited the basal sodium transport, whereas other serine proteases inhibitors (SBTI and {alpha}-1 antitrypsin) did not. The role of potent antithrombotic serine protease, such as activated protein C in the treatment of sepsis, emphasizes the importance of this kind of signaling cascade (52). The existence of an extracellular signaling transduction pathway is also postulated for the degenerins expressed in C. elegans, but the activation may be linked to the catalytic activity of the recently identified protein MEC-6 (53). MEC-6 encodes a single path membrane protein with some similarity to paraoxonases, which have been implicated in human coronary heart disease (54).
  4. What is the factor X that could trigger the signaling cascade? One interesting possibility is that sodium itself is controlling ENaC activity. In this context, the serine protease cascade would be part of a sodium-sensing mechanism. Such a sodium-sensing mechanism, also termed self-inhibition, has long been described as a phenomenon distinct from so-called feedback inhibition, which is related to intracellular sodium concentration and characterized by relatively long time courses (minutes or tens of minutes). Self-inhibition is a much faster phenomenon, with a time course of a few seconds, and is observed after a rapid increase in extracellular sodium concentration. Recently, a sodium self-inhibition of human epithelial sodium channel has been studied in the Xenopus expression system (55). Sodium self-inhibition was found to depend only on the extracellular sodium concentration. Interestingly, self-inhibition could be prevented by exposure to extracellular protease, suggesting that the mechanism had something in common with the membrane-bound signaling pathway described here.
  5. What are the hormones or factors that regulate the membrane-bound serine proteases? It has been proposed that prostasin, the human homolog of mCAP-1, is regulated by aldosterone in the kidney and in cultured cortical collecting duct cells (M1) (56), but this effect was not confirmed in another study (57).
  6. Are CAPs a limiting factors in vivo? Gene inactivation is going to be critical in order to answer these questions. Conditional gene activation of mCAP-1 has been already achieved (58). Tissue-specific inactivation (kidney, colon, airways, etc.) should soon provide more information about the role of mCAP-1 in the physiology or pathophysiology of sodium transport in epithelial cells.

mCAP-2 is the mouse ortholog of the human TMPRSS4. TMPRSS3 belongs to the same gene subfamily and, like mCAP-2, encodes a transmembrane serine protease that contains both low-density lipoprotein receptor and group A Scavenger receptor domains. TMPRSS3 is mutated in nonsyndromic autosomal recessive deafness (DFNB8–10) (59). Recently, it was shown that TMPRSS3 is expressed in the cells supporting the organ of Corti, the spiral ganglion, and the stria-vascularis. This cell expression matches perfectly the expression of ENaC subunits described by Gruender and coworker in the rat cochlea (60). In the Xenopus oocyte expression system, proteolytic processing of TMPRSS3 was associated with increased EnaC-mediated currents. In contrast, six mutants causing deafness failed to undergo proteolytic cleavage and activate ENaC. The data indicate that important signaling pathways in the inner ear are controlled by proteolytic cleavage and suggested (1) the existence of an autocatalytic processing by which TMPRSS3 would become active, and (2) that ENaC could be a a direct or indirect substrate of TMPRSS3 in the inner ear (59). Clearly, conditional gene inactivation of TMPRSS3 or TMPRSS4 should again help us in understanding the physiology or the pathophysiologic role of ENaC activation by membrane-bound serine proteases in different tissues.

mCAP3, also termed matriptase, was identified as a novel tumor-associated type 2 transmembrane serine protease that is highly expressed in epidermis, thymus, and other epithelia. Gene inactivation of matriptase has been observed to seriously compromise epidermal biofunction, leading to rapid and fatal dehydration and early death (61). The data so far published are therefore consistent with the facts that CAPs are important limiting factors in vivo, and membrane-bound serine proteases appear to display little redundancy.

In conclusion, interest in the function of membrane-bound serine proteases appears to be increasing rapidly, and many of the questions raised will likely be answered within the next few years. The membrane-bound serine proteases are obviously ideal targets for drugs: activation or inactivation of ENaC could be achieved by selective interaction with membrane-bound serine proteases or associated proteins (serine protease inhibitors or others).


   FOOTNOTES
 
Supported by Fonds National Suisse de la Recherche Scientifique grant 3100-61966.00.

(Received in original form June 19, 2003; accepted in final form October 30, 2003)


   REFERENCES
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 ABSTRACT
 ENAC IS A LIMITING...
 ENaC AND ITS ACTIVATION...
 CONCLUSIONS AND PERSPECTIVES
 REFERENCES
 

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