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The Epithelial Na⁺ Channel: Cell Surface Insertion and Retrieval in Na⁺ Homeostasis and Hypertension

Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52422

Correspondence: Address all correspondence and requests for reprints to: Peter M. Snyder, M.D., Department of Internal Medicine, University of Iowa College of Medicine, 371 EMRB, Iowa City, Iowa 52242. E-mail: psnyder{at}blue.weeg.uiowa.edu

	Abstract

Top Abstract I. Introduction II. Liddle’s Syndrome:... III. Trafficking of ENaC... IV. ENaC Polymorphisms:... V. Summary References

 
The epithelial Na⁺ channel (ENaC) forms the pathway for Na⁺ absorption in the kidney collecting duct and other epithelia. Dominant gain-of-function mutations cause Liddle’s syndrome, an inherited form of hypertension resulting from excessive renal Na⁺ absorption. Conversely, loss-of-function mutations cause pseudohypoaldosteronism type I, a disorder of salt wasting and hypotension. Thus, ENaC has a critical role in the maintenance of Na⁺ homeostasis and blood pressure control. Altered Na⁺ absorption in the lung may also contribute to the pathogenesis of cystic fibrosis. Epithelial Na⁺ absorption is regulated in large part by mechanisms that control the expression of ENaC at the cell surface. Nedd4, a ubiquitin protein ligase, binds to ENaC and targets the channel for endocytosis and degradation. Liddle’s syndrome mutations disrupt the interaction between ENaC and Nedd4, resulting in an increase in the number of ENaC channels at the cell surface. Aldosterone and vasopressin also regulate Na⁺ absorption to defend against hypotension and hypovolemia. Both hormones increase the expression of ENaC at the cell surface. The goal of this review is to summarize recent data on the regulation of ENaC expression at the cell surface.

I. Introduction

A. Molecular composition of the epithelial Na⁺ channel (ENaC)

B. Biophysical characteristics

C. DEG/ENaC ion channel family

D. Regulation of ENaC expression at the cell surface

II. Liddle’s Syndrome: Inherited Hypertension Resulting from Defective Internalization and Degradation of ENaC

A. Liddle’s syndrome mutations increase Na⁺ current

B. Mechanism(s) of increased Na⁺ current

C. Identification of PPPxYxxL motif

D. Similarity to internalization motifs

E. PY motif

F. Nedd4 binds to PY motifs and inhibits ENaC

G. Nedd4 structure/function

H. Working model

I. Na⁺-mediated inhibition of ENaC

J. Nedd4-related proteins

III. Trafficking of ENaC to the Cell Surface

A. Pseudohypoaldosteronism type 1 (PHA)

B. Aldosterone/serum and glucocorticoid-regulated kinase (SGK)

C. Vasopressin/cAMP

D. Potential role of syntaxins in ENaC exocytosis

IV. ENaC Polymorphisms: Potential Role in ENaC Surface Expression and Hypertension

A. ß_T594M polymorphism

B. Additional polymorphisms

V. Summary

	I. Introduction

Top Abstract I. Introduction II. Liddle’s Syndrome:... III. Trafficking of ENaC... IV. ENaC Polymorphisms:... V. Summary References

 
THE EPITHELIAL Na⁺ channel (ENaC) functions in the movement of Na⁺ across epithelial cells, a process of critical importance in a variety of tissues. For example, in the kidney collecting duct, regulated Na⁺ absorption functions in extracellular Na⁺ and volume homeostasis, and hence, in the control of blood pressure. Diseases that cause excessive Na⁺ absorption result in hypertension, whereas inadequate Na⁺ absorption causes hypotension (reviewed in Refs. 1 and 2). In the lung, Na⁺ absorption is required at the time of birth to convert the lung from a fluid-filled to an air-filled organ (3). After birth, Na⁺ absorption in the lung controls the quantity and Na⁺ content of the surface liquid that baths the apical membrane of the airway. This may play an important role in clearing airway secretions and in the function of naturally occurring airway antibiotics, including defensins (4). Excessive Na⁺ current in the airway may therefore contribute to the pathogenesis of cystic fibrosis (5).

ENaC is an integral component of the pathway for Na⁺ absorption. Epithelial cells are polarized, with an apical membrane that faces the lumen (urine in the case of the kidney) and basolateral membrane that faces the blood (Fig. 1A). The membranes are separated by tight junctions that limit the flow of ions and water between the cells. Na⁺ absorption therefore results from the movement of Na⁺ across the cell membranes in a two-step process. At the apical membrane, Na⁺ enters the cell through the pore of ENaC, moving down its electrochemical gradient (Fig. 1A and Refs. 6 and 7). Na⁺ entry is the rate-limiting step for Na⁺ absorption. At the basolateral membrane, Na⁺ is pumped out of the cell by the Na⁺-K⁺-ATPase. As a result, Na⁺ is absorbed from the lumen (e.g., urine) into the blood.

View larger version (16K): [in this window] [in a new window]   Figure 1. ENaC forms the pathway for epithelial Na+ absorption. A, Model of epithelial cells. ENaC is expressed at the apical membrane, where it forms the pathway for Na+ in the lumen to enter the cell. At the basolateral membrane, Na+ is pumped out of the cell by the Na+-K+-ATPase. B, Membrane topology of the ENaC subunits (, ß, and ). Each has two membrane-spanning segments, a large glycosylated extracellular domain, and cytoplasmic N and C termini. The PY motif and residues involved in selectivity, gating, and ubiquitination are indicated.

A. Molecular composition of the epithelial Na⁺ channel (ENaC)
ENaC is composed of three subunits (-, ß-, and ENaC) that share 30–35% sequence identity (Fig. 1A and Refs. 14, 15, 16, 17, 18, 19). The -subunit was cloned by virtue of its ability to generate a small Na⁺ current when expressed in Xenopus oocytes (14, 15). In contrast to ENaC, the ß- and -subunits do not form functional Na⁺ channels when expressed alone or in combination. However, ß- and ENaC greatly potentiated current when coexpressed with ENaC (16, 18). This results at least in part from an increase in ENaC expression at the cell surface (20), suggesting that all three subunits are required for the efficient synthesis, assembly, and trafficking of the channel complex. Thus, the functional ENaC channel contains all three subunits. A fourth subunit, ENaC, can substitute functionally for ENaC, although the physiological role of this subunit is not yet known (21).

The ENaC subunits have two membrane-spanning segments, cytoplasmic N and C termini, and a large glycosylated extracellular domain (Fig. 1B and Refs. 22, 23, 24). Fourteen conserved cysteines are thought to be important in the tertiary structure of this extracellular domain. Although it is clear that the -, ß-, and -subunits each contribute to the channel complex, the total number of subunits and stoichiometry remain uncertain. Functional and biochemical data have been consistent with either a complex of four subunits [₂, ß₁, ₁ (25, 26)] or nine subunits [₃, ß₃, ₃ (27)]. Recently, low-resolution images of channel particles were provided by freeze-fracture electron microscopy. The size and overall geometry of the complex suggested a channel of eight to nine subunits (28). Definitive determination will require higher-resolution structural information. Thus, the crystallization and x-ray analysis of ENaC is an important future goal. However, such work is complicated by the presence of a large cysteine-rich extracellular domain and the lack of a known related bacterial channel (to facilitate protein production).

B. Biophysical characteristics
ENaC has several defining characteristics (reviewed in Refs. 7 and 29). First, in contrast to the voltage-gated Na⁺ channels of muscle and neurons, ENaC is voltage independent; changes in membrane potential have minimal effect on ENaC Na⁺ currents. Second, ENaC is exquisitely selective for Na⁺ over K⁺; Na⁺ ions permeate the channel, but K⁺ does not. The consequence is a channel that allows Na⁺ to enter the cell by moving down its electrochemical gradient but does not allow K⁺ to leak out of the cell. This property is critical for the Na⁺-absorptive function of ENaC in epithelia. In addition, the channel is slightly more permeable to Li⁺ than to Na⁺. The selectivity properties of a channel are determined by the amino acids that line the channel pore. In ENaC, the pore is formed by residues from each of the three subunits (30, 31). The selectivity filter that discriminates between different cations is formed by a sequence motif (G/S-X-S) at the extracellular end of the second membrane-spanning segment (Fig. 1B and Refs. 30, 31, 32, 33, 34). Additional residues in this segment may also modulate ion conductance (35).

Third, ENaC is blocked by the diuretics amiloride (and amiloride analogs) and triamterene. Amiloride block was disrupted by mutation of a specific pore residue (30) or mutations in a segment of the extracellular domain (36). These residues may contribute to the binding site(s) for amiloride.

Fourth, ENaC opens and closes ("gates") with very slow kinetics, and the probability of the channel being open (P_O) varies widely between individual ENaC channels (37). Little is known about the mechanisms that underlie this heterogeneity in gating, or about the mechanisms that control channel gating. However, two channel segments appear to be important (Fig. 1B). Within the cytoplasmic N terminus, mutation of specific residues results in channels that are nearly always closed [low P_O (38, 39)]. Interestingly, a mutation in this domain causes PHA (12), consistent with an important functional role in vivo. A domain just extracellular to the second membrane-spanning segment is also involved in channel gating. In contrast to the N terminus, mutation of residues in this "DEG" domain increase Na⁺ current by locking the channel in a high P_O state (40, 41). These mutations introduce a bulky side chain, suggesting that steric hindrance might interfere with channel closing (amino acids with small side chains do not increase current). The mutations were named because, in related ion channels from Caenorhabditis elegans, an equivalent mutation causes swelling neurodegeneration (e.g., DEG-1, MEC-4, and MEC-10; Ref. 42). Interestingly, in ENaC, a mutation in this domain was identified in a patient with hypertension and renal disease (43). When expressed in heterologous cells, this mutation produced a large increase in Na⁺ current [compared with wild-type ENaC (40)]. However, the role of this mutation in hypertension is not yet known.

C. DEG/ENaC ion channel family
ENaC is related to ion channels with diverse functions (reviewed in Refs. 44 and 45). Members of this DEG/ENaC family are thought to function as receptors for taste, touch, and acidic pH. For example, mutations in MEC-4 and MEC-10 caused a defect in touch sensitivity in C. elegans (46, 47). Likewise, targeted disruption of BNC1 (48) [also known as ASIC2 (45), and BNaC1 (49)], a mammalian neuronal family member, caused a defect in a specific form of touch sensation in the mouse (50). BNC1 and the related channels ASIC (51) [also known as ASIC1 (45)], and BNaC2 (49) and DRASIC (52) [also known as ASIC3 (45)], are transiently activated by acidic pH, suggesting that these channels might be involved in nociception.

Could ENaC have functions in addition to Na⁺ absorption? In support of this hypothesis, ENaC is expressed in taste cells of the tongue, where it is thought to be a receptor for salt taste (53, 54, 55). ENaC subunits are also expressed in nonepithelial cells. For example, ENaC is expressed in baroreceptor neurons that innervate the carotid sinus and aortic arch (56). Interestingly, the baroreceptor reflex was inhibited by benzamil, a blocker of ENaC, suggesting that ENaC might be a component of the baroreceptor mechanotransducer (56). In addition, ß- and ENaC are expressed in specialized cutaneous sensory neurons (57). These findings suggest the possibility that ß- and ENaC function in mechanosensation, similar to related DEG/ENaC ion channels. Consistent with this hypothesis is evidence suggesting that ENaC function is altered by mechanical stress (58, 59, 60, 61). ENaC subunits are also expressed in B lymphocytes, where they have been proposed to generate amiloride-sensitive Na⁺ currents (62, 63, 64, 65, 66), and in skin keratinocytes (67). The function of ENaC in these locations is unknown.

D. Regulation of ENaC expression at the cell surface
To maintain Na⁺ homeostasis, epithelial Na⁺ absorption via ENaC must be tightly regulated. The rate of Na⁺ absorption varies widely to respond to conditions of Na⁺ deprivation and Na⁺ excess. Two hormones are primarily responsible, aldosterone and vasopressin, both of which increase renal Na⁺ absorption (7, 29).

Ion channels can be regulated by two fundamental mechanisms, changes in channel gating (P_O) or changes in the number of channels at the cell surface. Both mechanisms participate in the regulation of ENaC. Recent data indicate that mechanisms that control ENaC surface expression are critically important for the regulation of epithelial Na⁺ absorption. Two basic properties of ENaC hinted at such a possibility. First, in contrast with voltage-gated and ligand-gated channels, which require a stimulus for channel activity, ENaC is active in the absence of a known stimulus. When studied in native epithelia or expressed in heterologous cells, ENaC is constitutively active (16, 18, 68, 69). Second, epithelial Na⁺ absorption is regulated over a relatively slow time scale of minutes to hours, compatible with mechanisms that alter the expression of a protein at the cell surface (7, 29). This contrasts with the millisecond time scale of regulation for ion channels that participate in neural transmission and myocyte contraction (70). Most importantly, the investigation of inherited diseases confirmed the critical importance of mechanisms that control ENaC surface expression; ENaC mutations that alter surface expression result in abnormalities of Na⁺ homeostasis and blood pressure (20, 71, 72).

The number of ENaC channels at the cell surface is the net result of the movement of channels to the cell surface (synthesis, vesicle trafficking, and exocytosis) and the removal of channels from the cell surface [endocytosis and degradation (Fig. 2)]. This review will focus on the mechanisms that regulate the movement of ENaC to and from the cell surface, and how this regulation is disrupted by disease-causing mutations.

View larger version (33K): [in this window] [in a new window]   Figure 2. Regulation of ENaC surface expression. Epithelial Na+ absorption is controlled in part by the number of ENaC channels at the cell surface. This is the net result of channel synthesis and exocytosis, which are stimulated by aldosterone and vasopressin, and endocytosis and degradation, which are modulated by Nedd4.

	II. Liddle’s Syndrome: Inherited Hypertension Resulting from Defective Internalization and Degradation of ENaC

Top Abstract I. Introduction II. Liddle’s Syndrome:... III. Trafficking of ENaC... IV. ENaC Polymorphisms:... V. Summary References

View larger version (21K): [in this window] [in a new window]   Figure 3. C-terminal PPPxYxxL motif. Plot of amiloride-sensitive Na+ current (mean ± SEM, relative to wild type) when - and hENaC were coexpressed with the indicated ßENaC subunits in Xenopus oocytes. Schematics show C-terminal segment of ßENaC and the location of the mutations are indicated. Asterisk indicates P < 0.03.

The defect in Na⁺ homeostasis was also reconstituted in a mouse model of Liddle’s syndrome. A Liddle’s syndrome mutation (R556X) was introduced into the ßENaC gene by targeted gene replacement (77). Although mice homozygous for the Liddle’s syndrome mutation were not hypertensive under basal conditions, they did have excessive Na⁺ absorption in the distal colon and low plasma aldosterone levels. In addition, the mice developed hypertension when eating a high salt diet. Thus, the Liddle’s syndrome mutation partially reproduced the human phenotype.

B. Mechanism(s) of increased Na⁺ current
The mechanism responsible for increased Na⁺ current was somewhat surprising. Most previously described gain-of-function mutations in ion channels altered channel gating. For example, deletion of the N-terminal "ball" domain in Shaker K⁺ channels increased current by abolishing N-type inactivation (78). However, when ENaC bearing a Liddle’s syndrome mutation was studied at the single-channel level in cells, no significant alteration in channel gating could be detected; the P_O was similar to wild-type ENaC (71, 75). Likewise, there was no change in the single-channel conductance, which is a measure of the amount of current passing through a single open channel. By default, this suggested a novel mechanism, that Liddle’s syndrome mutations increased the number of ENaCs at the cell surface. Consistent with this mechanism, when the mutant channel was expressed in MDCK epithelia, there was significantly more ENaC immunofluorescence at the apical cell surface than with expression of wild-type ENaC (71). This finding has been confirmed in Xenopus oocytes (20, 79). Thus, Liddle’s syndrome mutations increase Na⁺ current at least in part by increasing the number of channels at the cell surface.

These findings suggest that the C terminus of ENaC plays an important role in controlling the number of channels at the cell surface. When the C terminus of ßENaC was fused onto an irrelevant membrane protein (the human histocompatibility leukocyte antigen protein A2), a Liddle’s-associated mutation increased expression of the chimeric protein at the cell surface (71). Thus, sequences within the C terminus are sufficient to function as a signal to control surface expression.

In addition to their effect on surface expression, it is possible that Liddle’s syndrome mutations also alter the gating of ENaC. When studied in lipid bilayers, a Liddle’s syndrome mutation altered channel gating (80). Although an increase in P_O was not detected in the intact cell, the highly variable gating of ENaC makes it difficult to exclude such a possibility. Moreover, a quantitative assay suggested that an increase in surface expression did not completely account for the increase in Na⁺ current; Liddle’s syndrome mutations increased ENaC surface expression to a lesser extent than Na⁺ current in Xenopus oocytes (20). However, this assay also has inherent problems; estimates of the number of channels at the cell surface did not correlate with predictions based on whole-cell currents and the measurement of P_O (20). Based on these findings, it has been suggested that Liddle’s syndrome mutations increase both surface expression and P_O, together resulting in an increase in renal Na⁺ absorption. Further work will be required to directly test whether Liddle’s syndrome mutations alter gating in the cell, and to elucidate the potential mechanism.

C. Identification of PPPxYxxL motif
How do Liddle’s syndrome mutations increase the surface expression of ENaC? Liddle’s syndrome mutations disrupt most of the cytoplasmic C terminus of the ß- or -subunits. However, smaller deletions were sufficient to increase Na⁺ current; deletion of the last 19 amino acids of ßENaC increased Na⁺ current to the same extent as a Liddle’s syndrome mutation that deleted most of the C terminus [ß_R566X (Fig. 3 and Ref. 71)]. By testing missense mutations in this segment, a sequence was identified (PPPxYxxL) that plays an important role in controlling the surface expression of ENaC (71, 76). Mutation of the three prolines or tyrosine increased Na⁺ current similar to deletion of the C terminus (Fig. 3). Mutation of the leucine also increased current, but to a lesser extent, and mutation of the other residues in this segment (indicated by "x") did not increase current. Also consistent with its critical importance, missense mutations have been identified within this sequence in patients with Liddle’s syndrome (10, 11, 81). Thus, Liddle’s syndrome results from the mutation or deletion of this C-terminal PPPxYxxL motif.

Although the three ENaC subunits share 30–35% sequence identity, there is little sequence conservation within the C terminus. However, the PPPxYxxL sequence is completely conserved in all three subunits, suggesting that it might also play an important role in - and ENaC. Consistent with this hypothesis, mutation of the tyrosine in - or ENaC increased Na⁺ current to the same extent as the tyrosine mutation in ßENaC (71). Why does mutation of the PPPxYxxL sequence in ENaC increase Na⁺ current, but deletion of the ENaC C terminus not increase current? This discrepancy suggests the presence of additional regulatory sequences in the C terminus of ENaC. Volk et al. (82) recently found that the C terminus of ENaC was required for the regulation of ENaC by a staurosporine-sensitive kinase. When the C terminus is deleted, the loss of such a regulatory sequence might counteract the effect of the loss of the PPPxYxxL sequence. Thus, the presence of additional regulatory sequences may explain why Liddle’s syndrome mutations have been identified in ß- and -, but not in ENaC.

D. Similarity to internalization motifs
The PPPxYxxL sequence resembles two different tyrosine-based motifs that function as signals for protein internalization (71). First, it is similar to the NPxY motif and similar motifs found in proteins including the low density lipoprotein receptor (83). Deletion of this motif, or mutation of the tyrosine, prevents receptor endocytosis. In the low density lipoprotein receptor, such mutations are responsible for some kindreds of familial hypercholesterolemia (83). Second, the PPPxYxxL sequence also fits the consensus of the YxxL/hydrophobic internalization motif found in proteins including the transferrin receptor (84). Once again, mutation of the tyrosine prevents receptor internalization. Thus, in both motifs, the tyrosine residue plays a key role.

It therefore seemed possible that the PPPxYxxL sequence functions as an internalization motif, and that defective internalization in Liddle’s syndrome results in an increase in ENaC surface expression (71). To test this hypothesis, the rate of endocytosis was determined for a chimeric protein derived from A2 and the C terminus of ßENaC (85). The protein was rapidly internalized within 5–8 min. Deletion of the PPPxYxxL sequence, or mutation of the tyrosine, abolished internalization. Thus, the PPPxYxxL sequence functions as an internalization motif in the context of a chimeric protein.

Does the PPPxYxxL sequence also function as an internalization motif in ENaC? There is suggestive evidence to support this notion. Shimkets et al. (86) expressed ENaC in Xenopus oocytes, and 2 d later they treated the cells with brefeldin A to block the intracellular trafficking of newly synthesized channels. For wild-type ENaC, brefeldin A decreased Na⁺ current with a half-time of about 4 h. A Liddle’s syndrome mutation delayed the decline in current. In this assay, current decay could result from a decrease in ENaC at the cell surface (the net result of channel endocytosis, recycling back to the cell surface, and degradation) and/or time-dependent changes in channel gating. Thus, Liddle’s syndrome mutations might disrupt any one of these steps.

One mechanism for the endocytosis of cell membrane proteins is via clathrin-coated pits. This pathway can be disrupted by a dominant negative mutation in dynamin (D44K), a GTP binding protein required for clathrin-mediated endocytosis (87). Expression of D44K increased Na⁺ current when coexpressed with wild-type ENaC, but not when the channel contained a Liddle’s syndrome mutation (86). The simplest interpretation of the data is that the D44K mutant increased Na⁺ current by inhibiting ENaC endocytosis. Moreover, if endocytosis is disrupted in Liddle’s syndrome, this might explain the failure of D44K to increase current when the channel contained a Liddle’s syndrome mutation. However, a limitation is that the rate of ENaC endocytosis was not directly measured. Thus, alternative explanations for the data cannot be excluded. Future work will be required to directly determine whether Liddle’s syndrome mutations disrupt ENaC endocytosis. Such studies have been hampered by the low abundance of channels such as ENaC at the cell surface.

E. PY motif
In addition to its resemblance to internalization motifs, the PPPxYxxL sequence fits the consensus of a motif involved in protein interactions— the "PY motif" [PPxY (88)]. This suggested the possibility that ENaC surface expression might be regulated through protein interactions with the PY motifs of -, ß-, and ENaC. To identify interacting proteins, Staub et al. (89) screened a rat kidney cDNA library using the yeast two-hybrid technique. They isolated Nedd4, a member of the ubiquitin protein-ligase family. Nedd4 was previously discovered and named as a neural precursor-expressed and developmentally down-regulated protein, although its function was unknown at that time (90).

F. Nedd4 binds to PY motifs and inhibits ENaC
Nedd4 binds to the sequence in ENaC that is disrupted in Liddle’s syndrome. In addition, Nedd4 is expressed in cells that express ENaC (91). Thus, it seemed likely that the interaction between ENaC and Nedd4 might be important in the regulation of Na⁺ absorption. To test the hypothesis that Nedd4 alters ENaC Na⁺ current, rat Nedd4 was coexpressed with ENaC in Xenopus oocytes. Nedd4 produced a dose-dependent decrease in Na⁺ current, consistent with a Nedd4-mediated inhibition of ENaC (92). In subsequent studies, it was found that Xenopus and human Nedd4 also inhibit ENaC (93, 94). An important observation was that Liddle’s syndrome mutations in ENaC disrupted the inhibition of the channel by Nedd4 (92, 94). Thus, the loss of regulation by Nedd4 likely contributes to the pathogenesis of Liddle’s syndrome.

Nedd4 could inhibit ENaC by altering channel gating, single-channel conductance, or the number of channels at the cell surface. To investigate the mechanism, the patch clamp technique was used to record single-channel currents in Xenopus oocytes coexpressing ENaC and Nedd4. Nedd4 did not decrease the P_O or single-channel conductance (92). Rather, Nedd4 decreased the cell surface expression of ENaC, as demonstrated by fluorescence staining or antibody binding to ENaC at the cell surface (92, 94). Importantly, a Liddle’s syndrome mutation in the PY motif abolished the decrease in surface expression, consistent with the effect of this mutation on the inhibition of Na⁺ current (92).

Nedd4 decreased the surface expression of ENaC at least in part by increasing channel degradation. Using a pulse-chase assay in cells expressing ENaC, coexpression of Nedd4 decreased the half-life of ENaC but did not alter the rate of channel synthesis (92). In addition, inhibitors of both the proteosome and lysosomes increased the half-life of ENaC protein (95). In A6 renal epithelia, an inhibitor of the proteosome (MG-132), but not a lysosomal inhibitor, increased ENaC subunit protein as well as Na⁺ current (96). These findings emphasize the importance of channel degradation in the control of epithelial Na⁺ absorption, and implicate a role for Nedd4.

G. Nedd4 structure/function
Nedd4 is a modular protein containing multiple domains that have homology to other proteins. At the N terminus is a calcium phospholipid binding (C2) domain, and the C terminus contains a ubiquitin ligase (HECT) domain. The intervening segment contains multiple WW domains, involved in protein interactions (Fig. 4).

View larger version (43K): [in this window] [in a new window]   Figure 4. Nedd4 and related WW domain proteins. A, Schematic representations of rat (r) and hNedd4, KIAA0439, WWP2, WWP1, and AIP-4, Smurf1 and 2, and NedL1. The locations of the C2 domain, WW domains, and ubiquitin ligase domain are indicated. B, Sequence lineup of the four WW domains of human Nedd4 and the WW domain of YAP. The conserved tryptophans are highlighted.

The interaction between the WW domains and PY motifs of ENaC is required for Nedd4 to inhibit the channel. This has been confirmed by two sets of experiments. First, mutation or deletion of the ENaC PY motifs abolished the Nedd4-mediated inhibition of ENaC (92, 94). Second, inhibition was also abolished by simultaneous mutation of all four Nedd4 WW domains (101). A critical question is which WW domains bind to the PY motifs of ENaC. In vitro, multiple WW domains have the capacity to bind to all three ENaC subunits. For example, when expressed individually, each of the three rat Nedd4 WW domains bound to the PY motif of ßENaC (89). The results differed slightly in other species; WW domain 1 of mouse (102) and human Nedd4 (93) did not bind to ENaC, but the other two (mouse) or three (human) WW domains bound similar to rat Nedd4. This highlights the potential importance of species differences in the regulation of ENaC.

However, the interaction between Nedd4 and ENaC in the intact cell is likely to be complex, because Nedd4 has multiple WW domains and the ENaC channel complex has multiple PY motifs. A combined biochemical and functional approach was used to investigate which WW domains are required for the binding and inhibition of ENaC. Mutation of all four WW domains simultaneously abolished both the binding of full-length human Nedd4 (hNedd4) to one of the ENaC subunits (ENaC) and the hNedd4-mediated inhibition of ENaC [in Xenopus oocytes and Fischer rat thyroid epithelia (101)]. Mutation of a single WW domain (WW domain 3) was sufficient to reproduce the disruption of binding and inhibition, but mutations of the other WW domains (1, 2, and 4) were not. Thus, WW domain 3 is required for the inhibition of ENaC by hNedd4. However, WW domain 3 alone was not sufficient; when WW domains 1, 2, and 4 were simultaneously mutated (leaving WW domain 3 intact), hNedd4 produced only a small decrease in Na⁺ current. This suggested a functional role for WW domains 2 and/or 4. Consistent with this hypothesis, mutation of WW domain 2 or 4 (but not WW domain 1) decreased the ability of hNedd4 to inhibit ENaC, although to a much smaller extent than mutation of WW domain 3. Together, the data indicate that multiple WW domains (2, 3, and 4) are required for hNedd4 to bind to ENaC and to inhibit Na⁺ current. WW domain 3, however, appears to most critical.

Further work will be required to determine whether these WW domains can bind to the PY motifs in any of three ENaC subunits, or whether they bind to a specific subunit(s). It is also possible that WW domains bind to PY motifs in other proteins within the channel complex. This may be particularly true for WW domain 1, which does not appear to bind to ENaC. Although the interaction between WW domain 3 and ENaC was most important for the inhibition of the channel, this domain is not present in rat or mouse Nedd4 (which contain only three WW domains); based on sequence similarity, the three WW domains of rat and mouse Nedd4 correspond to WW domains 1, 2, and 4 in hNedd4 (93). Thus, the interactions between Nedd4 and ENaC may differ between species.

2. C2 domain.
The N terminus of Nedd4 contains a C2 (calcium phospholipid binding) domain (Fig. 4B). Initially described in PKC, C2 domains have subsequently been identified in a large number of proteins in which they function in the Ca²⁺ and phospholipid modulation of protein function (103, 104). This provides a potential mechanism for the well described inhibition of ENaC by increased cytosolic Ca²⁺ (7). Consistent with this notion, increased cytosolic Ca²⁺ resulted in the redistribution of Nedd4 from the cytoplasm to the apical and lateral cell surface in MDCK epithelia (105). Redistribution was abolished by deletion of the Nedd4 C2 domain. In addition, the C2 domain bound to phospholipid vesicles in a Ca²⁺-dependent manner. These findings suggested that the C2 domain functioned in the Ca²⁺-dependent localization of Nedd4 to the cell surface through the binding of the C2 domain to the plasma membrane. However, there is currently no direct evidence implicating a role for Nedd4 in the Ca²⁺-dependent regulation of ENaC.

In addition to its association with lipid membranes, the C2 domain also mediates protein interactions. A C2-glutathione S-transferase fusion protein interacted with a 35- to 40-kDa protein in MDCK lysates. This protein was identified as annexin XIIIb by mass spectrometry (106). Annexin XIIIb is localized to the apical membrane, suggesting that Nedd4 might be targeted to the apical cell surface through its interaction with this protein.

Thus, the C2 domain may play an important role in the Nedd4-mediated inhibition of ENaC. Surprisingly, deletion of the C2 (hNedd4-C2) domain did not prevent inhibition (101). Rather, the mutant inhibited ENaC more potently than wild-type hNedd4. Thus, the C2 domain is not required for hNedd4 to interact with ENaC and decrease Na⁺ current. Perhaps deletion of the C2 domain allows Nedd4 to function independently of increases in cytosolic Ca²⁺. Interestingly, some recently identified Nedd4-related proteins lack C2 domains (107, 108).

3. Ubiquitin ligase domain.
Nedd4 is a member of the ubiquitin protein-ligase family; the C terminus contains a sequence homologous to E3 protein ligases (Fig. 4B and Ref. 109). E3 domains are involved in protein ubiquitination. Ubiquitin is transferred by an E2 protein to a cysteine in the E3 domain. The ubiquitin is then transferred from the E3 domain to lysines in target proteins. Sequential addition of ubiquitin polypeptides results in polyubiquitination, a signal that targets proteins for degradation in the proteosome and lysosome (110). In some proteins, monoubiquitination can function as a signal for protein internalization (111). Several findings support the hypothesis that Nedd4 inhibits ENaC by mediating ubiquitination of the channel.

a. Mutation of the ubiquitin ligase domain (C854A) prevented Nedd4 from inhibiting ENaC (92, 94).
The mutated cysteine is the site of ubiquitin conjugation (109) and is required for the enzymatic activity of this domain. Thus, the ubiquitin ligase activity of Nedd4 is required for the inhibition of ENaC.

b. ENaC subunits (- and ENaC) are substrates for ubiquitination when heterologously expressed (95).
Ubiquitination was decreased by mutation of specific lysine residues in the cytoplasmic N terminus of (K23, 27, 32, 47, 50R) and ENaC (K6, 8, 10, 12, 13R), suggesting that these lysines are sites for ubiquitin attachment (Fig. 5). However, further work will be required to demonstrate a direct role for Nedd4 in the ubiquitination of ENaC.

View larger version (9K): [in this window] [in a new window]   Figure 5. Nedd4 decreases ENaC surface expression – a working model. ENaC is part of a heteromultimeric complex. For simplicity, only a single subunit is shown. The binding of Nedd4 to the ENaC PY motifs results in ubiquitination of the channel complex. Ubiquitination is a signal that stimulates the internalization and degradation of ENaC, resulting in a decrease in the number of channels at the cell surface.

d. A dominant negative ubiquitin disrupted ENaC regulation.
Through a negative feedback mechanism, Na⁺ conductance is inhibited by increased intracellular Na⁺ in mouse mandibular duct cells (113). This will be discussed in more detail in Section II.I below. To test the role of ubiquitination in this process, cells were perfused with a mutant ubiquitin (K48R). This mutation prevents the formation of polyubiquitin chains, because Lys-48 is the site for ubiquitin attachment (114). The mutant ubiquitin abolished the Na⁺-mediated inhibition of ENaC, implicating a role for ubiquitination. In addition, the data suggest that monoubiquitination was not sufficient.

H. Working model
Figure 5 shows a working model for the inhibition of ENaC by Nedd4. Nedd4 interacts with ENaC through the binding of its WW domains to PY motifs in ENaC. The specificity of this interaction targets the activity of Nedd4 to ENaC and possibly additional proteins containing PY motifs. Although it seems likely that Nedd4 interacts with ENaC at the cell surface, it is also possible that these proteins interact at an intracellular location. After Nedd4 binds to ENaC, the ubiquitin ligase domain transfers ubiquitin to lysines in the N termini of - and ENaC. Ubiquitination triggers internalization and degradation of the channel complex, which decreases the number of channels at the cell surface. As a result, Nedd4 decreases Na⁺ current. In Liddle’s syndrome, the interaction between Nedd4 and ENaC is disrupted by mutation or deletion of a PY motif. The resulting decrease in internalization and degradation of ENaC generates increased Na⁺ absorption, and hence, hypertension.

I. Na⁺-mediated inhibition of ENaC
Increased Na⁺ entry through ENaC can result in an increase in the cytoplasmic Na⁺ concentration. As a result, ENaC activity is inhibited, providing negative feedback regulation of Na⁺ absorption (113, 115). Two findings implicate a potential role for Nedd4. First, in Xenopus oocytes, Liddle’s syndrome mutations reduced the inhibition of ENaC in response to elevated cytosolic Na⁺ (116). This suggests that the PY motif is required for Na⁺-mediated regulation. Second, in mouse mandibular salivary duct cells, Na⁺-mediated regulation was disrupted by interventions predicted to disrupt the interaction between Nedd4 and ENaC (117). Perfusion of the cell with a fusion protein containing the three WW domains of mouse Nedd4, or with an anti-Nedd4 antibody, prevented elevated cytoplasmic Na⁺ (72 mM) from decreasing Na⁺ conductance. Together, these results suggest that Nedd4, or a related WW domain protein, may play a role in the negative feedback regulation of ENaC by cytoplasmic Na⁺.

J. Nedd4-related proteins
The WW domain is an adapter sequence involved in protein interactions (98). It therefore seems possible that additional proteins with WW domains could bind to the ENaC PY motifs and modulate channel function. Two strategies have been used to identify such proteins: screening a cDNA library for proteins that bind to the PY motif, and searching sequence databases for homologous sequences. These approaches have revealed a large number of WW domain proteins (98, 107, 108, 118). Several have domain structures similar to Nedd4, with multiple WW domains, a ubiquitin ligase domain, and in some cases a C2 domain (Fig. 4B).

Do the Nedd4-related proteins alter the function of ENaC? One of these proteins, mouse Nedd4-2, was found to inhibit ENaC in Xenopus oocytes by decreasing the number of channels at the cell surface (108). Nedd4-2 is more similar to hNedd4, because it contains four WW domains rather than the three WW domains present in the originally identified mouse Nedd4. However, unlike mouse Nedd4 and hNedd4, Nedd4-2 lacks a C2 domain. This provides additional data that C2 domains are not necessary for the inhibition of ENaC. It has been suggested that Nedd4-2 may be the functionally relevant Nedd4 in mouse, because mouse Nedd4 did not inhibit ENaC in oocytes (108). However, the relative importance of these proteins in vivo awaits further investigation.

At least three human Nedd4-2 splice variants are present in GenBank. Nedd4La (also known as Nedd18) corresponds most closely to mouse Nedd4-2 (119). Two additional variants lack a 20-amino acid segment between WW domains 1 and 2 (KIAA0439), or the same 20 amino acids plus an additional 84 amino acids including WW domain 2 (DKFZp234p2422). Importantly, each splice form appears to be expressed in collecting duct epithelia (C. P. Thomas, personal communication), and all three inhibit ENaC (118, 119, 120).

WW domains could also mediate the interaction of ENaC with proteins having other functions. For example, YAP functions as an adapter that binds to PY motifs (Fig. 4A and Ref. 88). Thus, it seems possible that WW domain proteins with functions different from Nedd4 might bind to and regulate ENaC. These proteins might contain kinase or phosphatase domains, or they might modulate ENaC function by competing with Nedd4 for binding to the ENaC PY motifs. Such interactions could result in alterations in channel localization or phosphorylation or could have direct effects on channel function.

	III. Trafficking of ENaC to the Cell Surface

Top Abstract I. Introduction II. Liddle’s Syndrome:... III. Trafficking of ENaC... IV. ENaC Polymorphisms:... V. Summary References

 
The surface expression of ENaC can also be altered by changes in the trafficking of the channel to the cell surface (Fig. 2). Increases in the rate of channel synthesis and/or exocytosis result in enhanced Na⁺ absorption.

-, ß-, And ENaC are synthesized and glycosylated in the endoplasmic reticulum (121, 122, 123). The three subunits heteromultimerize before leaving the Golgi and then traffic together to the cell surface (123, 124). The subunits interact with one another through residues in the cytoplasmic N terminus and first transmembrane domain (121). Interestingly, recent data suggest that individual subunits may also traffic independently (125), although the functional relevance of this process is not yet known because biochemical and functional evidence indicate that the functional channel complex contains all three subunits (27, 30, 31). Mutations that disrupt ENaC trafficking result in PHA, a disorder of salt wasting and hypotension.

A. Pseudohypoaldosteronism type 1 (PHA)
Patients with PHA present as neonates with dehydration and hyponatremia resulting from salt wasting, hypotension, and life-threatening hyperkalemia (126). Serum aldosterone and renin levels are elevated, and the patients fail to respond to treatment with mineralocorticoids. PHA is an inherited disorder that can be transmitted with either an autosomal dominant or recessive pattern of inheritance. In some kindreds, the recessive form results from mutations in ENaC (12, 13). Mutations in all three ENaC subunits have been identified in patients with PHA. At least two PHA mutations disrupt the trafficking of ENaC to the cell surface.

One such mutation (_C133Y) lies within a highly conserved cysteine-rich domain of the extracellular loop. Through systematic mutation of each cysteine, it was found that four of the extracellular cysteines (including Cys¹³³) were required for channel function (127). Mutation of these cysteines decreased the expression of ENaC at the cell surface but did not alter subunit assembly or degradation. This suggests that residues in the extracellular cysteine-rich domains are required for the efficient trafficking of ENaC to the cell surface, and disruption of this trafficking causes one form of PHA. However, the mechanism is not yet known. The cysteines are likely to form disulfide bonds and to be important in the tertiary structure of the extracellular domain. Thus, perhaps cysteine mutations alter this structure, disrupting the folding of channel complex [analogous to some cystic fibrosis transmembrane conductance regulator (CFTR) mutations that cause cystic fibrosis (128)] or the recognition of the channel by proteins necessary for its trafficking to the cell surface.

PHA mutations in ENaC resulted in an apparent paradox: targeted disruption of ENaC in the mouse caused severe lung disease at the time of birth resulting from an inability to clear lung liquid, whereas patients with PHA mutations in both alleles of ENaC are born without apparent respiratory distress and seem to lack clinically important lung disease (3). One possible explanation is that the relative importance of ENaC in lung liquid clearance could differ between species. However, an alternative explanation is that in PHA patients, the mutant ENaC subunits could retain sufficient residual activity to prevent an apparent pulmonary phenotype. Recent data support such a mechanism; the _R508X mutation truncates the -subunit before the pore domain required for ion conduction. However, coexpression of _R508X with ß- and ENaC produced a small Na⁺ current (expression of ß- and ENaC alone did not), suggesting that the truncated -subunit might be sufficient to assemble with ß- and ENaC and participate in their trafficking to the cell surface [albeit inefficiently (129)].

B. Aldosterone/serum and glucocorticoid-regulated kinase (SGK)
The renin-angiotensin-aldosterone pathway is critical in the maintenance of Na⁺ homeostasis and the defense against hypovolemia and hypotension. ENaC is one of the distal targets of this pathway, where it is critically positioned to function in Na⁺ homeostasis and blood pressure control (Fig. 2). Increases in plasma aldosterone stimulate Na⁺ absorption in the kidney collecting duct and distal colon, in part by increasing the rate of Na⁺ entry through ENaC (7, 29). In addition, aldosterone increases the activity of the basolateral Na⁺-K⁺-ATPase, facilitating the exit of Na⁺ from the cell (130, 131).

Under Na⁺-replete conditions, when aldosterone levels are low, ENaC is mainly in an intracellular location; immunostaining of mouse cortical collecting duct with antibodies against ENaC revealed diffuse punctate labeling throughout the principal cells, consistent with localization of the channel in a vesicular pool (132). Na⁺ restriction (which increases serum aldosterone) or aldosterone infusion caused a dramatic redistribution of ENaC to the apical cell surface (132, 133). This was consistent with a previous patch clamp observation that Na⁺ restriction and aldosterone infusion both increased the number of functional Na⁺ channels at the apical surface of the rat collecting duct (134). Thus, aldosterone stimulates Na⁺ absorption by increasing the number of ENaC channels at the cell surface. Aldosterone may have additional effects on channel gating (135, 136). The response to aldosterone can be divided into two phases: an early phase over 1–3 h, and a late phase initiated over 6–24 h that can persist for several days. During both phases, the response to aldosterone is mediated by steroid receptors and is dependent on transcription and translation (7).

1. Early phase.
Increases in Na⁺ absorption in response to aldosterone begin before significant increases in ENaC mRNA are observed (7). Thus, the early phase does not result from increased ENaC transcription, suggesting that aldosterone might induce the transcription of proteins that modulate ENaC trafficking or function.

a. SGK.
One such aldosterone-induced protein is SGK, a member of the serine-threonine kinase family (Fig. 2). As the name suggests, SGK was initially discovered as a mRNA induced by glucocorticoids (137). In screens for proteins induced by aldosterone, two groups independently isolated SGK (138, 139). Dexamethasone and aldosterone increased SGK mRNA over 1–2 h in a Xenopus cortical collecting duct cell line (A6) and rabbit cortical collecting duct, and then levels decreased sharply. SGK protein also rapidly increased with maximal induction at 6 h. Similar to the mRNA, SGK protein then decreased at 24 h (138, 139). Two additional SGK homologs (SGK-2 and SGK-3) were recently identified, although they do not appear to be regulated by aldosterone (140).

To test the hypothesis that increased SGK mediates the early response to aldosterone, SGK was coexpressed with ENaC in Xenopus oocytes (138, 139). SGK produced a large increase in Na⁺ current, but it had no effect on an unrelated K⁺ channel (ROMK). Stimulation resulted from an increase in the number of ENaC channels at the cell surface (141).

The observation that SGK has sequence similarity to serine-threonine kinases suggests that SGK might increase ENaC surface expression by protein phosphorylation. SGK phosphorylates peptides containing the sequence RxRxxS/T, but the substrates phosphorylated by SGK in vivo are unknown (140, 142). The most straightforward hypothesis is that SGK phosphorylates ENaC. Consistent with such a hypothesis, aldosterone induced the phosphorylation of the C terminus of ß- and ENaC (143), although the functional consequence of this phosphorylation is unknown. However, recent work (144) suggests that ENaC is not phosphorylated by SGK. This finding suggests an indirect mechanism; SGK might phosphorylate one or more protein(s) involved in regulating the expression of ENaC at the cell surface.

The mechanism by which SGK increases ENaC surface expression is also unknown. It is possible that SGK increases the trafficking of ENaC to the cell surface (Fig. 2). In this regard, it is interesting to note that SGK is related to PKB/Akt, a kinase involved in the insulin-induced exocytosis of GLUT4, a glucose transporter (145). Thus, it is interesting to speculate that SGK might regulate ENaC through a similar mechanism. Alternatively, SGK might increase surface expression by decreasing ENaC endocytosis and/or degradation (Fig. 2).

b. SGK modulates Nedd4-2 function.
Recent data suggest that SGK regulates ENaC surface expression in part by modulating the function of Nedd4-2. First, SGK interacted with Nedd4-2; a PY motif in SGK mediated this interaction and was required for SGK to regulate epithelial Na⁺ absorption (Fig. 6 and Ref. 119). Second, SGK phosphorylated Nedd4-2, which contains three RxRxxS/T consensus sites (Fig. 6 and Ref. 119). Interestingly, Nedd4 lacks such consensus sites and was not phosphorylated by SGK. Thus, Nedd4 family members may be differentially regulated by SGK. Third, phosphorylation decreased the binding of Nedd4-2 to ENaC, and hence, reduced the inhibition of ENaC by Nedd4-2 (Fig. 6 and Ref. 119). The data suggest that aldosterone/SGK and Nedd4 family members converge to regulate ENaC surface expression through a common pathway. SGK increases ENaC surface expression in part by inhibiting the activity of Nedd4-2.

View larger version (15K): [in this window] [in a new window]   Figure 6. SGK modulates Nedd4-2 function. Nedd4-2 binding targets ENaC for endocytosis and degradation, reducing ENaC surface expression (left panel). Aldosterone increases transcription of SGK, which binds to Nedd4-2 (via SGK PY motif, right panel). SGK phosphorylates Nedd4-2, decreasing its binding to ENaC. As a result, Nedd4-2-mediated degradation of ENaC is reduced, leading to increased expression of ENaC at the cell surface.

d. K-Ras2.
Another potential mediator of the early aldosterone response is K-Ras2, a small G protein. In A6 epithelia, aldosterone increased K-Ras2 mRNA and protein 2- to 6-fold within 2.5–4 h (149, 150). Overexpression of K-Ras2 in either Xenopus oocytes (with ENaC) or A6 cells increased Na⁺ current (149, 150). Conversely, a K-Ras2 antisense oligonucleotide decreased K-Ras2 protein and aldosterone-induced Na⁺ current (150). However, similar to the studies on insulin, a role for K-Ras2 in mammalian epithelia has yet to established.

2. Late phase.
The late phase of aldosterone regulation results mainly from an increase in transcription of ENaC subunits (Fig. 2). In the kidney, aldosterone increased the abundance of ENaC mRNA and protein, but there was no change in the ß- and -subunits (151, 152, 153, 154). The simplest explanation is that increased ENaC transcription, in response to aldosterone, results in increased translation of ENaC subunits. Increased stability of the ENaC mRNA or protein could also explain the data. If synthesis of the -subunit is rate limiting for the assembly of ENaC channels, an increase in -protein would presumably increase the delivery of functional channels to the cell surface (151, 155). This hypothesis awaits further testing. Interestingly, the response to aldosterone is tissue dependent. In the rat colon, aldosterone produced large increases in mRNA for ß- and ENaC but produced only a small increase for ENaC (151, 153, 154). In the lung, glucocorticoids, but not mineralocorticoids, alter ENaC mRNA (151). It is likely that these differences in transcription result in heterogeneity in the regulation of Na⁺ absorption between different tissues.

In addition to increasing ENaC mRNA and protein in the kidney, it was recently reported that aldosterone (Na⁺ restriction) also produced a 15-kDa decrease (85–70 kDa) in the apparent molecular mass of ENaC (132). Glycosylation of the subunit appeared to be unchanged, suggesting that the shift in mass resulted from proteolytic cleavage. However, the 70-kDa band could also be an alternative splice form of ENaC. An antibody against the C terminus of ENaC was able to recognize the 70-kDa fragment, indicating that the C terminus had not been removed from the protein. Coupled with the size of the fragment, this observation suggests cleavage in the extracellular domain near the first membrane-spanning segment. It has been speculated that this proteolytic cleavage of ENaC is mediated by CAP1, a serine protease that stimulates ENaC (156). Perhaps aldosterone increases the expression or activity of CAP1 or a related protease. However, CAP1 is thought to stimulate ENaC through an effect on channel gating rather than a change in surface expression (156).

C. Vasopressin/cAMP
Vasopressin is also an important regulator of Na⁺ absorption (7). In response to hypovolemia, hypotension, or osmotic changes, vasopressin is released into the blood by the hypothalamus. In the kidney collecting duct, vasopressin binds to V2 receptors at the basolateral membrane, which activates adenylate cyclase, resulting in an increase in cellular cAMP (Fig. 2). Although it has long been known that cAMP increases ENaC-mediated Na⁺ current, the mechanism(s) have been uncertain. In native epithelia, most studies have implicated an increase in the number of channels at the cell surface (157, 158), whereas an increase in P_O was found when channels purified from epithelia [but not the cloned ENaC subunits (159)] were reconstituted in planar lipid bilayers (160). More recent studies of the cloned ENaC expressed in heterologous cells have also supported both mechanisms, as described in the next section.

1. cAMP increases ENaC surface expression by increasing channel exocytosis.
The low abundance of ENaC at the cell surface has provided a technical hurdle to determining whether cAMP alters channel surface expression. To overcome this limitation, an electrophysiological assay was used to measure changes in surface expression. The advantage of this approach is the sensitivity of electrophysiological techniques to detect the activity of a small number of channels at the cell surface. After expression of ENaC in epithelia, a cysteine in channels at the cell surface was covalently modified with [2(trimethylammonium)ethyl]methanethiosulfonate (MTSET), a methanethiosulfonate compound. Modification could be detected because it almost completely and irreversibly blocked Na⁺ current. Because MTSET does not permeate the cell membrane, intracellular channels were protected from modification. After washout of MTSET, the rate of appearance of unmodified (unblocked) channels at the cell surface was measured to quantify the rate of channel exocytosis. cAMP increased the rate of ENaC exocytosis [compared with cells not treated with cAMP agonists (Fig. 2 and Ref. 72).] Consistent with this interpretation, the increase in channel appearance was blocked by an intervention that blocks vesicle trafficking (15-C incubation).

To provide additional evidence that cAMP increased ENaC exocytosis, channels undergoing exocytosis were selectively labeled with a fluorescent probe. cAMP increased apical cell surface fluorescence (72). Thus, cAMP increases ENaC exocytosis, resulting in an increase in the number of channels at the cell surface (Fig. 2).

2. Mutation of the PY motif disrupts ENaC exocytosis.
To identify ENaC sequences required for cAMP-mediated exocytosis, the effect of C-terminal truncations in -, ß-, and ENaC were tested (72). Truncation of either ENaC (S594X) or ENaC (K576X) significantly decreased stimulation by cAMP. Truncation of ßENaC (R566X) had a more pronounced effect: cAMP produced a small decrease in Na⁺ current when the channel contained this mutation.

The data suggested that sequences in the C termini are required for the cAMP-mediated exocytosis of ENaC. By testing progressive truncations and missense mutations, a sequence was localized that was required for cAMP-mediated stimulation; mutations in the PY motif abolished stimulation, similar to truncation of the C terminus (72). Thus, the sequences targeted in Liddle’s syndrome were required for the cAMP-mediated exocytosis of ENaC. Three potential mechanisms could explain this requirement for the PY motif. First, the PY motif may function as a signal for exocytosis. Second, the PY motif might be involved in the retention of ENaC in an intracellular pool; loss of this sequence would result in the constitutive relocalization of ENaC to the cell surface in the absence of cAMP. Third, by disrupting ENaC endocytosis, PY motif mutations might deplete the cAMP-responsive intracellular pool of ENaC.

3. cAMP also increases P_O: potential role for CFTR.
Stutts et al. (161) expressed ENaC in fibroblasts and found that cAMP agonists increased Na⁺ current at least in part by an increase in P_O. Interestingly, the response to cAMP was altered by CFTR: cAMP stimulated Na⁺ current in the absence of CFTR, but not when CFTR was coexpressed (161, 162). Other studies have confirmed this inverse relationship between ENaC and CFTR in different experimental systems (163, 164), although it is unclear whether it results from a direct physical interaction or is indirect. Based on these data, it has been proposed that loss of CFTR function explains the increased Na⁺ current in lungs of patients with cystic fibrosis (162). However, the functional interaction between ENaC and CFTR is likely to be complex; recent work in another tissue produced quite different conclusions. In the sweat duct, CFTR was required for the cAMP-mediated stimulation of ENaC. cAMP agonists increased amiloride-sensitive Na⁺ current in normal sweat duct, but not in sweat duct from individuals with cystic fibrosis (165). Thus, the functional interaction appears to be tissue specific. Indeed, the regulation of ENaC by vasopressin/cAMP is also highly tissue specific, similar to the transcriptional regulation of ENaC by aldosterone; cAMP stimulates ENaC in some cells [e.g., kidney collecting duct (7)] but not in others [e.g., distal colon (7), lung (166), and Xenopus oocytes (159, 163)]. The presence or absence of CFTR does not explain this observation, suggesting that the tissue specific expression of another protein or factor is critical for the cAMP-dependent regulation of ENaC.

D. Potential role of syntaxins in ENaC exocytosis
The identification of proteins involved in ENaC exocytosis will be critical for understanding the regulation of epithelial Na⁺ absorption by hormones such as aldosterone and vasopressin. In turn, this may provide new candidate genes involved in hypertension. Likely candidates are the soluble N-ethylmaleimide sensitive factor attachment protein (SNAP) receptors (SNAREs), which mediate vesicle trafficking to the cell surface and between intracellular compartments. SNAREs were initially discovered for their role in synaptic vesicle exocytosis (167, 168, 169, 170). However, more recent data indicate that SNAREs are also involved in the exocytosis of nonneuronal proteins. For example, in adipocytes, SNAREs mediate the insulin-stimulated exocytosis of GLUT4 (171, 172, 173, 174). It therefore seems possible that SNAREs participate in the cell surface trafficking of ENaC. Figure 7 shows a hypothetical model.

View larger version (14K): [in this window] [in a new window]   Figure 7. Potential role of SNAREs in ENaC exocytosis. Vesicles containing ENaC bind and then fuse to the cell surface through the interaction of vesicle-associated VAMPs and plasma membrane- associated t-SNAREs (syntaxins and SNAPs). The interaction between VAMPs and t-SNAREs may be regulated by accessory proteins, such as Munc18.

The interaction between syntaxins and VAMPs can be regulated through the association of accessory proteins (Fig. 7). For example, Munc18c binds syntaxin 4, blocking the interaction between syntaxin 4 and VAMP2 in GLUT4- containing vesicles (176). Insulin reverses this inhibition by decreasing the binding of Munc18c to syntaxin 4. Recent work (177, 178) suggests that the interaction between syntaxins and v-SNAREs can also be regulated through conformational changes in syntaxin; syntaxin binds to v-SNAREs when in an "open," but not a "closed," conformation.

Two findings suggest that SNAREs may be involved in the exocytosis of ENaC. First, a number of SNAREs have been identified in epithelia that express ENaC, including principal cells of the kidney collecting duct (179, 180, 181, 182). Second, syntaxin 1A decreased Na⁺ current when expressed with ENaC in Xenopus oocytes (183, 184). Conversely, syntaxin 3 did not alter current. Interestingly, syntaxin 1A was coimmunoprecipitated by ENaC, suggesting a direct physical interaction between these proteins. It is not yet clear whether this interaction mediates the inhibition of ENaC, similar to the regulation of neuronal calcium channels and CFTR Cl^- channels by syntaxin 1A (185, 186, 187), or whether syntaxin 1A decreased Na⁺ current by disrupting channel exocytosis. Although the data implicate a role for syntaxins in the regulation of ENaC, it is important to note that syntaxin 1A may not be the specific syntaxin involved. Although low levels of syntaxin 1A are present in some epithelia (188), this syntaxin is principally expressed in neurons. In addition, there is considerable cross-reactivity between SNAREs (189); the overexpression of one syntaxin could disrupt the activity of another. Thus, future work will be required to identify the specific SNAREs involved in the regulation of ENaC trafficking and/or function.

	IV. ENaC Polymorphisms: Potential Role in ENaC Surface Expression and Hypertension

Top Abstract I. Introduction II. Liddle’s Syndrome:... III. Trafficking of ENaC... IV. ENaC Polymorphisms:... V. Summary References

 
ENaC plays a critical role of blood pressure control and hypertension. However, Liddle’s syndrome, and all of the known genetic causes combined, account for only a small fraction of hypertension (1, 2). Liddle’s syndrome provides a paradigm for mechanisms by which ENaC mutations alter the number of channels at the cell surface, resulting in an increase in Na⁺ absorption and hypertension. It therefore seems possible that additional mutations in ENaC, or in proteins that modulate ENaC function (e.g., Nedd4 and SGK), could also cause hypertension by increasing Na⁺ absorption. Such mutations might increase the expression of ENaC at the cell surface, either through a decrease in endocytosis and/or degradation, similar to Liddle’s syndrome mutations, or through an increase in exocytosis. Alternatively, mutations could alter ENaC gating. Indeed, recent work identified a sequence variation (_N530K) that increased the activity of ENaC (43, 40). The mechanism appeared to involve a change in channel gating, because a similar mutation in ßENaC (S520K) increased P_O. Interestingly, this sequence variation was found in a patient with hypertension and renal insufficiency (43), but additional investigation will be required to determine whether this mutation played a causative role.

A number of additional sequence variations (polymorphisms) have been identified in -, ß-, and ENaC (Fig. 8). However, the task of determining whether these polymorphisms are involved in hypertension has been difficult. If a polymorphism played a causative or contributing role, we would make two predictions. First, the sequence variation should be more common in hypertensive individuals than in normotensives. Second, the sequence variation should alter the function or regulation of ENaC.

View larger version (22K): [in this window] [in a new window]   Figure 8. ENaC polymorphisms. The location of polymorphisms in -, ß-, and hENaC are indicated. Also shown are the locations of the PY motifs and N-terminal ubiquitination sites.

A key question is whether the ß_T594M polymorphism alters the function or regulation of ENaC. Persu et al. (192) expressed ENaC containing this ß-variant in Xenopus oocytes and found no significant difference in Na⁺ current and Na⁺ flux compared with wild-type ENaC. As an alternative strategy, a group lead by Anil Menon and Raymond Pun (190) recorded amiloride-sensitive Na⁺ currents from human B lymphocytes obtained from either patients carrying the ß_T594M polymorphism or from normal subjects. The polymorphism did not alter basal amiloride-sensitive Na⁺ current, similar to results in Xenopus oocytes. However, cAMP increased Na⁺ current to a greater extent in cells carrying the polymorphism than in normal cells. In normal cells, PKC activators reversed this response to cAMP. In contrast, in lymphocytes from a patient homozygous for the ß_T594M polymorphism, PKC activators did not decrease Na⁺ current (64). Cells from heterozygous individuals had mixed responses. Thus, the data suggest that this single amino acid change alters the regulation of ENaC, supporting a possible causative role in hypertension. However, additional studies will be required to determine whether the amiloride-sensitive currents in B lymphocytes are an accurate model for ENaC regulation in the kidney collecting duct.

B. Additional polymorphisms
Ambrosius et al. (193) tested the potential functional effect of a different ENaC polymorphism (ß_G442V) by measuring the urine aldosterone/K⁺ ratio. An increase in ENaC activity would be predicted to decrease this ratio; excess Na⁺ absorption results in decreased aldosterone production and increased urinary K⁺ excretion. This prediction has been confirmed in patients with Liddle’s syndrome (74). When ENaC containing the ß_G442V polymorphism was expressed in Xenopus oocytes, current was not different from wild-type ENaC (192, 193). However, the urine aldosterone/K⁺ ratio was lower in subjects with the polymorphism than in normal subjects, consistent with increased ENaC activity. Although this polymorphism is more common in blacks (16%) than whites (<1%), there was no significant association between ß_G442V and hypertension. An additional polymorphism in the -subunit (T663A) was more common in whites and was associated with normotension in both blacks and whites. This sequence variation may protect against the development of hypertension. However, it did not alter ENaC function in Xenopus oocytes (193). Five additional polymorphisms have been identified in ßENaC, although none altered ENaC function in Xenopus oocytes (192).

Thus, the role of ENaC in essential hypertension is unclear. There have been several impediments to progress in this area. First, an ENaC polymorphism might predispose to hypertension but may not be sufficient on its own to cause hypertension. Such a polymorphism might produce a relatively small change in ENaC function or regulation. The contribution of this polymorphism to blood pressure might be difficult to detect by genetic linkage or functional assays. Second, a polymorphism might alter ENaC regulation but not the basal function of the channel. The ß_T594M polymorphism illustrates this principle, because it did not alter basal function but disrupted regulation by cAMP and PKC (190). Third, mutations/polymorphisms might be present in accessory proteins, such as Nedd4 or SGK. Thus, as proteins that modulate ENaC surface expression are identified, it will be important to test their role in hypertension.

	V. Summary

Top Abstract I. Introduction II. Liddle’s Syndrome:... III. Trafficking of ENaC... IV. ENaC Polymorphisms:... V. Summary References

 
The regulation of ENaC is critical for the maintenance of Na⁺ homeostasis and blood pressure control. Over the last few years, several themes have begun to emerge. First, epithelial Na⁺ absorption is regulated largely by mechanisms that control the expression of ENaC at the cell surface. Second, Nedd4 and related ubiquitin protein ligases play an important role in regulating ENaC surface expression by targeting the channel for endocytosis and degradation. Defects in this mechanism are responsible for Liddle’s syndrome, a genetic form of hypertension. Third, kinases including SGK, and cAMP-dependent protein kinase increase the expression of ENaC at the cell surface, resulting in increased Na⁺ absorption. Fourth, preliminary work has implicated a role for SNARE proteins in the trafficking of ENaC to the cell surface. Finally, a number of polymorphisms have been identified in -, ß-, and ENaC, suggesting that they might contribute to blood pressure variation in the population.

The work to date is important because it has discovered pathways that control ENaC surface expression and, in turn, regulate blood pressure. However, a great deal of work remains to put the pieces of the puzzle together. For example, it is not known which of the Nedd4-related proteins (e.g., Nedd4, Nedd4-2) modulate ENaC surface expression in native epithelia. It is possible that different members of this family function in different epithelia, or that multiple members work in concert within the same cell. Moreover, the mechanisms that regulate the expression and activity of these proteins are only beginning to emerge. Recent data suggest that SGK modulates the activity of a Nedd4 family member (Nedd4-2) by reducing its binding to ENaC. SGK and other kinases (e.g., PKA) might also phosphorylate other proteins that modulate ENaC surface expression. Identification of such substrates will be important because it will identify potential candidate genes for hypertension. Understanding the contribution of polymorphisms in ENaC, or in regulatory proteins, to essential hypertension is another important and exciting goal for the future. Thus, future work may help us to understand how multiple pathways are integrated to regulate ENaC surface expression, and how disruption of these pathways might lead to hypertension.

	Acknowledgments

 

	Footnotes

 
This work was supported by NHLBI Grants HL-58812, HL-03575, and HL-55006 and NIDDK Grant DK52617.

Abbreviations: CFTR, Cystic fibrosis transmembrane conductance regulator; ENaC, epithelial Na⁺ channel; h, human; MDCK, Madin-Darby canine kidney; MTSET, [2(trimethylammonium)ethyl]methanethiosulfonate; PHA, pseudohypoaldosteronism type 1; P_O, probability of the channel being open; SGK, serum and glucocorticoid-regulated kinase; SNAP, N-ethylmaleimide sensitive factor attachment protein; SNARE, SNAP receptor; YAP, Yes kinase-associated protein.

	References

Top Abstract I. Introduction II. Liddle’s Syndrome:... III. Trafficking of ENaC... IV. ENaC Polymorphisms:... V. Summary References

 

1. Gordon RD 1995 Heterogeneous hypertension. Nat Genet 11:6–9[CrossRef][Medline]
2. Lifton RP 1996 Molecular genetics of human blood pressure variation. Science 272:676–680[Abstract]
3. Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, Boucher R, Rossier BC 1996 Early death due to defective neonatal lung liquid clearance in -ENaC-deficient mice. Nat Genet 12:325–328[CrossRef][Medline]
4. Smith JJ, Travis SM, Greenberg EP, Welsh MJ 1996 Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85:229–236[CrossRef][Medline]
5. Boucher RC, Stutts MJ, Knowles MR, Cantley L, Gatzy JT 1986 Na⁺ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation. J Clin Invest 78:1245–1252
6. Benos DJ, Fuller CM, Shlyonsky VG, Berdiev BK, Ismailov II 1997 Amiloride-sensitive Na⁺ channels: insights and outlooks. News Phys Sci 12:55–61[Abstract/Free Full Text]
7. Garty H, Palmer LG 1997 Epithelial sodium channels: function, structure, and regulation. Physiol Rev 77:359–396[Abstract/Free Full Text]
8. Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, Gill Jr JR, Ulick S, Milora RV, Findling JW, Canessa CM, Rossier BC, Lifton RP 1994 Liddle’s syndrome: heritable human hypertension caused by mutations in the ß subunit of the epithelial sodium channel. Cell 79:407–414[CrossRef][Medline]
9. Hansson JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa C, Iwasaki T, Rossier B, Lifton RP 1995 Hypertension caused by a truncated epithelial sodium channel subunit: genetic heterogeneity of Liddle syndrome. Nat Genet 11:76–82[CrossRef][Medline]
10. Hansson JH, Schild L, Lu Y, Wilson TA, Gautschi I, Shimkets R, Nelson-Williams C, Rossier BC, Lifton RP 1995 A de novo missense mutation of the ß subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity. Proc Natl Acad Sci USA 92:11495–11499[Abstract/Free Full Text]
11. Tamura H, Schild L, Enomoto N, Matsui N, Marumo F, Rossier BC 1996 Liddle disease caused by a missense mutation of ß subunit of the epithelial sodium channel gene. J Clin Invest 97:1780–1784[Medline]
12. Chang SS, Grunder S, Hanukoglu A, Rosler A, Mathew PM, Hanukoglu I, Schild L, Lu Y, Shimkets RA, Nelson-Williams C, Rossier BC, Lifton RP 1996 Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 12:248–253[CrossRef][Medline]
13. Strautnieks SS, Thompson RJ, Gardiner RM, Chung E 1996 A novel splice-site mutation in the subunit of the epithelial sodium channel gene in three pseudohypoaldosteronism type 1 families. Nat Genet 13:248–250[CrossRef][Medline]
14. Canessa CM, Horisberger JD, Rossier BC 1993 Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361:467–470[CrossRef][Medline]
15. Lingueglia E, Voilley N, Waldmann R, Lazdunski M, Barbry P 1993 Expression cloning of an epithelial amiloride-sensitive Na⁺ channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett 318:95–99[CrossRef][Medline]
16. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, Rossier BC 1994 Amiloride-sensitive epithelial Na⁺ channel is made of three homologous subunits. Nature 367:463–467[CrossRef][Medline]
17. McDonald FJ, Snyder PM, McCray Jr PB, Welsh MJ 1994 Cloning, expression, and tissue distribution of a human amiloride-sensitive Na⁺ channel. Am J Physiol 266:L728–L734
18. McDonald FJ, Price MP, Snyder PM, Welsh MJ 1995 Cloning and expression of the ß- and -subunits of the human epithelial sodium channel. Am J Physiol 268:C1157–C1163
19. Voilley N, Lingueglia E, Champigny G, Mattei MG, Waldmann R, Lazdunski M, Barbry P 1994 The lung amiloride-sensitive Na⁺ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning. Proc Natl Acad Sci USA 91:247–251[Abstract/Free Full Text]
20. Firsov D, Schild L, Gautschi I, Merillat AM, Schneeberger E, Rossier BC 1996 Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci USA 93:15370–15375[Abstract/Free Full Text]
21. Waldmann R, Champigny G, Bassilana F, Voilley N, Lazdunski M 1995 Molecular cloning and functional expression of a novel amiloride-sensitive Na⁺ channel. J Biol Chem 270:27411–27414[Abstract/Free Full Text]
22. Renard S, Lingueglia E, Voilley N, Lazdunski M, Barbry P 1994 Biochemical analysis of the membrane topology of the amiloride-sensitive Na⁺ channel. J Biol Chem 269:12981–12986[Abstract/Free Full Text]
23. Snyder PM, McDonald FJ, Stokes JB, Welsh MJ 1994 Membrane topology of the amiloride-sensitive epithelial sodium channel. J Biol Chem 269:24379–24383[Abstract/Free Full Text]
24. Canessa CM, Merillat AM, Rossier BC 1994 Membrane topology of the epithelial sodium channel in intact cells. Am J Physiol 267:C1682–C1690
25. Firsov D, Gautschi I, Merillat AM, Rossier BC, Schild L 1998 The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO J 17:344–352[CrossRef][Medline]
26. Kosari F, Sheng S, Li J, Mak DO, Foskett JK, Kleyman TR 1998 Subunit stoichiometry of the epithelial sodium channel. J Biol Chem 273:13469–13474[Abstract/Free Full Text]
27. Snyder PM, Cheng C, Prince LS, Rogers JC, Welsh MJ 1998 Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits. J Biol Chem 273:681–684[Abstract/Free Full Text]
28. Eskandari S, Snyder PM, Kreman M, Zampighi GA, Welsh MJ, Wright EM 1999 Number of subunits comprising the epithelial sodium channel. J Biol Chem 274:27281–27286[Abstract/Free Full Text]
29. Benos DJ, Awayda MS, Ismailov II, Johnson JP 1995 Structure and function of amiloride-sensitive Na⁺ channels. J Membr Biol 143:1–18[Medline]
30. Schild L, Schneeberger E, Gautschi I, Firsov D 1997 Identification of amino acid residues in the , ß, and subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation. J Gen Physiol 109:15–26[Abstract/Free Full Text]
31. Snyder PM, Olson DR, Bucher DB 1999 A pore segment in DEG/ENaC Na⁺ channels. J Biol Chem 274:28484–28490[Abstract/Free Full Text]
32. Kellenberger S, Gautschi I, Schild L 1999 A single point mutation in the pore region of the epithelial Na⁺ channel changes ion selectivity by modifying molecular sieving. Proc Natl Acad Sci USA 96:4170–4175[Abstract/Free Full Text]
33. Kellenberger S, Hoffmann-Pochon N, Gautschi I, Schneeberger E, Schild L 1999 On the molecular basis of ion permeation in the epithelial Na⁺ channel. J Gen Physiol 114:13–30[Abstract/Free Full Text]
34. Sheng S, Li J, McNulty KA, Avery D, Kleyman TR 2000 Characterization of the selectivity filter of the epithelial sodium channel. J Biol Chem 275:8572–8581[Abstract/Free Full Text]
35. Langloh AL, Berdiev B, Ji HL, Keyser K, Stanton BA, Benos DJ 2000 Charged residues in the M2 region of -hENaC play a role in channel conductance. Am J Physiol Cell Physiol 278:C277–C291
36. Ismailov, II, Kieber-Emmons T, Lin C, Berdiev BK, Shlyonsky VG, Patton HK, Fuller CM, Worrell R, Zuckerman JB, Sun W, Eaton DC, Benos DJ, Kleyman TR 1997 Identification of an amiloride binding domain within the -subunit of the epithelial Na⁺ channel. J Biol Chem 272:21075–21083[Abstract/Free Full Text]
37. Palmer LG, Frindt G 1996 Gating of Na⁺ channels in the rat cortical collecting tubule: effects of voltage and membrane stretch. J Gen Physiol 107:35–45[Abstract/Free Full Text]
38. Grunder S, Firsov D, Chang SS, Jaeger NF, Gautschi I, Schild L, Lifton RP, Rossier BC 1997 A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel. EMBO J 16:899–907[CrossRef][Medline]
39. Grunder S, Jaeger NF, Gautschi I, Schild L, Rossier BC 1999 Identification of a highly conserved sequence at the N-terminus of the epithelial Na⁺ channel subunit involved in gating. Pflugers Arch 438:709–715[CrossRef][Medline]
40. Snyder PM, Bucher DB, Olson DR 2000 Gating induces a conformational change in the outer vestibule of ENaC. J Gen Physiol 116:781–790[Abstract/Free Full Text]
41. Sheng S, Li J, McNulty KA, Kieber-Emmons T, Kleyman TR 2001 Epithelial sodium channel pore region. Structure and role in gating. J Biol Chem 276:1326–1334[Abstract/Free Full Text]
42. Tavernarakis N, Driscoll M 1997 Molecular modeling of mechanotransduction in the nematode Caenorhabditis elegans. Annu Rev Physiol 59:659–689[CrossRef][Medline]
43. Melander O, Orho M, Fagerudd J, Bengtsson K, Groop PH, Mattiasson I, Groop L, Hulthen UL 1998 Mutations and variants of the epithelial sodium channel gene in Liddle’s syndrome and primary hypertension. Hypertension 31:1118–1124[Abstract/Free Full Text]
44. Mano I, Driscoll M 1999 DEG/ENaC channels: a touchy superfamily that watches its salt. Bioessays 21:568–578[CrossRef][Medline]
45. Waldmann R, Lazdunski M 1998 H⁺-gated cation channels: neuronal acid sensors in the NaC/DEG family of ion channels. Curr Opin Neurobiol 8:418–424[CrossRef][Medline]
46. Driscoll M, Chalfie M 1991 The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration. Nature 349:588–593[CrossRef][Medline]
47. Huang M, Chalfie M 1994 Gene interactions affecting mechanosensory transduction in Caenorhabditis elegans. Nature 367:467–470[CrossRef][Medline]
48. Price MP, Snyder PM, Welsh MJ 1996 Cloning and expression of a novel human brain Na⁺ channel. J Biol Chem 271:7879–7882[Abstract/Free Full Text]
49. Garcia-Anoveros J, Derfler B, Neville-Golden J, Hyman BT, Corey DP 1997 BNaC1 and BNaC2 constitute a new family of human neuronal sodium channels related to degenerins and epithelial sodium channels. Proc Natl Acad Sci USA 94:1459–1464[Abstract/Free Full Text]
50. Price MP, Lewin GR, McIlwrath SL, Cheng C, Xie J, Heppenstall PA, Stucky CL, Mannsfeldt AG, Brennan TJ, Drummond HA, Qiao J, Benson CJ, Tarr DE, Hrstka RF, Yang B, Williamson RA, Welsh MJ 2000 The mammalian sodium channel BNC1 is required for normal touch sensation. Nature 407:1007–1011[CrossRef][Medline]
51. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M 1997 A proton-gated cation channel involved in acid-sensing. Nature 386:173–177[CrossRef][Medline]
52. Waldmann R, Bassilana F, de Weille J, Champigny G, Heurteaux C, Lazdunski M 1997 Molecular cloning of a non-inactivating proton-gated Na⁺ channel specific for sensory neurons. J Biol Chem 272:20975–20978[Abstract/Free Full Text]
53. Li XJ, Blackshaw S, Snyder SH 1994 Expression and localization of amiloride-sensitive sodium channel indicate a role for non-taste cells in taste perception. Proc Natl Acad Sci USA 91:1814–1818[Abstract/Free Full Text]
54. Lin W, Finger TE, Rossier BC, Kinnamon SC 1999 Epithelial Na⁺ channel subunits in rat taste cells: localization and regulation by aldosterone. J Comp Neurol 405:406–420[CrossRef][Medline]
55. Boughter Jr JD, Gilbertson TA 1999 From channels to behavior: an integrative model of NaCl taste. Neuron 22:213–215[CrossRef][Medline]
56. Drummond HA, Price MP, Welsh MJ, Abboud FM 1998 A molecular component of the arterial baroreceptor mechanotransducer. Neuron 21:1435–1441[CrossRef][Medline]
57. Drummond HA, Abboud FM, Welsh MJ 2000 Localization of ß and subunits of ENaC in sensory nerve endings in the rat foot pad. Brain Res 884:1–12[CrossRef][Medline]
58. Awayda MS, Ismailov II, Berdiev BK, Benos DJ 1995 A cloned renal epithelial Na⁺ channel protein displays stretch activation in planar lipid bilayers. Am J Physiol 268:C1450–C1459
59. Ismailov II, Berdiev BK, Shlyonsky VG, Benos DJ 1997 Mechanosensitivity of an epithelial Na⁺ channel in planar lipid bilayers: release from Ca²⁺ block. Biophys J 72:1182–1192[Medline]
60. Kizer N, Guo XL, Hruska K 1997 Reconstitution of stretch-activated cation channels by expression of the -subunit of the epithelial sodium channel cloned from osteoblasts. Proc Natl Acad Sci USA 94:1013–1018[Abstract/Free Full Text]
61. Awayda MS, Subramanyam M 1998 Regulation of the epithelial Na⁺ channel by membrane tension. J Gen Physiol 112:97–111[Abstract/Free Full Text]
62. Bradford AL, Ismailov II, Achard JM, Warnock DG, Bubien JK, Benos DJ 1995 Immunopurification and functional reconstitution of a Na⁺ channel complex from rat lymphocytes. Am J Physiol 269:C601–C611
63. Bubien JK, Ismailov II, Berdiev BK, Cornwell T, Lifton RP, Fuller CM, Achard JM, Benos DJ, Warnock DG 1996 Liddle’s disease: abnormal regulation of amiloride-sensitive Na⁺ channels by ß-subunit mutation. Am J Physiol 270:C208–C213
64. Cui Y, Su YR, Rutkowski M, Reif M, Menon AG, Pun RY 1997 Loss of protein kinase C inhibition in the ß-T594M variant of the amiloride-sensitive Na⁺ channel. Proc Natl Acad Sci USA 94:9962–9966[Abstract/Free Full Text]
65. Oh Y, Warnock DG 1997 Expression of the amiloride-sensitive sodium channel ß subunit gene in human B lymphocytes. J Am Soc Nephrol 8:126–129[Abstract]
66. Bubien JK, Watson B, Khan MA, Langloh AL, Fuller CM, Berdiev B, Tousson A, Benos DJ 2000 Expression and regulation of normal and polymorphic ENaC by human lymphocytes. J Biol Chem 276:8557–8566[Abstract/Free Full Text]
67. Brouard M, Casado M, Djelidi S, Barrandon Y, Farman N 1999 Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation. J Cell Sci 112:3343–3352[Abstract]
68. Palmer LG, Frindt G 1986 Amiloride-sensitive Na⁺ channels from the apical membrane of the rat cortical collecting tubule. Proc Natl Acad Sci USA 83:2767–2770[Abstract/Free Full Text]
69. Hamilton KL, Eaton DC 1986 Single-channel recordings from two types of amiloride-sensitive epithelial Na⁺ channels. Membr Biochem 6:149–171[Medline]
70. Hille B 1992 Ionic channels of excitable membranes. Sunderland, MA: Sinauer Associates, Inc.
71. Snyder PM, Price MP, McDonald FJ, Adams CM, Volk KA, Zeiher BG, Stokes JB, Welsh MJ 1995 Mechanism by which Liddle’s syndrome mutations increase activity of a human epithelial Na⁺ channel. Cell 83:969–978[CrossRef][Medline]
72. Snyder PM 2000 Liddle’s syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na⁺ channel to the cell surface. J Clin Invest 105:45–53[Medline]
73. Liddle GW, Bledsoe T, Coppage WS 1963 A familial renal disorder simulating primary aldosteronism but with negligible aldosterone secretion. Trans Assoc Am Physicians 76:199–213
74. Botero-Velez M, Curtis JJ, Warnock DG 1994 Brief report: Liddle’s syndrome revisited – a disorder of sodium reabsorption in the distal tubule. New Engl J Med 330:178–181[Free Full Text]
75. Schild L, Canessa CM, Shimkets RA, Gautschi I, Lifton RP, Rossier BC 1995 A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci USA 92:5699–5703[Abstract/Free Full Text]
76. Schild L, Lu Y, Gautschi I, Schneeberger E, Lifton RP, Rossier BC 1996 Identification of a PY motif in the epithelial Na⁺ channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. EMBO J 15:2381–2387[Medline]
77. Pradervand S, Wang Q, Burnier M, Beermann F, Horisberger JD, Hummler E, Rossier BC 1999 A mouse model for Liddle’s syndrome. J Am Soc Nephrol 10:2527–2533[Abstract/Free Full Text]
78. Hoshi T, Zagotta WN, Aldrich RW 1990 Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250:533–538[Abstract/Free Full Text]
79. Awayda MS, Tousson A, Benos DJ 1997 Regulation of a cloned epithelial Na⁺ channel by its ß- and -subunits. Am J Physiol 273:C1889–C1899
80. Ismailov, II, Shlyonsky VG, Serpersu EH, Fuller CM, Cheung HC, Muccio D, Berdiev BK, Benos DJ 1999 Peptide inhibition of ENaC. Biochemistry 38:354–363[CrossRef][Medline]
81. Uehara Y, Sasaguri M, Kinoshita A, Tsuji E, Kiyose H, Taniguchi H, Noda K, Ideishi M, Inoue J, Tomita K, Arakawa K 1998 Genetic analysis of the epithelial sodium channel in Liddle’s syndrome. J Hypertens 16:1131–1135[CrossRef][Medline]
82. Volk KA, Husted RF, Snyder PM, Stokes JB 2000 Kinase regulation of hENaC mediated through a region in the COOH-terminal portion of the -subunit. Am J Physiol 278:C1047–C1054
83. Hobbs HH, Russell DW, Brown MS, Goldstein JL 1990 The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu Rev Genet 24:133–170[CrossRef][Medline]
84. Collawn JF, Stangel M, Kuhn LA, Esekogwu V, Jing SQ, Trowbridge IS, Tainer JA 1990 Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell 63:1061–1072[CrossRef][Medline]
85. Goulet CC, Adams CM, Volk KA, Stokes JB, Welsh MJ, Snyder PM 1997 Regulation of ENaC by the C-terminus. Physiologist 40:265 (Abstract)
86. Shimkets RA, Lifton RP, Canessa CM 1997 The activity of the epithelial sodium channel is regulated by clathrin-mediated endocytosis. J Biol Chem 272:25537–25541[Abstract/Free Full Text]
87. Damke H, Baba T, Warnock DE, Schmid SL 1994 Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol 127:915–934[Abstract/Free Full Text]
88. Chen HI, Sudol M 1995 The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. Proc Natl Acad Sci USA 92:7819–7823[Abstract/Free Full Text]
89. Staub O, Dho S, Henry P, Correa J, Ishikawa T, McGlade J, Rotin D 1996 WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na⁺ channel deleted in Liddle’s syndrome. EMBO J 15:2371–2380[Medline]
90. Kumar S, Tomooka Y, Noda M 1992 Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem Biophys Res Commun 185:1155–1161[CrossRef][Medline]
91. Staub O, Yeger H, Plant PJ, Kim H, Ernst SA, Rotin D 1997 Immunolocalization of the ubiquitin-protein ligase Nedd4 in tissues expressing the epithelial Na⁺ channel (ENaC). Am J Physiol 272:C1871–C1880
92. Goulet CC, Volk KA, Adams CM, Prince LS, Stokes JB, Snyder PM 1998 Inhibition of the epithelial Na⁺ channel by interaction of Nedd4 with a PY motif deleted in Liddle’s syndrome. J Biol Chem 273:30012–30017[Abstract/Free Full Text]
93. Farr TJ, Coddington-Lawson SJ, Snyder PM, McDonald FJ 2000 Human Nedd4 interacts with the human epithelial Na⁺ channel: WW3 but not WW1 binds to Na⁺-channel subunits. Biochem J 345:503–509
94. Abriel H, Loffing J, Rebhun JF, Pratt JH, Schild L, Horisberger JD, Rotin D, Staub O 1999 Defective regulation of the epithelial Na⁺ channel by Nedd4 in Liddle’s syndrome. J Clin Invest 103:667–673[Medline]
95. Staub O, Gautschi I, Ishikawa T, Breitschopf K, Ciechanover A, Schild L, Rotin D 1997 Regulation of stability and function of the epithelial Na⁺ channel (ENaC) by ubiquitination. EMBO J 16:6325–6336[CrossRef][Medline]
96. Malik B, Schlanger L, Al-Khalili O, Bao HF, Yue G, Price SR, Mitch WE, Eaton DC 2001 ENac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway. J Biol Chem 276:12903–12910[Abstract/Free Full Text]
97. Bork P, Sudol M 1994 The WW domain: a signalling site in dystrophin? Trends Biochem Sci 19:531–533[CrossRef][Medline]
98. Sudol M 1996 Structure and function of the WW domain. Prog Biophys Mol Biol 65:113–132[Medline]
99. Macias MJ, Hyvonen M, Baraldi E, Schultz J, Sudol M, Saraste M, Oschkinat H 1996 Structure of the WW domain of a kinase-associated protein complexed with a proline-rich peptide. Nature 382:646–649[CrossRef][Medline]
100. Kanelis V, Rotin D, Forman-Kay JD 2001 Solution structure of a Nedd4 WW domain-ENaC peptide complex. Nat Struct Biol 8:407–412[CrossRef][Medline]
101. Snyder PM, Olson DR, McDonald FJ, Bucher DB 2001 Multiple WW domains, but not the C2 domain, are required for the inhibition of ENaC by human Nedd4. J Biol Chem 276:28321–28326[Abstract/Free Full Text]
102. Harvey KF, Dinudom A, Komwatana P, Jolliffe CN, Day ML, Parasivam G, Cook DI, Kumar S 1999 All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na⁺. J Biol Chem 274:12525–12530[Abstract/Free Full Text]
103. Nishizuka Y 1988 The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334:661–665[CrossRef][Medline]
104. Nalefski EA, Falke JJ 1996 The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci 5:2375–2390[Medline]
105. Plant PJ, Yeger H, Staub O, Howard P, Rotin D 1997 The C2 domain of the ubiquitin protein ligase Nedd4 mediates Ca²⁺- dependent plasma membrane localization. J Biol Chem 272:32329–32336[Abstract/Free Full Text]
106. Plant PJ, Lafont F, Lecat S, Verkade P, Simons K, Rotin D 2000 Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb. J Cell Biol 149:1473–1484[Abstract/Free Full Text]
107. Pirozzi G, McConnell SJ, Uveges AJ, Carter JM, Sparks AB, Kay BK, Fowlkes DM 1997 Identification of novel human WW domain-containing proteins by cloning of ligand targets. J Biol Chem 272:14611–14616[Abstract/Free Full Text]
108. Kamynina E, Debonneville C, Bens M, Vandewalle A, Staub O 2001 A novel mouse Nedd4 protein suppresses the activity of the epithelial Na⁺ channel. FASEB J 15:204–214[Abstract/Free Full Text]
109. Huibregtse JM, Scheffner M, Beaudenon S, Howley PM 1995 A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc Natl Acad Sci USA 92:5249[Free Full Text]
110. Ciechanover A 1994 The ubiquitin-proteasome proteolytic pathway. Cell 79:13–21[CrossRef][Medline]
111. Hicke L, Riezman H 1996 Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell 84:277–287[CrossRef][Medline]
112. Chalfant ML, Denton JS, Langloh AL, Karlson KH, Loffing J, Benos DJ, Stanton BA 1999 The NH₂ terminus of the epithelial sodium channel contains an endocytic motif. J Biol Chem 274:32889–32896[Abstract/Free Full Text]
113. Komwatana P, Dinudom A, Young JA, Cook DI 1996 Cytosolic Na⁺ controls and epithelial Na⁺ channel via the Go guanine nucleotide-binding regulatory protein. Proc Natl Acad Sci USA 93:8107–8111[Abstract/Free Full Text]
114. Chau V, Tobias JW, Bachmair A, Marriott D, Ecker DJ, Gonda DK, Varshavsky A 1989 A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243:1576–1583[Abstract/Free Full Text]
115. Awayda MS 1999 Regulation of the epithelial Na⁺ channel by intracellular Na⁺. Am J Physiol 277:C216–C224
116. Kellenberger S, Gautschi I, Rossier BC, Schild L 1998 Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system. J Clin Invest 101:2741–2750[Medline]
117. Dinudom A, Harvey KF, Komwatana P, Young JA, Kumar S, Cook DI 1998 Nedd4 mediates control of an epithelial Na⁺ channel in salivary duct cells by cytosolic Na⁺. Proc Natl Acad Sci USA 95:7169–7173[Abstract/Free Full Text]
118. Harvey KF, Dinudom A, Cook DI, Kumar S 2001 The Nedd4-like protein KIAA0439 is a potential regulator of the epithelial sodium channel. J Biol Chem 276:8597–8601[Abstract/Free Full Text]
119. Snyder PM, Olson DR, Thomas BC 2002 Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na⁺ channel. J Biol Chem 277:5–8[Abstract/Free Full Text]
120. Kamynina E, Tauxe C, Staub O 2001 Distinct characteristics of two human Nedd4 proteins with respect to epithelial Na⁺ channel regulation. Am J Physiol Renal Physiol 281:F469–F477
121. Adams CM, Snyder PM, Welsh MJ 1997 Interactions between subunits of the human epithelial sodium channel. J Biol Chem 272:27295–27300[Abstract/Free Full Text]
122. Valentijn JA, Fyfe GK, Canessa CM 1998 Biosynthesis and processing of epithelial sodium channels in Xenopus oocytes. J Biol Chem 273:30344–30351[Abstract/Free Full Text]
123. Prince LS, Welsh MJ 1998 Cell surface expression and biosynthesis of epithelial Na⁺ channels. Biochem J 336:705–710
124. Cheng C, Prince LS, Snyder PM, Welsh MJ 1998 Assembly of the epithelial Na⁺ channel evaluated using sucrose gradient sedimentation analysis. J Biol Chem 273:22693–22700[Abstract/Free Full Text]
125. Weisz OA, Wang JM, Edinger RS, Johnson JP 2000 Non-coordinate regulation of endogenous epithelial sodium channel (ENaC) subunit expression at the apical membrane of A6 cells in response to various transporting conditions. J Biol Chem 275:39886–39893[Abstract/Free Full Text]
126. Dillon MJ, Leonard JV, Buckler JM, Ogilvie D, Lillystone D, Honour JW, Shackleton CH 1980 Pseudohypoaldosteronism. Arch Dis Child 55:427–434[Abstract/Free Full Text]
127. Firsov D, Robert-Nicoud M, Gruender S, Schild L, Rossier BC 1999 Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC). Identification of cysteines essential for channel expression at the cell surface. J Biol Chem 274:2743–2749[Abstract/Free Full Text]
128. Welsh MJ, Smith AE 1993 Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73:1251–1254[CrossRef][Medline]
129. Bonny O, Chraibi A, Loffing J, Jaeger NF, Grunder S, Horisberger JD, Rossier BC 1999 Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na⁺ channel lacking the pore-forming region of its subunit. J Clin Invest 104:967–974[Medline]
130. Verrey F, Schaerer E, Zoerkler P, Paccolat MP, Geering K, Kraehenbuhl JP, Rossier BC 1987 Regulation by aldosterone of Na⁺,K⁺-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells. J Cell Biol 104:1231–1237[Abstract/Free Full Text]
131. Beron J, Mastroberardino L, Spillmann A, Verrey F 1995 Aldosterone modulates sodium kinetics of Na, K-ATPase containing an 1 subunit in A6 kidney cell epithelia. Mol Biol Cell 6:261–271[Abstract]
132. Masilamani S, Kim GH, Mitchell C, Wade JB, Knepper MA 1999 Aldosterone-mediated regulation of ENaC , ß, and subunit proteins in rat kidney. J Clin Invest 104:R19–23
133. Loffing J, Zecevic M, Feraille E, Kaissling B, Asher C, Rossier BC, Firestone GL, Pearce D, Verrey F 2001 Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK. Am J Physiol Renal Physiol 280:F675–F682
134. Palmer LG, Antonian L, Frindt G 1994 Regulation of apical K and Na channels and Na/K pumps in rat cortical collecting tubule by dietary K. J Gen Physiol 104:693–710[Abstract/Free Full Text]
135. Kemendy AE, Kleyman TR, Eaton DC 1992 Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia. Am J Physiol 263:C825–C837
136. Kleyman TR, Coupaye-Gerard B, Ernst SA 1992 Aldosterone does not alter apical cell-surface expression of epithelial Na⁺ channels in the amphibian cell line A6. J Biol Chem 267:9622–9628[Abstract/Free Full Text]
137. Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL 1993 Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 13:2031–2040[Abstract/Free Full Text]
138. Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, Pearce D 1999 Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 96:2514–2519[Abstract/Free Full Text]
139. Naray-Fejes-Toth A, Canessa C, Cleaveland ES, Aldrich G, Fejes-Toth G 1999 sgk Is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial Na⁺ channels. J Biol Chem 274:16973–16978[Abstract/Free Full Text]
140. Kobayashi T, Deak M, Morrice N, Cohen P 1999 Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem J 344:189–197
141. Alvarez de la Rosa D, Zhang P, Naray-Fejes-Toth A, Fejes-Toth G, Canessa CM 1999 The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes. J Biol Chem 274:37834–37839[Abstract/Free Full Text]
142. Park J, Leong ML, Buse P, Maiyar AC, Firestone GL, Hemmings BA 1999 Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J 18:3024–3033[CrossRef][Medline]
143. Shimkets RA, Lifton R, Canessa CM 1998 In vivo phosphorylation of the epithelial sodium channel. Proc Natl Acad Sci USA 95:3301–3305[Abstract/Free Full Text]
144. Wang J, Barbry P, Maiyar AC, Rozansky DJ, Bhargava A, Leong M, Firestone GL, Pearce D 2001 SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport. Am J Physiol Renal Physiol 280:F303–F313
145. Cong LN, Chen H, Li Y, Zhou L, McGibbon MA, Taylor SI, Quon MJ 1997 Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 11:1881–1890[Abstract/Free Full Text]
146. Record RD, Froelich LL, Vlahos CJ, Blazer-Yost BL 1998 Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells. Am J Physiol 274:E611–E617
147. Shepherd PR, Withers DJ, Siddle K 1998 Phosphoinositide 3- kinase: the key switch mechanism in insulin signalling. Biochem J 333:471–490
148. Fidelman ML, May JM, Biber TU, Watlington CO 1982 Insulin stimulation of Na⁺ transport and glucose metabolism in cultured kidney cells. Am J Physiol 242:C121–C123
149. Spindler B, Verrey F 1999 Aldosterone action: induction of p21(ras) and fra-2 and transcription-independent decrease in myc, jun, and fos. Am J Physiol 276:C1154–C1161
150. Stockand JD, Spier BJ, Worrell RT, Yue G, Al-Baldawi N, Eaton DC 1999 Regulation of Na⁺ reabsorption by the aldosterone- induced small G protein K-Ras2A. J Biol Chem 274:35449–35454[Abstract/Free Full Text]
151. Stokes JB, Sigmund RD 1998 Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue, and steroid heterogeneity. Am J Physiol 274:C1699–C1707
152. Ono S, Kusano E, Muto S, Ando Y, Asano Y 1997 A low-Na⁺ diet enhances expression of mRNA for epithelial Na⁺ channel in rat renal inner medulla. Pflugers Arch 434:756–763[CrossRef][Medline]
153. Asher C, Wald H, Rossier BC, Garty H 1996 Aldosterone-induced increase in the abundance of Na⁺ channel subunits. Am J Physiol 271:C605–C611
154. Escoubet B, Coureau C, Bonvalet JP, Farman N 1997 Noncoordinate regulation of epithelial Na channel and Na pump subunit mRNAs in kidney and colon by aldosterone. Am J Physiol 272:C1482–C1491
155. May A, Puoti A, Gaeggeler HP, Horisberger JD, Rossier BC 1997 Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel subunit in A6 renal cells. J Am Soc Nephrol 8:1813–1822[Abstract]
156. Vallet V, Chraibi A, Gaeggeler HP, Horisberger JD, Rossier BC 1997 An epithelial serine protease activates the amiloride-sensitive sodium channel. Nature 389:607–610[CrossRef][Medline]
157. Marunaka Y, Eaton DC 1991 Effects of vasopressin and cAMP on single amiloride-blockable Na channels. Am J Physiol 260:C1071–C1084
158. Frindt G, Silver RB, Windhager EE, Palmer LG 1995 Feedback regulation of Na channels in rat CCT. III. Response to cAMP. Am J Physiol 268:F480–F489
159. Awayda MS, Ismailov II, Berdiev BK, Fuller CM, Benos DJ 1996 Protein kinase regulation of a cloned epithelial Na⁺ channel. J Gen Physiol 108:49–65[Abstract/Free Full Text]
160. Ismailov II, McDuffie JH, Benos DJ 1994 Protein kinase A phosphorylation and G protein regulation of purified renal Na⁺ channels in planar bilayer membranes. J Biol Chem 269:10235–10241[Abstract/Free Full Text]
161. Stutts MJ, Rossier BC, Boucher RC 1997 Cystic fibrosis transmembrane conductance regulator inverts protein kinase A-mediated regulation of epithelial sodium channel single channel kinetics. J Biol Chem 272:14037–14040[Abstract/Free Full Text]
162. Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, Boucher RC 1995 CFTR as a cAMP-dependent regulator of sodium channels. Science 269:847–850[Abstract/Free Full Text]
163. Mall M, Hipper A, Greger R, Kunzelmann K 1996 Wild type but not F508 CFTR inhibits Na⁺ conductance when coexpressed in Xenopus oocytes. FEBS Lett 381:47–52[CrossRef][Medline]
164. Ismailov II, Awayda MS, Jovov B, Berdiev BK, Fuller CM, Dedman JR, Kaetzel M, Benos DJ 1996 Regulation of epithelial sodium channels by the cystic fibrosis transmembrane conductance regulator. J Biol Chem 271:4725–4732[Abstract/Free Full Text]
165. Reddy MM, Light MJ, Quinton PM 1999 Activation of the epithelial Na⁺ channel (ENaC) requires CFTR Cl- channel function. Nature 402:301–304[CrossRef][Medline]
166. Mall M, Bleich M, Greger R, Schreiber R, Kunzelmann K 1998 The amiloride-inhibitable Na⁺ conductance is reduced by the cystic fibrosis transmembrane conductance regulator in normal but not in cystic fibrosis airways. J Clin Invest 102:15–21[Medline]
167. Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman JE 1993 SNAP receptors implicated in vesicle targeting and fusion. Nature 362:318–324[CrossRef][Medline]
168. Calakos N, Scheller RH 1996 Synaptic vesicle biogenesis, docking, and fusion: a molecular description. Physiol Rev 76:1–29[Abstract/Free Full Text]
169. Bennett MK 1997 Ca²⁺ and the regulation of neurotransmitter secretion. Curr Opin Neurobiol 7:316–322[CrossRef][Medline]
170. Goda Y 1997 SNAREs and regulated vesicle exocytosis. Proc Natl Acad Sci USA 94:769–772[Free Full Text]
171. Olson AL, Knight JB, Pessin JE 1997 Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes. Mol Cell Biol 17:2425–2435[Abstract/Free Full Text]
172. Rea S, Martin LB, McIntosh S, Macaulay SL, Ramsdale T, Baldini G, James DE 1998 Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface. J Biol Chem 273:18784–18792[Abstract/Free Full Text]
173. Volchuk A, Wang Q, Ewart HS, Liu Z, He L, Bennett MK, Klip A 1996 Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport. Mol Biol Cell 7:1075–1082[Abstract]
174. Cheatham B, Volchuk A, Kahn CR, Wang L, Rhodes CJ, Klip A 1996 Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins. Proc Natl Acad Sci USA 93:15169–15173[Abstract/Free Full Text]
175. Hanson PI, Heuser JE, Jahn R 1997 Neurotransmitter release – four years of SNARE complexes. Curr Opin Neurobiol 7:310–315[CrossRef][Medline]
176. Thurmond DC, Ceresa BP, Okada S, Elmendorf JS, Coker K, Pessin JE 1998 Regulation of insulin-stimulated GLUT4 translocation by Munc18c in 3T3L1 adipocytes. J Biol Chem 273:33876–33883[Abstract/Free Full Text]
177. Dulubova I, Sugita S, Hill S, Hosaka M, Fernandez I, Sudhof TC, Rizo J 1999 A conformational switch in syntaxin during exocytosis: role of munc18. EMBO J 18:4372–4382[CrossRef][Medline]
178. Misura KM, Scheller RH, Weis WI 2000 Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex. Nature 404:355–362[CrossRef][Medline]
179. Nielsen S, Marples D, Birn H, Mohtashami M, Dalby NO, Trimble M, Knepper M 1995 Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels. J Clin Invest 96:1834–1844
180. Mandon B, Chou CL, Nielsen S, Knepper MA 1996 Syntaxin-4 is localized to the apical plasma membrane of rat renal collecting duct cells: possible role in aquaporin-2 trafficking. J Clin Invest 98:906–913[Medline]
181. Mandon B, Nielsen S, Kishore BK, Knepper MA 1997 Expression of syntaxins in rat kidney. Am J Physiol 273:F718–F730
182. Inoue T, Nielsen S, Mandon B, Terris J, Kishore BK, Knepper MA 1998 SNAP-23 in rat kidney: colocalization with aquaporin-2 in collecting duct vesicles. Am J Physiol 275:F752–F760
183. Qi J, Peters KW, Liu C, Wang JM, Edinger RS, Johnson JP, Watkins SC, Frizzell RA 1999 Regulation of the amiloride-sensitive epithelial sodium channel by syntaxin 1A. J Biol Chem 274:30345–30348[Abstract/Free Full Text]
184. Saxena S, Quick MW, Tousson A, Oh Y, Warnock DG 1999 Interaction of syntaxins with the amiloride-sensitive epithelial sodium channel. J Biol Chem 274:20812–20817[Abstract/Free Full Text]
185. Bezprozvanny I, Scheller RH, Tsien RW 1995 Functional impact of syntaxin on gating of N-type and Q-type calcium channels. Nature 378:623–626[CrossRef][Medline]
186. Sheng ZH, Rettig J, Cook T, Catterall WA 1996 Calcium-dependent interaction of N-type calcium channels with the synaptic core complex. Nature 379:451–454[CrossRef][Medline]
187. Naren AP, Nelson DJ, Xie W, Jovov B, Pevsner J, Bennett MK, Benos DJ, Quick MW, Kirk KL 1997 Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms. Nature 390:302–305[CrossRef][Medline]
188. Naren AP, Di A, Cormet-Boyaka E, Boyaka PN, McGhee JR, Zhou W, Akagawa K, Fujiwara T, Thome U, Engelhardt JF, Nelson DJ, Kirk KL 2000 Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl^- currents. J Clin Invest 105:377–386[Medline]
189. Yang B, Gonzalez Jr L, Prekeris R, Steegmaier M, Advani RJ, Scheller RH 1999 SNARE interactions are not selective. Implications for membrane fusion specificity. J Biol Chem 274:5649–5653[Abstract/Free Full Text]
190. Su YR, Rutkowski MP, Klanke CA, Wu X, Cui Y, Pun RY, Carter V, Reif M, Menon AG 1996 A novel variant of the ß-subunit of the amiloride-sensitive sodium channel in African Americans. J Am Soc Nephrol 7:2543–2549[Abstract]
191. Baker EH, Dong YB, Sagnella GA, Rothwell M, Onipinla AK, Markandu ND, Cappuccio FP, Cook DG, Persu A, Corvol P, Jeunemaitre X, Carter ND, MacGregor GA 1998 Association of hypertension with T594M mutation in ß subunit of epithelial sodium channels in black people resident in London. Lancet 351:1388–1392[CrossRef][Medline]
192. Persu A, Barbry P, Bassilana F, Houot AM, Mengual R, Lazdunski M, Corvol P, Jeunemaitre X 1998 Genetic analysis of the ß subunit of the epithelial Na⁺ channel in essential hypertension. Hypertension 32:129–137[Abstract/Free Full Text]
193. Ambrosius WT, Bloem LJ, Zhou L, Rebhun JF, Snyder PM, Wagner MA, Guo C, Pratt JH 1999 Genetic variants in the epithelial sodium channel in relation to aldosterone and potassium excretion and risk for hypertension. Hypertension 34:631–637[Abstract/Free Full Text]

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				J. A. McCormick, V. Bhalla, A. C. Pao, and D. Pearce SGK1: A Rapid Aldosterone-Induced Regulator of Renal Sodium Reabsorption Physiology, April 1, 2005; 20(2): 134 - 139. [Abstract] [Full Text] [PDF]

				R. Zhou and P. M. Snyder Nedd4-2 Phosphorylation Induces Serum and Glucocorticoid-regulated Kinase (SGK) Ubiquitination and Degradation J. Biol. Chem., February 11, 2005; 280(6): 4518 - 4523. [Abstract] [Full Text] [PDF]

				C.-T. Chang, M. Bens, E. Hummler, S. Boulkroun, L. Schild, J. Teulon, B. C. Rossier, and A. Vandewalle Vasopressin-stimulated CFTR Cl- currents are increased in the renal collecting duct cells of a mouse model of Liddle's syndrome J. Physiol., January 1, 2005; 562(1): 271 - 284. [Abstract] [Full Text] [PDF]

				M. B. Butterworth, R. S. Edinger, J. P. Johnson, and R. A. Frizzell Acute ENaC Stimulation by cAMP in a Kidney Cell Line is Mediated by Exocytic Insertion from a Recycling Channel Pool J. Gen. Physiol., December 28, 2004; 125(1): 81 - 101. [Abstract] [Full Text] [PDF]

				A. Naray-Fejes-Toth, P. M. Snyder, and G. Fejes-Toth The kidney-specific WNK1 isoform is induced by aldosterone and stimulates epithelial sodium channel-mediated Na+ transport PNAS, December 14, 2004; 101(50): 17434 - 17439. [Abstract] [Full Text] [PDF]

				P. M. Snyder, D. R. Olson, R. Kabra, R. Zhou, and J. C. Steines cAMP and Serum and Glucocorticoid-inducible Kinase (SGK) Regulate the Epithelial Na+ Channel through Convergent Phosphorylation of Nedd4-2 J. Biol. Chem., October 29, 2004; 279(44): 45753 - 45758. [Abstract] [Full Text] [PDF]

				J. Lebowitz, R. S. Edinger, B. An, C. J. Perry, S. Onate, T. R. Kleyman, and J. P. Johnson I{kappa}B Kinase-{beta} (IKK{beta}) Modulation of Epithelial Sodium Channel Activity J. Biol. Chem., October 1, 2004; 279(40): 41985 - 41990. [Abstract] [Full Text] [PDF]

				S. J. Ramminger, K. Richard, S. K. Inglis, S. C. Land, R. E. Olver, and S. M. Wilson A regulated apical Na+ conductance in dexamethasone-treated H441 airway epithelial cells Am J Physiol Lung Cell Mol Physiol, August 1, 2004; 287(2): L411 - L419. [Abstract] [Full Text] [PDF]

				S. Sheng, C. J. Perry, and T. R. Kleyman Extracellular Zn2+ Activates Epithelial Na+ Channels by Eliminating Na+ Self-inhibition J. Biol. Chem., July 23, 2004; 279(30): 31687 - 31696. [Abstract] [Full Text] [PDF]

				L. M. Shearwin-Whyatt, D. L. Brown, F. G. Wylie, J. L. Stow, and S. Kumar N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicular bodies and the Golgi, and has a potential role in protein trafficking J. Cell Sci., July 15, 2004; 117(16): 3679 - 3689. [Abstract] [Full Text] [PDF]

				C. A. Hinojos and P. A. Doris Altered Subcellular Distribution of Na+,K+-ATPase in Proximal Tubules in Young Spontaneously Hypertensive Rats Hypertension, July 1, 2004; 44(1): 95 - 100. [Abstract] [Full Text] [PDF]

				J. Murdaca, C. Treins, M.-N. Monthouel-Kartmann, R. Pontier-Bres, S. Kumar, E. Van Obberghen, and S. Giorgetti-Peraldi Grb10 Prevents Nedd4-mediated Vascular Endothelial Growth Factor Receptor-2 Degradation J. Biol. Chem., June 18, 2004; 279(25): 26754 - 26761. [Abstract] [Full Text] [PDF]

				F. F. Samaha, R. C. Rubenstein, W. Yan, M. Ramkumar, D. I. Levy, Y. J. Ahn, S. Sheng, and T. R. Kleyman Functional Polymorphism in the Carboxyl Terminus of the {alpha}-Subunit of the Human Epithelial Sodium Channel J. Biol. Chem., June 4, 2004; 279(23): 23900 - 23907. [Abstract] [Full Text] [PDF]

				B. K. Berdiev, B. Jovov, W. C. Tucker, A. P. Naren, C. M. Fuller, E. R. Chapman, and D. J. Benos ENaC subunit-subunit interactions and inhibition by syntaxin 1A Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1100 - F1106. [Abstract] [Full Text] [PDF]

				R. Sepehrdad, P. N. Chander, G. Singh, and C. T. Stier Jr Sodium transport antagonism reduces thrombotic microangiopathy in stroke-prone spontaneously hypertensive rats Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1185 - F1192. [Abstract] [Full Text] [PDF]

				S. Sheng, J. B. Bruns, and T. R. Kleyman Extracellular Histidine Residues Crucial for Na+ Self-inhibition of Epithelial Na+ Channels J. Biol. Chem., March 12, 2004; 279(11): 9743 - 9749. [Abstract] [Full Text] [PDF]

				S. B. Condliffe, H. Zhang, and R. A. Frizzell Syntaxin 1A Regulates ENaC Channel Activity J. Biol. Chem., March 12, 2004; 279(11): 10085 - 10092. [Abstract] [Full Text] [PDF]

				W. Biasio, T. Chang, C. J. McIntosh, and F. J. McDonald Identification of Murr1 as a Regulator of the Human {delta} Epithelial Sodium Channel J. Biol. Chem., February 13, 2004; 279(7): 5429 - 5434. [Abstract] [Full Text] [PDF]

				P. M. Snyder, J. C. Steines, and D. R. Olson Relative Contribution of Nedd4 and Nedd4-2 to ENaC Regulation in Epithelia Determined by RNA Interference J. Biol. Chem., February 6, 2004; 279(6): 5042 - 5046. [Abstract] [Full Text] [PDF]

				O. A. Itani, J. R. Campbell, J. Herrero, P. M. Snyder, and C. P. Thomas Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains Am J Physiol Renal Physiol, November 1, 2003; 285(5): F916 - F929. [Abstract] [Full Text] [PDF]

				Y. Berthiaume Long-term stimulation of alveolar epithelial cells by {beta}-adrenergic agonists: increased Na+ transport and modulation of cell growth? Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L798 - L801. [Full Text] [PDF]

				J. B. Bruns, B. Hu, Y. J. Ahn, S. Sheng, R. P. Hughey, and T. R. Kleyman Multiple epithelial Na+ channel domains participate in subunit assembly Am J Physiol Renal Physiol, October 1, 2003; 285(4): F600 - F609. [Abstract] [Full Text] [PDF]

				R. E. Booth and J. D. Stockand Targeted degradation of ENaC in response to PKC activation of the ERK1/2 cascade Am J Physiol Renal Physiol, May 1, 2003; 284(5): F938 - F947. [Abstract] [Full Text] [PDF]

				S. B. Condliffe, M. D. Carattino, R. A. Frizzell, and H. Zhang Syntaxin 1A Regulates ENaC via Domain-specific Interactions J. Biol. Chem., April 4, 2003; 278(15): 12796 - 12804. [Abstract] [Full Text] [PDF]

				J. Loffing and B. Kaissling Sodium and calcium transport pathways along the mammalian distal nephron: from rabbit to human Am J Physiol Renal Physiol, April 1, 2003; 284(4): F628 - F643. [Abstract] [Full Text] [PDF]

				A. S. Leonard, O. Yermolaieva, A. Hruska-Hageman, C. C. Askwith, M. P. Price, J. A. Wemmie, and M. J. Welsh cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1 PNAS, February 18, 2003; 100(4): 2029 - 2034. [Abstract] [Full Text] [PDF]

				S. Sheng, C. J. Perry, and T. R. Kleyman External Nickel Inhibits Epithelial Sodium Channel by Binding to Histidine Residues within the Extracellular Domains of alpha and gamma Subunits and Reducing Channel Open Probability J. Biol. Chem., December 13, 2002; 277(51): 50098 - 50111. [Abstract] [Full Text] [PDF]

				E. Hendron, P. Patel, M. Hausenfluke, N. Gamper, M. S. Shapiro, R. E. Booth, and J. D. Stockand Identification of Cytoplasmic Domains within the Epithelial Na+ Channel Reactive at the Plasma Membrane J. Biol. Chem., September 6, 2002; 277(37): 34480 - 34488. [Abstract] [Full Text] [PDF]

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