The gamma -subunit of ENaC is more important for channel surface expression than the beta -subunit

Angelos-Aristeidis Konstas1 and Christoph Korbmacher1,2

1 University Laboratory of Physiology, University of Oxford, Oxford OX1 3PT, United Kingdom; and 2 Institut für Zelluläre und Molekulare Physiologie, Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and is composed of three homologous subunits: alpha , beta , and gamma . Only heteromultimeric channels made of alpha beta gamma ENaC are efficiently expressed at the cell surface, resulting in maximally amiloride-sensitive currents. To study the relative importance of various regions of the beta - and gamma -subunits for the expression of functional ENaC channels at the cell surface, we constructed hemagglutinin (HA)-tagged beta -gamma -chimeric subunits composed of beta - and gamma -subunit regions and coexpressed them with HA-tagged alpha beta - and alpha gamma -subunits in Xenopus laevis oocytes. The whole cell amiloride-sensitive sodium current (Delta Iami) and surface expression of channels were assessed in parallel using the two-electrode voltage-clamp technique and a chemiluminescence assay. Because coexpression of alpha gamma ENaC resulted in larger Delta Iami and surface expression compared with coexpression of alpha beta ENaC, we hypothesized that the gamma -subunit is more important for ENaC trafficking than the beta -subunit. Using chimeras, we demonstrated that channel activity is largely preserved when the highly conserved second cysteine rich domains (CRD2) of the beta - and gamma -subunits are exchanged. In contrast, exchanging the whole extracellular loops of the beta - and the gamma -subunits largely reduced ENaC currents and ENaC expression in the membrane. This indicates that there is limited interchangeability between molecular regions of the two subunits. Interestingly, our chimera studies demonstrated that the intracellular termini and the two transmembrane domains of gamma ENaC are more important for the expression of functional channels at the cell surface than the corresponding regions of beta ENaC.

epithelial sodium channel domains; chimeras; whole cell amiloride-sensitive current; surface expression


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE AMILORIDE-SENSITIVE EPITHELIAL NA+ channel (ENaC) is the rate-limiting step for sodium absorption in a variety of epithelia, including the renal collecting duct (2, 19). The appropriate regulation of this channel is essential for the maintenance of renal sodium balance and, hence, for long-term regulation of arterial blood pressure (39). Indeed, the analysis of two human genetic diseases, Liddle's syndrome and pseudohypoladosteronism type 1 (PHA-1), has provided direct evidence that molecular dysfunction of ENaC has severe effects on arterial blood pressure. Although loss-of-function mutations of ENaC cause urinary sodium loss, hyperkalemia, and low blood pressure in patients with PHA-1 (10), gain-of-function mutations in Liddle's syndrome result in increased sodium reabsorption, hypokalemia, and severe arterial hypertension (43).

ENaC is composed of three subunits called alpha , beta , and gamma  (7). The expression of the alpha -subunit in Xenopus oocytes generated small amiloride-sensitive currents, whereas expression of the beta - and/or gamma -subunits generated no currents. Coexpression of either beta ENaC or gamma ENaC with alpha ENaC resulted in three- to fivefold larger currents than those seen with alpha ENaC expressed on its own. When all three subunits were coexpressed, the currents were at least 100-fold larger than those observed with alpha ENaC alone (7). By using a quantitative approach to measure the surface expression of ENaC, it was demonstrated that the increase in currents was paralleled by a similar increase in channel surface expression (17). Because the alpha -subunit can apparently form functional channels when expressed on its own, it is thought to play a key role in pore formation. In contrast, the beta - and the gamma -subunits are thought to be important for efficient trafficking of the heteromeric channel to the plasma membrane. This interpretation is consistent with the proposed ENaC stoichiometry of two alpha -, one beta -, and one gamma -subunit (alpha 2beta gamma ) (15, 29) and with biochemical studies demonstrating that alpha -subunits, but not beta - or gamma -subunits, could assemble to homomeric channel complexes (11). However, it has to be acknowledged that the stoichiometry of ENaC is still a matter of debate and that in addition to a tetrameric model, a channel arrangement of eight or nine subunits has been proposed (14, 44).

Amino acid sequence analysis and biochemical studies suggest that the ENaC subunits have cytoplasmic NH2 and COOH termini, two hydrophobic transmembrane domains (M1 and M2), and a large extracellular domain (6, 7, 37, 45). Recent studies have focused on the function of the different domains of ENaC. The extracellular loop contains two cysteine-rich domains (CRD1 and CRD2) important for the efficient transport of assembled subunits to the plasma membrane (16). The region immediately preceding the second transmembrane domain (pre-M2) contains an amiloride-binding site and determines ion selectivity, suggesting that it forms part of the channel pore (41), together with the M2 domain (18). The NH2 terminus contains two key regions, one with an endocytic motif important for channel retrieval from the plasma membrane (8) and a second one, just before the M1 (pre-M1) domain, involved in channel gating (20). The COOH terminus contains a proline-rich PPXY motif, which is believed to be important for interaction with the ubiquitin-protein ligases Nedd4 and Nedd4-2, promoting the endocytosis of the channel (23, 42, 47, 48). The importance of these motifs is illustrated in Liddle's syndrome, in which mutations and/or deletions of the PPXY motif in beta - or gamma ENaC reduce the endocytic retrieval of ENaC from the membrane (1, 46). This results in an increase in the number of ENaC channels in the membrane, which causes hyperabsorption of Na+ and hypertension (17, 24).

Interestingly, the beta - and gamma -subunits share a higher degree of amino acid homology between them than with alpha ENaC (7, 51). As mentioned above, beta  and gamma  have common functional domains. However, the finding that the simultaneous presence of both subunits is required for maximal ENaC surface expression (17) suggests that they have similar but complementary functions. The aim of the present study was to investigate the distinct roles of the beta - and gamma -subunits for the expression of functional ENaC channels at the cell surface.

To elucidate the relative contributions of various regions of the beta - and gamma -subunits to ENaC trafficking, we constructed hemagglutinin (HA)-tagged beta -gamma -chimeric subunits composed of molecular regions of the beta - and gamma -subunits and coexpressed them with HA-tagged alpha beta - and alpha gamma -subunits in Xenopus laevis oocytes. ENaC-mediated whole cell currents and ENaC surface expression were assessed using the two-electrode voltage-clamp technique and a recently established chemiluminescence assay (26, 52). Our results demonstrate that exchanging the whole extracellular loop of the beta - and the gamma -subunits results in an inefficient surface expression of the ENaC complex. On the other hand, some well-conserved regions, like the CRD2, can be exchanged between the beta - and the gamma -subunits, with most of the channel activity being preserved. Moreover, our results suggest that the intracellular termini and the two transmembrane domains of gamma ENaC are more important than the corresponding beta ENaC regions to promote the trafficking of the ENaC complex to the plasma membrane.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Isolation of oocytes and injection of cRNA. Xenopus laevis oocytes were prepared and injected as described (27). Defolliculated stage V-VI oocytes were injected with 1 ng of cRNA of each ENaC subunit and/or chimera. Injected oocytes were kept in modified Barth's saline [in mM: 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.3 Ca(NO3)2,, 0.41 CaCl2, 0.82 MgSO4, and 15 HEPES, adjusted to pH 7.6 with Tris] containing 2 µM amiloride to reduce sodium loading of the oocytes.

Two-electrode voltage-clamp experiments. Oocytes were studied 2 days after injection using the two electrode voltage-clamp technique as previously described (27). Oocytes were clamped at a holding potential of -60 mV. The amiloride-sensitive whole cell current (Delta Iami) was determined by subtracting the corresponding current value measured in the presence of 2 µM amiloride from that measured before the application of amiloride in a NaCl solution (in mM: 95 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, adjusted to pH 7.4 with Tris).

Surface labeling of oocytes. Experiments were performed as recently described (25, 26, 28, 52). Oocytes were incubated for 30 min in ND96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, adjusted to pH 7.4 with Tris) with 1% bovine serum albumin (BSA) at 4°C to block nonspecific binding of the antibodies. Oocytes were then incubated for 60 min at 4°C with 1 µg/ml rat monoclonal anti-HA antibody (3F10, Boehringer, in 1% BSA/ND96), washed eight times at 4°C with 1% BSA/ND96, and incubated with 2 µg/ml horseradish peroxidase (HRP)-coupled secondary antibody (goat anti-rat Fab fragments; Jackson ImmunoResearch) in 1% BSA/ND96 for 40 min. Oocytes were washed thoroughly, initially in 1% BSA/ND96 (4°C, 60 min), and then in ND96 without BSA (4°C, 60 min). Individual oocytes were placed in 50 µl of Power Signal Elisa solution (Pierce, Chester, UK) and, after an equilibration period of about 10 s, chemiluminescence was quantified in a Turner TD-20/20 luminometer (Sunnyvale, CA) by integrating the signal over a period of 15 s. Results are given in relative light units (RLU).

Construction of chimeras and cRNA synthesis. The alpha -, beta -, and gamma -subunits of rat ENaC were extracellularly HA tagged according to the FLAG-epitope placement into the rat ENaC sequence (17). The HA epitope (YPYDVPDYA) was inserted as follows: 191NSSYYPYDVPDYASSR206 in alpha ENaC, 135NTTSYPYDVPDYATLN141 in beta ENaC, and 139EAGSYPYDVPDYAPRF154 in gamma ENaC. HA-tagged constructs were subcloned into the pGEMHE vector (gifts from Dr. Blanche Schwappach, Heidelberg, Germany). To facilitate the construction of the beta -gamma -chimeras, unique restriction sites were created in the beta - and gamma -subunits, resulting in the following alterations in the amino acid sequences: in beta ENaC A383I, I455T and M457L (beta A383I,I455T,M457L), in gamma ENaC G617E and a silent mutation at Y107 (gamma G617E,Y107). Point mutations were generated using the QUICKCHANGE XL site-directed mutagenesis protocol (Stratagene, La Jolla, CA). To confirm that the point mutations did not affect channel function, the mutant subunits were expressed in oocytes and Delta Iami and channel surface expression were determined. In the same batch of oocytes, Delta Iami averaged 1.21 ± 0.18 µA (n = 7) in alpha beta gamma control oocytes, 1.32 ± 0.20 µA (n = 7) in alpha beta A383I,I455T,M457Lgamma oocytes, 1.33 ± 0.12 µA (n = 7) alpha beta gamma G617E,Y107 oocytes, and 1.29 ± 0.21 µA (n = 7) in alpha beta A383I,I455T,M457Lgamma G617E,Y107 oocytes. Similarly, the chemiluminescence signals obtained in the surface expression assay averaged 16.1 ± 3.1 RLU (n = 10) in alpha beta gamma -control oocytes, 15.9 ± 2.8 RLU (n = 10) in alpha beta A383I,I455T,M457Lgamma oocytes, 16.8 ± 2.4 RLU (n = 10) alpha beta gamma G617E,Y107 oocytes, and 15.9 ± 3.0 RLU (n = 10) in alpha beta A383I,I455T,M457Lgamma G617E,Y107 oocytes. Thus the beta A383I,I455T,M457L and gamma G617E,Y107 were deemed suitable for the construction of chimeras; they were used throughout the experiments instead of the wild-type subunits, and, for simplicity, we refer to them as "beta " and "gamma ". The name and exact amino acid constitution of each chimera (E) is shown in Table 1. Table 2 indicates the percent amino acid identity of the swapped regions. All constructs were verified by DNA sequencing. Capped cRNAs from linearized cDNAs were synthesized in vitro using the T7 mMESSAGEmMACHINE (Ambion, Austin, TX), according to the provider's instructions.

                              
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Table 1.   Description of the 14 ENaC chimeras constructed and tested


                              
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Table 2.   Percent amino acid identity of portions of beta - and gamma ENaC subunits swapped in epithelial sodium channel(s) ENaC chimeras

Western blot analysis. Oocytes were homogenized, separated by SDS electrophoresis, and transferred to nitrocellulose filters. Primary rat anti-HA monoclonal antibody (100 ng/ml) and secondary peroxidase-conjugated goat anti-rat antibody (160 ng/ml) were diluted in TBS-blocking solution. Detection was performed with the enhanced luminol reagent NEN (NEN, Boston, MA).

Data analysis. To summarize data from different batches of oocytes and to account for the batch-to-batch variability of overall expression levels, individual Delta Iami and surface expression values were normalized to the average Delta Iami of at least five control alpha beta gamma ENaC oocytes from the same batch and to the average surface expression of at least 10 control oocytes, respectively. Delta Iami and surface expression were assessed in 14 different batches of oocytes for alpha beta gamma -, alpha beta -, and alpha gamma ENaC and in at least two different batches of oocytes for each chimera. Data are given as mean values ± SE, n indicates the number of oocytes, and N indicates the number of different batches of oocytes used. Significance was evaluated by the appropriate version of Student's t-test using values from individual oocytes.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha gamma ENaC travels to the membrane more efficiently than the alpha beta ENaC. Expression of alpha beta gamma ENaC in Xenopus laevis oocytes resulted in Delta Iami averaging 5.60 ± 0.79 µA (n = 70, N = 14) at a holding potential of -60 mV and also in high chemiluminescence signals in the surface expression assay consistent with previously reported data (25, 26, 28). In contrast, Delta Iami and surface expression levels were very low in alpha beta ENaC- or alpha gamma ENaC-expressing oocytes (Fig. 2C). Normalized to the values obtained in oocytes expressing alpha beta gamma ENaC, Delta Iami averaged 1.1 ± 0.2% (n = 70, N = 14) or 3.1 ± 0.4% (n = 70, N = 14) in oocytes expressing alpha beta ENaC or alpha gamma ENaC, respectively. Similarly, the surface expression averaged 1.5 ± 0.2% (n = 144, N = 14) in alpha beta ENaC oocytes and 3.0 ± 0.4% (n = 146, N = 14) in alpha gamma ENaC oocytes (Fig. 2C). Interestingly, both the Delta Iami and the surface expression values for alpha gamma ENaC were significantly higher than those for alpha beta ENaC (P < 0.001). These results confirm the well-known finding that all three ENaC subunits are required to form a fully functional channel (7, 17). In addition, they suggest that the alpha gamma -heteromer travels more efficiently to the plasma membrane than the alpha beta -heteromer.

The Western blot analysis shown in Fig. 1 confirmed that all the beta -gamma -constructs (E1-E14) could be expressed in the oocyte system. To assess the function of the various chimeras, Delta Iami and the surface expression values obtained from oocytes coexpressing a construct E with either alpha beta or alpha gamma were compared with the baseline values determined in alpha beta -, alpha gamma -, and alpha beta gamma ENaC-expressing oocytes (see above). For example, if, in alpha beta E oocytes, Delta Iami and surface expression values were similar to those in alpha beta -oocytes, then this particular chimera E could not functionally substitute for gamma  and could not stimulate the trafficking of alpha beta to the plasma membrane. On the other hand, if Delta Iami and surface expression values in alpha beta E oocytes were similar to those measured in alpha beta gamma ENaC oocytes, then the chimera E could functionally substitute for gamma  and efficiently assemble with alpha beta to promote trafficking of alpha beta E heteromers to the cell surface. Additional information regarding the importance of channel regions for channel trafficking or for ion permeation could be gained from chimeras that were able to stimulate surface expression in the absence of a corresponding increase in Delta Iami.


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Fig. 1.   Similar protein expression of all beta -gamma -chimeras. Western blot analysis to detect hemagglutinin (HA)-tagged protein was performed using total membrane protein from homogenates of oocytes expressing different beta -gamma -chimeras (E1 to E14). Each group of oocytes was injected with only one of the chimeric cRNAs, and 25 oocytes/group were homogenized 2 days after cRNA injection. The bands at ~70-75 kDa indicate that similar levels of HA-tagged chimeric proteins are expressed in all 14 groups of oocytes. The band was absent from noninjected control oocytes, confirming the specificity of the blot.

CRD2 substitution chimeras. The CRD2 domain is highly conserved between the ENaC subunits and is thought to play an essential role in the efficient transport of assembled ENaC channels to the plasma membrane (16). Thus we wondered whether functional ENaC channels could be formed when the CRD2 domains were swapped between beta - and gamma -subunits. Figure 2A illustrates chimeras E9 and E10, in which the beta ENaC CRD2 was substituted by the gamma ENaC CRD2 and vice versa.


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Fig. 2.   A: in chimeras E9 and E10, the beta -epithelial sodium channel (beta ENaC) CRD2 was substituted by the gamma ENaC CRD2 and vice versa. B: representative whole cell current traces, recorded at -60 mV holding potential, are shown from 3 individual oocytes expressing alpha beta gamma , alpha beta E10, and alpha gamma E9, respectively. Amiloride (2 µM) was present in the bath solution during the periods indicated. C: in this and in subsequent figures, the amiloride-sensitive whole cell currents (Delta Iami, open bars) were assessed in 10-70 oocytes from each group, and surface expression (filled bars) was assessed in 19-146 oocytes from each group, from at least 2 different batches of oocytes. Delta Iami and surface expression values are normalized for each group according to the values obtained for alpha beta gamma ENaC control oocytes.

Representative whole cell current recordings from oocytes expressing alpha beta gamma , alpha beta E10, and alpha gamma E9 are shown in Fig. 2B. In alpha beta E10 oocytes, Delta Iami and surface expression averaged 97.2 ± 9.1% (n = 10, N = 2) and 105.6 ± 11.1% (n = 22, N = 2), respectively, of the corresponding values obtained in alpha beta gamma ENaC oocytes (Fig. 2C). These results suggest that beta CRD2 can fully substitute for gamma CRD2. In contrast, in alpha gamma E9 oocytes, Delta Iami and surface expression averaged 30.7 ± 7.2% (n = 10, N = 2) and 74.1 ± 5.1% (n = 21, N = 2), respectively. This indicates that the chimera E9 efficiently assembles with alpha gamma and that the alpha gamma E9 heteromer is transported to the plasma membrane almost as well as alpha beta gamma ENaC. However, the currents mediated by the alpha gamma E9 heteromeric channel are significantly smaller than those observed with alpha beta gamma ENaC (P < 0.01), and there appears to be a dissociation between the ability of E9 to promote channel trafficking and current flow through the channel (Fig. 2C). Although replacing beta CRD2 by gamma CRD2 had little effect on channel trafficking, it apparently reduced the ability of the expressed channel complex to carry sodium currents. Theoretically, a reduced Delta Iami may also be due to a decrease of the amiloride sensitivity of the alpha gamma E9 channel complex. However, as illustrated in Fig. 2B, the amiloride-insensitive whole cell current measured in alpha gamma E9 oocytes in the presence of 2 µM amiloride was slightly smaller than that measured in alpha beta gamma -control oocytes averaging 0.10 ± 0.01 µA (n = 10, N = 2) and 0.20 ± 0.02 µA (n = 10, N = 2), respectively. This essentially rules out the possibility that the E9 chimera alters the amiloride sensitivity of the channel complex. Taken together, these findings indicate that the presence of the beta CRD2 region within the channel complex may be important for ion permeation.

Coexpression of alpha beta E9 or of alpha gamma E10 resulted in similar Delta Iami and surface expression values as observed with alpha beta or alpha gamma , respectively (Fig. 2C). This indicates that E9 cannot functionally substitute for the gamma -subunit and that E10 cannot substitute for the beta -subunit. This is plausible, because with the exception of the CRD2 domains, the structure of E9 corresponds to that of the beta -subunit, whereas the structure of E10 corresponds to that of the gamma -subunit (Fig. 2A).

Extracellular loop substitution chimeras. In the next pair of chimeras, the complete extracellular loop, except the pre-M2 domain of beta ENaC, was substituted by the corresponding loop of gamma ENaC (E1) and vice versa (E2) (Fig. 3A). As shown in Fig. 3B, the Delta Iami and the surface expression resulting from the coexpression of each chimera with either alpha beta or alpha gamma were very low and similar to the values obtained for alpha beta and alpha gamma alone.


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Fig. 3.   Extracellular loop substitution chimeras. A: chimeras had the extracellular loop of beta ENaC substituted by the corresponding loop of gamma ENaC (E1) and vice versa (E2). The other two chimeras (E11 and E12) had similar extracellular loop substitutions as E1 and E2, respectively, but the CRD2 region of the loop was not included in the substitution domain. B: Delta Iami (open bars) and surface expression (filled bars) are illustrated for each group of oocytes tested.

Chimeras E11 and E12 had similar extracellular loop substitutions as E1 and E2, respectively, except that the CRD2 region of the loop was not included in the substitution (Fig. 3A). The results obtained with E11 and E12 chimeras were essentially identical to those obtained with E1 and E2 (Fig. 3B). These findings indicate that the extracellular loops (with or without the CRD2 domains) cannot be swapped between the beta - and the gamma -subunits without loss of function. This indicates that their precise localization within the ENaC channel complex is functionally important for the assembly and trafficking of the complex.

M2 and COOH termini substitution chimeras. Chimeras E7 and E8 (Fig. 4A) had the pre-M2 domain, the M2 domain, and the COOH terminus of beta ENaC substituted by the corresponding regions of gamma ENaC (E7) and vice versa (E8). In alpha beta E8 oocytes, Delta Iami and surface expression averaged 16.5 ± 1.5% (n = 10, N = 2) and 28.7 ± 4.6% (n = 19, N = 2), respectively (Fig. 4B). This indicates that E8 can at least partially substitute for gamma ENaC. In alpha gamma E7 oocytes, Delta Iami and surface expression averaged 75.9 ± 10.7% (n = 10, N = 2) and 86.9 ± 4.7% (n = 23, N = 2), respectively, reaching similar levels as observed in alpha beta gamma ENaC oocytes. This suggests that the E7 chimera can almost fully substitute for the function of the beta -subunit.


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Fig. 4.   M2 and COOH termini substitution chimeras. A: chimeras E7 and E8 had the pre-M2 domain, the M2, and the COOH terminus of beta ENaC substituted by the corresponding regions of gamma ENaC and vice versa. B: Delta Iami (open bars) and surface expression (filled bars) are illustrated for each group of oocytes tested.

It is surprising that although E8 and E7 are converse chimeras (Fig. 4A), their ability to functionally substitute for the gamma - and beta -subunits, respectively, is remarkably different (Fig. 4B). This could be due to a different ability of the chimeras to promote channel delivery to the plasma membrane or to a different effect of the chimeras on channel retrieval rate. Indeed, because the COOH termini of the beta - and gamma -subunits have been reported to be essential for an efficient retrieval of ENaC channels from the plasma membrane (24, 42), arrangements with a duplication of the gamma - or the beta -COOH termini (as in alpha gamma E7 and alpha beta E8) may alter the rate of channel retrieval.

To assess the rate of channel retrieval in alpha beta gamma , alpha beta E8, and alpha gamma E7 oocytes, delivery of new channels to the plasma membrane was inhibited by adding 18 µM brefedin A (BFA) to oocytes 2 days after cRNA injection. BFA is a fungal metabolite that inhibits the secretory pathway of newly synthesized proteins without affecting clathrin-mediated endocytosis (36). Figure 5 illustrates the effect of BFA on Delta Iami, in alpha beta gamma -, alpha beta E8, and alpha gamma E7 oocytes. In alpha beta gamma -oocytes, Delta Iami decreased by about 85% within 4 h after addition of BFA (Fig. 5A). This BFA-induced decline of Delta Iami reflects the rate of channel retrieval and is in good agreement with previously reported data (42). In nontreated oocytes, Delta Iami continued to increase throughout the 20-h period examined, suggesting that in nontreated oocytes, channel insertion exceeded channel retrieval during this period. BFA had essentially the same effect on Delta Iami in alpha beta E8 and alpha gamma E7 oocytes (Fig. 5, B and C, respectively). The similar susceptibility of Delta Iami to BFA in alpha beta E8 and alpha gamma E7 oocytes indicates that alpha beta E8 and alpha gamma E7 have similar retrieval rates as alpha beta gamma ENaC. Hence, the higher surface expression detected in alpha gamma E7 oocytes compared with that in alpha beta E8 oocytes has to be due to enhanced delivery of alpha gamma E7 heteromeric channels to the plasma membrane.


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Fig. 5.   Effect of brefedin A (BFA) on Delta Iami in alpha beta gamma -control oocytes (A), in alpha beta E8 oocytes (B), and alpha gamma E7 oocytes (C). Two days after cRNA injection, oocytes were divided into a control group (open circles) and a BFA-treated group (filled circles). BFA (18 µM) was added at time zero as indicated by the arrow pointing down, and Delta Iami was subsequently assessed in 4- and 12-h intervals. Each value represents the mean Delta Iami of 10 oocytes.

In alpha beta E7 oocytes, which are assumed to express channels with a predicted stoichiometry of 2alpha :1beta :1E7, the beta -subunit is essentially duplicated in each channel complex except for the pre-M2 domain, the M2 domain, and the COOH terminus of E7, which are derived from gamma ENaC (Fig. 4A). Therefore, it is not surprising that in alpha beta E7 oocytes, the Delta Iami and surface expression values were similar to those in alpha beta -oocytes expected to form 2alpha :2beta -complexes (Fig. 4B). In analogy to the situation with alpha beta E7, coexpression of alpha gamma E8 is expected to result in a quasi-duplication of the gamma -subunit within the channel complex (Fig. 4B). Indeed, in alpha gamma E8 oocytes, Delta Iami was found to be as low as that detected in alpha gamma -oocytes likely to express channel complexes with 2alpha :2gamma -subunits. However, in alpha gamma E8 oocytes, a substantial surface expression was observed averaging 43.9 ± 4.7% (n = 22, N = 2) of that of alpha beta gamma -oocytes (Fig. 4B). This high level of surface expression in the presence of a low Delta Iami indicates that alpha gamma E8 heteromeric complexes are expressed at the cell surface but do not show proper ion channel function. An alternative explanation for a low Delta Iami would be a reduced sensitivity to amiloride of the alpha gamma E8 channel complex. However, this explanation can be ruled out because the inward currents observed in the presence of amiloride were very similar in alpha gamma E8 oocytes and in alpha gamma matched control oocytes averaging 23 ± 3 nA (n = 10, N = 2) and 21 ± 4 nA (n = 10, N = 10), respectively .

The high level of surface expression of alpha gamma E8 is of particular interest because it suggests that even in an arrangement where the alpha -subunit is supplemented with a gamma -subunit and a chimera that contains large parts of the gamma -subunit, the resulting heteromeric channel complex can be transported efficiently to the plasma membrane. This is not the case with the alpha beta E7 complex containing mainly beta -subunit components. This suggests that the gamma -subunit has a more potent role than the beta -subunit in assembly and/or trafficking the ENaC complexes to the plasma membrane. However, in terms of its ion-conducting property, the function of the alpha gamma E8 heteromeric complex is poor, which suggests an important role of the missing beta -subunit regions for ion permeation.

The permissive substitutions of CRD2 in E9 and E10 (Fig. 2) and of the M2 and COOH termini in E7 and E8 (Fig. 4B) pointed to the possibility that substitutions of all the above domains may still result in functional channels. To test this hypothesis, we constructed E3 and E4 that had the CRD2, the pre-M2 domain, M2, and the COOH terminus of beta ENaC substituted by the corresponding regions of gamma ENaC and vice versa. However, the Delta Iami and the surface expression of E3 and E4 chimeras, when coexpressed with either alpha beta or alpha gamma , were very low and similar to the values obtained for alpha beta and alpha gamma alone (data not shown). This indicates that beta -gamma -chimeras with such large substituted regions, including the CRD2 domain, the pre-M2 domain, the M2 domain, and the COOH terminus, cannot functionally replace the native beta - or gamma -subunits.

NH2 termini and M1 substitution chimeras. In chimeras E5 and E6 (Fig. 6A), the NH2 terminus and M1 domain of beta ENaC were substituted by the corresponding regions of gamma ENaC (E5) and vice versa (E6). In alpha gamma E5 oocytes, Delta Iami and surface expression averaged 52.3 ± 11.5% (n = 10, N = 2) and 53.3 ± 10.1% (n = 20, N = 2), respectively. This indicates that E5 can replace beta ENaC function to a large extent (Fig. 6B). In contrast, in alpha beta E6 oocytes, Delta Iami and surface expression were very low and not significantly different from the value measured in alpha beta oocytes (Fig. 6B). Hence, although the NH2 terminus and the M1 region of beta  can be substituted by the corresponding regions of gamma , the reverse substitution fails to produce functional channels.


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Fig. 6.   NH2 termini and M1 substitution chimeras. A: chimeras E5 and E6 had the NH2 terminus and M1 of beta ENaC substituted by the corresponding regions of gamma ENaC and vice versa. B: Delta Iami (open bars) and surface expression (filled bars) are illustrated for each group of oocytes tested.

NH2 and COOH termini substitution chimeras. Both the NH2 and COOH termini of beta - and gamma -subunits have been shown to be important for the retrieval/endocytosis of the ENaC channels from the plasma membrane (8, 42). We speculated that chimeras with both intracellular beta - or gamma -termini substituted by the corresponding gamma - or beta -termini, respectively, may still be efficiently expressed at the plasma membrane when coexpressed with alpha gamma or alpha beta . These chimeras are illustrated in Fig. 7A, and are named E13 and E14. Figure 7B shows that the Delta Iami and the surface expression of each chimera when coexpressed with either alpha beta or alpha gamma were very low and similar to the values obtained for alpha beta and alpha gamma alone. Therefore, it is apparently not possible to efficiently transport an ENaC complex to the plasma membrane when all intracellular termini are contributed by the alpha beta - or alpha gamma -subunits. This finding, in combination with the results in Figs. 4 and 6, suggests that at least one of the intracellular termini must be contributed from beta  and at least another one from gamma  in order for the ENaC complex to be transported to the plasma membrane.


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Fig. 7.   NH2 and COOH termini substitution chimeras. A: chimeras E13 and E14 had the intracellular beta ENaC termini substituted by the corresponding gamma ENaC termini and vice versa. B: Delta Iami (open bars) and surface expression (filled bars) are illustrated for each group of oocytes tested.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we used a chimera approach to investigate the relative importance of several domains of the beta - and gamma -subunits to promote the expression of functional ENaC channels in the plasma membrane of Xenopus laevis oocytes. The main findings of the present study are the following: 1) replacing the extracellular loop of beta ENaC by that of gamma ENaC or vice versa results in a very inefficient surface expression of ENaC, 2) the beta CRD2 region and the gamma CRD2 region can efficiently substitute for each other, 3) the M2 and COOH terminus of gamma ENaC have a more potent role than the corresponding regions of beta ENaC in the assembly/trafficking of the ENaC complex and its expression at the plasma membrane, and 4) the NH2 terminus and the M1 region of gamma ENaC are more important for the formation of a functional ENaC channel than the corresponding regions of beta ENaC.

Functional role of extracellular domains of the beta - and gamma -subunits of ENaC. The inability of the chimeras E1 and E2 to increase the surface expression of alpha gamma and alpha beta , respectively, suggests that the extracellular loops of both the beta - and the gamma -subunits have to be simultaneously present to allow a fully functional ENaC channel complex to form. An alternative interpretation is the possibility that chimeras with an inability to increase surface expression do not fold properly due to some misfit between the extracellular, intracellular, and transmembrane domains and that this causes their retention in the ER (i.e., an "intrasubunit" problem rather than an "intersubunit" problem). At present, the functional role of the large extracellular loops of ENaC and the relative importance of the alpha -, beta -, and gamma -subunit loops are still poorly understood (5). It is possible that a distinct glycosylation pattern of the beta - and gamma -subunits is essential for the efficient assembly of the channel and its translocation to the plasma membrane. It has also been suggested that the extracellular loops may contain important target sites for ENaC-activating proteases (12, 50). Our study provides evidence that the extracellular loops of the beta - and gamma -subunits each have unique and distinct functional features because both loops are required for proper channel function.

The two highly conserved cysteine-rich domains (CRD1 and CRD2) spanning nearly 50% of the extracellular loop are a distinguishing feature of the extracellular loop of ENaC (7). Chimeras E11 and E12 with partial substitutions of the extracellular loops not affecting the CRD2 region also failed to promote the trafficking of alpha gamma and alpha beta , respectively, to the plasma membrane. This result suggests that there are important extracellular domains before the CRD2 region and including the CRD1 region that cannot be exchanged between the beta - and gamma -subunits without loss of function of the subunits to promote expression of a heteromeric ENaC channel complex.

Interestingly, ENaC channels were efficiently translocated to the cell surface when the CRD2 was exchanged between the beta - and gamma -subunits in E9 and E10. It has been proposed that cysteines in a CRD domain form disulfide bonds with cysteines in another CRD domain on the same subunit (intrasubunit bonds) (16). Thus our results suggest that the CRD2 regions in the beta - and gamma -subunits have such a similar arrangement of cysteines that the gamma CRD2 can form successful intrasubunit disulfide bonds with the beta CRD1 in E9 and the beta CRD2 with gamma CRD1 in E10. The finding that, in alpha beta E9 oocytes, Delta Iami was relatively small compared with the substantial level of ENaC surface expression suggests that the substitution of the beta CRD2 by the gamma CRD2 alters the ion conducting properties of the channel complex. This indicates a possible involvement of the CRD2 region in the function of the pore region or the gating mechanism of the channel.

Preferential role of the gamma -subunit in the trafficking of ENaC. A series of results suggests that several domains of the gamma ENaC have a more potent role in trafficking the channel complex to the plasma membrane than the corresponding regions of beta ENaC. First, expression of alpha gamma E5 resulted in surface expression and Delta Iami values that were substantially greater than those obtained with alpha gamma , whereas alpha beta E6 failed to increase expression levels above those observed with alpha beta . Hence, the NH2 terminus and the M1 region of gamma  can successfully replace the corresponding regions of the beta -subunit, whereas the reverse substitution fails to produce fully functional channels. Second, alpha gamma E7 produced higher Delta Iami and surface expression values than alpha beta E8. This indicates that the combined pre-M2 region, M2 domain, and COOH terminus of gamma ENaC can substitute for the corresponding regions of beta ENaC more efficiently than the combined pre-M2 region, M2 domain, and COOH terminus of beta ENaC can substitute for the corresponding regions of gamma ENaC. Third, alpha gamma E8, which essentially represents a duplication of most of the gamma -subunit except the pre-M2 region, M2 domain, and the COOH terminus of E8 that are from beta ENaC, can be transported efficiently to the plasma membrane. This is not the case with alpha beta E7 representing a similar duplication of most of the beta -subunit, indicating once more the primacy of the gamma -subunit over the beta -subunit in promoting the formation of ENaC channel complexes that can efficiently traffic to the plasma membrane.

The interpretation that regions of the gamma -subunit are more important for ENaC trafficking than corresponding regions of the beta -subunit is consistent with our finding that the alpha gamma -heteromer (probably as 2alpha :2gamma ) travels more efficiently to the plasma membrane than the alpha beta -heteromer and produces larger Delta Iami. However, conflicting results have been reported on this issue. Two studies reported similar Delta Iami and surface expression values for alpha beta and alpha gamma (7, 17), but more recent studies found higher values for alpha gamma compared with alpha beta (9, 34), consistent with our results. The failure to detect a significant difference between alpha gamma and alpha beta expression levels in some of the earlier studies may be due to the limited number of oocytes used. We examined 70 matched oocytes for each of the two groups from 14 different batches of oocytes. Thus we are confident that there is a small but significant difference between the Delta Iami and surface expression levels of alpha beta ENaC and alpha gamma ENaC. The higher values obtained with alpha gamma ENaC support the conclusion form our chimera studies that the gamma -subunit plays a particularly important role for the assembly and/or trafficking of ENaC.

Physiological significance of the present results. Interestingly, the BFA experiments demonstrated that alpha gamma E7 and alpha beta E8 have a rapid retrieval rate similar to that of alpha beta gamma ENaC controls. This result is of significance because it indicates that the PPXY motifs of the beta - and gamma -COOH termini are recognized equally well from the endocytic machinery (i.e., Nedd4/Nedd4-2). As a result, channel complexes are internalized efficiently even in subunit arrangements in which there are two beta ENaC PPXY motifs and not a single gamma ENaC PPXY motif present and vice versa. Therefore, the COOH-terminal PPXY motifs are apparently functionally interchangeable between the beta - and the gamma -subunits. This conclusion is in good agreement with a recent study suggesting that each of the second and the third WW motifs of Nedd4 binds both to beta - and gamma -COOH termini (21). It is probably the absence of any preference of the binding of these two WW motifs to the beta - and gamma -COOH termini that makes the COOH termini interchangeable between the two subunits and, on the other hand, also explains why truncation of both beta - and gamma -COOH termini has an additive effect regarding the hyperactivity of the channel (40).

Aldosterone stimulates the functional expression of ENaC in several different tissues (19). So far, the majority of the results have suggested that the alpha ENaC mRNA has a different pattern of response to aldosterone compared with beta - and gamma ENaC mRNAs that share a similar profile. Aldosterone administration markedly elevated alpha ENaC mRNA and protein expression in the distal nephron, whereas neither beta - nor gamma ENaC mRNA expression was altered (3, 13, 31, 35). In contrast, in the distal colon, the abundance of beta - and gamma ENaC mRNA was increased by aldosterone, whereas alpha ENaC mRNA was not changed (3, 13, 35, 38). A recent study suggested that aldosterone enhanced ENaC activity in mouse endometrial endothelium by upregulating only the gamma ENaC subunit (49). Thus the regulation of the expression of the three subunits seems to vary between tissues. The situation is further complicated by the fact that changes observed at the mRNA level may not necessarily reflect concomitant changes at the protein level and vice versa (32). Moreover, a differential subcellular distribution of ENaC subunits and redistribution of all three subunits to the apical region in response to aldosterone stimulation has been reported (30, 31). At present, it is not quite clear which of the subunit is rate limiting for the assembly of fully functional ENaC complexes and for their efficient trafficking to the plasma membrane. The results of the present study suggest that gamma ENaC makes a particularly important contribution to ENaC trafficking and that some of its regions are essential and cannot be substituted by corresponding regions of the beta -subunit.

Studies of ENaC subunit knockout mice provide additional support for the conclusion that gamma ENaC is of key importance for ENaC trafficking. alpha ENaC knockout neonates failed to clear their lungs of liquid and died within 40 h after birth from respiratory distress (22). In contrast, beta ENaC-deficient mice did not show a lung phenotype but died within 2 days after birth because of an acute PHA1 phenotype, most likely of hyperkalemia (33). Hence, beta ENaC is essential for ENaC function in the renal collecting duct but, in contrast to alpha ENaC, does not seem to be required for the transition from a liquid-filled to an air-filled lung. Newborn gamma ENaC knockout mice also exhibited a PHA 1 phenotype but, in addition, had an impaired lung water clearance (4). The above results indicate that the presence of gamma ENaC is probably more important for neonatal lung liquid clearance than the presence of the beta ENaC. This is consistent with our results that the alpha gamma dimer travels to the plasma membrane more efficiently than the alpha beta -dimer. Thus it is likely that the beta ENaC knockout newborn mice have a higher residual ENaC activity in respiratory epithelia than the gamma ENaC knockouts. Consequently, beta ENaC knockout mice can clear lung liquid more efficiently that gamma ENaC knockouts.

In conclusion, the results of this study suggest that although both beta ENaC and gamma ENaC are essential in the translocation of the channel complex to the plasma membrane, gamma ENaC has a more potent role than beta ENaC in assembling and trafficking the complex to the surface.


    ACKNOWLEDGEMENTS

We thank Dr. S. J. Tucker for expert advice regarding the construction of the chimeras.


    FOOTNOTES

This work was supported by grants from the Wellcome Trust and the National Kidney Research Fund. A.-A. Konstas is a recipient of a National Kidney Research Fund studentship.

Address for reprint requests and other correspondence: C. Korbmacher, Institut für Zelluläre und Molekulare Physiologie, Universität Erlangen-Nürnberg, Waldstr. 6, D-91054 Erlangen, Germany (E-mail: christoph.korbmacher{at}physiologie2.med.uni-erlangen.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00385.2002

Received 26 August 2002; accepted in final form 23 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abriel, H, Loffing J, Rebhun JF, Pratt JH, Schild L, Horisberger JD, Rotin D, and Staub O. Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome. J Clin Invest 103: 667-673, 1999[Abstract/Free Full Text].

2.   Alvarez de la Rosa, D, Canessa CM, Fyfe GK, and Zhang P. Structure and regulation of amiloride-sensitive sodium channels. Annu Rev Physiol 62: 573-594, 2000[ISI][Medline].

3.   Asher, C, Wald H, Rossier BC, and Garty H. Aldosterone-induced increase in the abundance of Na+ channel subunits. Am J Physiol Cell Physiol 271: C605-C611, 1996[Abstract/Free Full Text].

4.   Barker, PM, Nguyen MS, Gatzy JT, Grubb B, Norman H, Hummler E, Rossier B, Boucher RC, and Koller B. Role of gamma ENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism. J Clin Invest 102: 1634-1640, 1998[Abstract/Free Full Text].

5.   Benos, DJ, and Stanton BA. Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion channels. J Physiol 520: 631-644, 1999[Abstract/Free Full Text].

6.   Canessa, CM, Merillat AM, and Rossier BC. Membrane topology of the epithelial sodium channel in intact cells. Am J Physiol Cell Physiol 267: C1682-C1690, 1994[Abstract/Free Full Text].

7.   Canessa, CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, and Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994[ISI][Medline].

8.   Chalfant, ML, Denton JS, Langloh AL, Karlson KH, Loffing J, Benos DJ, and Stanton BA. The NH2 terminus of the epithelial sodium channel contains an endocytic motif. J Biol Chem 274: 32889-32896, 1999[Abstract/Free Full Text].

9.   Chalfant, ML, Denton JS, Langloh AL, Karlson KH, Loffing J, Benos DJ, and Stanton BA. The NH2 terminus of the epithelial sodium channel contains an endocytic motif. J Biol Chem 274: 32889-32896, 1999[Abstract/Free Full Text].

10.   Chang, SS, Gründer S, Hanukoglu A, Rosler A, Mathew PM, Hanukoglu I, Schild L, Lu Y, Shimkets RA, Nelson Williams C, Rossier BC, and Lifton RP. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 12: 248-253, 1996[ISI][Medline].

11.   Cheng, C, Prince LS, Snyder PM, and Welsh MJ. Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis. J Biol Chem 273: 22693-22700, 1998[Abstract/Free Full Text].

12.   Chraibi, A, Vallet V, Firsov D, Hess SK, and Horisberger JD. Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes. J Gen Physiol 111: 127-138, 1998[Abstract/Free Full Text].

13.   Escoubet, B, Coureau C, Bonvalet JP, and Farman N. Noncoordinate regulation of epithelial Na+ channel and Na+ pump subunit mRNAs in kidney and colon by aldosterone. Am J Physiol Cell Physiol 272: C1482-C1491, 1997[Abstract/Free Full Text].

14.   Eskandari, S, Snyder PM, Kreman M, Zampighi GA, Welsh MJ, and Wright EM. Number of subunits comprising the epithelial sodium channel. J Biol Chem 274: 27281-27286, 1999[Abstract/Free Full Text].

15.   Firsov, D, Gautschi I, Merillat AM, Rossier BC, and Schild L. The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO J 17: 344-352, 1998[Abstract/Free Full Text].

16.   Firsov, D, Robert Nicoud M, Gründer S, Schild L, and Rossier BC. 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, 1999[Abstract/Free Full Text].

17.   Firsov, D, Schild L, Gautschi I, Merillat AM, Schneeberger E, and Rossier BC. 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, 1996[Abstract/Free Full Text].

18.   Fyfe, GK, Zhang P, and Canessa CM. The second hydrophobic domain contributes to the kinetic properties of epithelial sodium channels. J Biol Chem 274: 36415-36421, 1999[Abstract/Free Full Text].

19.   Garty, H, and Palmer LG. Epithelial sodium channels: function, structure, and regulation. Physiol Rev 77: 359-396, 1997[Abstract/Free Full Text].

20.   Gründer, S, Jaeger NF, Gautschi I, Schild L, and Rossier BC. Identification of a highly conserved sequence at the N-terminus of the epithelial Na+ channel alpha  subunit involved in gating. Pflügers Arch 438: 709-715, 1999[ISI][Medline].

21.   Harvey, KF, Dinudom A, Komwatana P, Jolliffe CN, Day ML, Parasivam G, Cook DI, and Kumar S. 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, 1999[Abstract/Free Full Text].

22.   Hummler, E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, Boucher R, and Rossier BC. Early death due to defective neonatal lung liquid clearance in alpha -ENaC-deficient mice. Nat Genet 12: 325-328, 1996[ISI][Medline].

23.   Kamynina, E, Debonneville C, Bens M, Vandewalle A, and Staub O. A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel. FASEB J 15: 204-214, 2001[Abstract/Free Full Text].

24.   Kellenberger, S, Gautschi I, Rossier BC, and Schild L. Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system. J Clin Invest 101: 2741-27450, 1998[Abstract/Free Full Text].

25.   Konstas, AA, Koch JP, Tucker SJ, and Korbmacher C. CFTR-dependent upregulation of Kir1.1 (ROMK) renal K+ channels by the epithelial sodium channel (ENaC). J Biol Chem 277: 21346-21351, 2002[Abstract/Free Full Text].

26.   Konstas, AA, Bielfeld-Ackermann A, and Korbmacher C. Sulfonylurea receptors inhibit the epithelial sodium channel (ENaC) by reducing surface expression. Pflügers Arch 442: 752-761, 2001[ISI][Medline].

27.   Konstas, AA, Mavrelos D, and Korbmacher C. Conservation of pH sensitivity in the epithelial sodium channel (ENaC) with Liddle's syndrome mutation. Pflügers Arch 441: 341-350, 2000[ISI][Medline].

28.   Konstas, AA, Shearwin-Whyatt LM, Fotia AB, Degger B, Riccardi D, Cook DI, Korbmacher C, and Kumar S. Regulation of the epithelial sodium channel by N4WBP5A, a novel Nedd4/Nedd4-2-interacting protein. J Biol Chem 277: 29406-29416, 2002[Abstract/Free Full Text].

29.   Kosari, F, Sheng S, Li J, Mak DO, Foskett JK, and Kleyman TR. Subunit stoichiometry of the epithelial sodium channel. J Biol Chem 273: 13469-13474, 1998[Abstract/Free Full Text].

30.   Loffing, J, Pietri L, Aregger F, Bloch-Faure M, Ziegler U, Meneton P, Rossier BC, and Kaissling B. Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and low-Na+ diets. Am J Physiol Renal Physiol 279: F252-F258, 2000[Abstract/Free Full Text].

31.   Masilamani, S, Kim GH, Mitchell C, Wade JB, and Knepper MA. Aldosterone-mediated regulation of ENaC alpha , beta , and gamma  subunit proteins in rat kidney. J Clin Invest 104: R19-R23, 1999[ISI][Medline].

32.   May, A, Puoti A, Gaeggeler HP, Horisberger JD, and Rossier BC. Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel alpha  subunit in A6 renal cells. J Am Soc Nephrol 8: 1813-1822, 1997[Abstract].

33.   McDonald, FJ, Yang B, Hrstka RF, Drummond HA, Tarr DE, McCray PB, Jr, Stokes JB, Welsh MJ, and Williamson RA. Disruption of the beta  subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype. Proc Natl Acad Sci USA 96: 1727-1731, 1999[Abstract/Free Full Text].

34.   McNicholas, CM, and Canessa CM. Diversity of channels generated by different combinations of epithelial sodium channel subunits. J Gen Physiol 109: 681-692, 1997[Abstract/Free Full Text].

35.   Ono, S, Kusano E, Muto S, Ando Y, and Asano Y. A low-Na+ diet enhances expression of mRNA for epithelial Na+ channel in rat renal inner medulla. Pflügers Arch 434: 756-763, 1997[ISI][Medline].

36.   Pelham, HR. Multiple targets for brefeldin A. Cell 67: 449-451, 1991[ISI][Medline].

37.   Renard, S, Lingueglia E, Voilley N, Lazdunski M, and Barbry P. Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel. J Biol Chem 269: 12981-12986, 1994[Abstract/Free Full Text].

38.   Renard, S, Voilley N, Bassilana F, Lazdunski M, and Barbry P. Localization and regulation by steroids of the alpha , beta  and gamma  subunits of the amiloride-sensitive Na+ channel in colon, lung and kidney. Pflügers Arch 430: 299-307, 1995[ISI][Medline].

39.   Rossier, BC, Pradervand S, Schild L, and Hummler E. Epithelial sodium channel and the control of sodium balance: interaction between genetic and environmental factors. Annu Rev Physiol 64: 877-897, 2002[ISI][Medline].

40.   Schild, L, Canessa CM, Shimkets RA, Gautschi I, Lifton RP, and Rossier BC. 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, 1995[Abstract].

41.   Schild, L, Schneeberger E, Gautschi I, and Firsov D. Identification of amino acid residues in the alpha , beta , and gamma  subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation. J Gen Physiol 109: 15-26, 1997[Abstract/Free Full Text].

42.   Shimkets, RA, Lifton RP, and Canessa CM. The activity of the epithelial sodium channel is regulated by clathrin-mediated endocytosis. J Biol Chem 272: 25537-25541, 1997[Abstract/Free Full Text].

43.   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, and Lifton RP. Liddle's syndrome: heritable human hypertension caused by mutations in the beta  subunit of the epithelial sodium channel. Cell 79: 407-414, 1994[ISI][Medline].

44.   Snyder, PM, Cheng C, Prince LS, Rogers JC, and Welsh MJ. Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits. J Biol Chem 273: 681-684, 1998[Abstract/Free Full Text].

45.   Snyder, PM, McDonald FJ, Stokes JB, and Welsh MJ. Membrane topology of the amiloride-sensitive epithelial sodium channel. J Biol Chem 269: 24379-24383, 1994[Abstract/Free Full Text].

46.   Staub, O, Abriel H, Plant P, Ishikawa T, Kanelis V, Saleki R, Horisberger JD, Schild L, and Rotin D. Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination. Kidney Int 57: 809-815, 2000[ISI][Medline].

47.   Staub, O, Dho S, Henry P, Correa J, Ishikawa T, McGlade J, and Rotin D. 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, 1996[Abstract].

48.   Staub, O, Gautschi I, Ishikawa T, Breitschopf K, Ciechanover A, Schild L, and Rotin D. Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. EMBO J 16: 6325-6336, 1997[Abstract/Free Full Text].

49.   Tsang, LL, Chan LN, Wang XF, So SC, Yuen JP, Fiscus RR, and Chan HC. Enhanced epithelial Na+ channel (ENaC) activity in mouse endometrial epithelium by upregulation of gamma ENaC subunit. Jpn J Physiol 51: 539-543, 2001[ISI][Medline].

50.   Vallet, V, Pfister C, Loffing J, and Rossier BC. Cell-surface expression of the channel activating protease xCAP-1 is required for activation of ENaC in the Xenopus oocyte. J Am Soc Nephrol 13: 588-594, 2002[Abstract/Free Full Text].

51.   Waldmann, R, Champigny G, Bassilana F, Voilley N, and Lazdunski M. Molecular cloning and functional expression of a novel amiloride-sensitive Na+ channel. J Biol Chem 270: 27411-27414, 1995[Abstract/Free Full Text].

52.   Zerangue, N, Schwappach B, Jan YN, and Jan LY. A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane KATP channels. Neuron 22: 537-548, 1999[ISI][Medline].


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