INTRODUCTION

TOP 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 , , and  (7). The expression of the -subunit in Xenopus oocytes generated small amiloride-sensitive currents, whereas expression of the - and/or -subunits generated no currents. Coexpression of either ENaC or ENaC with ENaC resulted in three- to fivefold larger currents than those seen with ENaC expressed on its own. When all three subunits were coexpressed, the currents were at least 100-fold larger than those observed with 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 -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 - and the -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 -, one -, and one -subunit (₂) (15, 29) and with biochemical studies demonstrating that -subunits, but not - or -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 - or 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 - and -subunits share a higher degree of amino acid homology between them than with ENaC (7, 51). As mentioned above,  and  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 - and -subunits for the expression of functional ENaC channels at the cell surface.

To elucidate the relative contributions of various regions of the - and -subunits to ENaC trafficking, we constructed hemagglutinin (HA)-tagged --chimeric subunits composed of molecular regions of the - and -subunits and coexpressed them with HA-tagged - and -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 - and the -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 - and the -subunits, with most of the channel activity being preserved. Moreover, our results suggest that the intracellular termini and the two transmembrane domains of ENaC are more important than the corresponding ENaC regions to promote the trafficking of the ENaC complex to the plasma membrane.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 (I_ami) 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 -, -, and -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 ENaC, ¹³⁵NTTSYPYDVPDYATLN¹⁴¹ in ENaC, and ¹³⁹EAGSYPYDVPDYAPRF¹⁵⁴ in ENaC. HA-tagged constructs were subcloned into the pGEMHE vector (gifts from Dr. Blanche Schwappach, Heidelberg, Germany). To facilitate the construction of the --chimeras, unique restriction sites were created in the - and -subunits, resulting in the following alterations in the amino acid sequences: in ENaC A383I, I455T and M457L (_{A383I,I455T,M457L}), in ENaC G617E and a silent mutation at Y107 (_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 I_ami and channel surface expression were determined. In the same batch of oocytes, I_ami averaged 1.21 ± 0.18 µA (n = 7) in control oocytes, 1.32 ± 0.20 µA (n = 7) in _{A383I,I455T,M457L} oocytes, 1.33 ± 0.12 µA (n = 7) _G617E,Y107 oocytes, and 1.29 ± 0.21 µA (n = 7) in _{A383I,I455T,M457LG617E,Y107} oocytes. Similarly, the chemiluminescence signals obtained in the surface expression assay averaged 16.1 ± 3.1 RLU (n = 10) in -control oocytes, 15.9 ± 2.8 RLU (n = 10) in _{A383I,I455T,M457L} oocytes, 16.8 ± 2.4 RLU (n = 10) _G617E,Y107 oocytes, and 15.9 ± 3.0 RLU (n = 10) in _{A383I,I455T,M457LG617E,Y107} oocytes. Thus the _{A383I,I455T,M457L} and _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 "" and "". 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.

View this table: [in this window] [in a new window]   Table 2.   Percent amino acid identity of portions of - and 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 I_ami and surface expression values were normalized to the average I_ami of at least five control ENaC oocytes from the same batch and to the average surface expression of at least 10 control oocytes, respectively. I_ami and surface expression were assessed in 14 different batches of oocytes for -, -, and 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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ENaC travels to the membrane more efficiently than the ENaC. Expression of ENaC in Xenopus laevis oocytes resulted in I_ami 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, I_ami and surface expression levels were very low in ENaC- or ENaC-expressing oocytes (Fig. 2C). Normalized to the values obtained in oocytes expressing ENaC, I_ami averaged 1.1 ± 0.2% (n = 70, N = 14) or 3.1 ± 0.4% (n = 70, N = 14) in oocytes expressing ENaC or ENaC, respectively. Similarly, the surface expression averaged 1.5 ± 0.2% (n = 144, N = 14) in ENaC oocytes and 3.0 ± 0.4% (n = 146, N = 14) in ENaC oocytes (Fig. 2C). Interestingly, both the I_ami and the surface expression values for ENaC were significantly higher than those for 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 -heteromer travels more efficiently to the plasma membrane than the -heteromer.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Similar protein expression of all --chimeras. Western blot analysis to detect hemagglutinin (HA)-tagged protein was performed using total membrane protein from homogenates of oocytes expressing different --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 - and -subunits. Figure 2A illustrates chimeras E9 and E10, in which the ENaC CRD2 was substituted by the ENaC CRD2 and vice versa.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A: in chimeras E9 and E10, the -epithelial sodium channel (ENaC) CRD2 was substituted by the ENaC CRD2 and vice versa. B: representative whole cell current traces, recorded at 60 mV holding potential, are shown from 3 individual oocytes expressing , E10, and 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 (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. Iami and surface expression values are normalized for each group according to the values obtained for ENaC control oocytes.

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

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Extracellular loop substitution chimeras. A: chimeras had the extracellular loop of ENaC substituted by the corresponding loop of 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: Iami (open bars) and surface expression (filled bars) are illustrated for each group of oocytes tested.

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 ENaC substituted by the corresponding regions of ENaC (E7) and vice versa (E8). In E8 oocytes, I_ami 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 ENaC. In E7 oocytes, I_ami 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 ENaC oocytes. This suggests that the E7 chimera can almost fully substitute for the function of the -subunit.

View larger version (24K): [in this window] [in a new window]   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 ENaC substituted by the corresponding regions of ENaC and vice versa. B: Iami (open bars) and surface expression (filled bars) are illustrated for each group of oocytes tested.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effect of brefedin A (BFA) on Iami in -control oocytes (A), in E8 oocytes (B), and 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 Iami was subsequently assessed in 4- and 12-h intervals. Each value represents the mean Iami of 10 oocytes.

NH₂ termini and M1 substitution chimeras. In chimeras E5 and E6 (Fig. 6A), the NH₂ terminus and M1 domain of ENaC were substituted by the corresponding regions of ENaC (E5) and vice versa (E6). In E5 oocytes, I_ami 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 ENaC function to a large extent (Fig. 6B). In contrast, in E6 oocytes, I_ami and surface expression were very low and not significantly different from the value measured in oocytes (Fig. 6B). Hence, although the NH₂ terminus and the M1 region of  can be substituted by the corresponding regions of , the reverse substitution fails to produce functional channels.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   NH2 termini and M1 substitution chimeras. A: chimeras E5 and E6 had the NH2 terminus and M1 of ENaC substituted by the corresponding regions of ENaC and vice versa. B: 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 - and -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 - or -termini substituted by the corresponding - or -termini, respectively, may still be efficiently expressed at the plasma membrane when coexpressed with or . These chimeras are illustrated in Fig. 7A, and are named E13 and E14. Figure 7B shows that the I_ami and the surface expression of each chimera when coexpressed with either or were very low and similar to the values obtained for and 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 - or -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  and at least another one from  in order for the ENaC complex to be transported to the plasma membrane.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   NH2 and COOH termini substitution chimeras. A: chimeras E13 and E14 had the intracellular ENaC termini substituted by the corresponding ENaC termini and vice versa. B: 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 - and -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 ENaC by that of ENaC or vice versa results in a very inefficient surface expression of ENaC, 2) the CRD2 region and the CRD2 region can efficiently substitute for each other, 3) the M2 and COOH terminus of ENaC have a more potent role than the corresponding regions of 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 ENaC are more important for the formation of a functional ENaC channel than the corresponding regions of ENaC.

Functional role of extracellular domains of the - and -subunits of ENaC. The inability of the chimeras E1 and E2 to increase the surface expression of and , respectively, suggests that the extracellular loops of both the - and the -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 -, -, and -subunit loops are still poorly understood (5). It is possible that a distinct glycosylation pattern of the - and -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 - and -subunits each have unique and distinct functional features because both loops are required for proper channel function.

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

Physiological significance of the present results. Interestingly, the BFA experiments demonstrated that E7 and E8 have a rapid retrieval rate similar to that of ENaC controls. This result is of significance because it indicates that the PPXY motifs of the - and -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 ENaC PPXY motifs and not a single ENaC PPXY motif present and vice versa. Therefore, the COOH-terminal PPXY motifs are apparently functionally interchangeable between the - and the -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 - and -COOH termini (21). It is probably the absence of any preference of the binding of these two WW motifs to the - and -COOH termini that makes the COOH termini interchangeable between the two subunits and, on the other hand, also explains why truncation of both - and -COOH termini has an additive effect regarding the hyperactivity of the channel (40).