Regulation of a cloned epithelial Na+ channel by its beta - and gamma -subunits

Mouhamed S. Awayda1, Albert Tousson2, and Dale J. Benos3

1 Department of Medicine and Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112; and 2 Department of Cell Biology and 3 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35223

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Using the Xenopus oocyte expression system, we examined the mechanisms by which the beta - and gamma -subunits of an epithelial Na+ channel (ENaC) regulate alpha -subunit channel activity and the mechanisms by which beta -subunit truncations cause ENaC activation. Expression of alpha -ENaC alone produced small amiloride-sensitive currents (-43 ± 10 nA, n = 7). These currents increased >30-fold with the coexpression of beta - and gamma -ENaC to -1,476 ± 254 nA (n = 20). This increase was accompanied by a 3.1- and 2.7-fold increase of membrane fluorescence intensity in the animal and vegetal poles of the oocyte, respectively, with use of an antibody directed against the alpha -subunit of ENaC. Truncation of the last 75 amino acids of the beta -subunit COOH terminus, as found in the original pedigree of individuals with Liddle's syndrome, caused a 4.4-fold (n = 17) increase of the amiloride-sensitive currents compared with wild-type alpha beta gamma -ENaC. This was accompanied by a 35% increase of animal pole membrane fluorescence intensity. Injection of a 30-amino acid peptide with sequence identity to the COOH terminus of the human beta -ENaC significantly reduced the amiloride-sensitive currents by 40-50%. These observations suggest a tonic inhibitory role on the channel's open probability (Po) by the COOH terminus of beta -ENaC. We conclude that the changes of current observed with coexpression of the beta - and gamma -subunits or those observed with beta -subunit truncation are likely the result of changes of channel density in combination with large changes of Po.

oocyte expression; immunofluorescence; Liddle's syndrome; channel activation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE GENE CODING for the alpha -subunit of an epithelial Na+ channel (ENaC) was cloned from rat colon in 1993 by Canessa et al. (5) and Lingueglia et al. (17). Expression of this subunit by itself in Xenopus oocytes generated amiloride-sensitive Na+ currents. Expression of this subunit in an in vitro translation system and its subsequent incorporation into planar lipid bilayers also confirmed that it was capable of producing an Na+-selective channel (2, 11). Despite the ability of the subunit to induce Na+ currents in both the oocyte and the lipid bilayer systems, the currents attributed to alpha -ENaC expressed in Xenopus oocytes were smaller than those obtained in oocytes injected with total mRNA from Na+-transporting epithelia. This observation has lead to the cloning of two additional "auxiliary" subunits (beta  and gamma ) that, when coexpressed with the alpha -subunit, cause a large increase of currents by more than 20-fold (6, 24).

The presence of ENaC homologues in many native and cultured epithelial and nonepithelial tissues has been well documented (6-8, 14, 16, 18, 24, 25). In these tissues, the protein or message levels for all three subunits were highly variable, such that not all tissues expressed equal levels of these subunits and some tissues even expressed only alpha -ENaC. Moreover, in some experiments, the beta - and gamma -subunits were found to be preferentially upregulated in response to hormones such as aldosterone (1). These observations may indicate that the changes in "auxiliary" subunit mRNA or protein levels are potential intrinsic mechanisms by which tissues regulate their rates of Na+ transport.

Another mechanism by which the beta - and gamma -subunits regulate ENaC activity involves the COOH termini of these subunits. Mutations that truncate the COOH termini of either the beta - or the gamma -subunit, as observed in Liddle's syndrome (10, 22), cause an increase in channel activity. This was experimentally confirmed in experiments in which alpha - and gamma -ENaC were coexpressed with a truncated beta -ENaC in Xenopus oocytes and the finding of elevated amiloride-sensitive whole cell currents compared with those observed with the coexpression of all three wild-type subunits (21, 23).

A lack of consensus exists as to how beta -subunit truncations activate ENaC. Ismailov et al. (13) have observed that the channel purified from Liddle's syndrome-affected human lymphocytes is constitutively activated when incorporated into planar lipid bilayers. This activated channel was inhibited (~25%) by application of a 30-amino acid peptide derived from the COOH-terminal region of the beta -subunit. They concluded that the observed increase of activity is likely because of large (4- to 5-fold) changes of open probability (Po) and that this increase of Po is mediated, at least in part, by relief of the inhibitory actions of the COOH-terminal region of the beta -subunit. Snyder et al. (23) reported that the changes of activity likely result from an increase of channel density (NT) alone. On the other hand, a recent report by Firsov et al. (9) indicated that ENaC activation after subunit truncation is the result of a combination of increases in NT and Po.

We used a combination of an immunofluorescence assay and dual-electrode voltage recording of amiloride-sensitive ENaC currents in the Xenopus oocyte expression system to examine the mechanisms by which expression of beta - and gamma -ENaC alter the induced amiloride-sensitive currents. We also examined the differences in plasma membrane channel levels between oocytes that express a normal beta -subunit and those that mimic Liddle's syndrome by expression of a truncated beta -subunit. Electrophysiological measurements were carried out on the same batch of oocytes as were the immunofluorescent measurements. We report that coexpression of the beta - and gamma -subunits caused a threefold increase of membrane-bound alpha -ENaC levels accompanied by a 34-fold increase of amiloride-sensitive whole cell currents. On the other hand, only a 35% increase of membrane fluorescence levels was observed when beta -ENaC was truncated. This was accompanied by a 4.4-fold increase of whole cell current. Moreover, intracellular injection of a peptide derived from the beta -subunit COOH-terminal sequence (final concentration ~300 µM) resulted in a 40-50% inhibition of ENaC currents. We conclude that beta - and gamma -ENaC and the COOH termini of these subunits affect channel activity by altering both NT and Po of the resulting Na+ channel.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Antibody production. A rabbit anti-alpha -ENaC antibody was produced as previously described (12). Briefly, 200 µg of bovine alpha -ENaC full-size fusion protein (dissolved in 1 ml of sterile water) were injected into multiple subcutaneous dorsal sites in a New Zealand White rabbit. This was repeated every 3 wk until an acceptable antibody titer was obtained in an enzyme-linked immunosorbent assay using the fusion protein as substrate. After five boosts, the rabbit was killed and serum was collected. Immunoglobin G (IgG) was purified from serum with the use of protein A-coupled Sepharose beads (Bio-Rad, Richmond, CA) and was stored at -20°C until further use. Preimmune serum was treated in the same manner as the immune serum. This antibody cross-reacts with the rat alpha -ENaC homologue, as assessed using both immunofluorescence methods and Western blotting.

Chimera construction. To circumvent potential problems with the coexpression of rat and human subunits, a situation that by itself may cause changes of the magnitude of amiloride-sensitive currents (18) and potentially influence membrane fluorescence intensity, we used a beta -subunit chimera (rat + human) that only contained a small portion of human ENaC sequences. This chimera was constructed by replacing the COOH-terminal region (last 125 amino acids) of the rat beta -ENaC with a polymerase chain reaction (PCR)-generated human COOH-terminal sequence, using cDNAs prepared from peripheral blood lymphocytes either from a normotensive individual or from an individual from the original Liddle's kindred in the amplification procedure. The PCR primers were designed so that the products contained an artificially introduced Xho I (5') and Acc I (3') sites. These products were digested with both Xho I and Acc I and were subsequently ligated into rat beta -ENaC (beta -rENaC) contained in pSPORT that was predigested with these two enzymes. The Xho I site in beta -rENaC was created at the corresponding site of the human beta -subunit by PCR-directed mutagenesis. An extra Acc I site in beta -rENaC at nucleotide position 2 (numbered according to GenBank X77932) was removed by restriction with Sal I followed by blunt-end ligation.

Two different chimeras containing different human beta -COOH-terminal sequences were used: 1) a control chimera containing normal human beta -sequences (beta chim) and 2) a truncated chimera containing a premature stop codon as it was found in the original Liddle's pedigree that encoded a C to T mutation at Arg-564, deleting the last 75 amino acids from the COOH terminus of the beta -subunit (&bgr;′<SUB>chim</SUB>). The control chimera was found to behave in an identical manner to the full-length rat beta -subunit in both electrophysiological and immunofluorescent assays (see RESULTS). The validity of the sequence of both chimeras was confirmed by restriction analysis and DNA sequencing.

RNA synthesis. RNA synthesis was as previously described (3). The plasmid containing either alpha -, beta -, or gamma -rENaC (a gift of B. Rossier) or the chimeric constructs (a gift of Y. Oh and D. Warnock) was linearized with the appropriate 3' restriction enzyme. Linearized plasmid DNA was purified using the Geneclean kit (Bio 101, Vista, CA). Sense RNA was synthesized from purified plasmid DNA using T7 RNA polymerase according to the manufacturer's instructions (Promega, Madison, WI). RNA was in vitro synthesized in the presence of methyl guanosine cap analog m7G(5')ppp(5')G (NEB, Beverly, MA) in threefold excess to GTP. This stabilizes the cRNA and enhances its translational efficiency (15, 19). After two rounds of phenol-chloroform extraction and ethanol precipitation, RNA was quantitated by measuring optical density at 260 nm and stored at -80°C.

Oocyte expression and recording. Toads were obtained from Xenopus I (Ann Arbor, MI) and were kept in dechlorinated tap water at 18°C. Oocytes were surgically removed from anesthetized toads and were processed as previously described (3). All injection volumes consisted of 50 nl of nuclease-free water containing cRNAs at various concentrations. alpha -rENaC-expressing oocytes were injected with 12.5 ng of cRNA, whereas alpha beta gamma -rENaC-expressing oocytes were injected with 2.5 ng of each cRNA. Both concentrations have been shown to result in subsaturating levels of ENaC expression (21). Injected oocytes were incubated at 18°C for 2-3 days until their recording or processing for immunofluorescence measurements. All recordings were performed at 19-21°C.

In experiments that required the direct injection of peptide, oocytes were first impaled with the injecting electrode and then impaled with the two recording microelectrodes as previously described (3). The injecting electrode was filled with the appropriate peptide. The tip of the electrode was filled with air (~10-nl volume) so that the peptide did not leak into the oocyte immediately after impalement. This allowed us to determine a control period with a relatively constant baseline before the injection of any material into the oocyte. A 30-amino acid peptide with a sequence identical to that of the COOH-terminal region of the human beta -ENaC and a 26-amino acid peptide of random sequence were both synthesized by Research Genetics (Huntsville, AL), followed by high-performance liquid chromatography purification. The 10-amino acid uncharged peptide was synthesized in-house as described by Ismailov et al. (13). All peptides were dissolved in water at a stock concentration of 5 mM. Attempts to use other peptides (a charged 10-amino acid peptide; see Ref. 13) were unsuccessful because these peptides were dissolved in dimethyl sulfoxide (DMSO) and resulted in DMSO concentrations that were incompatible with the oocyte expression system.

Solutions and chemicals. The pH of all oocyte recording solutions was adjusted to 7.5. Oocytes were defoliculated in Ca2+-free Ringer of the following composition (in mM): 84 NaCl, 1 MgCl2, 2 KCl, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The oocyte culture medium was one-half strength L-15 medium (Sigma, St. Louis, MO) supplemented with 15 mM HEPES and 0.5% of a 10,000 U/ml solution of penicillin-streptomycin (GIBCO, Grand Island, NY). The recording medium ND-96 contained (in mM) 96 NaCl, 1 MgCl2, 2 KCl, 1.8 CaCl2, and 5 HEPES. Oocytes were continuously perfused with solution at the rate of 2 ml/min or ~2 chamber volumes/min.

Data acquisition and analysis. The program pCLAMP version 5.5 (Axon Instruments, Foster City, CA) was used for data acquisition and analysis as previously described (3). Oocyte holding voltage was clamped to 0 mV at all times except during acquisition of whole cell currents at various voltages. During periods of data acquisition, the membrane voltage was stepped for 450 ms from -100 mV to +80 mV in increments of 20 mV. Currents at -100 mV were summarized from the averaged value of the last five current points at the end of each voltage episode. By convention, inward flow of cations is designated as inward current (negative current), and all voltages are reported with respect to ground or bath. All data are reported as means ± SE.

Oocyte fixation. All procedures were carried out at room temperature except where noted. Oocytes were fixed in 3% formaldehyde (EM grade; Tousimis, Rockville, MD) in ND-96 for 2 h at room temperature. Fixed oocytes were partially dehydrated by incubation in ND-96 containing 95% ethanol for 45 min, with a new solution change every 15 min. This was followed by complete dehydration in 100% ethanol, followed by xylene for 45 min, each with three bath solution changes. Oocytes were then infiltrated with liquid paraffin at 60°C for 1 h with two solution changes and were allowed to cool before sectioning. To facilitate sectioning, paraffin-embedded samples were reembedded in larger blocks. These blocks were sectioned with a microtome at a section thickness of ~5 µm. Sections were air dried, followed by an overnight incubation in a 60°C oven.

Sections were deparaffinized by a 15-min incubation in xylene, with three solution changes. Sections were subsequently rehydrated by a series of 15-min incubations in ethanol ranging from 100 to 0% in ND-96. This was followed by a 15-min postfixation in 3% formaldehyde.

Immunofluorescence. Postfixed sections were incubated in tris(hydroxymethyl)aminomethane-NaCl (pH 8.2, 220 mosM) for 30 min to quench any unreacted aldehydes. Nonspecific immunoreactivity was blocked using 20% normal goat serum (NGS; Sigma) in ND-96 for 15-30 min. Sections were then incubated for 1 h with either the primary antibody (antialpha -bENaC antibody) or with preimmune IgG at the same final concentration of 0.2 mg/ml in 20% NGS. Unbound primary antibody was washed away with four solution changes within 15 min. Sections were blocked with 5% NGS in ND-96 for 15 min and were then incubated with the secondary antibody for 1 h at 37°C at a final concentration of 0.05 mg/ml in 5% NGS in ND-96. This antibody was a goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (Boehringer Mannheim). After 1 h, sections were washed in ND-96 and then were mounted in 0.1% p-phenylenediamine in 9:1 glycerol to ND-96.

Photography and fluorescence quantification. Micrographs were prepared with a Leitz Orthoplan fluorescence microscope equipped with a Vario-orthomat II camera system and a digital photometer. The microscope was outfitted with epifluorescence and phase-contrast optics. Images were recorded on Kodak T-Max 400 film or Ektachrome p1600 (Kodak, Rochester, NY) push processed to 800 ASA. High-contrast prints were made on polycontrast III RC glossy paper using a no. 4 contrast filter (Kodak) on a Beseler CB7 enlarger (East Orange, NJ). All experimental and negative control trials were photographed with the same exposure times.

Fluorescence intensity was measured using a digital photometer attached to the microscope. The photometer was standardized using the InSpeck Green (490- and 515-nm absorption and emission wavelengths, respectively) microscope image intensity calibration kit (Molecular Probes, Eugene, OR). Fluorescence intensity of individual calibration beads was measured by spot metering. The reciprocity compensation control was adjusted so that the measured bead intensity would most closely match the standard relative bead intensity set by the manufacturer. With the camera set for spot metering, the zoom lens was adjusted to 5×, a 50× fluotar objective was swiveled into place, and the intensity of a 30-µm segment of cell membrane surface was measured. All measurements were done in triplicate and were averaged and treated as a single value. Values of fluorescence intensity were >90% accurate as assessed from the average triplicate variability. Reported values were corrected for background cytoplasmic fluorescence because it was not possible to eliminate its contribution. We assumed that this background signal contributed equally to the raw data because it was observed in all groups of oocytes, including those treated with the secondary antibody alone.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of coexpression of beta - and gamma -ENaC. Previous reports have indicated that expression of alpha -ENaC produces currents that are much smaller than those observed with alpha beta gamma -ENaC expression. As shown in the representative example in Fig. 1, the whole cell currents and the amiloride-sensitive currents were much smaller in oocytes expressing alpha -ENaC alone. This was observed despite a fivefold higher concentration of RNA in oocytes injected with the alpha -subunit by itself.


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Fig. 1.   Expression of beta - and gamma -rat epithelial Na+ channel (rENaC) with alpha -rENaC causes a marked increase of amiloride-sensitive whole cell currents. Representative whole cell currents (Im) in oocytes expressing alpha -rENaC (A) and alpha beta gamma -rENaC (C) and corresponding changes of these currents with 10 µM amiloride (B and D). Currents were recorded in both groups of oocytes for >= 30 min (until a stable value was observed), followed by addition of 10 µM amiloride for 10 min.

The values of whole cell currents in water-injected oocytes and in oocytes expressing alpha -rENaC and alpha beta gamma -rENaC before and after amiloride treatment are summarized in Table 1. The ratio of whole cell currents at -100 mV was 16-fold higher in oocytes expressing alpha beta gamma -rENaC than in those expressing alpha -rENaC. Moreover, the ratio of the amiloride-sensitive currents was 34-fold higher. Consistent with previous reports (18, 26), water-injected oocytes exhibited amiloride-sensitive currents. These currents averaged -3 nA. These values of endogenous currents are small compared with the microampere-level currents observed with expression of all three subunits but are important to note because of the presence of a Xenopus ENaC homologue (20) and the levels of spontaneous membrane fluorescence in water-injected oocytes.

                              
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Table 1.   Whole cell currents in Xenopus oocytes expressing alpha -rENaC and alpha beta gamma -rENaC and their subsequent inhibition by amiloride

With the assumption that the conductance of the channel formed by the alpha -subunit alone and that of the channel formed by all three subunits are similar (see DISCUSSION), the differences of whole cell currents imply a 34-fold difference of channel activity, or NT · Po (the product of total NT and the single-channel Po). Because currents are very low in oocytes expressing alpha -ENaC and because of the large size of oocytes, it would be difficult to determine the origin of the differences between these two channels using patch-clamp analysis. Instead, we resorted to an immunofluorescence approach to estimate differences of membrane ENaC content in oocytes expressing these two channel complexes.

Whole oocytes, because of their thickness, are difficult to examine intracellularly by routine fluorescence microscopy. The protocol using paraffin embedding and sectioning (see MATERIALS AND METHODS) circumvented this problem and yielded the best overall oocyte preservation. Figure 2 shows a low-magnification view of paraffin-embedded oocytes, as imaged by phase contrast microscopy. The absence of large membrane or cytoplasmic gaps was taken as an indication that paraffin embedding did not disrupt the overall distribution of cytosolic and membrane-bound proteins.


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Fig. 2.   Formaldehyde fixation and paraffin embedding preserve oocyte structure. Representative phase-contrast image of formaldehyde-fixed, paraffin-embedded oocytes (see MATERIALS AND METHODS for details). Note absence of any large gaps in cytosol or plasma membrane. Also note dark-pigmented segment of membrane (animal pole) and light-pigmented segment (vegetal pole). Scale bar, 500 µm.

Under the conditions stated in MATERIALS AND METHODS, the protein A-purified, preimmune IgG did not exhibit any membrane surface immunoreactivity with control noninjected oocytes (data not shown) or with oocytes expressing alpha beta gamma -rENaC (Fig. 3). There was an appreciable amount of yolk-related fluorescence that was observed in these oocytes. Moreover, this nonspecific fluorescence was always limited to the cytoplasm and was not observed at the plasma membrane. As such, this limited our studies to an examination of the specific fluorescence at the plasma membrane.


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Fig. 3.   Specificity of alpha -subunit antibody used in present studies. This antibody did not show any appreciable immunoreactivity in oocytes expressing either of the beta - or gamma -subunits (A and B, respectively). Moreover, preimmune serum did not cross-react with any of the 3 subunits (C). Arrowheads indicate position of plasma membrane. Scale bar, 50 µm.

Before assessment of the differences between oocytes expressing the alpha -subunit alone and those expressing all three subunits, it was important to determine by immunofluorescence whether this antibody showed any appreciable cross-reactivity with the beta - and gamma -subunits. Two representative oocytes that have been incubated with the anti-alpha -ENaC antibody and are expressing beta - and gamma -rENaC, respectively, are shown in Fig. 3, A and B. There was no detectable membrane-associated fluorescence in these oocytes. Given the observation by Firsov et al. (9) of similar membrane-bound expression levels of beta - and gamma -ENaC compared with alpha -ENaC when expressed by themselves in Xenopus oocytes (see Fig. 1B in Ref. 9), we can surmise that our antibody was likely specific for the alpha -subunit over the beta - and gamma -subunits. This is not altogether surprising given the low homology at the amino acid level among the three subunits (6, 18).

An example of membrane immunofluorescence in water-injected oocytes and those expressing alpha -rENaC and alpha beta gamma -rENaC is shown in Fig. 4. Membrane fluorescence in control oocytes was almost absent. The oocyte shown in Fig. 4A was intentionally chosen to reflect the highest amount of membrane fluorescence observed in this group. This membrane-associated fluorescence was still much smaller than that observed in oocytes expressing alpha -rENaC and alpha beta gamma -rENaC, shown in the two typical examples in Fig. 4, B and C, respectively. A punctate pattern of membrane staining was the typical observation, indicating a possible grouping or clustering of this Na+ channel in the membrane. As seen from the accompanying bright-field images, these examples of membrane segments originated from the animal pole of the oocyte, because the dark-pigmented background allowed a better delineation of membrane fluorescence. A very similar pattern of membrane-associated fluorescence was observed in the vegetal pole.


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Fig. 4.   Membrane-surface fluorescence is increased in oocytes expressing all 3 ENaC subunits. Control oocytes (A) exhibited a small amount of endogenous membrane labeling. alpha -rENaC-expressing oocytes exhibited higher membrane immunofluorescence (B) that is further increased by expression of beta - and gamma -subunits (C). Right panels show corresponding phase-contrast images. Note discrete and punctate staining of plasma membrane in all 3 examples. Conditions are same as in Fig. 3. Arrowheads indicate position of plasma membrane. Scale bar, 50 µm.

Values of membrane fluorescence in all three groups of oocytes are summarized in Table 2. These values are in arbitrary units and are inversely related to the exposure times and directly related to the fluorescence intensity. Moreover, these values are corrected for the unavoidable contribution of background cytoplasmic fluorescence.

                              
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Table 2.   Relative plasma membrane fluorescence intensity of Xenopus oocytes expressing alpha -rENaC and alpha beta gamma -rENaC probed by an anti-alpha -subunit antibody

To determine whether there was any differential partitioning of alpha -rENaC and alpha beta gamma -rENaC to either the animal or vegetal poles, data from both parts of the membrane were summarized. The values of fluorescence intensity in control oocytes were not different from either hemisphere. However, it is clear that there was a higher distribution of the Na+ channel in the animal pole in the experimental groups of oocytes. This was especially evident in oocytes expressing alpha beta gamma -rENaC, in which the fluorescence intensity attributed to exogenous ENaC (corrected for the values measured in control oocytes) was 2.3-fold higher in the animal pole. The differences in membrane fluorescence intensity between the two hemispheres in alpha -ENaC and alpha beta gamma -ENaC-expressing oocytes cannot be attributed to differences in the contribution of background fluorescence, because values of membrane autofluorescence were the same in both hemispheres.

Oocytes expressing the alpha -subunit alone exhibited higher fluorescence intensity than control oocytes. This was observed in both the animal and vegetal poles but was more pronounced in the animal pole. Oocytes expressing alpha beta gamma -rENaC exhibited higher fluorescence intensity than oocytes exhibiting alpha -rENaC alone. Likewise, the differences were more pronounced between these two groups of oocytes in the animal pole, where the amount of staining in alpha beta gamma -rENaC-expressing oocytes was 3.1-fold higher than that in alpha -rENaC oocytes. Thus these values alone cannot account for the change of amiloride-sensitive macroscopic currents that were >30-fold. Instead, it is likely that a combination of increase of NT and of Po best explains the increase of current with beta - and gamma -subunit expression.

Effect of truncation of the beta -subunit. The COOH termini of the beta - and gamma -subunits also control ENaC activity, whereby truncation of the COOH terminus of these subunits has been reported to stimulate ENaC currents when expressed in Xenopus oocytes. To examine the mechanisms by which beta -subunit truncation causes Na+ hyperabsorption and Liddle's syndrome, we expressed the truncated beta -subunit &bgr;′<SUB>chim</SUB> with alpha - and gamma -rENaC. As shown in the representative example in Fig. 5, truncation of the terminal 75 amino acids of the beta -subunit construct caused a large activation of whole cell currents over those observed with the nontruncated beta -subunit rat-plus-human construct beta chim. The majority of whole cell currents in both oocytes was blocked by 10 µM amiloride. Moreover, as observed in Fig. 6, there were no detectable differences at the level of whole cell currents or amiloride sensitivity (data not shown) between the channels formed with wild-type beta -subunits and those formed with beta chim.


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Fig. 5.   Expression of a truncated beta -subunit (&bgr;′<SUB>chim</SUB>) causes a marked increase of whole cell currents. Representative whole cell currents in oocytes expressing alpha beta chimgamma -rENaC (A) and alpha &bgr;′<SUB>chim</SUB>gamma -rENaC (B).


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Fig. 6.   Whole cell currents in oocytes coexpressing control chimeric beta -subunits (B) were indistinguishable from those observed in oocytes coexpressing rat beta -subunit (A).

Amiloride-sensitive currents averaged -1.48 ± 0.25 µA (n = 20) in wild-type alpha beta gamma -rENaC and -1.39 ± 0.31 µA (n = 14) in control chimeric alpha beta chimgamma -rENaC. Thus it appears that the channel formed with the control nontruncated rat-plus-human chimera behaved in an identical manner to that observed with the beta -subunit of rat origin. On the other hand, currents in oocytes expressing the truncated chimeric beta -subunit were much larger than control and averaged -6.50 ± 0.92 µA (n = 17). This resulted in a 4.4-fold activation of amiloride-sensitive current and is consistent with observations from other laboratories (21, 23).

As shown in the representative example in Fig. 7, the membrane fluorescence intensity in an oocyte expressing alpha &bgr;′<SUB>chim</SUB>gamma -rENaC was similar to that of an oocyte expressing alpha beta chimgamma -rENaC. Moreover, the pattern and magnitude of membrane fluorescence were indistinguishable between alpha beta gamma -ENaC- and alpha beta chimgamma -ENaC-expressing oocytes, and the values from these two control groups were combined. To obtain a better estimate of small changes of membrane fluorescence intensity, we limited our analysis to the animal pole of the oocytes, where the largest signal was observed. In this hemisphere and after we corrected for the background fluorescence signal of control water-injected oocytes, oocytes expressing alpha beta gamma -rENaC or alpha beta chimgamma -rENaC exhibited intensity values of 6.28 ± 0.03 (n = 50), whereas those expressing alpha &bgr;′<SUB>chim</SUB>gamma -rENaC exhibited a fluorescence intensity of 8.45 ± 0.09 (n = 50). Thus beta -subunit truncation resulted in a 35% increase of fluorescence intensity compared with oocytes without the truncated beta -subunits. The differences between these two groups of oocytes were essentially similar in the vegetal pole in that there was only a small change observed between oocytes with a normal beta -subunit and &bgr;′<SUB>chim</SUB> (data not shown). This finding indicates that the activation of ENaC observed with Liddle's syndrome is the result of a combination of small changes of NT and larger changes of Po.


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Fig. 7.   Truncation of beta -subunit COOH terminus results in a small increase of Na+ channel membrane-associated immunofluorescence. Membrane fluorescence in control oocytes was essentially absent (C). Membrane fluorescence intensity was slightly higher in oocytes expressing truncated beta -subunit (A) compared with oocytes expressing chimeric nontruncated beta -subunit (B). This small increase of signal alone could not explain 4.4-fold increase of amiloride-sensitive currents (see Fig. 5 and associated text). In same group of oocytes there were no detectable differences between expression of alpha - and gamma -rENaC with chimeric beta -subunit or full rat beta -subunit (compare, for example, Fig. 4C and Fig. 5B). Arrowheads point to cell surface. Bar, 50 µm.

To further demonstrate the importance of the COOH terminus of the beta -subunit in the regulation of ENaC activity, we injected a 30-amino acid peptide corresponding to the human beta -subunit COOH terminus into either the alpha beta chimgamma -rENaC- or the alpha &bgr;′<SUB>chim</SUB>gamma -rENaC-expressing oocytes (Figs. 8 and 9). Thirty to forty nanoliters of the 30-amino acid peptide solution (5 mM) were injected into the oocytes, resulting in an approximate final concentration of 300-400 µM in the oocyte cytosol (assuming that an average oocyte cytosol volume is 500 nl). Injection of this 30-amino acid peptide into oocytes caused an inhibition of whole cell currents within minutes. This inhibition reached a plateau at ~8-10 min in both groups of ENaC-expressing oocytes (data not shown). On a paired basis, i.e., comparing the values of amiloride-sensitive currents from the same oocyte before and 10 min after the injection of the peptide, we observed a 38% decrease of current in &agr;&bgr;′<SUB>chim</SUB>gamma -rENaC-expressing oocytes (from -5.65 ± 1.11 to -3.52 ± 0.73 µA, n = 10). A similar ratio of inhibition was observed in alpha beta chimgamma -rENaC-expressing oocytes as currents decreased by 47% of control from -1.35 ± 0.24 to -0.72 ± 0.14 µA (n = 12).


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Fig. 8.   Representative effect of peptide injection into ENaC-expressing oocytes. After establishment of a control period, peptide was injected using a third pipette into alpha beta chimgamma -rENaC- (A) and alpha &bgr;′<SUB>chim</SUB>gamma -rENaC (B)-expressing oocytes, and whole cell currents were measured every minute for 10 min. Inhibitory effect of peptide reached a plateau at 7-10 min, and data shown for inhibition of alpha beta chimgamma -rENaC- (C) and &agr;&bgr;′<SUB>chim</SUB>gamma -rENaC (D)-expressing oocytes are at plateau time point. E and F demonstrate effect of 10 µM amiloride on alpha beta chimgamma - and alpha &bgr;′<SUB>chim</SUB>gamma -rENaC, respectively.


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Fig. 9.   Summary of inhibitory effects of peptide injection in ENaC-expressing oocytes. In all groups, amiloride-sensitive whole cell currents (Iamil) are summarized at a holding voltage of -100 mV. beta hC30, 30-mer peptide derived from last 30 amino acids of human beta -subunit.

Conducted as a control for the peptide experiments, injection of isotonic KCl or a 10-amino acid peptide corresponding to the last 10 amino acids of the rat beta -subunit COOH terminus, with its four positively charged amino acids substituted with neutral glycine (NH2-MGSGSGVGAI-COOH), did not result in any appreciable change of whole cell currents (n = 8; see Fig. 10). Moreover, injection of the random 26-amino acid peptide NH2-NAHNFPLDLASGEQAPVALTAPAVNG-COOH resulted in a small (<10%) inhibition of whole cell currents (n = 6). Thus, despite the presence of multiple charged residues, this 26-mer failed to display the same inhibitory effects as the more specific 30-mer with sequence identity to the beta -subunit COOH terminus.


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Fig. 10.   Lack of effect of 10-amino acid uncharged peptide on currents in ENaC-expressing oocytes. After establishment of a control period, peptide was injected as described. A: currents in a representative oocyte expressing alpha &bgr;′<SUB>chim</SUB>gamma -rENaC were unchanged after 10 min of injection of this peptide (B). Data representative of 8 such experiments. Lack of an inhibitory action of this peptide was also observed in alpha beta chimgamma -rENaC-expressing oocytes (n = 6).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We used a rabbit primary antibody directed against a nearly full-size alpha -ENaC fusion protein as previously described (12) to determine the changes of alpha -subunit density at the plasma membrane subsequent to coexpression with full-length beta - and gamma -subunits or after coexpression with a full-length gamma -subunit and a truncated beta -subunit. We conclude that the changes of whole cell currents produced by expression of alpha -subunits with beta - and gamma -subunits are likely the result of a combination of a threefold change of NT and a 10-fold change of Po. Likewise, the increase of current observed with beta -subunit truncation is also attributed to a combination of a 1.35-fold increase of NT and approximately threefold increase of Po. In both cases, it appears that the potential changes of NT are small in comparison to the potential changes of Po.

Because this antibody is directed against a full-length fusion protein, it is expected that it would recognize a complete and functional alpha -subunit as well as a partially degraded and electrically nonfunctional alpha -subunit. This is not unique to this antibody, and this possibility could be envisioned with any site-specific and even monoclonal antibody. This is a shortcoming of determining the changes of NT with antibodies irrespective of their specific recognition site in that an electrically silent or partially degraded channel could still contain antigenic sites. However, the issues of changes of NT and Po cannot be easily answered using patch- clamp methodologies, because translation of changes of channel activity (NT · Po) in multiple-channel patches to changes of either NT or Po is very difficult. This situation is further exacerbated in low-Po patches and in cases in which the channel activity varies enormously, as with oocytes expressing the alpha -subunit alone and those expressing alpha beta gamma -subunits. Moreover, this situation is also complicated by the potential presence of subconductance states (11) that render the task of determining channel number in multiple-channel patches a very formidable task. Therefore, the methods used in the current report represent an alternative approach to estimate changes of NT.

It is unlikely that any observed differences in fluorescence intensity result from differences in the affinity of the channel to the antibody. This could be a potential pitfall if the antibody were recognizing a single antigenic site. However, because this is a polyclonal antibody recognizing multiple antigenic sites, it is unlikely that the affinity of the antibody to all of these sites is altered in the same manner. Moreover, the relatively high concentration of antibody used in immunofluorescence detection assays is likely to represent a saturating concentration that is relatively unaffected by small changes of the antigen-antibody binding rate constants or affinity.

Does alpha -subunit alone form a functional channel? We have previously reported that alpha -ENaC by itself is capable of forming an amiloride-sensitive Na+ channel when incorporated into planar lipid bilayers (2). These experiments were carried out with alpha -rENaC obtained by way of an in vitro translation system. Because the control reaction contained no channel activity, it was straightforward to attribute the channel activity to the newly translated channel protein that contained the alpha -subunit alone. Moreover, in mammalian cells transfected with alpha -rENaC alone, an amiloride-sensitive Na+ channel is observed (14), indicating that alpha -ENaC alone can form a functional channel. The observation that there is membrane-bound distribution of alpha -rENaC when expressed by itself in oocytes may be indicative of its potential to form a channel. This is especially true given the finite ionic current observed in oocytes expressing the alpha -subunit. This current is probably not a result of the presence of endogenous beta - and gamma -ENaC, because injection of beta - or gamma -ENaC individually or of beta - and gamma -ENaC together in the absence of alpha -ENaC does not result in any current over that observed in water-injected oocytes.

Mechanism by which beta - and gamma -subunits increase current. The origin of the changes of current simplifies to changes in NT, Po, or Na+ single-channel conductance (gNa). It seems unlikely that the increase of macroscopic current is the result of an increase of gNa for the channel formed with alpha -, beta -, and gamma -subunits, because we have previously reported that the single-channel conductance is actually higher in the absence of beta - and gamma -subunits when tested in planar lipid bilayers (11). A similar observation of a relatively high-conductance channel formed by alpha -rENaC was made by Kizer et al. (14) who, after transfection of mammalian fibroblasts with the alpha -subunit cDNA, observed a 24-pS channel that displayed short burstlike activity punctuated by long closed intervals. These observations of higher gNa between alpha - and alpha beta gamma -ENaC would tend to underestimate the changes of NT or Po that are needed to account for the observed increase of whole cell currents and thus would not affect the present conclusions.

Another possibility to consider is that a different number of alpha -subunits are needed to form a functional channel in the presence of the beta - and gamma -subunits. This situation is impossible to evaluate without knowledge of the subunit stoichiometry of the channel. It would seem unlikely that the differences in the number of subunits can be large enough to account for the almost 10-fold difference between macroscopic current activation (34-fold) and membrane fluorescence activation (3- to 4-fold).

Truncated beta -subunit. Truncation of the beta -subunit caused a 4.4-fold increase of current. Data from the ENaC channel reconstituted into planar lipid bilayers (13) and from patch clamping of Xenopus oocytes expressing ENaC (21, 23) are in agreement that the single-channel conductance is unaltered. This activation of current must then be mediated via an increase of NT or Po. Our observations are consistent with the conclusion that only a small increase of the alpha -subunit density is observed with beta -subunit truncation. The remaining 3- to 3.5-fold increase in activation must be attributed to changes of Po. These conclusions are in partial agreement with the recent report by Firsov et al. (9), who used a similar approach to that used in this report except that they used a monoclonal tag-specific antibody. The degree of activation attributed to a potential increase of NT is in disagreement between the two reports (35 vs. 93%). However, the absolute values of these numbers are complicated with the potential presence of electrically silent channels, the potential differences in the stoichiometry between the wild-type and Liddle's channel, and other problems associated with quantification of membrane immunofluorescence (see above).

Peptide block. To understand the mechanisms by which the truncation in the beta -subunit brings about the elevated whole cell currents, we carried out experiments with injection of beta -subunit COOH-terminal peptide. Consistent with previous reports (4, 13), we demonstrated that ENaC can be blocked by a 30-amino acid beta -subunit COOH-terminal peptide. Nearly equal percentages of inhibition with injections that resulted in an approximate intracellular concentration of 300 µM of this peptide were observed for both the &agr;&bgr;′<SUB>chim</SUB>gamma -rENaC- and alpha beta chimgamma -rENaC-expressing oocytes (see Fig. 9). This finding has several implications. First, it supports the conclusion that a large fraction of the elevated currents observed in alpha &bgr;′<SUB>chim</SUB>gamma -rENaCexpressing oocytes results from an increase in Po. Second, this finding further documents the importance of the COOH terminus of the beta - (and gamma -) subunit in controlling ENaC activity in part by a direct blocking of normal ENaC currents. The observations of similar values of peptide block of alpha &bgr;′<SUB>chim</SUB>gamma -rENaC and alpha beta chimgamma -rENaC in combination with the specificity of this peptide to block currents (see RESULTS) argue in favor of the conclusion that we were injecting a saturating concentration of this peptide. Thus the observed 40-50% inhibition with this peptide may represent the maximal in vivo inhibition that can be exerted directly by the COOH terminus of the beta -subunit on ENaC.

Conclusions. We conclude that coexpression of the beta - and gamma -subunits with the alpha -subunit increases NT and Po. Likewise, truncation of the beta -subunit COOH terminus also increases NT and Po. Our data indicate that the majority of changes are mediated via effects on the Po of the channel. However, it is possible that the contribution of changes of NT and Po to channel activation may be different in various native ENaC-expressing tissues, because other local factors may determine the activity of the channel complex. In comparing immunolocalization data with functional assays, our work offers a convenient approach for assessing the relative importance of changes in channel surface expression and Po in regulating Na+ channel activity.

    ACKNOWLEDGEMENTS

We thank David L. Stetson (The Ohio State University) for helpful suggestions with the fixation procedures and Cathy Guy for help in typing the manuscript. We also thank Bernard Rossier (University of Lausanne) for the gift of ENaC and Young Oh and David Warnock (University of Alabama at Birmingham) for the truncated beta -ENaC constructs.

    FOOTNOTES

This work was initiated while M. S. Awayda was a fellow at the University of Alabama at Birmingham and was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-37206 and DK-07545 to D. J. Benos and by a Louisiana American Heart Grant-In Aid to M. S. Awayda.

Address for reprint requests: M. S. Awayda, Dept. of Medicine, SL35, Tulane Univ. School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112.

Received 5 June 1997; accepted in final form 21 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(6):C1889-C1899
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