INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

THE GENE CODING for the -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 -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 ( and ) that, when coexpressed with the -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 -ENaC. Moreover, in some experiments, the - and -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 - and -subunits regulate ENaC activity involves the COOH termini of these subunits. Mutations that truncate the COOH termini of either the - or the -subunit, as observed in Liddle's syndrome (10, 22), cause an increase in channel activity. This was experimentally confirmed in experiments in which - and -ENaC were coexpressed with a truncated -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 -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 -subunit. They concluded that the observed increase of activity is likely because of large (4- to 5-fold) changes of open probability (P_o) and that this increase of P_o is mediated, at least in part, by relief of the inhibitory actions of the COOH-terminal region of the -subunit. Snyder et al. (23) reported that the changes of activity likely result from an increase of channel density (N_T) 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 N_T and P_o.

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 - and -ENaC alter the induced amiloride-sensitive currents. We also examined the differences in plasma membrane channel levels between oocytes that express a normal -subunit and those that mimic Liddle's syndrome by expression of a truncated -subunit. Electrophysiological measurements were carried out on the same batch of oocytes as were the immunofluorescent measurements. We report that coexpression of the - and -subunits caused a threefold increase of membrane-bound -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 -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 -subunit COOH-terminal sequence (final concentration ~300 µM) resulted in a 40-50% inhibition of ENaC currents. We conclude that - and -ENaC and the COOH termini of these subunits affect channel activity by altering both N_T and P_o of the resulting Na⁺ channel.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Antibody production. A rabbit anti--ENaC antibody was produced as previously described (12). Briefly, 200 µg of bovine -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 -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 -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 -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 -ENaC (-rENaC) contained in pSPORT that was predigested with these two enzymes. The Xho I site in -rENaC was created at the corresponding site of the human -subunit by PCR-directed mutagenesis. An extra Acc I site in -rENaC at nucleotide position 2 (numbered according to GenBank X77932) was removed by restriction with Sal I followed by blunt-end ligation.

RNA synthesis. RNA synthesis was as previously described (3). The plasmid containing either -, -, or -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. -rENaC-expressing oocytes were injected with 12.5 ng of cRNA, whereas -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.

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.

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 (anti-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.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Effect of coexpression of - and -ENaC. Previous reports have indicated that expression of -ENaC produces currents that are much smaller than those observed with -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 -ENaC alone. This was observed despite a fivefold higher concentration of RNA in oocytes injected with the -subunit by itself.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Expression of - and -rat epithelial Na+ channel (rENaC) with -rENaC causes a marked increase of amiloride-sensitive whole cell currents. Representative whole cell currents (Im) in oocytes expressing -rENaC (A) and -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.

View this table: [in this window] [in a new window]   Table 1.   Whole cell currents in Xenopus oocytes expressing -rENaC and -rENaC and their subsequent inhibition by amiloride

View larger version (64K): [in this window] [in a new window]   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.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Specificity of -subunit antibody used in present studies. This antibody did not show any appreciable immunoreactivity in oocytes expressing either of the - or -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.

View larger version (50K): [in this window] [in a new window]   View larger version (92K): [in this window] [in a new window]   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. -rENaC-expressing oocytes exhibited higher membrane immunofluorescence (B) that is further increased by expression of - and -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.

View this table: [in this window] [in a new window]   Table 2.   Relative plasma membrane fluorescence intensity of Xenopus oocytes expressing -rENaC and -rENaC probed by an anti--subunit antibody

Effect of truncation of the -subunit. The COOH termini of the - and -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 -subunit truncation causes Na⁺ hyperabsorption and Liddle's syndrome, we expressed the truncated -subunit with - and -rENaC. As shown in the representative example in Fig. 5, truncation of the terminal 75 amino acids of the -subunit construct caused a large activation of whole cell currents over those observed with the nontruncated -subunit rat-plus-human construct _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 -subunits and those formed with _chim.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Expression of a truncated -subunit () causes a marked increase of whole cell currents. Representative whole cell currents in oocytes expressing chim-rENaC (A) and -rENaC (B).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Whole cell currents in oocytes coexpressing control chimeric -subunits (B) were indistinguishable from those observed in oocytes coexpressing rat -subunit (A).

View larger version (49K): [in this window] [in a new window]   Fig. 7.   Truncation of -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 -subunit (A) compared with oocytes expressing chimeric nontruncated -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 - and -rENaC with chimeric -subunit or full rat -subunit (compare, for example, Fig. 4C and Fig. 5B). Arrowheads point to cell surface. Bar, 50 µm.

View larger version (41K): [in this window] [in a new window]   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 chim-rENaC- (A) and -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 chim-rENaC- (C) and -rENaC (D)-expressing oocytes are at plateau time point. E and F demonstrate effect of 10 µM amiloride on chim- and -rENaC, respectively.

View larger version (52K): [in this window] [in a new window]   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. hC30, 30-mer peptide derived from last 30 amino acids of human -subunit.

View larger version (19K): [in this window] [in a new window]   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 -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 chim-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 -ENaC fusion protein as previously described (12) to determine the changes of -subunit density at the plasma membrane subsequent to coexpression with full-length - and -subunits or after coexpression with a full-length -subunit and a truncated -subunit. We conclude that the changes of whole cell currents produced by expression of -subunits with - and -subunits are likely the result of a combination of a threefold change of N_T and a 10-fold change of P_o. Likewise, the increase of current observed with -subunit truncation is also attributed to a combination of a 1.35-fold increase of N_T and approximately threefold increase of P_o. In both cases, it appears that the potential changes of N_T are small in comparison to the potential changes of P_o.

Because this antibody is directed against a full-length fusion protein, it is expected that it would recognize a complete and functional -subunit as well as a partially degraded and electrically nonfunctional -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 N_T 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 N_T and P_o cannot be easily answered using patch- clamp methodologies, because translation of changes of channel activity (N_T · P_o) in multiple-channel patches to changes of either N_T or P_o is very difficult. This situation is further exacerbated in low-P_o patches and in cases in which the channel activity varies enormously, as with oocytes expressing the -subunit alone and those expressing -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 N_T.

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 -subunit alone form a functional channel? We have previously reported that -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 -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 -subunit alone. Moreover, in mammalian cells transfected with -rENaC alone, an amiloride-sensitive Na⁺ channel is observed (14), indicating that -ENaC alone can form a functional channel. The observation that there is membrane-bound distribution of -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 -subunit. This current is probably not a result of the presence of endogenous - and -ENaC, because injection of - or -ENaC individually or of - and -ENaC together in the absence of -ENaC does not result in any current over that observed in water-injected oocytes.

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

Truncated -subunit. Truncation of the -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 N_T or P_o. Our observations are consistent with the conclusion that only a small increase of the -subunit density is observed with -subunit truncation. The remaining 3- to 3.5-fold increase in activation must be attributed to changes of P_o. 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 N_T 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 -subunit brings about the elevated whole cell currents, we carried out experiments with injection of -subunit COOH-terminal peptide. Consistent with previous reports (4, 13), we demonstrated that ENaC can be blocked by a 30-amino acid -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 -rENaC- and _chim-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 -rENaCexpressing oocytes results from an increase in P_o. Second, this finding further documents the importance of the COOH terminus of the - (and -) subunit in controlling ENaC activity in part by a direct blocking of normal ENaC currents. The observations of similar values of peptide block of -rENaC and _chim-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 -subunit on ENaC.

Conclusions. We conclude that coexpression of the - and -subunits with the -subunit increases N_T and P_o. Likewise, truncation of the -subunit COOH terminus also increases N_T and P_o. Our data indicate that the majority of changes are mediated via effects on the P_o of the channel. However, it is possible that the contribution of changes of N_T and P_o 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 P_o in regulating Na⁺ channel activity.