Am J Physiol Renal Physiol 294: F47-F52, 2008.
First published November 21, 2007; doi:10.1152/ajprenal.00399.2007
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Aldosterone and Epithelial Na+ Channels
Defining an inhibitory domain in the 
-subunit of the epithelial sodium channel
Marcelo D. Carattino,1
Christopher J. Passero,1
Carlos A. Steren,2
Ahmad B. Maarouf,1
Joseph M. Pilewski,3
Mike M. Myerburg,3
Rebecca P. Hughey,1,4 and
Thomas R. Kleyman1,4
1Renal-Electrolyte Division and 3Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, and 4Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania; and 2NMR Facility, Department of Chemistry, University of Tennessee, Knoxville, Tennessee
Submitted 29 August 2007
; accepted in final form 19 November 2007
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ABSTRACT
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Epithelial sodium channels (ENaC) are processed by proteases as they transit the biosynthetic pathway. We recently observed that furin-dependent processing of the 
-subunit of ENaC at two sites within its extracellular domain is required for channel activation due to release of a 26-residue inhibitory domain. While channels with -subunits lacking the furin sites are not cleaved and have very low activity, channels lacking the furin consensus sites as well as the tract between these sites (D206–R231) are active. We analyzed channels with a series of deletions in the tract D206–R231 and lacking the -subunit furin consensus sites in Xenopus laevis oocytes. We found an eight-residue tract that, when deleted, restored channel activity to the level found in oocytes expressing wild-type ENaC. A synthetic peptide, LPHPLQRL, representing the tract L211–L218, inhibited wild-type ENaC expressed in oocytes with an IC50 of 0.9 µM, and inhibited channels expressed in collecting duct cells and human primary airway epithelial cells with an IC50s of between 
50 and 100 µM. Analyses of peptides with deletions within this inhibitory tract indicate that eight residues is the minimal backbone length that is required for ENaC inhibition. Analyses of 8-mer peptides with conserved and nonconserved substitutions suggest that L1, P2, H3, P4, and L8 are required for inhibitory activity. Our findings suggest that this eight-residue tract is a key conserved inhibitory domain that provides epithelial cells with a reserve of inactive channels that can be activated as required by proteases.
peptide inhibitors; proteases; furin
EPITHELIAL SODIUM CHANNELS (ENaCs) are constituted by three structurally similar subunits, termed , β, and 
, and are expressed in the apical membrane of Na+-transporting epithelia including the distal nephron, airways, and distal colon. ENaC subunits have intracellular NH2 and COOH termini and two membrane-spanning domains connected by large extracellular loops (21). In the distal nephron, ENaC constitutes the limiting step for Na+ absorption and hence has an important role in extracellular fluid volume homeostasis and control of blood pressure.
In airway epithelia, ENaC has an important role in regulating the volume of airway surface liquids and mucociliary clearance. Knockout of the -subunit in mice leads to premature death as a consequence of defective lung liquid clearance, indicating that ENaC activity is required for reabsorption of fluids from the alveolar space at the time of birth (13). Conversely, overexpression of the β-subunit in mouse airway produces a phenotype similar to cystic fibrosis (CF) with airway surface liquid volume depletion, mucus obstruction, goblet cell metaplasia, neutrophil inflammation, and poor bacterial clearance (17).
We previously reported that - and -ENaC subunits are processed within their extracellular domains by furin, a serine protease that resides primarily in the trans-Golgi network (11). When expressed in Xenopus laevis oocytes, Chinese hamster ovary (CHO) cells, or Madin-Darby canine kidney (MDCK) cells, the -subunit is cleaved twice immediately following R205 and R231, and at a single site within the -subunit after R143 (11). Channels with noncleaved -subunits or with -subunits that are cleaved at only one furin site are inactive and display a low channel open probability. However, channels lacking both -subunit furin cleavage sites as well as the tract between these sites (D206–R231) are active when expressed in oocytes, suggesting that the release of an inhibitory tract (D206–R231) from the extracellular domain of the -subunit is required for channel activation (5). A synthetic 26-mer peptide corresponding to the tract D206–R231 (-26) reversibly inhibited ENaCs expressed in X. laevis oocytes, as well as endogenous Na+ channels expressed in a mammalian collecting duct cell line and primary cultures of human airway cells (5). The IC50 for amiloride block of ENaC was not affected by the presence of -26, indicating that the inhibitory peptide does not bind to or interact with the amiloride-binding site (5). Although the peptide has an overall positive charge at neutral pH, peptide block of ENaC was not voltage dependent. Furthermore, channels carrying mutations in the -subunit furin cleavage sites were not inhibited by the -26 mer peptide. These results suggest that the peptide binds to a site within the extracellular domain of the -subunit (5).
We have now identified a limited, eight-residue tract (L211–L218) within the previously described inhibitory tract D206–R231 that, when deleted from an -subunit with mutated furin cleavage sites, results in active Na+ channels. Synthetic peptides, based on the sequence of this eight-residue tract, were generated and structural requirements for ENaC inhibition were determined. This eight-residue tract appears to be a key inhibitory domain within the subunit.
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EXPERIMENTAL PROCEDURES
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Oocyte expression.
cRNAs for mouse -, β-, and -ENaC (wild-type and mutant) subunits were synthesized with a T3 mMessage mMachine (Ambion, Austin, TX). Stage V-VI X. laevis oocytes pretreated with 1.5 mg/ml type II collagenase were injected with 1–2 ng of cRNA/subunit. Injected oocytes were maintained as previously described (4).
Site-directed mutagenesis.
Mutations of mouse ENaC subunits were generated by site-directed mutagenesis with the sequential PCR method using Pfu DNA polymerase (Stratagene, La Jolla, CA) (20). Mutations were confirmed by DNA sequencing.
Peptides.
Peptides with acetylated NH2 termini and amidated COOH termini were synthesized and HPLC purified (purity >90%) by GenScript (Piscataway, NJ).
Two-electrode voltage clamp.
Two-electrode voltage clamp (TEV) was performed as previously described (4). The extracellular solution (TEV solution) contained (in mM) 110 NaCl, 2 KCl, 1.54 CaCl2, and 10 HEPES, pH 7.4. The ENaC-independent component of the whole-cell Na+ current was determined by bath perfusion with TEV solution supplemented with 10 µM amiloride. ENaC-mediated whole-cell Na+ currents, at –60 mV, were defined as the amiloride-sensitive component of the current.
mpkCCDc14 cell culture.
mpkCCDc14 cells derived from mouse cortical collecting duct were grown and subcultured onto permeable filter supports coated with collagen [0.4-µm pore size, 1-cm2 surface area Snapwell filters (Corning, Acton, MA)] as previously reported (5). Cells were maintained on filters for at least 4 days in defined medium (2) that was changed every second day.
Primary cultures of human airway epithelial cells.
Human airway epithelial (HAE) cells were obtained from excess pathological tissue remaining after lung transplantation or from organ donor lungs deemed not suitable for transplantation under a protocol approved by the University of Pittsburgh Institutional Review Board. All cells were isolated from second through sixth generation bronchi and grown on permeable Transwell supports (Corning), as previously described (8).
Short-circuit current measurements.
Cell culture inserts were mounted in modified Ussing chambers (Harvard Apparatus, Holliston, MA). The monolayers were continuously short-circuited with a voltage/current clamp system (Physiologic Instruments, San Diego, CA), as previously described (5). A 10-mV bipolar pulse was applied periodically to measure the resistance of the monolayer. The bath solution contained (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose. The pH of this solution was 7.4 when gassed with a mixture of 95% O2-5% CO2 at 37°C.
Solution-state nuclear magnetic resonance studies.
The eight-amino acid synthetic peptide LPHPLQRL was characterized by solution-state nuclear magnetic resonance (NMR). The sample was dissolved in a 90% H2O/10% D2O solution. The pH of the solution was 2.7. Experiments were performed on a 600-MHz Varian VNMRS spectrometer equipped with an inverse triple-resonance (1H, 13C, and 15N) probe. Two-dimensional (2D)-1H homonuclear watergate DQF-COSY, watergate TOCSY (60-ms mixing time), presat NOESY (300-ms mixing time), and watergate ROESY (300-ms mixing time) spectra were acquired. Chemical shift values were referenced to dioxane (dH = 3.75) that was added to the solution. Temperature was set to 297°K for all the experiments. Spectra were processed with NMRPIPE (7) and analyzed with SPARKY (Goddard TD and Kneller DG, SPARKY V3.1, University of California, San Francisco, CA).
Data and statistical analyses.
The IC50 is expressed as the mean with a 95% confidence interval (CI); otherwise, data are expressed as means ± SE (n), where n equals the number of independent experiments analyzed. The IC50 was estimated from normalized currents plotted as a function of the peptide concentration fitted with the following equation, y = t 
, where y is the response variable, X is the concentration of peptide, and IC50 is the concentration of peptide that provokes a response halfway between the baseline (t) and maximum response. For statistical analyses, the normality and equality of the SDs of the data were tested. Based on these results, parametric or nonparametric tests were used. Fitting and statistical comparisons were performed with GraphPad 3.0 (GraphPad Software, San Diego, CA).
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RESULTS
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We previously reported that ENaC requires furin-dependent proteolysis of the - and -ENaC subunits for activation in endogenous and heterologous expression systems. Mutations at the furin cleavages sites within the -subunit (R205A and R231A) prevent proteolytic processing, leading to a large (85%) decrease in channel activity (11). Channels carrying a mutation in the first furin cleavage site as well as a deletion of the tract D206–R231 between both furin cleavage sites (R205A,
D206–R231) display similar activity to wild-type channels (5), suggesting that removal of the intervening tract between both furin cleavage sites is required for channel activation. We speculated that the entire tract D206–R231 was not required to inhibit ENaC activity and that there was a limited region between D206 and R231 that conferred the inhibitory property. To identify a limited inhibitory region, we generated a series of deletions within the tract D206–R231 in -subunits carrying mutations in both furin cleavages sites (R205A, R231A). We reasoned that when the inhibitory region is deleted from this mutant -subunit, channel activity should be restored to a level similar to that of wild-type ENaC. Amiloride-sensitive whole-cell Na+ currents were measured in oocytes expressing either wild-type or mutant ENaCs (Fig. 1). Oocytes expressing channels with furin site mutations (R205A,R231Aβ) displayed 13.9 ± 1.7% of the amiloride-sensitive current of oocytes expressing wild-type ENaC. Deletion of residues D206–A210, R219–R228, or S229–R231 did not relieve inhibition. In contrast, deletion of the tracts D206–P224, D206–L218, and L211–L218 restored channel activity, suggesting that the tract L211–L218 contained an inhibitory region.
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Fig. 1. Identification of a minimal inhibitory domain within the tract D206–R231. Two-electrode voltage clamp (TEV) was performed in oocytes expressing either wild-type or mutant epithelial sodium channels (ENaCs). Amiloride-sensitive currents from oocytes expressing mutant ENaCs were normalized to the currents of oocytes expressing wild-type channels (I/IWT). Statistically significant differences in amiloride-sensitive currents were observed between β control vs. R205A,R231Aβ, R205A,R231A,206–210β, R205A,R231A,219–228β, and R205A,R231A,229–231β [P < 0.001, Kruskal-Wallis test (nonparametric ANOVA) followed by Dunn's multiple comparisons posttest]. Experiments were performed with 23–56 oocytes for each group. Black bars within the illustrations on the left indicate residues that are either deleted within the tract D206–R231, or are excised by furin in the wild-type -subunit. Hatched bars indicate residues that are retained within the tract D206–R231.
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We examined the inhibitory properties of the synthetic peptide LPHPLQRL corresponding to the eight-residue inhibitory tract L211–L218. This 8-mer peptide inhibited ENaC expressed in oocytes with an IC50 of 8.7 x 10–7 M (CI, 7.1 x 10–7 M to 10.7 x 10–7 M) (n = 6–9), similar to the IC50 of the -26 peptide of 2.8 x 10–6 M that we previously reported (Fig. 2) (5). In the cortical collecting duct cell line mpkCCDc14 that endogenously expresses ENaCs, the IC50 was 4.5 x 10–5 M (CI, 3.8 x 10–5 M to 5.3 x 10–5 M) (n = 8–10) (Fig. 3). The 8-mer peptide inhibited ENaC in primary cultures of HAE cells from non-CF and CF donors with an IC50 of 10.6 x 10–5 M (CI, 4.4 x 10–5 M to 25.9 x 10–5 M) and 7.6 x 10–5 M (CI, 2.5 x 10–5 M to 22.8 x 10–5 M), respectively (n = 5–7) (Fig. 4). These results are also similar to the IC50 values of the -26 peptide in mpkCCDc14 (10 x 10–5 M) and HAE cells derived from non-CF donors (4.6 x 10–5 M) that we have observed (5).
Based on the sequence of the 8-mer tract, additional peptides were synthesized bearing changes or deletions of specific residues to study structural requirements for inhibition. The extent of inhibition was estimated as the ratio of the amiloride-sensitive component of the current following 3 min of perfusion with the peptides (10 µM) to the basal currents (I/I0) in oocytes expressing wild-type channels. The 8-mer wild type peptide (LPHPLQRL) reduced amiloride-sensitive currents by 82.0 ± 0.7%, whereas a scrambled peptide (QLHLLPPR) had no effect on ENaC activity (Table 1). We investigated the role of backbone length on the inhibitory properties of the peptide. Removal of residues from the NH2 and/or COOH terminal (L1 and L8) led to a significant reduction in the efficacy of the peptide (Table 1). Furthermore, substitution of the L1 or L8 with N, which has a hydrophilic side chain with similar accessible surface area (6), or with A, which has a small aliphatic side chain with a smaller accessible surface area, led to a significant lost of inhibition (Table 1). Based on the alignment of the region spanning the furin cleavage sites of mammalian -subunits (see Fig. 5), we substituted L1 by W or F, which are the homologous residues found in Canis familiaris and Rattus norvegicus, respectively, and L8 by I, which is the homologous residue found in Cavia porcellus. We found no difference in the efficacy of inhibition of wild-type ENaC by WPHPLQRL, FPHPLQRL, or LPHPLQRI compared with the 8-mer peptide LPHPLQRL (Mus musculus) (Table 1).
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Fig. 5. Alignment of the region spanning the furin cleavage sites in mammalian -ENaC subunits. Identical residues are highlighted in dark gray, whereas conserved residues are highlighted in dark gray with white lettering. Key R residues within the furin consensus cleavage sites are highlighted in black.
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Additional conservative and nonconservative substitutions were introduced in the PHP4 tract and the QR7 tract within the 8-mer peptide. Substitutions of the H3 by E, which contains a similar surface area but a negative charge, or K, which contains a similar surface area and a positive charge, significantly reduced the efficacy of inhibition by the 8-mer peptide. However, a peptide with a substitution of H3 by Y, which is the homologous residue found in the -subunit of C. porcellus (see Fig. 5), showed a similar efficacy to the 8-mer peptide LPHPLQRL (Table 1). Substitutions of P2 and P4 by G, which is highly flexible, or by F, which has a larger surface area than P, also significantly reduced peptide inhibition of ENaC, suggesting that rigidity provided by P to the peptide is required for inhibition. Conserved and non-conserved substitutions at Q6 and R7 had only modest effects on the efficacy of inhibition, suggesting that this region is relatively permissive with regard to amino acid side chains.
Solution-state NMR spectroscopy was performed to examine the structure of the 8-mer peptide. COSY and TOCSY spectra were used to assign the spin systems, and NOESY and ROESY spectra to find the sequential placement of the residues. The six HN/Ha cross-peaks corresponding to the amino acids other than P residues, their side chains, and the two P residues of the peptide were identified from the TOCSY spectrum. Table 2 summarizes the chemical shift assignments for the eight residues. NOESY and ROESY spectra show few NOEs, those assigned corresponding to intraresidue or sequential interactions. No evidence of secondary or tertiary structure was observed. This indicates a random coil conformation, a conclusion that is further supported by the low dispersion of the measured chemical shifts.
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DISCUSSION
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We identified an eight-residue tract (L211–L218) within the -subunit that encompasses a key inhibitory domain. Channels bearing mutations in both -subunit furin cleavage sites (R205A, R231A) and a deletion of this eight-residue tract (L211–L218) retained activity. A synthetic peptide, LPHPLQRL, corresponding to the tract (L211–L218), inhibited ENaCs expressed in oocytes (IC50 = 0.9 µM) as well as in mpkCCDc14 monolayers (IC50 = 45 µM), and primary cultures of HAE cells from non-CF and CF donors (IC50 = 106 µM and IC50 = 76 µM, respectively). The decrease in efficacy of the 8-mer peptide in these cells endogenously expressing ENaC compared with oocytes heterologously expressing mouse ENaC is similar to that previously observed with the -26 peptide (corresponding to the tract D206–R231) (5). The reason for this difference in efficacy remains to be resolved.
A sequence alignment of mammalian -subunits showed that, within the region between the furin cleavages sites (corresponding to mouse D206–R231), the 8-mer tract is highly conserved (Fig. 5). Interestingly, this region is not conserved in amphibian species such as X. laevis (LPYLLELL) and Rana catesbeiana (LPFPLEKV). The high degree of conservation of this tract suggests that this region provided an advantage in the evolution of mammalians.
Structure-activity analyses of the inhibitory peptide revealed that eight is the minimal backbone length required to achieve inhibition. Deletions or substitutions (A or N) of L1 or L8 lead to a significant reduction in the efficacy of the ENaC block. Comparisons of the mammalian sequences suggested that nonpolar aliphatic or aromatic residues are likely substitutions at positions L1 and L8 (Fig. 5). We found no difference between the efficacy of peptides derived from the sequence of the -subunit of C. familiaris (WPHPLQRL), R. norvegicus (FPHPLQRL) and C. porcellus (LPHPLQRI) compared with the homologous peptide from M. musculus (LPHPLQRL). Our results suggest that hydrophobic interactions occur between the side chain of these nonpolar residues and the channel. Alternatively, these residues may be required either to stabilize the structure of the peptide or to stabilize the interaction of the peptide with the channel. The PHP4 tract appears to be essential for the peptide's inhibitory activity. At neutral pH, the imidazole group may be largely uncharged. We observed that substitution of H3 by Y, which is the homologous residue found in the subunit of C. porcellus, did not modify the inhibitory properties of the peptide. While our data raise the possibility that the uncharged imidazole group interacts with the channel, we cannot exclude the possibility that amino acid substitutions within the peptide alter its conformation and indirectly affect its binding to the channel. Solution-state NMR studies provide no evidence of secondary or tertiary structure, suggesting that the peptide in solution has a random coil conformation.
The recently revealed crystal structure of chicken ASIC1, an ENaC/Deg family member, showed that this family of ion channels have a chalice-like shape, with a large extracellular domain protruding from the plane of the membrane (14). The high degree of identity between ENaC and ASIC suggests that the core of the protein is probably conserved. The transmembrane-extracellular domain junction and extracellular domain resemble a wrist and clenched hand, respectively. The extracellular region is organized in discrete domains, rich in -helices and β-sheets, including four distinct domains referred to as the palm, knuckle, finger, and thumb. Alignment of the -subunit of ENaC and ASIC1 suggests that the region spanning the furin sites is located in the finger domain, which is one of the most accessible regions of the channel. The structure of the finger region of -ENaC and ASIC likely differs since this region is not conserved. We propose that the peptide likely interacts with a region of the channel within or close to the finger domain.
Inhibition of ENaC activity in the airway has been proposed as a rational approach to increase airway surface liquid volume and mucus clearance in CF and chronic bronchitis (10). Amiloride is a prototypic ENaC inhibitor and has been used for many years to block ENaC activity in the nephron, resulting in both natriuresis and antikaliuresis. However, the use of aerosolized amiloride in the treatment of CF lung disease has provided inconclusive results (1, 3, 9, 15, 16, 18, 19). This, in part, may reflect its limited potency and rapid clearance from the airway. While the 8-mer peptide is a relatively low-affinity blocker in mammalian cells endogenously expressing ENaC, analyses of derivatives may provide a basis to design higher affinity blockers of ENaC that could serve as an alternative to amiloride and related compounds to inhibit airway Na+ channels.
In conclusion, we identified an eight-residue inhibitory tract in the extracellular domain of the -subunits that is highly conserved in mammals. Since both pools of immature (with immature N-glycans and non-proteolytically processed) and mature subunits (with mature N-glycans and proteolytically processed) are found at the plasma membrane (12), this inhibitory domain likely has a role in reducing the activity of noncleaved channels at the plasma membrane.
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GRANTS
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This work was supported by grants from the National Institutes of Health (R01-DK-065161, P30-DK-072506, and P50-DK-54690) and the Cystic Fibrosis Foundation (R883CR02 and MYERBU07). The mpkCCDc14 cells were a gift from Alain Vandewalle (Paris, France). C. J. Passero and A. B. Maarouf are recipients of postdoctoral fellowship awards from the American Heart Association.
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FOOTNOTES
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Address for reprint requests and other correspondence: M. D. Carattino, Renal-Electrolyte Div., Univ. of Pittsburgh, S931 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (e-mail: carattinom{at}dom.pitt.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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V. Bhalla and K. R. Hallows
Mechanisms of ENaC Regulation and Clinical Implications
J. Am. Soc. Nephrol.,
October 1, 2008;
19(10):
1845 - 1854.
[Abstract]
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Copyright © 2008 by the American Physiological Society.
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ABSTRACT
EXPERIMENTAL PROCEDURES
) or the scrambled peptide (QLHLLPPR; 
) in mpkCCDc14 monolayers. I/I0 represents the ratio of the amiloride-sensitive current 2 min following and before the addition of peptide. Endogenously expressed ENaCs in mpkCCDc14 monolayers were inhibited by the 8-mer peptide with an IC50 of 4.5 x 10–5 M (CI, 3.8 x 10–6 M to 5.3 x 10–5 M) (n = 8–10). B: representative recordings of the effect of the 8-mer (top) or the scrambled peptide (bottom) on mpkCCDc14 monolayers. Arrows indicate the addition of peptides in the micromolar range or amiloride (amil) at 10 µM. Experiments were performed with 9–10 mpkCCDc14 monolayers per group. ISC, short-circuit current.