Cloning and functional expression of the mouse epithelial sodium channel

Yoon J. Ahn, David R. Brooker, Farhad Kosari, Brian J. Harte, Jinqing Li, Scott A. Mackler, and Thomas R. Kleyman

Departments of Medicine and Physiology, University of Pennsylvania, and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104-6144


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The epithelial sodium channel (ENaC) plays a major role in the transepithelial reabsorption of sodium in the renal cortical collecting duct, distal colon, and lung. ENaCs are formed by three structurally related subunits, termed alpha -, beta -, and gamma ENaC. We previously isolated and sequenced cDNAs encoding a portion of mouse alpha -, beta -, and gamma ENaC (alpha -, beta -, and gamma mENaC). These cDNAs were used to screen an oligo-dT-primed mouse kidney cDNA library. Full-length beta mENaC and partial-length alpha - and gamma mENaC clones were isolated. Full-length alpha - and gamma mENaC cDNAs were subsequently obtained by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Injection of mouse alpha -, beta -, and gamma ENaC cRNAs into Xenopus oocytes led to expression of amiloride-sensitive (Ki = 103 nM), Na+-selective currents with a single-channel conductance of 4.7 pS. Northern blots revealed that alpha -, beta -, and gamma mENaC were expressed in lung and kidney. Interestingly, alpha mENaC was detected in liver, although transcript sizes of 9.8 kb and 3.1 kb differed in size from the 3.2-kb message observed in other tissues. A partial cDNA clone was isolated from mouse liver by 5'-RACE PCR. Its sequence was found to be nearly identical to alpha mENaC. To begin to identify regions within alpha mENaC that might be important in assembly of the native heteroligomeric channel, a series of functional experiments were performed using a construct of alpha mENaC encoding the predicted cytoplasmic NH2 terminus. Coinjection of wild-type alpha -, beta -, and gamma mENaC with the intracellular NH2 terminus of alpha mENaC abolished amiloride-sensitive currents in Xenopus oocytes, suggesting that the NH2 terminus of alpha mENaC is involved in subunit assembly, and when present in a 10-fold excess, plays a dominant negative role in functional ENaC expression.

cloning; Xenopus oocytes; structure-function relationship


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ORGANIZATION of plasma membrane proteins in the epithelial cell layer of polarized cells is a necessary requirement for vectorial transport of solutes. Apical and basolateral plasma membranes differ in protein composition and contain Na+-selective transport proteins that facilitate the movement of Na+ across the epithelium in a directed fashion (5, 33, 38, 49). Epithelial Na+ channels are expressed in apical plasma membranes of principal cells in the distal nephron, airway and alveolar epithelia in the lung, surface cells in the distal colon, urinary bladder epithelia, and other tissues including ducts of salivary and sweat glands (5, 13, 39, 50). These channels mediate Na+ transport across polarized epithelia (5, 17, 39, 49) and are selectively inhibited by submicromolar concentrations of amiloride (28). Renal epithelial Na+ channels are aldosterone responsive and are the rate-limiting step for distal Na+ reabsorption from the uriniferous space. Na+ crosses the apical surface of the cortical collecting duct principal cell via ENaC and reenters the bloodstream via Na+-K+-ATPase located on the basolateral membrane. Although precise gating mechanisms have not been fully elucidated, the up- or downregulation of ENaCs in the collecting tubule is manifest in perturbations of total body sodium homeostasis, extracellular fluid volume status, and blood pressure control (5, 18, 58). ENaC gain of function mutations has been identified in patients with Liddle's disease, a disorder characterized by volume expansion and hypertension (24, 48); conversely, ENaC loss of function mutations have been noted in patients with type I pseudohypoaldosteronism, a disorder characterized by volume depletion and hypotension (10).

The epithelial Na+ channel was initially cloned from rat distal colon (7, 9, 30, 59). An expression cloning technique led to the identification of one cDNA, termed alpha ENaC, whose cRNA induced expression of amiloride-sensitive Na+ currents in Xenopus oocytes (7, 30). However, Na+ current levels were considerably lower than expected. Two subsequent cDNA clones were isolated on the basis of their ability to complement alpha ENaC cRNA in the expression of amiloride-sensitive Na+ currents in Xenopus oocytes, and these were termed beta ENaC and gamma ENaC (9). Coinjection of the three cRNA species into Xenopus oocytes led to expression of Na+ channels with characteristics nearly identical to those observed in Na+ channels of renal cortical collecting tubules and in A6 cells (9, 23, 35). The three subunits share limited (~30% to 40%) sequence similarity, suggesting that they are derived from a common ancestral gene. ENaCs have subsequently been cloned from human lung and kidney (31, 32, 59), Xenopus kidney (36), avian distal intestine (21), and bovine kidney (alpha -subunit) (16). We have isolated cDNA clones encoding mouse alpha -, beta -, and gamma ENaC and have examined their tissue distribution and functional characteristics. To begin to identify regions within alpha mENaC that might be important in assembly of the native channel, a deletion construct of alpha mENaC encoding the cytoplasmic NH2 terminus was synthesized. We report that coinjection of wild-type alpha -, beta -, and gamma mENaC with the intracellular amino terminus of alpha mENaC inhibited expression of amiloride-sensitive currents in oocytes, suggesting that the NH2 terminus of alpha mENaC is involved in subunit assembly.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents were purchased from Sigma Chemical (St. Louis, MO), unless otherwise specified. For molecular biology protocols, standard procedures were followed (2, 41, 57). DNA sequencing and oligonucleotide syntheses were performed by the University of Pennsylvania DNA Core Facility.

Generation of probes and library screen. cDNAs partially encoding mouse alpha -, beta -, and gamma ENaC were isolated as previously described (6). These cDNAs were radiolabeled by random priming (Prime-It II random primer labeling kit; Stratagene, La Jolla, CA) with [alpha -32P]dATP (ICN, Costa Mesa, CA). A mouse kidney cDNA library cloned into the Lambda Uni-ZAP XR vector (Stratagene) and transformed bacteria were plated, and colonies were transferred to Hybond-N nylon filters (Amersham, Arlington Heights, IL). For primary screening, the filters were hybridized overnight to the labeled probes in hybridization solution [6× standard saline citrate (SSC), 20 mM NaH2PO4, 0.5% (wt/vol) SDS, and 500 µg/ml denatured, sonicated salmon sperm DNA] at 55°C. Filters were washed with 2× SSC + 0.1% SDS at room temperature, twice with 1× SSC + 0.1% SDS at 50°C, and once with 0.1× SSC + 0.1% SDS at 50°C. Filters were exposed overnight to Kodak X-Omat AR film at -70°C. Positive colonies on duplicate filters were selected and rescreened. Colonies were isolated by sib selection, subjected to plasmid rescue, excision to determine insert size, and partial nucleotide sequencing of the 5' end of the inserts.

Generation of full-length alpha - and gamma mENaC subunits. As no full-length alpha mENaC or gamma mENaC clones were obtained in the library screen, 5'-rapid amplification of cDNA ends (5'-RACE) PCR using gene-specific antisense primers based on known sequence (5'-TGGAAGACATCCAGAGATTG-3' for alpha - and 5'-CCACCAGTTTCTTCGACTCAT-3' for gamma mENaC) was performed to obtain the missing upstream fragments of the alpha - and gamma mENaC subunits. PCR conditions were as follows: initial denaturation at 94°C for 2 min, 30 amplification cycles (94°C for 10 s, 55°C for 20 s, 68°C for 2 min), and final elongation at 68°C for 10 min. The PCR products were subcloned into pCR2 (Invitrogen, Carlsbad, CA). The upstream (PCR products) and downstream (cDNAs from library screen) fragments of alpha - and gamma mENaC were ligated following restriction enzyme digestion to generate the full-length alpha - or gamma mENaC. Full-length clones were sequenced in both directions by the method of Sanger et al. (42).

Cloning of NH2-terminal alpha mENaC and ectodomain alpha mENaC. The cDNA residues encoding the NH2-terminal domain of alpha mENaC (corresponding to amino acid residues M1-F81) were PCR amplified and subcloned into pBS SK(-) (Stratagene). cDNA encoding the ectodomain of alpha mENaC (corresponding to amino acid residues Y166-P568) was PCR amplified and subcloned into pBK-CMV (Stratagene). Sequences were confirmed by Sanger dideoxynucleotide sequence analysis.

Northern blots. A commercial mouse multiple tissue Northern blot containing equal quantities (2 µg) of purified poly(A)+ RNA in each lane was used (Clontech, Palo Alto, CA) to examine mENaC tissue distribution. cDNA fragments1 of alpha ENaC (G1365-T1755), beta ENaC (T811-C1018), gamma ENaC (G958-C1205), and mouse beta -actin were 32P labeled (14) and individually hybridized with the membrane overnight at 50°C following the manufacturer's protocol. The blots were washed at high stringency (final wash, 0.1× SSC at 65°C) and exposed to film or to a phosphor screen for imaging (Molecular Dynamics, Sunnyvale, CA).

Oocyte expression and electrophysiology. cRNAs were generated from mENaC cDNA inserts in pBS SK(-) or pBK-CMV vector using a T3 cRNA synthesis kit (m-MESSAGE MACHINE; Ambion, Austin, TX) following the manufacturer's protocol. Prior to transcription all cDNA constructs were linearized by restriction digestion with Xho I except for gamma mENaC where Kpn I was used. Oocytes were isolated from Xenopus laevis females (Nasco, Fort Atkinson, WI), and stage V-VI oocytes were selected for collagenase treatment following standard protocols (57). Oocytes were injected with full-length mouse alpha -, beta -, and gamma ENaC cRNAs at a concentration of 2 ng · subunit-1 · oocyte-1. NH2-terminal alpha ENaC cRNA was injected at concentrations of 20 ng, 6 ng, or 2 ng per oocyte. All cRNAs were injected in a volume of 50 nl/oocyte. Following injection, oocytes were incubated at 18°C in modified Barth's saline [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 15 mM HEPES, 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 10 µg/ml penicillin, 10 µg/ml streptomycin sulfate, 100 µg/ml gentamycin sulfate, and 10 µg/ml nystatin; pH 7.2] and then assayed 18-48 h postinjection. In selected experiments, oocytes were incubated in a low-Na+ modified Barth's saline (the 88 mM NaCl was replaced with 88 mM KCl) to prevent the cells from loading with Na+ prior to voltage clamping. Whole cell currents were measured using the two-electrode voltage clamp technique (TEV) at a holding potential of -100 mV (with reference to bath) for 500 ms and then 450 ms at 0 mV. During recordings, oocytes were bathed in a sodium gluconate buffer [100 mM sodium gluconate, 2 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, 5 mM BaCl2, 10 mM tetraethylammonium chloride (TEA-Cl2), pH 7.2]. Cation selectivity measurements were performed in sodium gluconate or potassium gluconate (100 mM potassium gluconate, 2 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, 5 mM BaCl2, and 10 mM TEA-Cl2, pH 7.2) buffers. Amiloride-sensitive currents were determined by subtracting currents measured in oocytes perfused with sodium gluconate (or potassium gluconate) buffers supplemented with 100 µM amiloride from baseline currents in sodium gluconate (or potassium gluconate) buffers. TEV was performed under continuous flow (~4 ml/min) of buffers. Single-channel recordings were performed in the cell-attached configuration. All data were collected at room temperature and were filtered at 300 Hz. The applied voltage to the membrane patch represents the voltage deflection from the resting membrane potential. Inward Na+ currents were represented by downward deflections in single-channel recordings. Measurements of single-channel conductance were performed with a buffer containing 100 mM NaCl, 1.8 mM CaCl2, 2 mM KCl, and 10 mM HEPES, pH 7.2, in the patch pipette and in the bath. Statistical analyses were performed with pCLAMP software (Axon Instruments) or MatLab (MathWorks).

Statistics. Results are expressed as means ± SE. Statistical significance was determined by Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of full-length ENaC subunits. Labeled probes for alpha -, beta -, and gamma mENaC were synthesized as previously described (6) and used to screen mouse kidney oligo-dT-primed cDNA lambda libraries. Primary screening yielded 8 positive colonies for alpha mENaC, 9 positive colonies for beta mENaC, and 12 positive colonies for gamma mENaC. All alpha mENaC, 2 beta mENaC, and 11 gamma mENaC colonies were successfully isolated by sib selection, subjected to plasmid rescue, and excision to determine insert size, followed by partial nucleotide sequencing of the 5' end of the inserts. Two full-length beta mENaC clones were isolated (clones B1 and B2), but no full-length alpha mENaC or gamma mENaC clones were obtained (9). The alpha mENaC clone with the longest insert (clone A2) apparently lacked 978 residues of the 5' end of the open-reading frame compared with rat alpha ENaC (9). The gamma mENaC clones with the longest inserts (clones G5 and G11) appeared to lack 428 and 388 residues, respectively, at the 5' end of the open-reading frame compared with gamma rENaC (9). Clones A2, B1, and G5, and G11 were sequenced. The sequence of G11 was ~1 kb longer than both the gamma mENaC clone G5 and gamma rENaC in the 3'-untranslated region (data not shown). On the basis of sequence analysis, this 1-kb addition likely represented a cloning artifact.

5'-RACE was performed to obtain cDNAs encoding the 5' regions of alpha - and gamma mENaC. Sequence analyses confirmed that we had obtained the 5' regions of alpha - and gamma mENaC. The RACE products and the partial-length alpha - and gamma mENaC cDNAs were subjected to restriction digestion and ligation to obtain full-length alpha - and gamma mENaC cDNAs.

The full-length mENaC clones were sequenced. The deduced amino acid sequences of the mouse alpha -, beta -, and gamma ENaCs were 95%, 96%, and 97% identical to rat alpha -, beta -, and gamma ENaC, respectively, and 87%, 88%, and 88% identical to human alpha -, beta -, and gamma ENaC. Sequence comparisons illustrate the high degree of identity between rat and mouse ENaC, as depicted in Fig. 1. Of particular interest was a 15-nucleotide insertion present in gamma mENaC encompassing residues T399-C413, which is absent in rat. This insertion did not occur in close proximity to defined rat genomic intron-exon junctions (55) and likely did not arise as a result of alternative mRNA splicing. PCR analysis of mouse genomic DNA using primers flanking the 15-bp insertion (sense 5'-GATGTTCTAGACAGTACACCTCGGAAA-3'; antisense 5'- AAATCCCACCAGTTTCTTCGACTCATG-3') was performed. The 235-bp PCR product was sequenced and confirmed that the 15-bp insertion represented a species-specific phenomenon at the genomic level.




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Fig. 1.   Deduced amino acid sequences of mouse alpha -, beta -, and gamma -subunits of the epithelial sodium channel (ENaC). Sequence comparisons with rat ENaCs (7, 9) are included. Amino acid identity is indicated by a dash. Putative membrane-spanning domains (M1 and M2) are indicated by bold underscore.

Tissue distribution. Tissue distribution of alpha -, beta -, and gamma mENaC was examined by Northern blot analyses. As expected, all three mENaCs were expressed in mouse lung and kidney, with the alpha -, beta -, and gamma mENaC probes recognizing mRNAs of 3.7, 2.6, and 3.2 kb, respectively (Fig. 2). alpha mENaC recognized two mRNA species in mouse liver, of ~9.8 and 3.1 kb. Hybridization to liver mRNAs was observed under high-stringency conditions, but the signal was relatively weak. These results suggested that alpha mENaC is expressed in liver. To further confirm this observation, 5'-RACE PCR using a 3' gene-specific primer (5'-CGGAACCTGTGCAGTAACATGATGAG-3') and a 5' adaptor primer was performed on liver cDNA (Clontech). A PCR product of 702 bp was obtained. Its sequence was nearly identical to alpha mENaC (sequence not shown), providing additional evidence that alpha mENaC or an alpha mENaC isoform is expressed in mouse liver.


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Fig. 2.   Mouse alpha -, beta -, and gamma ENaC tissue distribution. A mouse multiple tissue Northern blot containing equal quantities (2 µg) of poly(A)+ RNA per lane was hybridized consecutively with 32P-labeled alpha -, beta -, or gamma mENaC or mouse actin probes as described in the MATERIALS AND METHODS. Bound probe was visualized by autoradiography or phosphorimager. All three mENaCs were expressed in mouse lung and kidney, with the alpha -, beta -, and gamma mENaC probes recognizing mRNAs of 3.7, 2.6, and 3.2 kb, respectively. Sk muscle, skeletal muscle.

Functional expression of mENaCs. The Xenopus oocyte expression system was used to examine the functional properties of mouse ENaCs. Whole cell amiloride-sensitive currents obtained in oocytes injected with alpha -, beta -, and gamma mENaC cRNAs by the TEV technique are illustrated in Figs. 3 and 4. Amiloride inhibited the Na+ current with an IC50 of 103 ± 16 nM (Fig. 3, n = 7). The Na+-to-K+ selectivity ratio was greater than 80:1 at a holding potential of -100 mV (Fig. 4, n = 5). Analyses of Na+ channel characteristics by cell-attached patch clamp revealed long open and closed times (on the order of seconds) (Fig. 5) and a linear current/voltage relationship with a slope conductance of 4.7 pS (Fig. 6, n = 4-6). These functional characteristics of mouse ENaCs are in agreement with the characteristics of the cloned rat, human, and X. laevis ENaCs (9, 31, 36).


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Fig. 3.   Expression of mouse alpha -, beta -, and gamma ENaC in oocytes: amiloride dose-response relationship. Oocytes were injected with alpha -, beta -, and gamma mENaC cRNAs and maintained in modified Barth's saline. Currents were measured in oocytes bathed in sodium gluconate in presence of increasing concentrations of amiloride using the two-electrode voltage clamp technique with a holding potential of -100 mV. Currents shown were normalized to the current measured in absence of amiloride. IC50 for amiloride was 103 ± 16 nM (n = 7).



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Fig. 4.   Expression of mouse alpha -, beta -, and gamma ENaC in oocytes: whole cell current/voltage (I/V) relationships and cation selectivity. Oocytes were injected with alpha -, beta -, and gamma mENaC cRNAs and maintained in a low-Na+ modified Barth's solution. Currents were measured while varying the holding potential between -100 and +60 mV (20 mV steps), using the TEV technique. Oocytes were bathed in a buffer containing either sodium gluconate () or potassium gluconate (open circle ) in presence or absence of 100 µM amiloride. Amiloride-sensitive currents are shown. Currents were normalized to the value obtained at a holding potential of -100 mV in Na+ bath (n = 5).



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Fig. 5.   Expression of mouse alpha -, beta -, and gamma ENaC in oocytes: single-channel recording. Analyses of Na+ channels by cell-attached patch clamp. Na+ channels present in the patch exhibited long (>1 s) open (O1, O2) and closed (C) states. Pipette solution contained 100 mM NaCl. A -120-mV potential was applied to the patch.



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Fig. 6.   Expression of mouse alpha -, beta -, and gamma ENaC in oocytes: single-channel I/V relationship. Oocytes were injected with alpha -, beta -, and gamma mENaC cRNAs and maintained in modified Barth's saline. Currents were measured while varying the holding potential between -120 and +60 mV (20-mV steps), using a cell-attached patch clamp. Pipette and oocyte bath solutions contained 100 mM NaCl. A linear I/V relationship was observed. Slope conductance was 4.7 pS. Reversal potential was ~0 mV, suggesting that the oocytes were loaded with Na+ prior to patch clamp analyses; n = 4-6 different oocytes for each data point.

Amino-terminal alpha mENaC has a dominant negative effect on ENaC expression in Xenopus oocytes. We examined whether coexpression of the NH2-terminal domain of alpha mENaC with wild-type alpha -, beta -, and gamma mENaC would inhibit the formation of functionally competent channels in Xenopus oocytes. Coinjection of wild-type alpha -, beta -, and gamma mENaC cRNAs with a 10-fold excess (weight basis) of alpha mENaC NH2-terminal cRNA in Xenopus oocytes inhibited amiloride-sensitive current compared with injection with wild-type cRNAs alone (Fig. 7, n = 20). Furthermore, this effect was dose dependent. A partial inhibition of amiloride-sensitive current was observed at coinjection ratios of 3:1 and 1:1 (Fig. 7, n = 6). Oocytes coinjected with wild-type mENaC cRNAs and a 10-fold excess of a control cRNA encoding for the ectodomain of alpha mENaC produced Na+-selective currents commensurate with those seen in oocytes injected with wild type alone (Fig. 8, n = 7). These data suggest that the NH2 terminus of alpha mENaC is involved in subunit assembly, and when present in a 10-fold excess, has a dominant negative role in functional ENaC expression in Xenopus oocytes.


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Fig. 7.   NH2-terminal alpha mENaC cRNA coinjected with wild-type mENaC cRNAs inhibits amiloride-sensitive Na+ currents in Xenopus oocytes in a dose-dependent fashion. Xenopus oocytes were coinjected with wild-type (wt) alpha -, beta -, and gamma mENaC (2 ng · subunit-1 · oocyte-1) cRNAs alone or in conjunction with an excess of the NH2-terminal (nt) alpha mENaC cRNA construct (cRNA subunit weight ratios of 10:1, 3:1, or 1:1), as described under MATERIALS AND METHODS. Whole cell TEV assays were performed 18-48 h postinjection. Na+ currents were measured at a holding potential of -100 mV in absence or presence of 10 µM amiloride. Normalized amiloride-sensitive currents are illustrated. Values were normalized to the amiloride-sensitive current measured in oocytes expressing wild-type alpha -, beta -, and gamma mENaC alone. Coinjection of wild-type mENaC cRNAs with a 10-fold excess of the alpha mENaC NH2-terminal cRNA in Xenopus oocytes almost completely abolished amiloride-sensitive current (0.10 ± 0.04, P < 0.001, n = 20) compared with oocytes injected with wild-type mENaCs alone (1 ± 0.10, n = 20). Coinjection of wild-type mENaC cRNAs with a 3-fold excess (3:1 injection) or equal amounts (1:1 injection) of the alpha mENaC NH2-terminal cRNA significantly reduced amiloride-sensitive currents (3:1 injection, 0.26 ± 0.08, P < 0.001, n = 6; 1:1 injection, 0.46 ± 0.08, P < 0.005, n = 6).



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Fig. 8.   alpha mENaC ectodomain cRNA coinjected with wild-type mENaC cRNAs does not inhibit amiloride-sensitive Na+ currents in Xenopus oocytes. Xenopus oocytes were coinjected with wild-type alpha -, beta -, and gamma mENaCs (wt) cRNAs alone or in conjunction with a 10-fold excess of alpha mENaC ectodomain (ecto) cRNA. Whole cell TEV assays were performed 18-48 h postinjection. Na+ currents were measured at a holding potential of -100 mV in absence or presence of 10 µM amiloride. Normalized amiloride-sensitive currents are depicted. Values were normalized to the amiloride-sensitive current measured in oocytes expressing wild-type alpha -, beta -, and gamma mENaC alone. Oocytes coinjected with wild-type mENaC cRNAs and alpha mENaC ectodomain cRNA expressed currents (0.86 ± 0.15, n = 7) commensurate with those injected with wild-type mENaC alone (1 ± 0.20; P = 0.6, n = 7).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The epithelial sodium channel is a heteroligomeric protein comprising three homologous subunits, alpha -, beta -, and gamma ENaC (9). We obtained full-length cDNAs encoding mouse alpha -, beta -, and gamma ENaC. The deduced amino acid sequences of the mouse ENaCs are nearly identical to rat alpha -, beta -, and gamma ENaC, respectively (Fig. 1) (7, 9). Dagenais et al. (12) have reported a partial cDNA clone of mouse alpha ENaC, corresponding to amino acid residues H445-L558 of our full-length mouse alpha ENaC. There are three residues that differ from our sequence near the extreme NH2 and COOH termini, which may reflect sequence polymorphisms. Regions within ENaCs that have defined functions are conserved. For example, each subunit has two putative membrane-spanning domains (M1 and M2) that are amphipathic and predicted to assume an alpha -helical structure (Fig. 1). A predominantly hydrophobic domain, previously termed H2 by Canessa and coworkers (9), precedes the second membrane-spanning domain of each subunit and may contribute to the channel pore and selectivity filter (9, 37), as mutations within this region alter cation selectivity, amiloride-sensitivity, and single-channel conductance (29, 37, 44, 45, 60). The region between the two hydrophobic membrane-spanning domains comprising approximately two-thirds of the mass of each subunit is extracellular (8, 52). Sequence analysis of this large extracellular domain reveals multiple N-linked glycosylation sites (6, 12, and 5 N-linked glycosylation sites for alpha -, beta -, and gamma -subunits, respectively) and cysteine-rich domains, features conserved between the three subunits as well as other members of the ENaC/mec/deg superfamily (11, 19). The relatively short cytoplasmic NH2- and COOH-termini have consensus sites for phosphorylation by protein kinase A and protein kinase C. Recent studies support the notion that phosphorylation of the beta - and gamma -subunits of the channel may have a role in the regulation of the channel by forskolin, insulin, aldosterone, and phorbol esters (47). Proline-rich regions and PXY motifs have defined roles in the binding of alpha -spectrin and the ubiquitin ligase Nedd4 to the channel (40, 54). A gating domain has been identified within the NH2 terminus of the alpha -subunit (22). These regions are conserved within the mouse ENaCs reported here.

Several groups have reported that the subunit stoichiometry of ENaC is alpha 2,beta 1,gamma 1 (15, 29), although one group has reported a subunit stoichiometry of alpha 3,beta 3,gamma 3 (51). When the three subunits are expressed in Xenopus oocytes, they oligomerize to form a channel with properties similar to that observed in native tissues (9, 19, 31). We also observed that mouse ENaCs, when expressed in Xenopus oocytes, are highly selective for sodium (Na+ >>> K+) (Fig. 4), demonstrate slow gating kinetics (long open and closed times on the order of several seconds) (Fig. 5), low single-channel conductance (4.7 pS) (Fig. 6), and are blocked by amiloride with a Ki of 103 nM (Fig. 3).

Liver alpha mENaC gene product may serve as an amiloride-sensitive Na+ channel. Mouse ENaC mRNA was expressed in sodium-absorptive epithelia (Fig. 2), including kidney and lung. Mouse alpha ENaC, like human alpha ENaC (31), was expressed in liver. Interestingly, the beta - and gamma -subunits were not detected in liver. The expression of the alpha -subunit alone raises the question of whether functional Na+ channels are expressed in the liver, either composed solely of alpha -subunits or a heteroligomer of alpha -subunits with related ENaC subunits that have yet to be identified. Na+-conductive pathways have an important role in hepatocyte volume regulation (61). Rat hepatocytes in confluent primary cultures respond to hypertonic stress with a considerable increase in cell membrane Na+ conductance. These adaptations (the regulatory volume increase, the increases in Na+ conductance and intracellular Na+ concentration, as well as the activation of Na+-K+-ATPase) were completely blocked by 10 µM amiloride (61). At this concentration, amiloride had no effect on osmotically induced cell alkalinization via Na+/H+ exchange (61). These data imply that an amiloride-sensitive Na+ channel is expressed in hepatocytes and serves as the conduit for Na+ influx. Activation of Na+ channels, in conjunction with activation of Na+-K+-ATPase, results in increases in intracellular K+ and Na+, and these are the major ionic mechanisms responsible for regulatory volume increases in hepatocytes. Our observation that alpha mENaC was expressed in liver suggested that alpha ENaC (and possibly as yet unidentified related subunits) may function as a mechanosensitive cation channel and play an important role in hepatocyte cell volume regulation.

The question of whether ENaC is a mechanosensitive ion channel has been examined by several laboratories. Differing results have been reported. Channels composed of all three ENaC subunits were mechanosensitive when expressed in lipid bilayers (25). Xenopus oocytes expressing alpha -, beta -, and gamma ENaC responded to cell swelling with either no change (3) or a decrease (26) in Na+ conductance and responded to cell shrinkage with an increase (26) or decrease (3) in Na+ conductance. Na+ channels in the collecting tubule responded in a variable manner to an increase in membrane tension by altering the hydrostatic pressure in a patch pipette (34); an increase in open probability was reported in 6 of 22 patches. Although controversy exists in the literature as to the effects of changes in cell volume on functional alpha -, beta -, and gamma ENaC expression, these data do not exclude the possibility that channels formed by alpha -subunits alone are mechanosensitive.

The biophysical properties of Na+ channels composed of alpha -subunits differ from alpha -, beta -, and gamma ENaC with regard to single-channel conductance and cation selectivity (9, 31, 32). Application of a hydrostatic pressure gradient across lipid bilayers increases the open probability of alpha ENaC (4). When expressed in fibroblasts, alpha ENaCs were activated in response to increases in the negative hydrostatic pressure applied to patch pipettes (27). These data suggest that alpha -subunits may oligomerize to form channels that are mechanosensitive and participate in hepatocyte volume regulation.

Functional effect of coexpression of NH2-terminal alpha mENaC with wild-type Na+ channels. The putative cytoplasmic domains of each ENaC subunit comprise a small fraction of the total mass of the channel protein and contain regions critical for ENaC activity. For example, regulatory motifs in the COOH termini of beta - and gamma ENaC including proline-rich domains and tyrosine-based internalization signals have been implicated in ENaC regulation and protein-protein interactions. Mutations or deletions of internalization signals or sites of interaction with Nedd4 have been associated with increases in functional ENaC activity (43, 46, 53, 54). A recent study suggested that the NH2 terminus of gamma hENaC participates in subunit-subunit interactions and in subunit assembly (1). Studies of other ion channels, such as voltage-gated K+ channels, suggest that NH2 termini are important sites of subunit-subunit interaction (56). We examined whether the NH2 terminus of alpha mENaC might be a site of subunit-subunit interactions. Our data suggest that the NH2 terminus of alpha mENaC may serve as an intersubunit association domain, as coexpression of the NH2 terminus of alpha mENaC with wild-type alpha -, beta -, and gamma mENaC subunits in Xenopus oocytes inhibited amiloride-sensitive current. The mechanisms of suppression of ENaC expression are unclear, but the data are consistent with a disruption in normal assembly by formation of a heteroligomeric complex between the NH2 terminus of alpha mENaC and wild-type alpha -, beta -, and/or gamma mENaC subunits.

Type 1 pseudohypoaldosteronism (PHA1) is a disorder exhibiting Mendelian inheritance that is characterized by salt wasting, metabolic acidosis, and hyperkalemia. Our data suggest that the NH2-terminal domain of alpha ENaC functions as a dominant negative mutant when coexpressed with wild-type subunits, perhaps by inhibiting assembly of functional Na+ channels. Our dose-response experiments highlight the fact that inhibition of ENaC function was most profound when an excess of NH2-terminal alpha mENaC was expressed with wild-type alpha mENaC (Fig. 7). Genotyping and single-strand chain polymorphism analysis of several autosomal recessive PHA1 kindreds have identified frame-shift mutations within human alpha - and beta ENaC prior to the first transmembrane domain resulting in truncated subunits (10). To date, ENaC mutations associated with autosomal dominant kindreds have not been described (20). It is interesting to speculate that heterozygotes expressing both truncated and wild-type alpha hENaC (or gamma hENaC) may have reduced levels of ENaC expression. However, the PHA1 phenotype might be observed only under conditions of stress, such as in the immediate postpartum period, in states of profound salt/water depletion, and in association with increased potassium intake.


    ACKNOWLEDGEMENTS

We thank Dr. Bruce Stanton for providing sequence information for the upstream portion of alpha mENaC.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-51391, DK-54354, HL-07027, and DK-07006. Y. J. Ahn was a recipient of postdoctoral fellowship awards from the American Heart Association, Southeastern Pennsylvania Affiliate, and from the Cystic Fibrosis Foundation. B. J. Harte was a recipient of a medical student research award from the American Heart Association.

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. §1734 solely to indicate this fact.

1 Refer to GenBank accession numbers AF112185, AF112186, and AF112187 for full nucleotide sequence. cDNA nucleotide residues referred to in this manuscript are numbered from the initiation ATG of the open-reading frame.

Address for reprint requests and other correspondence: Y. J. Ahn, Renal Electrolyte Hypertension Division, Univ. of Pennsylvania, 700 Clinical Research Bldg., 422 Curie Boulevard, Philadelphia, PA 19104-6144 (E-mail: yahn{at}mail.med.upenn.edu).

Received 17 December 1998; accepted in final form 24 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adams, C., P. Snyder, and M. Welsh. Interactions between subunits of the human epithelial sodium channel. J. Biol. Chem. 272: 27295-27300, 1997[Abstract/Free Full Text].

2.   Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. Current Protocols in Molecular Biology. New York: Wiley, 1995.

3.   Awayda, M., and M. Subramanyam. Regulation of the epithelial Na channel by membrane tension. J. Gen. Physiol. 112: 97-111, 1998[Abstract/Free Full Text].

4.   Awayda, M. S., I. I. Ismailov, B. K. Berdiev, and D. J. Benos. A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers. Am. J. Physiol. 268 (Cell Physiol. 37): C1450-C1459, 1995[Abstract/Free Full Text].

5.   Benos, D. J., M. S. Awayda, I. I. Ismailov, and J. P. Johnson. Structure and function of amiloride-sensitive Na+ channels. J. Membr. Biol. 143: 1-18, 1995[Medline].

6.   Brooker, D. R., C. A. Kozak, and T. R. Kleyman. Epithelial sodium channel genes Scnn1b and Scnn1g are closely linked on distal mouse chromosome 7. Genomics 29: 784-786, 1995[Medline].

7.   Canessa, C. M., J.-D. Horisberger, and B. C. Rossier. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467-470, 1993[Medline].

8.   Canessa, C. M., A. M. Merillat, and B. C. Rossier. Membrane topology of the epithelial sodium channel in intact cells. Am. J. Physiol. 267 (Cell Physiol. 36): C1682-C1690, 1994[Abstract/Free Full Text].

9.   Canessa, C. M., L. Schild, G. Buell, B. Thorens, I. Gautschl, J.-D. Horisberger, and B. C. Rossier. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994[Medline].

10.   Chang, S. S., S. Grunder, A. Hanukoglu, A. Rosler, P. M. Mathew, I. Hanukoglu, L. Schild, Y. Lu, R. A. Shimkets, C. N. Nelson-Williams, B. C. Rossier, and R. P. Lifton. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalemic acidosis, pseudohypoaldosteronism type 1. Nat. Genet. 12: 248-253, 1996[Medline].

11.   Corey, D. P., and J. Garcia-Anoveros. Mechanosensation and the DEG/ENaC ion channels. Science 273: 323-324, 1996[Medline].

12.   Dagenais, A., R. Kothary, and Y. Berthiaume. The alpha subunit of the epithelial sodium channel in the mouse: developmental regulation of its expression. Pediatr. Res. 42: 327-334, 1997[Abstract].

13.   Duc, C., N. Farman, C. M. Canessa, J. P. Bonvalet, and B. C. Rossier. Cell-specific expression of epithelial sodium channel alpha , beta , and gamma  subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J. Cell Biol. 127: 1907-1921, 1994[Abstract].

14.   Feinberg, A. P., and B. Vogelstein. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13, 1983[Medline].

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

16.   Fuller, C. M., M. S. Awayda, M. P. Arrate, A. L. Bradford, R. G. Morris, C. M. Canessa, B. C. Rossier, and D. J. Benos. Cloning of a bovine renal epithelial Na+ channel subunit. Am. J. Physiol. 269 (Cell Physiol. 38): C641-C654, 1995[Abstract].

17.   Garty, H. Molecular properties of epithelial, amiloride-blockable Na+ channels. FASEB J. 8: 522-528, 1994[Abstract/Free Full Text].

18.   Garty, H. Regulation of Na+ permeability by aldosterone. Semin. Nephrol. 12: 24-29, 1992[Medline].

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

20.   Geller, D. S., J. Rodriguez-Soriano, A. Vallo Boado, S. Schifter, M. Bayer, S. S. Chang, and R. P. Lifton. Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism type I. Nat. Genet. 19: 279-281, 1998[Medline].

21.   Goldstein, O., C. Asher, and H. Garty. Cloning and induction by low NaCl intake of avian intestine Na+ channel subunits. Am. J. Physiol. 272 (Cell Physiol. 41): C270-C277, 1997[Abstract/Free Full Text].

22.   Grunder, S., D. Firsov, S. S. Chang, N. F. Jaeger, I. Gautschi, L. Schild, R. P. Lifton, and B. C. Rossier. A mutation causing pseudohypoaldosteronism type-1 identifies a conserved glycine that is involved in the gating of the epithelial sodium-channel. EMBO J. 16: 899-907, 1997[Abstract/Free Full Text].

23.   Hamilton, K. L., and D. C. Eaton. Single channel recordings from amiloride-sensitive epithelial sodium channel. Am. J. Physiol. 249 (Cell Physiol. 18): C200-C207, 1985[Abstract].

24.   Hansson, J. H., C. Nelson-Williams, H. Suzuki, L. Schild, R. Shimkets, Y. Lu, C. Canessa, T. Iwasaki, B. Rossier, and R. P. Lifton. Hypertension caused by a truncated epithelial sodium channel gamma-subunit: Genetic-heterogeneity of Liddle syndrome. Nat. Genet. 11: 76-82, 1995[Medline].

25.   Ismailov, I. I., M. S. Awayda, B. K. Berdiev, J. K. Bubien, J. E. Lucas, C. M. Fuller, and D. J. Benos. Triple-barrel organization of ENaC, a cloned epithelial Na+ channel. J. Biol. Chem. 271: 807-816, 1996[Abstract/Free Full Text].

26.   Ji, H. L., C. M. Fuller, and D. J. Benos. Osmotic pressure regulates alpha beta gamma -rENaC expressed in Xenopus oocytes. Am. J. Physiol. 275 (Cell Physiol. 44): C1182-C1190, 1998[Abstract/Free Full Text].

27.   Kizer, N., X. L. Guo, and K. Hruska. Reconstitution of stretch-activated cation channels by expression of the a-subunit of the epithelial sodium channel cloned from osteoblasts. Proc. Natl. Acad. Sci. USA 94: 1013-1018, 1997[Abstract/Free Full Text].

28.   Kleyman, T. R., and E. J. Cragoe, Jr. Cation transport probes: the amiloride series. In: Methods in Enzymology. Cellular and Subcellular Transport: Epithelial Cell, edited by S. Fleischer, and B. Fleischer. Orlando: Academic, 1990, p. 739-755.

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

30.   Lingueglia, E., N. Voilley, R. Waldmann, M. Lazdunski, and P. Barbry. Expression cloning of an epithelial amiloride-sensitive Na+ channel. FEBS Lett. 318: 95-99, 1993[Medline].

31.   McDonald, F. J., M. P. Price, P. M. Snyder, and M. J. Welsh. Cloning and expression of the beta - and gamma -subunits of the human epithelial sodium channel. Am. J. Physiol. 268 (Cell Physiol. 37): C1157-C1163, 1995[Abstract/Free Full Text].

32.   McDonald, F. J., P. M. Snyder, P. B. McCray, Jr., and M. J. Welsh. Cloning, expression, and tissue distribution of a human amiloride-sensitive Na+ channel. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L728-L734, 1994[Abstract/Free Full Text].

33.   Nelson, W. J. Regulation of cell surface polarity from bacteria to mammals. Science 258: 948-955, 1992[Medline].

34.   Palmer, L. G., and G. Frindt. Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch. J. Gen. Physiol. 107: 35-45, 1996[Abstract].

35.   Palmer, L. G. Epithelial Na channels: function and diversity. Annu. Rev. Physiol. 54: 51-66, 1992[Medline].

36.   Puoti, A., A. May, C. M. Canessa, J. D. Horisberger, L. Schild, and B. C. Rossier. The highly selective low-conductance epithelial Na channel of Xenopus laevis A6 kidney cells. Am. J. Physiol. 269 (Cell Physiol. 38): C188-C197, 1995[Abstract/Free Full Text].

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

38.   Rodriguez-Boulan, E., and W. J. Nelson. Morphogenesis of the polarized epithelial cell phenotype. Science 245: 718-725, 1989[Medline].

39.   Rossier, B. C., C. M. Canessa, L. Schild, and J. D. Horisberger. Epithelial sodium channels. Curr. Opin. Nephrol. Hypertens. 3: 487-496, 1994[Medline].

40.   Rotin, D., D. Barsagi, O. B. H. J. Merilainen, V. P. Lehto, C. M. Canessa, B. C. Rossier, and G. P. Downey. An SH3 binding region the epithelial Na+ channel (alpha-rENaC) mediates its localization at the apical membrane. EMBO J. 13: 4440-4450, 1994[Abstract].

41.   Sambrook, J., E. F. Fritsch, and T. Maniatis. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989.

42.   Sanger, F. G., S. Nicklen, and A. R. Coulson. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977[Abstract].

43.   Schild, L., X. Lu, I. Gautschi, E. Schneeberger, R. P. Lifton, and B. C. Rossier. Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. EMBO J. 15: 2381-2387, 1996[Abstract].

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

45.   Sheng, S., J. Li, and T. R. Kleyman. Epithelial sodium channel cation selectivity is altered by a mutation of a tryptophan residue (Abstract). J. Am. Soc. Nephrol. 9: 45, 1998.

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

47.   Shimkets, R., R. Lifton, and C. Canessa. In vivo phosphorylation of the epithelial sodium channel. Proc. Natl. Acad. Sci. USA 95: 3301-3305, 1998[Abstract/Free Full Text].

48.   Shimkets, R. A., D. G. Warnock, C. M. Bositis, C. Nelson-Williams, J. H. Hansson, M. Schambelan, J. R. Gill, Jr., S. Ulick, R. V. Milora, J. W. Findling, C. M. Canessa, B. C. Rossier, and R. P. Lifton. Liddle's syndrome: heritable human hypertension caused by mutations in the beta  subunit of the epithelial Na channel. Cell 79: 407-414, 1994[Medline].

49.   Smith, P. R., and D. J. Benos. Epithelial Na+ channels. Annu. Rev. Physiol. 53: 509-530, 1991[Medline].

50.   Smith, P. R., S. A. Mackler, P. C. Weiser, D. R. Brooker, Y. J. Ahn, B. J. Harte, K. A. McNulty, and T. R. Kleyman. Expression and localization of epithelial sodium channel in mammalian urinary bladder. Am. J. Physiol. 274 (Renal Physiol. 43): F91-F96, 1998[Abstract/Free Full Text].

51.   Snyder, P. M., C. Cheng, L. S. Prince, J. C. Rogers, and J. M. Welsh. Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits. J. Biol. Chem. 273: 681-684, 1998[Abstract/Free Full Text].

52.   Snyder, P. M., F. J. McDonald, J. B. Stokes, and M. J. Welsh. Membrane topology of the amiloride-sensitive epithelial sodium channel. J. Biol. Chem. 269: 24379-24383, 1994[Abstract/Free Full Text].

53.   Snyder, P. M., M. P. Price, F. J. McDonald, C. M. Adams, K. A. Volk, B. G. Zeiher, J. B. Stokes, and M. J. Welsh. Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na channel. Cell 83: 969-978, 1995[Medline].

54.   Staub, O., S. Dho, P. C. Henry, J. Correa, T. Ishikawa, J. McGlade, and D. Rotin. WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome. EMBO J. 15: 2371-2380, 1996[Abstract].

55.   Thomas, C. P., S. D. Auerbach, C. Zhang, and J. B. Stokes. The structure of the rat amiloride-sensitive epithelial sodium channel gamma subunit gene and functional analysis of its promoter. Gene 228: 111-122, 1999[Medline].

56.   Tu, L. W., V. Santarelli, Z. F. Sheng, W. Skach, D. Pain, and C. Deutsch. Voltage-gated K+ channels contain multiple intersubunit association sites. J. Biol. Chem. 271: 18904-18911, 1996[Abstract/Free Full Text].

57.   Tymms, M. In vitro transcription and translation protocols. In: Methods in Molecular Biology. Totowa, NJ: Humana, 1995.

58.   Verrey, F. Transcriptional control of sodium transport in tight epithelia by adrenal steroids. J. Membr. Biol. 143: 93-110, 1995.

59.   Voilley, N., E. Lingueglia, G. Champigny, M. G. Mattei, R. Waldmann, M. Lazdunski, and P. Barbry. The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning. Proc. Natl. Acad. Sci. USA 91: 247-251, 1994[Abstract].

60.   Waldmann, R., G. Champigny, and M. Lazdunski. Functional degenerin-containing chimeras identify residues essential for amiloride-sensitive Na+ channel function. J. Biol. Chem. 270: 11735-11737, 1995[Abstract/Free Full Text].

61.   Wehner, F., H. Sauer, and R. K. Kinne. Hypertonic stress increases the Na+ conductance of rat hepatocytes in primary culture. J. Gen. Physiol. 105: 507-535, 1995[Abstract].


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