The epithelial sodium channel (ENaC): Mediator of the aldosterone response in the vascular endothelium?

In the kidney the epithelial sodium channel (ENaC) is regulated by the mineralocorticoid hormone aldosterone, which is essential for long-term blood pressure control. Evidence has accumulated showing that ENaC is expressed in endothelial cells. Moreover, its activity modifies the biomechanical properties of the endothelium. Therefore, the vascular system is also an important target for aldosterone and responds to the hormone with an increase in cell volume, surface area, and mechanical stiffness. These changes occur in a concerted fashion from minutes to hours and can be prevented by the specific sodium channel blocker amiloride and the mineralocorticoid receptor (MR) blocker spironolactone. Aldosterone acts on cells of the vascular system via genomic and non-genomic pathways. There is evidence that the classical cytosolic MR could mediate both types of response. Using a nanosensor covalently linked to aldosterone, binding sites at the plasma membrane were identified by atomic force microscopy. The interaction of aldosterone and this newly identified surface receptor could precede the slow classic genomic aldosterone response resulting in fast activation of endothelial ENaC. Recent data suggest that aldosterone-induced ENaC activation initiates a sequence of cellular events leading to a reduced release of vasodilating nitric oxide. We propose a model in which ENaC is the key mediator of aldosterone-dependent blood pressure control in the vascular endothelium.     

An inhibitory peptide derived from the α-subunit of the epithelial sodium channel (ENaC) shows a helical conformation

Proteolytic activation of the heteromeric epithelial sodium channel (ENaC) is thought to involve the release of inhibitory peptides from the extracellular domains of its α- and γ-subunit. Recently, we demonstrated that an α-13-mer peptide, corresponding to a putative inhibitory region within the extracellular domain of human αENaC, inhibits human αβγENaC. The aim of the present study was to investigate the structural basis of the inhibitory effect of this α-13-mer peptide. Analysis of the peptide by replica exchange molecular dynamics method, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations suggested that a helical turn at the carboxy-terminus is the preferred conformational state of the α-13-mer peptide. From this we predicted that a specific mutation (leucine 188 to alanine) should have a strong effect on the conformational preferences of the peptide. To functionally test this, we compared the effect of the wild-type α-13-mer with that of a mutant α-L188A-13-mer on ENaC currents in Xenopus laevis oocytes heterologously expressing human αβγENaC. We demonstrated that replacing the leucine 188 by alanine abolished the inhibitory effect of the α-13-mer peptide on ENaC. These findings suggest that a helical conformation in its carboxyterminal part is functionally important to mediate ENaC inhibition by the α-13-mer peptide. However, high resolution structural information on the complex of the inhibitory αENaC peptide and the channel are needed to confirm this conclusion.     

Novel indole and benzothiophene ring derivatives showing differential modulatory activity against human epithelial sodium channel subunits,ENaC β and γ

The epithelial sodium channel (ENaC) plays a pivotal role in sodium homeostasis, and the development of drugs that modulate ENaC activity is of great potential therapeutic relevance. We screened 6100 chemicals for their ability to activate sodium permeability of ENaC. We used a two-step strategy: a high throughput cell-based assay and an electrophysiological assay. Five compounds were identified showing common structural features including an indole or benzothiophene ring. ENaC consists of three subunits: α, β, and γ. Changing the heteromeric combination of human and mouse ENaC αβγ subunits, we found that all five compounds activated the human β subunit but not the mouse subunit. However, four of them exhibited lower activity when the human γ subunit was substituted by the mouse γ subunit. Our findings provide a structural basis for designing human ENaC activity modulators.

Abbreviations: ENaC: Epithelial sodium channel; ΔRFU: delta relative fluorescence units; EC₅₀: Half-maximal effective concentration; E_max: maximum effect value.     

Activation of the epithelial sodium channel by the metalloprotease meprin β subunit

The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin β (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin β or α/β in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin β, but not the α subunit. Meprin β promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin β regulates the activity of ENaC in a metalloprotease-dependent fashion.     

Nadph oxidase regulates alveolar epithelial sodium channel activity and lung fluid balance in vivo via O⁻₂ signaling

To define roles for reactive oxygen species (ROS) and epithelial sodium channel (ENaC) in maintaining lung fluid balance in vivo, we used two novel whole animal imaging approaches. Live X-ray fluoroscopy enabled quantification of air space fluid content of C57BL/6J mouse lungs challenged by intratracheal (IT) instillation of saline; results were confirmed by using conventional lung wet-to-dry weight ratios and Evans blue as measures of pulmonary edema. Visualization and quantification of ROS produced in lungs was performed in mice that had been administered a redox-sensitive dye, hydro-Cy7, by IT instillation. We found that inhibition of NADPH oxidase with a Rac-1 inhibitor, NSC23766, resulted in alveolar flooding, which correlated with a decrease in lung ROS production in vivo. Consistent with a role for Nox2 in alveolar fluid balance, Nox2(-/-) mice showed increased retention of air space fluid compared with wild-type controls. Interestingly, fluoroscopic analysis of C57BL/6J lungs IT instilled with LPS showed an acute stimulation of lung fluid clearance and ROS production in vivo that was abrogated by the ROS scavenger tetramethylpiperidine-N-oxyl (TEMPO). Acute application of LPS increased the activity of 20 pS nonselective ENaC channels in rat type 1 cells; the average number of channel and single-channel open probability (NPo) increased from 0.14 ± 0.04 to 0.62 ± 0.23. Application of TEMPO to the same cell-attached recording caused an immediate significant decrease in ENaC NPo to 0.04 ± 0.03. These data demonstrate that, in vivo, ROS has the capacity to stimulate lung fluid clearance by increasing ENaC activity.     

Smurf1 protein negatively regulates interferon-γ signaling through promoting STAT1 protein ubiquitination and degradation

Interferons are important cytokines that mediate antiviral, antiproliferative, antitumor, and immunoregulatory activities. However, uncontrolled IFN signaling may lead to autoimmune diseases. Here we identified Smurf1 as a negative regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation. Smurf1 interacted with STAT1 through the WW domains of Smurf1 and the PY motif in STAT1 and catalyzed K48-linked polyubiquitination of STAT1. Interestingly, the Smurf1-mediated ubiquitination and degradation did not require STAT1 tyrosine and serine phosphorylation. Subsequently, overexpression of Smurf1 attenuated IFN-γ-mediated STAT1 activation and antiviral immune responses, whereas knockdown of Smurf1 enhanced IFN-γ-mediated STAT1 activation, expression of STAT1 target genes, and antiviral immune responses. Furthermore, IFN-γ stimulation led to enhanced expression of Smurf1. Therefore, our results demonstrate that Smurf1 is a negative feedback regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation.     

The TNFα receptor TNFRSF1A and genes encoding the amiloride-sensitive sodium channel ENaC as modulators in cystic fibrosis

The CFTR mutations in cystic fibrosis (CF) lead to ion transport anomalities which predispose to chronic infection and inflammation of CF airways as the major determinants for morbidity and mortality in CF. Discordant clinical phenotypes of siblings with identical CFTR mutations and the large variability of clinical manifestations of patients who are homozygous for the most common mutation F508del suggest that both environment and genes other than CFTR contribute substantially to CF disease. The prime candidates for genetic modifiers in CF are elements of host defence such as the TNFα receptor and of ion transport such as the amiloride-sensitive epithelial sodium channel ENaC, both of which are encoded side by side on 12p13 (TNFRSF1A, SCNN1A) and 16p12 (SCNN1B, SCNN1G). Thirty-seven families with F508del-CFTR homozygous siblings exhibiting extreme clinical phenotypes that had been selected from the 467 pairs of the European CF Twin and Sibling Study were genotyped at 12p13 and 16p12 markers. The ENaC was identified as a modulator of CF by transmission disequilibrium at SCNN1G and association with CF phenotype intrapair discordance at SCNN1B. Family-based and case-control analyses and sequencing of SCNN1A and TNFRSF1A uncovered an association of the TNFRSF1A intron 1 haplotype with disease severity. Carriers of risk haplotypes were underrepresented suggesting a strong impact of both loci on survival. The finding that TNFRSF1A, SCNN1B and SCNN1G are clinically relevant modulators of CF disease supports current concepts that the depletion of airway surface liquid and inadequate host inflammatory responses trigger pulmonary disease in CF.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the γ subunit of the human epithelial sodium channel

Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its γ subunit in a critical region targeted by several proteases. Our aim was to identify cleavage sites in this region that are functionally important for activation of human ENaC by plasmin and chymotrypsin. Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) αβγENaC and αβγ_K189AENaC in Xenopus laevis oocytes. The γ_K189A mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (γ_RKRK178AAAA) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γ_{RKRK178AAAA;K189A}) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189, the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wt or double mutant ENaC. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near-silent channels was confirmed in single-channel recordings from outside-out patches. Mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. This indicates that chymotrypsin preferentially cleaves at FF174. The close proximity of FF174 to the prostasin site may explain why mutating the prostasin site impedes channel activation by chymotrypsin. In conclusion, this study supports the concept that different proteases have distinct preferences for certain cleavage sites in γENaC, which may be relevant for tissue-specific proteolytic ENaC activation.     

Differential N termini in epithelial Na+ channel δ-subunit isoforms modulate channel trafficking to the membrane

The epithelial Na(+) channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na(+) reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, β, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ(1) and δ(2) , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ(1) or δ(2) , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ(1) in Xenopus oocytes and HEK293 cells produces higher current levels than δ(2) . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of β- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ(2)-N terminus. Analysis of δ(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αβγ-channels.     

β-Catenin regulates melanocyte dendricity through the modulation of PKCζ and PKCδ

The Wnt/β-catenin signaling pathway is involved in the melanocyte differentiation and melanoma development. However, the effect of β-catenin for dendrite formation has not been clearly elucidated yet in normal human epidermal melanocytes (NHEM). To investigate the effect of β-catenin, we transduced NHEM with recombinant adenovirus expressing β-catenin. Forced expression of β-catenin led to the dramatic morphological changes of NHEM, including the reduction of dendrite length and enlargement of cell body. Concomitantly with, the protein levels for dendrite formation-related molecules, such as Rac1 and Cdc42, were markedly decreased. In addition, phosphorylation of p38 MAPK was significantly reduced by β-catenin, potentiating its inhibitory role for dendrite formation. Interestingly, overexpression of β-catenin led to the increase of protein kinase C ζ (PKCζ) level, while protein kinase C δ (PKCδ) was decreased by β-catenin, suggesting that those PKCs were β-catenin-downstream modulators in NHMC. When PKCζ was overexpressed, dendrites were shortened, with the reduced protein levels for Rac1 and Cdc42. In contrast, PKCδ overexpression led to the elongation of dendrites, with the increased levels for Rac1 and Cdc42. These results suggest that β-catenin play an inhibitory role for dendrite formation through the modulation of PKCζ and PKCδ.     

Structural organisation of the gene encoding the α-subunit of the human amiloride-sensitive epithelial sodium channel

The human amiloride-sensitive epithelial sodium channel (ENaC) is a member of the degenerin/ENaC family of ion channels and regulates fluid and electrolyte absorption across a number of epithelia, including kidney, colon and lung. Native ENaC has been shown to be a multimer made up of at least three homologous subunits (α, β, γ) and mutations affecting the channel complex have been identified in various human diseases. “Gain of function” mutations in one of the three ENaC subunits have been found to cause pseudoaldosteronism (Liddle’s syndrome) and ENaC “reduction of function” mutations are found in patients affected with the recessive form of pseudohypoaldosteronism (PHA) type 1. In this report, we describe the genomic organisation of the humanαENaC gene. Human αENaC consists of 13 exons spanning 17 kb on chromosome 12p13 and contains at least eight Alu sequences. In addition to the intron/exon boundaries, we have deciphered almost all the intron sequences and 475 bp of the CCAAT-less and TATA-less 5′ flanking region. Received: 23 December 1997 / Accepted: 5 March 1998     

βTrCP regulates BMI1 protein turnover via ubiquitination and degradation

The polycomb group protein BMI1 has been linked to proliferation, senescence, cancer progression and stem cell phenotype. At present, very little is known about its regulation. Here, we report that BMI1 contains a functional recognition motif for the F box protein βTrCP, which regulates ubiquitination and proteasome-mediated degradation of various proteins. We show that overexpression of wild-type βTrCP but not the ΔF mutant of it promotes BMI1 ubiquitination and degradation, and knockdown of βTrCP results in increased expression of BMI1. Furthermore, a mutant of BMI1 with an altered βTrCP recognition motif is much more stable than wild-type BMI1. We also show that wild-type BMI1 but not the mutant BMI1 interacts with βTrCP. Accordingly, compared to wild-type BMI1, mutant protein exhibited increased pro-oncogenic activity. In summary, our findings suggest that βTrCP regulates turnover of BMI1 and its function relevant to oncogenesis, cellular senescence and aging.     

βTrCP regulates BMI1 protein turnover via ubiquitination and degradation

The polycomb group protein BMI1 has been linked to proliferation, senescence, cancer progression and stem cell phenotype. At present, very little is known about its regulation. Here, we report that BMI1 contains a functional recognition motif for the F box protein βTrCP, which regulates ubiquitination and proteasome-mediated degradation of various proteins. We show that overexpression of wild-type βTrCP but not the ΔF mutant of it promotes BMI1 ubiquitination and degradation, and knockdown of βTrCP results in increased expression of BMI1. Furthermore, a mutant of BMI1 with an altered βTrCP recognition motif is much more stable than wild-type BMI1. We also show that wild-type BMI1 but not the mutant BMI1 interacts with βTrCP. Accordingly, compared to wild-type BMI1, mutant protein exhibited increased pro-oncogenic activity. In summary, our findings suggest that βTrCP regulates turnover of BMI1 and its function relevant to oncogenesis, cellular senescence and aging.Key words: BMI1, βTrCP, polycomb group proteins, senescence, breast cancer     

Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling

As a Ca²⁺ binding protein, calreticulin (CRT) has many functions and plays an important role in a variety of tumors. The role of CRT in TGF-β1-induced EMT is unknown. In this study, we demonstrated in vitro that TGF-β1-induced EMT elevated the expression of CRT in A549 lung cancer cells. Subsequently, we confirmed that overexpression CRT had no capacity to induce A549 cells EMT alone, but successfully enhanced TGF-β1-induced-EMT. Furthermore, knockdown of CRT in A549 cells significantly suppressed changes of EMT marks expression induced by TGF-β1. On treatment with TGF-β1, overexpression of CRT could enhance the phosphorylation of both Smad2 and Smad3. Consistently, the knockdown of CRT by siRNA-CRT could inhibit Smad signaling pathway activated by TGF-β1. These results indicated that CRT regulates EMT induced by TGF-β1 through Smad signaling pathway. Finally, TGF-β1-induced-EMT enhanced store-operated Ca²⁺ influx in A549 cells. CRT knockdown was able to abolish the effect of TGF-β1 on thapsigargin (TG) −induced Ca²⁺ release, but had failed to reduce store-operated Ca²⁺ influx. The alteration of intracellular Ca²⁺ concentration by TG or BAPTA-AM was able to regulate EMT induced by TGF-β1 through Smad signaling pathway. Together, these data identify that CRT regulates TGF-β1-induced-EMT through modulating Smad signaling. Furthermore, TGF-β1-induced-EMT is highly calcium-dependent, CRT was partly involved in it.     

Binding modes of μ-conotoxin to the bacterial sodium channel (NaVAb)

Polypeptide toxins isolated from the venom of cone snails, known as μ-conotoxins, block voltage-gated sodium channels by physically occluding the ion-conducting pathway. Using molecular dynamics, we show that one subtype of μ-conotoxins, PIIIA, effectively blocks the bacterial voltage-gated sodium channel Na_VAb, whose crystal structure has recently been elucidated. The spherically shaped toxin, carrying a net charge of +6 e with six basic residues protruding from its surface, is attracted by the negatively charged residues on the vestibular wall and the selectivity filter of the channel. The side chain of each of these six arginine and lysine residues can wedge into the selectivity filter, whereas the side chains of other basic residues form electrostatic complexes with two acidic residues on the channel. We construct the profile of potential of mean force for the unbinding of PIIIA from the channel, and predict that PIIIA blocks the bacterial sodium channel with subnanomolar affinity.