Epithelial Na(+) Channel Subunits Research Articles

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 NH2-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.

Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains

The epithelial Na(+) channel (ENaC) mediates the rate-limiting step in transepithelial Na(+) transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.

The Cystic Fibrosis Transmembrane Conductance Regulator Impedes Proteolytic Stimulation of the Epithelial Na+ Channel

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl(-) secretion in CF airways causes poorly hydrated airway surfaces in CF patients, and this condition is exacerbated by excessive Na(+) absorption. The mechanistic link between missing CFTR and increased Na(+) absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na(+) channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its α- and γ-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and found that ENaC associated with wild-type CFTR was protected from proteolytic cleavage and stimulation of open probability. In contrast, ΔF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full-length to total α-ENaC protein signal was consistently reduced compared with normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down-regulates Na(+) absorption.

Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na(+). A His residue within the extracellular domain of gammaENaC (gammaHis(239)) was found to have a critical role in Na(+) self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na(+) self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na(+) self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na(+) self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the gammaH239F mutant showed that, in the absence of Na(+) self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that gammaHis(239) functions within an extracellular allosteric regulatory subdomain of the gamma subunit that has an important role in conferring the response of the channel to external Na(+).

Effects of central sodium on epithelial sodium channels in rat brain

We evaluated the effects of intracerebroventricular (icv) infusion of Na(+)-rich artificial cerebrospinal fluid (aCSF), with or without the mineralocorticoid receptor (MR) blocker spironolactone, on epithelial Na(+) channel (ENaC) subunits and regulators, such as MR, serum/glucocorticoid-inducible kinase 1, neural precursor cells expressed developmentally downregulated 4-like gene, 11beta-hydroxylase, and aldosterone synthase, in brain regions of Wistar rats. The effects of icv infusion of the amiloride analog benzamil on brain tissue and CSF Na(+) concentration ([Na(+)]) were also assessed. In the choroid plexus and ependyma of the anteroventral third ventricle, ENaC subunits are present in apical and basal membranes. Na(+)-rich aCSF increased beta-ENaC mRNA and immunoreactivity in the choroid plexus and increased alpha- and beta-ENaC immunoreactivities in the ependyma. Na(+)-rich aCSF increased alpha- and beta-ENaC-gold-labeled particles in the microvilli of the choroid plexus and in basolateral membranes of the ependyma. Spironolactone only prevented the increase in beta-ENaC immunoreactivity in the choroid plexus and ependyma. In the supraoptic nucleus, paraventricular nucleus, and subfornical organ, Na(+)-rich aCSF did not affect mRNA expression levels of the studied genes. Benzamil significantly increased CSF [Na(+)] in the control, but not Na(+)-rich, aCSF group. In contrast, benzamil prevented the increase in hypothalamic tissue [Na(+)] by Na(+)-rich aCSF. These results suggest that CSF Na(+) upregulates ENaC expression in the brain epithelia, but not in the neurons of hypothalamic nuclei. ENaC in the choroid plexus and ependyma appear to contribute to regulation of Na(+) homeostasis in the brain.

Epithelial Sodium Channel Gating is Modulated by Cysteine‐Palmitoylation

The epithelial Na+ channel (ENaC) serves as the final site of renal Na+ absorption in the distal nephron, where the fine tuning of its activity regulates extracellular fluid volume and blood pressure. The three homologous subunits of ENaC (alpha, beta and gamma) have a similar topology with two transmembrane domains, a large extracellular loop, and cytoplasmic N‐ and C‐termini. While various lipids are known regulators of ENaC activity, cytoplasmic Cys‐palmitoylation of ENaC has not been previously described. Palmitoylation of individual epitope‐tagged subunits was assessed with fatty acid‐exchange chemistry, whereby palmitate is replaced by biotin and biotinylated subunits were analyzed by immunoblotting. We observed that only the beta and gamma subunits were modified. The function of palmitoylation adjacent to the membrane was studied by expressing ENaC containing a beta C43,557A mutant in Xenopus oocytes. These mutant channels exhibited significantly enhanced Na+ self inhibition and reduced open probability, when compared with wild type ENaC. In contrast, normal membrane trafficking was observed. Our results indicate that palmitoylation of the beta subunit enhances ENaC gating. (Funding from DK065161, DK065521 and AHA).

Renal epithelial Na channel protein expression is reduced in H,K‐ATPase knockout mice

The importance of the epithelial Na+ channel (ENaC) to Na+ reabsorption is highlighted by genetic gain of function mutations in ENaC that lead to severe hypertension (Liddle's Syndrome). Spicer et al (Am J Phys, 2001) demonstrated that colonic ENaC activity was reduced in HKα2−/− mice, suggesting that H+,K+‐ATPases are required for complete ENaC function. Given the importance of functional ENaC to renal Na+ transport, we investigated whether double knockout of H+,K+‐ATPase α subunits in mice (HKα1,2−/−) affected ENaC expression. Real time quantitative PCR was used to evaluate the mRNA expression of α‐, β‐, and γ‐ENaC subunits in cortex and medulla from wild type (WT) and HKα1,2−/− mice. Cortical α‐ and β‐ENaC mRNA expression was slightly decreased (~20–25%) in HKα1,2−/− mice compared to WT (p=0.10 and 0.08, respectively). Importantly, we observed a 45% reduction in medullary α‐ENaC protein expression (n=3, p<0.05) in HKα1,2−/− mice vs WT, as determined by densitometry of immunoblots. In summary, these data suggest that renal H+,K+‐ATPases are needed for appropriate expression of ENaC subunits and are important for renal Na+ adaptation.Studies were funded by NIH RO1‐DK‐049750 to C.S.W and 5T32DK007518‐22 to M.M.G.

The Molecular Chaperones, ERdj3 and ERdj4, Two Hsp40s, Select ENaC for ER Associated Degradation (ERAD)

The epithelial Na+ channel (ENaC) is a vital channel and mutations in ENaC may contribute to hyper‐ and hypotension and cystic fibrosis. ENaC consists of 3 homologous subunits (α, β, γ) that assemble in the ER and traffic to the plasma membrane. ENaC assembly is inefficient with <20% reaching the plasma membrane, and a large fraction of ENaC subunits are targeted for ER‐associated degradation (ERAD). Commonly, selection of ERAD substrates occurs via the chaperones Hsp70s and Hsp40s; however, little is known about the chaperones involved in the ERAD of ENaC. We therefore employed a yeast expression system and identified two Hsp40s, Jem1 and Scj1, that function as ENaC chaperones. The mammalian homologs of these Hsp40s are ERdj3 and ERdj4, and our hypothesis was that ERdj3 and ERdj4 over‐expression would increase ENaC degradation and cause a decrease in ENaC function‐expression. Using a Xenopus oocyte expression system and two‐electrode voltage clamp (TEV), we discovered that ENaC current was reduced >30% by co‐expression of ERdj3 or ERdj4. This reduction in current was proteosome‐mediated and correlated with a decrease in ENaC surface expression. However, these Hsp40s neither affected the rate of ENaC endocytosis nor exocytosis. These data support the hypothesis that Hsp40s are important in the ERAD of ENaC, and that data obtained in our yeast system can be recapitulated in a vertebrate‐expression system. Funded by NIH: GM75061, DK65161, and DK79307

Intact Cytoskeleton Is Required for Small G Protein Dependent Activation of the Epithelial Na+ Channel

BackgroundThe Epithelial Na+ Channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is regulated, in part, by cortical cytoskeleton. Besides, the cytoskeleton is an established target for small G proteins signaling. Here we studied whether ENaC activity is modulated by changes in the state of the cytoskeleton and whether cytoskeletal elements are involved in small G protein mediated increase of ENaC activity.Methods and FindingsFirst, the functional importance of the cytoskeleton was established with whole-cell patch clamp experiments recording ENaC reconstituted in CHO cells. Pretreatment with Cytochalasin D (CytD; 10 µg/ml; 1–2 h) or colchicine (500 µM; 1–3 h) to disassembly F-actin and destroy microtubules, respectively, significantly decreased amiloride sensitive current. However, acute application of CytD induced rapid increase in macroscopic current. Single channel measurements under cell-attached conditions revealed similar observations. CytD rapidly increased ENaC activity in freshly isolated rat collecting duct, polarized epithelial mouse mpkCCDc14 cells and HEK293 cells transiently transfected with ENaC subunits. In contrast, colchicine did not have an acute effect on ENaC activity. Small G proteins RhoA, Rac1 and Rab11a markedly increase ENaC activity. 1–2 h treatment with colchicine or CytD abolished effects of these GTPases. Interestingly, when cells were coexpressed with ENaC and RhoA, short-term treatment with CytD decreased ENaC activity.ConclusionsWe conclude that cytoskeleton is involved in regulation of ENaC and is necessary for small G protein mediated increase of ENaC activity.

Surface expression of sodium channels and transporters in rat kidney: effects of dietary sodium

The abundance of Na transport proteins in the luminal membrane of the rat kidney was assessed using in situ biotinylation and immunoblotting. When animals were fed an Na-deficient diet for 1 wk, the amounts of epithelial Na channel (ENaC) beta-subunit (beta-ENaC) and gamma-subunit (gamma-ENaC) and Na-Cl cotransporter (NCC) protein in the surface fraction increased relative to controls by 1.9-, 3.5-, and 1.5-fold, respectively. The amounts of the luminal Na/H exchanger (NHE3) and the luminal Na-K-2Cl cotransporter (NKCC2) did not change significantly. The increases in ENaC subunits were mimicked by administration of aldosterone for 1 wk, but the increase in NCC was not. When the animals were fed a high-Na (5% NaCl) diet for 1 wk, the surface expression of beta-ENaC increased by 50%, whereas that of the other membrane proteins did not change, relative to controls. The biochemical parameter most strongly affected by dietary Na was the abundance of the 65-kDa cleaved form of gamma-ENaC at the surface. This increased by 8.5-fold with Na depletion and decreased by 40% with Na loading. The overall 14-fold change reflected regulation of the total abundance of the subunit as well as the fraction of the subunit protein in the cleaved form. We conclude that cleavage of gamma-ENaC and its expression at the apical surface play a major role in the regulation of renal Na reabsorption.

Intrinsic Voltage Dependence of the Epithelial Na+ Channel Is Masked by a Conserved Transmembrane Domain Tryptophan

Tryptophan residues critical to function are frequently located at the lipid-water interface of transmembrane domains. All members of the epithelial Na+ channel (ENaC)/Degenerin (Deg) channel superfamily contain an absolutely conserved Trp at the base of their first transmembrane domain. Here, we test the importance of this conserved Trp to ENaC/Deg function. Targeted substitution of this Trp in mouse ENaC and rat ASIC subunits decrease channel activity. Differential substitution with distinct amino acids in alpha-mENaC shows that it is loss of this critical Trp rather than introduction of residues having novel properties that changes channel activity. Surprisingly, Trp substitution unmasks voltage sensitivity. Mutant ENaC has increased steady-state activity at hyperpolarizing compared with depolarizing potentials associated with transient activation and deactivation times, respectively. The times of activation and deactivation change 1 ms/mV in a linear manner with rising and decreasing slopes, respectively. Increases in macroscopic currents at hyperpolarizing potentials results from a voltage-dependent increase in open probability. Voltage sensitivity is not influenced by divalent cations; however, it is Na+-dependent with a 63-mV decrease in voltage required to reach half-maximal activity per log increase in [Na+]. Mutant channels are particularly sensitive to intracellular [Na+] for removing this sodium abolishes voltage dependence. We conclude that the conserved Trp at the base of TM1 in ENaC/Deg channels protects against voltage by masking an inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.

Effects of dietary Na+ deprivation on epithelial Na+ channel (ENaC), BDNF, and TrkB mRNA expression in the rat tongue

BackgroundIn rodents, dietary Na+ deprivation reduces gustatory responses of primary taste fibers and central taste neurons to lingual Na+ stimulation. However, in the rat taste bud cells Na+ deprivation increases the number of amiloride sensitive epithelial Na+ channels (ENaC), which are considered as the "receptor" of the Na+ component of salt taste. To explore the mechanisms, the expression of the three ENaC subunits (α, β and γ) in taste buds were observed from rats fed with diets containing either 0.03% (Na+ deprivation) or 1% (control) NaCl for 15 days, by using in situ hybridization and real-time quantitative RT-PCR (qRT-PCR). Since BDNF/TrkB signaling is involved in the neural innervation of taste buds, the effects of Na+ deprivation on BDNF and its receptor TrkB expression in the rat taste buds were also examined.ResultsIn situ hybridization analysis showed that all three ENaC subunit mRNAs were found in the rat fungiform taste buds and lingual epithelia, but in the vallate and foliate taste buds, only α ENaC mRNA was easily detected, while β and γ ENaC mRNAs were much less than those in the fungiform taste buds. Between control and low Na+ fed animals, the numbers of taste bud cells expressing α, β and γ ENaC subunits were not significantly different in the fungiform, vallate and foliate taste buds, respectively. Similarly, qRT-PCR also indicated that Na+ deprivation had no effect on any ENaC subunit expression in the three types of taste buds. However, Na+ deprivation reduced BDNF mRNA expression by 50% in the fungiform taste buds, but not in the vallate and foliate taste buds. The expression of TrkB was not different between control and Na+ deprived rats, irrespective of the taste papillae type.ConclusionThe findings demonstrate that dietary Na+ deprivation does not change ENaC mRNA expression in rat taste buds, but reduces BDNF mRNA expression in the fungiform taste buds. Given the roles of BDNF in survival of cells and target innervation, our results suggest that dietary Na+ deprivation might lead to a loss of gustatory innervation in the mouse fungiform taste buds.

Novel Determinants of Epithelial Sodium Channel Gating within Extracellular Thumb Domains

Activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na(+). We searched for determinants of Na(+) self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at gammaMet(438) altered the Na(+) self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na(+) self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na(+) self-inhibition. Mutations at the homologous site in the alpha subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na(+) self-inhibition. Mutations at the homologous betaAla(422) resulted in minimal or no change in Na(+) self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing alphaG481Mbetagamma, alphabetagammaM438V, and alphaG481M/betagammaM438V, respectively. This pair of residues map to thealpha5 helix in the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both alphaG481Cbetagamma and alphabetagammaM438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that alphaGly(481) and gammaMet(438) are functional determinants of Na(+) self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.

Functional Regulation of the Epithelial Na+ Channel by IκB Kinase-β Occurs via Phosphorylation of the Ubiquitin Ligase Nedd4-2

We have previously shown that IkappaB kinase-beta (IKKbeta) interacts with the epithelial Na+ channel (ENaC) beta-subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKKbeta-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells, as RNA interference-mediated knockdown of endogenous IKKbeta in these cells by approximately 50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKKbeta binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKKbeta in vitro. Because IKKbeta stimulation of ENaC activity occurs through enhanced channel surface expression and the ubiquitin-protein ligase Nedd4-2 has emerged as a central locus for ENaC regulation at the plasma membrane, we tested the role of Nedd4-2 in this regulation. IKKbeta-dependent phosphorylation of Xenopus Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity. 32P labeling studies utilizing wild-type or mutant forms of Xenopus Nedd4-2 demonstrated that Ser-444, a key SGK1 and protein kinase A-phosphorylated residue, is also an important IKKbeta phosphorylation target. ENaC stimulation by IKKbeta was preserved in oocytes expressing wild-type Nedd4-2 but blocked in oocytes expressing either a dominant-negative (C938S) or phospho-deficient (S444A) Nedd4-2 mutant, suggesting that Nedd4-2 function and phosphorylation by IKKbeta are required for IKKbeta regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKKbeta-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.

Inactivation of Channel-Activating Protease 1 in mouse distal lung impairs ENaC-mediated lung fluid clearance

Transepithelial sodium (Na + ) transport via amiloride-sensitive epithelial Na + channels (ENaC) expressed in the apical membrane of alveolar epithelial cells provides the driving force for removal of fluid from the alveolar space. In vitro , the membrane-anchored channel-activating serine protease 1 (CAP1) strongly activates ENaC channels at the cell surface by increasing their open probability through proteolytic cleavage of γ-ENaC subunit, but the physiological relevance of this process in vivo is unknown. We investigated whether inactivation of CAP1 gene in mouse distal lung epithelium would affect in vivo transepithelial alveolar sodium transport and alveolar fluid clearance. Mice lacking CAP1 in the alveolar epithelium were generated by CreLoxP-recombination inducible in utero by doxycycline (Tet-ON system). Specificity and efficiency of Cre-recombination as well as expression of ENaC subunits were examined by PCR, real-time RT-PCR and Western-blotting in freshly isolated alveolar epithelial cells. Alveolar fluid clearance was measured in vivo in an in situ lung model under basal and β 2 -agonist-stimulated conditions using radiolabeled albumin as a tracer of alveolar fluid volume. Cre-recombination was restricted to alveolar epithelial cells by PCR and led to a 95% decrease in CAP1 mRNA expression by quantitative RT-PCR, without affecting ENaC mRNA and protein levels. CAP1-deficient mice had normal growth and survival rate, and normal lung histology. Basal alveolar fluid clearance was reduced by 30% in CAP1-deficient mice as compared with control littermates (p<0.01), due to a 50%-decrease in amiloride-sensitive clearance reflecting ENaC activity (p<0.001). β 2 -agonists failed to stimulate alveolar fluid clearance in CAP1-deficient mice. Basal alveolar fluid clearance and stimulation by β 2 -agonists were fully restored by addition in the alveolar instillate of human neutrophil elastase, a serine protease that reproduces the effect of CAP1 on ENaC. Conditional inactivation of CAP1 in mouse alveolar epithelium impairs ENaC-mediated alveolar fluid clearance, and abolishes the response to β 2 -agonists. These data provide in vivo evidence that epithelium-derived CAP1 is a potent activator of ENaC in mouse distal lung. Funded by Inserm, SPLF, Fondation du Legs Poix and Swiss National Foundation

Expression and Regulation of Epithelial Na+ Channels by Nucleotides in Pleural Mesothelial Cells

Pleural effusions are commonly clinical disorders, resulting from the imbalance between pleural fluid turnover and reabsorption. The mechanisms underlying pleural fluid clearance across the mesothelium remain to be elucidated. We hypothesized that epithelial Na(+) channel (ENaC) is expressed and forms the molecular basis of the amiloride-sensitive resistance in human mesothelial cells. Our RT-PCR results showed that three ENaC subunits, namely, alpha, beta, gamma, and two delta ENaC subunits, are expressed in human primary pleural mesothelial cells, a human mesothelioma cell line (M9K), and mouse pleural tissue. In addition, Western blotting and immunofluorescence microscopy studies revealed that alpha, beta, gamma, and delta ENaC subunits are expressed in primary human mesothelial cells and M9K cells at the protein level. An amiloride-inhibitable short-circuit current was detected in M9K monolayers and mouse pleural tissues when mounted in Ussing chambers. Whole-cell patch clamp recordings showed an ENaC-like channel with an amiloride concentration producing 50% inhibition of 12 microM in M9K cells. This cation channel has a high affinity for extracellular Na+ ions (K(m): 53 mM). The ion selectivity of this channel to cations follows the same order as ENaC: Li+ > Na+ > K+. The unitary Li(+) conductance was 15 pS in on-cell patches. Four ENaC subunits form a functional Na+ channel when coinjected into Xenopus oocytes. Furthermore, we found that both forskolin and cGMP increased the short-circuit currents in mouse pleural tissues. Taken together, our data demonstrate that the ENaC channels are biochemically and functionally expressed in human pleural mesothelial cells, and can be up-regulated by cyclic AMP and cyclic GMP.

AICAR activates AMPK and alters PIP2 association with the epithelial sodium channel ENaC to inhibit Na+ transport in H441 lung epithelial cells

Changes in amiloride-sensitive epithelial Na(+) channel (ENaC) activity (NP(o)) in the lung lead to pathologies associated with dysregulation of lung fluid balance. UTP activation of purinergic receptors and hydrolysis of PIP(2) via activation of phospholipase C (PLC) or AICAR activation of AMP-activated protein kinase (AMPK) inhibited amiloride-sensitive Na(+) transport across human H441 epithelial cell monolayers. Neither treatment altered alpha, beta or gamma ENaC subunit abundance (N) in the apical membrane indicating that the mechanism of inhibition was via a change in channel open state probability (P(o)). We found that UTP depleted PIP(2) abundance in the apical membrane whilst activation of AMPK prevented the binding of beta and gamma ENaC subunits to PIP(2.) The association of PIP(2) with the ENaC subunits is required to maintain channel activity via P(o). Thus, these data show for the first time that AICAR activation of AMPK inhibits Na(+) transport via a mechanism that perturbs the PIP(2)-ENaC channel interaction to alter P(o). In addition, we show that dissociation of PIP(2) from ENaC together with activation of AMPK further reduced Na(+) transport by a secondary effect that correlated with ENaC subunit internalization. Thus, when PIP(2)-ENaC subunit interactions were compromised, ENaC protein retrieval was initiated, indicating that AMPK can modulate ENaC P(o) and N.

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2

Nedd4-2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na+ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na+ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaCl-sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder.

Surface Expression of Epithelial Na Channel Protein in Rat Kidney

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface β and γENaC subunits were assayed by immunoblot. Surface αENaC was assessed by membrane fractionation. Most of the γENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.

GENDER DIFFERENCES IN RISK FACTOR CONTROL OF PATIENTS ON LIPID LOWERING DRUGS, A NATIONAL PRIMARY HEALTH CARE STUDY

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is highly expressed in the kidney, where its function remains unclear. In vitro data suggested that PCSK9 could impair the trafficking of the epithelial Na channel (ENaC). Here, we aimed at determining the consequences of PCSK9-deficiency on blood pressure, sodium balance and ENaC function in vivo in mice.Blood pressure was measured using non-invasive tail-cuff system or radiotelemetry under basal conditions in male and female PCSK9+/+ and PCSK9−/− mice, as well as in models of hypertension: l-NAME (2 mg/kg/day), angiotensin II (1 mg/kg/day) and deoxycorticosterone acetate (DOCA)-salt in male mice only. Plasma and urine electrolytes (Na+, K+, Cl−) were collected under basal conditions, after DOCA-salt and amiloride treatment. Renal expression of ENaC subunits was assessed by western blotting.PCSK9-deficiency did not alter both basal blood pressure and its increase in salt-insensitive (l-NAME) and salt-sensitive (Ang-II and DOCA-salt) hypertension models. Plasma PCSK9 concentrations were increased by 2.8 fold in DOCA-salt-induced hypertension. The relative expression of the cleaved, active, 30-kDa αENaC subunit was significantly increased by 32% in kidneys of PCSK9−/− mice under basal, but not under high-Na+ diet or DOCA-salt conditions. Amiloride increased urinary Na+ excretion to similar level in both genotypes, indicating that ENaC activity was not affected by PCSK9-deficiency.Despite an increase of cleaved αENaC under basal condition, PCSK9−/− mice display normal sodium balance and blood pressure regulation. Altogether, these data are reassuring regarding the development of PCSK9 inhibitors in hypercholesterolemia.