Calreticulin positively regulates the expression and function of epithelial sodium channel

Epithelial sodium channel (ENaC) is a heteromultimeric Na⁺ channel at the apical membrane in the kidney, colon, and lung. Because ENaC plays a crucial role in regulating Na⁺ absorption and extracellular fluid volume, its dysregulation causes severe phenotypes including hypertension, hypokalemia, and airway obstruction. Despite the importance of ENaC, its protein quality control mechanism remains less established. Here we firstly show the role of calreticulin (CRT), a lectin-like molecular chaperone in the endoplasmic reticulum (ER), on the regulation of ENaC. Overexpression and knockdown analyses clearly indicated that CRT positively affects the expression of each ENaC subunit (α, β and γ). CRT overexpression also up-regulated the cell surface expression of α-, β- and γ-ENaC. Moreover, we found that CRT directly interacts with each ENaC subunit. Although CRT knockdown did not affect the de novo synthesis of ENaC subunits, CRT overexpression decreased α-, β- and γ-ENaC expression in the detergent (RIPA)-insoluble fraction, suggesting that CRT enhanced the solubility of ENaC subunits. Consistent with the increased intracellular and cell surface expression of ENaC subunits, increased channel activity of ENaC was also observed upon overexpression of CRT. Our study thus identifies CRT as an ER chaperone that regulates ENaC expression and function.     

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.     

Oxygen regulation of the epithelial Na channel in the collecting duct

The PO(2) within the kidney changes dramatically from cortex to medulla. The present experiments examined the effect of changing PO(2) on epithelial Na channel (ENaC)-mediated Na transport in the collecting duct using the mpkCCD-c14 cell line. Decreasing ambient O(2) concentration from 20 to 8% decreased ENaC activity by 40%; increasing O(2) content to 40% increased ENaC activity by 50%. The O(2) effect required several hours to develop and was not mimicked by the acid pH that developed in monolayers incubated in low-O(2) medium. Corticosteroids increased ENaC activity at each O(2) concentration; there was no interaction. The pathways for O(2) and steroid regulation of ENaC are different since O(2) did not substantially affect Sgk1, α-ENaC, Gilz, or Usp2-45 mRNA levels, genes involved in steroid-mediated ENaC regulation. The regulation of ENaC activity by these levels of O(2) appears not to be mediated by changes in hypoxia-inducible factor-1α or -2α activity or a change in AMP kinase activity. Changes in O(2) concentration had minimal effect on α- or γ-ENaC mRNA and protein levels; there were moderate effects on β-ENaC levels. However, 40% O(2) induced substantially greater total β- and γ-ENaC on the apical surface compared with 8% O(2); both subunits demonstrated a greater increase in the mature forms. The α-ENaC subunit was difficult to detect on the apical surface, perhaps because our antibodies do not recognize the major mature form. These results identify a mechanism of ENaC regulation that may be important in different regions of the kidney and in responses to changes in dietary NaCl.     

Acute cholesterol-induced anti-natriuretic effects: role of epithelial Na+ channel activity, protein levels, and processing

The epithelial Na(+) channel (ENaC) is modulated by membrane lipid composition. However, the effect of an in vivo change of membrane composition is unknown. We examined the effect of a 70-day enhanced cholesterol diet (ECD) on ENaC and renal Na(+) handling. Rats were fed a standard chow or one supplemented with 1% cholesterol and 0.5% cholic acid (ECD). ECD animals exhibited marked anti-diuresis and anti-natriuresis (40 and 47%), which peaked at 1-3 weeks. Secondary compensation returned urine output and urinary Na(+) excretion to control levels by week 10. During these initial changes, there were no accompanying effects on systolic blood pressure, serum creatinine, or urinary creatinine excretion, indicating that the these effects of ECD preceded those which modify renal filtration and blood pressure. The effects of ECD on ENaC were evaluated by measuring the relative protein content of α, β, and γ subunits. α and γ blots were further examined for subunit cleavage (a process that activates ENaC). No significant changes were observed in α and β levels throughout the study. However, levels of cleaved γ were elevated, suggesting that ENaC was activated. The changes of γ persisted at week 10 and were accompanied by additional subunit fragments, indicating potential changes of γ-cleaving proteases. Enhanced protease activity, and specifically that which could act on the second identified cleavage site in γ, was verified in a newly developed urinary protease assay. These results predict enhanced ENaC activity, an effect that was confirmed in patch clamp experiments of principal cells of split open collecting ducts, where ENaC open probability was increased by 40% in the ECD group. These data demonstrate a complex series of events and a new regulatory paradigm that is initiated by ECD prior to the onset of elevated blood pressure. These events lead to changes of renal Na(+) handling, which occur in part by effects on extracellular γ-ENaC cleavage.     

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.     

K(+) channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

Active Na(+) absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and K(ATP) K(+) channel activities exerts sustained control in Na(+) transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K(+) channels, and 2) to determine the physiological impact of K(+) channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and K(ATP) channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24h) increased α-ENaC expression, similarly to K(ATP) activation by pinacidil. Conversely, pharmacological KvLQT1 and K(ATP) inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K(+) channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and K(ATP) activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K(+) channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K(+) channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.     

Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric alpha gamma beta channel architecture

The extracellular domain of the epithelial Na(+) channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl(-) inhibits ENaC activity. To identify sites involved in Cl(-) inhibition, we mutated residues in the extracellular domain of α-, β-, and γENaC that are homologous to the Cl(-) binding site in acid-sensing ion channel 1a and tested the effect of Cl(-) on the activity of ENaC expressed in Xenopus oocytes. We identified two Cl(-) inhibitory sites in ENaC. One is formed by residues in the thumb domain of αENaC and the palm domain of βENaC. Mutation of residues at this interface decreased Cl(-) inhibition and decreased Na(+) self-inhibition. The second site is formed by residues at the interface of the thumb domain of βENaC and the palm domain of γENaC. Mutation of these residues also decreased Cl(-) inhibition yet had no effect on Na(+) self-inhibition. In contrast, mutations in the thumb domain of γENaC and palm of αENaC had little or no effect on Cl(-) inhibition or Na(+) self-inhibition. The data demonstrate that Cl(-) inhibits ENaC activity by two distinct Na(+)-dependent and Na(+)-independent mechanisms that correspond to the two functional Cl(-) inhibitory sites. Furthermore, based on the effects of mutagenesis on Cl(-) inhibition, the additive nature of mutations, and on differences in the mechanisms of Cl(-) inhibition, the data support a model in which ENaC subunits assemble in an αγβ orientation (listed clockwise when viewed from the top).     

Epithelial Na⁺ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei

The epithelial Na? channels (ENaCs) are present in kidney and contribute to Na? and water homeostasis. All three ENaC subunits (α, β, and γ) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the α-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for α-ENaC. In contrast, immunoreactivity to β- and γ-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT.     

Second transmembrane domain modulates epithelial sodium channel gating in response to shear stress

Na(+) absorption and K(+) secretion in the distal segments of the nephron are modulated by the tubular flow rate. Epithelial Na(+) channels (ENaC), composed of α-, β-, and γ-subunits respond to laminar shear stress (LSS) with an increase in open probability. Higher vertebrates express a δ-ENaC subunit that is functionally related to the α-subunit, while sharing only 35% of sequence identity. We investigated the response of δβγ channels to LSS. Both the time course and magnitude of activation of δβγ channels by LSS were remarkably different from those of αβγ channels. ENaC subunits have similar topology, with an extracellular region connected by two transmembrane domains with intracellular N and C termini. To identify the specific domains that are responsible for the differences in the response to flow of αβγ and δβγ channels, we generated a series of α-δ chimeras and site-specific α-subunit mutants and examined parameters of activation by LSS. We found that specific sites in the region encompassing and just preceding the second transmembrane domain were responsible for the differences in the magnitude and time course of channel activation by LSS.     

K+ channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

Active Na⁺ absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and K_ATP K⁺ channel activities exerts sustained control in Na⁺ transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K⁺ channels, and 2) to determine the physiological impact of K⁺ channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and K_ATP channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24 h) increased α-ENaC expression, similarly to K_ATP activation by pinacidil. Conversely, pharmacological KvLQT1 and K_ATP inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K⁺ channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and K_ATP activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K⁺ channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K⁺ channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.     

Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis

Epithelial sodium channels (ENaC) are composed of three homologous subunits whose extracellular domains (ECD) form a funnel that directs ions from the lumen into the pore of ENaC. To examine the roles of conserved charged residues (Asp, Glu, Arg, and Lys) on ECD, we mutated 16 residues in human α-ENaC to alanine. The modified cRNAs were expressed in Xenopus laevis oocytes together with wild-type β- and γ-ENaC. The effect of each mutation was examined on three parameters: amiloride-sensitive Na(+) conductance (assayed by the two-electrode voltage-clamp method), Na(+)-dependent self-inhibition of ENaC, and oocyte cell surface expression of ENaC (quantitated by confocal microscopy of yellow fluorescent protein linked to γ-ENaC). Mutation of 13 of 16 residues reduced the ENaC Na(+) conductance (to 40-80% of WT). Mutation of only six residues showed a significant effect on the Na(+) self-inhibition time constant (τ). All 16 mutants showed a strong correlation between ENaC activity and oocyte surface expression (r = 0.62). Exclusion of four mutants showing the greatest effect on self-inhibition kinetics (Glu250 and Arg350 with τ = ~30% of WT, and Asp393 and Glu530 with τ = ~170% of WT) increased the correlation to r = 0.87. In the ASIC1 homotrimeric model, the homologs of α-ENaC Asp400 and Asp446 are exposed on the protein surface far from the other two chains. The mutations of these two residues showed the strongest effect on cell surface expression but had no effect on self-inhibition. Control mutations to a homologous charged residue (e.g., Asp to Glu) did not significantly affect ENaC activity. Changes in the two parameters, Na(+) self-inhibition and oocyte surface expression level, accounted for the magnitude of reduction in ENaC activity as a result of the mutation to Ala. These results establish that while some conserved charged residues are part of the structure responsible for Na(+) self-inhibition, most are essential for transport to the oocyte cell surface.     

COMMD1 regulates the delta epithelial sodium channel (δENaC) through trafficking and ubiquitination

The delta subunit of the epithelial sodium channel (δENaC) is a member of the ENaC/degenerin family of ion channels. δENaC is distinct from the related α-, β- and γENaC subunits, known for their role in sodium homeostasis and blood pressure control, as δENaC is expressed in brain neurons and activated by external protons. COMMD1 (copper metabolism Murr1 domain 1) was previously found to associate with and downregulate δENaC activity. Here, we show that COMMD1 interacts with δENaC through its COMM domain. Co-expression of δENaC with COMMD1 significantly reduced δENaC surface expression, and led to an increase in δENaC ubiquitination. Immunocytochemical and confocal microscopy studies show that COMMD1 promoted localization of δENaC to the early/recycling endosomal pool where the two proteins were localized together. These results suggest that COMMD1 downregulates δENaC activity by reducing δENaC surface expression through promoting internalization of surface δENaC to an intracellular recycling pool, possibly via enhanced ubiquitination.     

External Cu2+ inhibits human epithelial Na+ channels by binding at a subunit interface of extracellular domains

Epithelial Na(+) channels (ENaCs) play an essential role in the regulation of body fluid homeostasis. Certain transition metals activate or inhibit the activity of ENaCs. In this study, we examined the effect of extracellular Cu(2+) on human ENaC expressed in Xenopus oocytes and investigated the structural basis for its effects. External Cu(2+) inhibited human αβγ ENaC with an estimated IC(50) of 0.3 μM. The slow time course and a lack of change in the current-voltage relationship were consistent with an allosteric (non pore-plugging) inhibition of human ENaC by Cu(2+). Experiments with mixed human and mouse ENaC subunits suggested that both the α and β subunits were primarily responsible for the inhibitory effect of Cu(2+) on human ENaC. Lowering bath solution pH diminished the inhibition by Cu(2+). Mutations of two α, two β, and two γ His residues within extracellular domains significantly reduced the inhibition of human ENaC by Cu(2+). We identified a pair of residues as potential Cu(2+)-binding sites at the subunit interface between thumb subdomain of αhENaC and palm subdomain of βhENaC, suggesting a counterclockwise arrangement of α, β, and γ ENaC subunits in a trimeric channel complex when viewed from above. We conclude that extracellular Cu(2+) is a potent inhibitor of human ENaC and binds to multiple sites within the extracellular domains including a subunit interface.     

Co-expression of γ2 Subunits Hinders Processing of N-Linked Glycans Attached to the N104 Glycosylation Sites of GABAA Receptor β2 Subunits

GABA_A receptors, the major mediators of fast inhibitory neuronal transmission, are heteropentameric glycoproteins assembled from a panel of subunits, usually including α and β subunits with or without a γ2 subunit. The α1β2γ2 receptor is the most abundant GABA_A receptor in brain. Co-expression of γ2 with α1 and β2 subunits causes conformational changes, increases GABA_A receptor channel conductance, and prolongs channel open times. We reported previously that glycosylation of the three β2 subunit glycosylation sites, N32, N104 and N173, was important for α1β2 receptor channel gating. Here, we examined the hypothesis that steric effects or conformational changes caused by γ2 subunit co-expression alter the glycosylation of partnering β2 subunits. We found that co-expression of γ2 subunits hindered processing of β2 subunit N104 N-glycans in HEK293T cells. This γ2 subunit-dependent effect was strong enough that a decrease of γ2 subunit expression in heterozygous GABRG2 knockout (γ2^+/?) mice led to appreciable changes in the endoglycosidase H digestion pattern of neuronal β2 subunits. Interestingly, as measured by flow cytometry, γ2 subunit surface levels were decreased by mutating each of the β2 subunit glycosylation sites. The β2 subunit mutation N104Q also decreased GABA potency to evoke macroscopic currents and reduced conductance, mean open time and open probability of single channel currents. Collectively, our data suggested that γ2 subunits interacted with β2 subunit N-glycans and/or subdomains containing the glycosylation sites, and that γ2 subunit co-expression-dependent alterations in the processing of the β2 subunit N104 N-glycans were involved in altering the function of surface GABA_A receptors.     

The epithelial sodium channel δ-subunit: new notes for an old song

Amiloride-sensitive epithelial Na(+) channels (ENaCs) can be formed by different combinations of four homologous subunits, named α, β, γ, and δ. In addition to providing an apical entry pathway for transepithelial Na(+) reabsorption in tight epithelia such as the kidney distal tubule and collecting duct, ENaCs are also expressed in nonepithelial cells, where they may play different functional roles. The δ-subunit of ENaC was originally identified in humans and is able to form amiloride-sensitive Na(+) channels alone or in combination with β and γ, generally resembling the canonical kidney ENaC formed by α, β, and γ. However, δ differs from α in its tissue distribution and channel properties. Despite the low sequence conservation between α and δ (37% identity), their similar functional characteristics provide an excellent model for exploring structural correlates of specific ENaC biophysical and pharmacological properties. Moreover, the study of cellular mechanisms modulating the activity of different ENaC subunit combinations provides an opportunity to gain insight into the regulation of the channel. In this review, we examine the evolution of ENaC genes, channel subunit composition, the distinct functional and pharmacological features that δ confers to ENaC, and how this can be exploited to better understand this ion channel. Finally, we briefly consider possible functional roles of the ENaC δ-subunit.     

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.     

Hypotonic stress upregulates β- and γ-ENaC expression through suppression of ERK by inducing MKP-1

We investigated a physiological role for ERK, a member of the MAPK family, in the hypotonic stimulation of epithelial Na(+) channel (ENaC)-mediated Na(+) reabsorption in renal epithelial A6 cells. We show that hypotonic stress causes a major dephosphorylation of ERK following a rapid transient phosphorylation. PD98059 (a MEK inhibitor) increases dephosphorylated ERK and enhances the hypotonic-stress-stimulated Na(+) reabsorption. ERK dephosphorylation is mediated by MAPK phosphatase (MKP). Hypotonic stress activates p38, which in turn induces MKP-1 and to a lesser extent MKP-3 mRNA expression. Inhibition of p38 suppresses MKP-1 induction, preventing hypotonic stress from dephosphorylating ERK. Inhibition of MKP-1 and -3 by the inhibitor NSC95397 also suppresses the hypotonicity-induced dephosphorylation of ERK. NSC95397 reduces both β- and γ-ENaC mRNA expression and ENaC-mediated Na(+) reabsorption stimulated by hypotonic stress. In contrast, pretreatment with PD98059 significantly enhances mRNA and protein expression of β- and γ-ENaC even under isotonic conditions. However, PD98059 only stimulates Na(+) reabsorption in response to hypotonic stress, suggesting that ERK inactivation by itself (i.e., under isotonic conditions) is not sufficient to stimulate Na(+) reabsorption, even though ERK inactivation enhances β- and γ-ENaC expression. Based on these results, we conclude that hypotonic stress stimulates Na(+) reabsorption through at least two signaling pathways: 1) induction of MKP-1 that suppresses ERK activity and induces β- and γ-ENaC expression, and 2) promotion of translocation of the newly synthesized ENaC to the apical membrane.     

α-ENaC in bullfrog embryo: expression in cement gland, gills and skin

The epithelial sodium channel (ENaC) is involved in Na⁺ responses such as Na⁺ absorption and salt taste. The alpha ENaC subunit (α-ENaC) is expressed in the skin of both the adult and larval (tadpole) bullfrog. α-ENaC expression in the developing bullfrog embryo has not been previously investigated. In this study, the expression of α-ENaC at various stages (Sts.) of bullfrog embryonic development is assessed by western blot and immunofluorescence analysis. Bullfrog α-ENaC (α-fENaC) protein was detected by western blot in embryos at Sts. (Gosner/Shumway) 19, 21 and 25. Immunofluorescence studies indicate that α-fENaC was localized to the embryonic cement glands at St. 18 (muscular response), St. 19 (heart beat) and St. 21 (mouth open and/or cornea transparent), to the external gills at St. 21 and to the outermost cell-layer of the skin at St. 25 (operculum complete). The function(s) of ENaC in these embryonic structures remain to be elucidated.     

Epithelial sodium channel enhanced osteogenesis via cGMP/PKGII/ENaC signaling in rat osteoblast

The amiloride-sensitive epithelial sodium channel (ENaC) is a major contributor to intracellular sodium homeostasis. In addition to epithelial cells, osteoblasts (Obs) express functional ENaCs. Moreover, a correlation between bone Na content and bone disease has been reported, suggesting that ENaC-mediated Na⁺ regulation may influence osteogenesis. Obs were isolated and cultured by enzyme digestion. Cell proliferation and differentiation were evaluated by WST-8 assay kit and AKP assay kit respectively. PKGII expression was silenced by siRNA. The mRNA expression was investigated by semi-quantitative PCR and the protein expression was determined by Western-blot. The cell-permeable cGMP analog 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) increased α-ENaC channel expression in primary rat Obs as indicated by RT-PCR. In addition, 8-pCPT-cGMP stimulation enhanced expression of the mRNA encoding cGMP-dependent protein kinases II (PKGII). The cGMP analog also promoted osteoblast proliferation, differentiation and induced the expression of several osteogenic genes, including core binding factor al, osteocalcin, alkaline phosphatase, collagen type I, and osteopontin. Furthermore, the expression of α-ENaC, the main functional subunit of ENaC, was reduced when a small interfering RNA specific for PKGII was introduced into Obs. Treatment with 8-pCPT-cGMP in cells transfected with the siRNA for PKGII partially reversed downregulated α-ENaC mRNA expression. Our results suggest that 8-pCPT-cGMP stimulates proliferation, differentiation, and osteogenic gene expression in Obs through cGMP/PKGII-dependent regulation of ENaC channel expression. The cGMP/PKGII signaling pathway is a potential target for pharmaceutical interventions to treat metabolic bone diseases.     

Interaction of ASIC1 and ENaC subunits in human glioma cells and rat astrocytes

Glioblastoma multiforme (GBM) is the most common and aggressive of the primary brain tumors. These tumors express multiple members of the epithelial sodium channel (ENaC)/degenerin (Deg) family and are associated with a basally active amiloride-sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test the hypothesis that ASIC1 interacts with αENaC and γENaC at the cellular level, we have used total internal reflection fluorescence microscopy (TIRFM) in live rat astrocytes transiently cotransfected with cDNAs for ASIC1-DsRed plus αENaC-yellow fluorescent protein (YFP) or ASIC1-DsRed plus γENaC-YFP. TIRFM images show colocalization of ASIC1 with both αENaC and γENaC. Furthermore, using TIRFM in stably transfected D54-MG cells, we also found that ASIC1 and αENaC both localize to a submembrane region following exposure to pH 6.0, similar to the acidic conditions found in the core of a glioblastoma lesion. Using high-resolution clear native gel electrophoresis, we found that ASIC1 forms a complex with ENaC subunits which migrates at ≈480 kDa in D54-MG glioma cells. These data suggest that different ENaC/Deg subunits interact and could combine to form a hybrid channel that likely underlies the amiloride-sensitive current seen in human glioma cells.