Proteases, cystic fibrosis and the epithelial sodium channel (ENaC)

Proteases perform a diverse array of biological functions. From simple peptide digestion for nutrient absorption to complex signaling cascades, proteases are found in organisms from prokaryotes to humans. In the human airway, proteases are associated with the regulation of the airway surface liquid layer, tissue remodeling, host defense and pathogenic infection and inflammation. A number of proteases are released in the airways under both physiological and pathophysiological states by both the host and invading pathogens. In airway diseases such as cystic fibrosis, proteases have been shown to be associated with increased morbidity and airway disease progression. In this review, we focus on the regulation of proteases and discuss specifically those proteases found in human airways. Attention then shifts to the epithelial sodium channel (ENaC), which is regulated by proteolytic cleavage and that is considered to be an important component of cystic fibrosis disease. Finally, we discuss bacterial proteases, in particular, those of the most prevalent bacterial pathogen found in cystic fibrosis, Pseudomonas aeruginosa.     

Regulation of the epithelial sodium channel (ENaC) by membrane trafficking

The epithelial Na⁺ channel (ENaC) is a major regulator of salt and water reabsorption in a number of epithelial tissues. Abnormalities in ENaC function have been directly linked to several human disease states including Liddle syndrome, psuedohypoaldosteronism, and cystic fibrosis and may be implicated in salt-sensitive hypertension. ENaC activity in epithelial cells is regulated both by open probability and channel number. This review focuses on the regulation of ENaC in the cells of the kidney cortical collecting duct by trafficking and recycling. The trafficking of ENaC is discussed in the broader context of epithelial cell vesicle trafficking. Well-characterized pathways and protein interactions elucidated using epithelial model cells are discussed, and the known overlap with ENaC regulation is highlighted. In following the life of ENaC in CCD epithelial cells the apical delivery, internalization, recycling, and destruction of the channel will be discussed. While a number of pathways presented still need to be linked to ENaC regulation and many details of the regulation of ENaC trafficking remain to be elucidated, knowledge of these mechanisms may provide further insights into ENaC activity in normal and disease states.     

Thiol-reactive compounds from garlic inhibit the epithelial sodium channel (ENaC)

The epithelial sodium channel (ENaC) is a key factor in the transepithelial movement of sodium, and consequently salt and water homeostasis in various organs. Dysregulated activity of ENaC is associated with human diseases such as hypertension, the salt-wasting syndrome pseudohypoaldosteronism type 1, cystic fibrosis, pulmonary oedema or intestinal disorders. Therefore it is important to identify novel compounds that affect ENaC activity. This study investigated if garlic (Allium sativum) and its characteristic organosulfur compounds have impact on ENaCs. Human ENaCs were heterologously expressed in Xenopus oocytes and their activity was measured as transmembrane currents by the two-electrode voltage-clamp technique. The application of freshly prepared extract from 5g of fresh garlic (1% final concentration) decreased transmembrane currents of ENaC-expressing oocytes within 10 min. This effect was dose-dependent and irreversible. It was fully sensitive to the ENaC-inhibitor amiloride and was not apparent on native control oocytes. The effect of garlic was blocked by dithiothreitol and l-cysteine indicating involvement of thiol-reactive compounds. The garlic organosulsur compounds S-allylcysteine, alliin and diallyl sulfides had no effect on ENaC. By contrast, the thiol-reactive garlic compound allicin significantly inhibited ENaC to a similar extent as garlic extract. These data indicate that thiol-reactive compounds which are present in garlic inhibit ENaC.     

Phosphatidylinositol 4-phosphate 5-kinase reduces cell surface expression of the epithelial sodium channel (ENaC) in cultured collecting duct cells

Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.     

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits.     

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa

Background

The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na⁺-K⁺-ATPase.

Methods

Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na⁺-K⁺-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.

Results

The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α₁Na⁺-K⁺-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.

Conclusions

These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.     

Anionic phospholipids regulate native and expressed epithelial sodium channel (ENaC).

Using patch clamp techniques, we found that the epithelial sodium channel (ENaC) activity in the apical membrane of A6 distal nephron cells showed a sudden rundown beginning at 4 min after forming the inside-out configuration. This sudden rundown was prevented by addition of anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidylserine (PS) to the "cytoplasmic" bath. Conversely, chelation of endogenous PIP(2) with anti-PIP(2) antibody, hydrolysis of PIP(2) with either exogenous phospholipase C (PLC) or activation of endogenous PLC by extracellular ATP, or application of the positively charged molecule, poly-L-lysine, accelerated channel rundown. However, neutral phosphatidylcholine had no effect on ENaC activity. By two-electrode voltage clamp recordings, we demonstrated that PIP(2) and PIP(3) significantly increased amiloride-sensitive current in Xenopus oocytes injected with cRNAs of rat alpha-, beta-, and gamma-ENaC. However, PIP(2) and PIP(3) did not affect surface expression of ENaC, indicating that PIP(2) and PIP(3) regulate ENaC at the level of the inner plasma membrane through a mechanism that is independent of ENaC trafficking. These data suggest that anionic phospholipids may mediate the regulation of ENaC by PLC- or phosphoinositide 3-kinase-coupled receptors.     

Atomic force microscopy reveals the architecture of the epithelial sodium channel (ENaC)

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (α, β, and γ); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the α-, β- and γ-subunits. We show that for α-, β- and γ-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that αβγ-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits.     

Mechanism of epithelial sodium channel (ENaC) regulation by cortactin: involvement of dynamin

We have recently shown that epithelial sodium channels (ENaC) are regulated by the actin-binding protein cortactin via the Arp2/3 protein complex. However, it has been also demonstrated that GTPase, dynamin, which is known to regulate clathrin-mediated endocytosis, can as well initiate signaling cascades regulated by cortactin. This study was designed to investigate the involvement of dynamin into cortactin-mediated regulation of ENaC. Initially, a recently described inhibitor of dynamin, dynasore, was used. However, use of this inhibitor seemed to be inappropriate due to discovered side effects. F. i., treatment of mpkCCD(c14) cells monolayers with dynasore (in concentrations of 10 and 100 microM) resulted in a decrease in ENaC-mediated transepithelial currents. Besides, the same concentrations of dynasore caused reduced currents in CHO cells transfected with ENaC subunits. Therefore, the data demonstrated that dynasore down regulates both native and overexpressed channel's activity and is not suitable for studies of a role of dynamin in the clathrin-mediated endocytosis of ENaC. This effect is most likely caused either by dynasore's toxic effect upon the cells or by enhanced endocytosis of ENaC-activating proteins. In the following experiments designed to study the role of dynamin different plasmids encoding mutant forms of dynamin and cortactin were used. Dominant negative dynamin K44A transfected into CHO cells together with ENaC subunits significantly increased amiloride-sensitive current density compared to cells transfected with ENaC subunits only (control); additional transfection of cortactin in this system resulted in current density restitution back to the control level. Moreover, ENaC overexpression with the SH3 domain of cortactin, which is responsible for dynamin binding, caused a decrease if ENaC current. Thus, we have shown in this study that cortactin can mediate ENaC activity not only via the Arp2/3 complex, but apart from that dynamin and related processes also might be involved into ENaC regulation by cortactin.     

Mechanisms of epithelial sodium channel (ENaC) regulation by cortactin: Involvement of dynamin

We have recently shown that epithelial sodium channels (ENaCs) are regulated by the actin-binding protein cortactin via the Arp2/3 protein complex. It has been also demonstrated that a GTPase dynamin, which is known to regulate clathrin-mediated endocytosis, can as well initiate signaling cascades regulated by cortactin. This study was designed to investigate the involvement of dynamin into cortactin-mediated regulation of ENaC. Initially, a recently described inhibitor of dynamin, dynasore, was used. However, use of this inhibitor seemed to be inappropriate due to discovered side effects. Thus, treatment of mpkCCD_c14 cells monolayers with dynasore (in concentrations of 10 and 100 μM) resulted in a decrease in ENaC-mediated transepithelial currents. Besides, dynasore caused reduced amiloride-sensitive currents in CHO cells transfected with ENaC subunits. Therefore, the data demonstrated that dynasore down regulates both native and overexpressed channel’s activity and use of this drug is not appropriate for studies of ENaC endocytosis. We hypothesize that this effect is most likely caused either by dynasore’s toxic actions upon the cells or by enhanced endocytosis of ENaC-activating proteins. In the following experiments plasmids encoding mutant forms of dynamin and cortactin were used. Dominant negative dynamin (K44A) transfected into CHO cells together with ENaC subunits significantly increased amiloride-sensitive current density compared to cells transfected with ENaC only (control); additional transfection of cortactin together with the K44A dynamin resulted in current density restitution back to the control level. Moreover, ENaC overexpression with the SH3 domain of cortactin, which is responsible for dynamin binding, caused a decrease of ENaC current. Thus, we have shown in this study that cortactin can mediate ENaC activity not only via the Arp2/3 complex, but also through the dynamin-mediated processes.     

Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC)

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na(+)) transport. The greatest reduction in Na(+) transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na(+) transport in mpkCCD cells.     

Decreased decidual epithelial sodium channel (ENaC) alpha and gamma subunits in recurrent pregnancy loss (RPL)

正Dear Editor,Recurrent pregnancy loss (RPL) in early pregnancy is a devastating problem for couples who want to become parents and a difficult challenge for their physician. RPL is also referred to as recurrent miscarriage or habitual abortion. It is defined as two or more consecutive clinical miscarriages(CMs) before 20 weeks of gestation (Practice Committee of     

Angiotensin II increases activity of the epithelial Na+ channel (ENaC) in distal nephron additively to aldosterone

Dietary salt intake controls epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron by affecting status of the renin-angiotensin-aldosterone system (RAAS). Whereas regulation of ENaC by aldosterone is generally accepted, little is known about whether other components of RAAS, such as angiotensin II (Ang II), have nonredundant to aldosterone-stimulatory actions on ENaC. We combined patch clamp electrophysiology and immunohistochemistry in freshly isolated split-opened distal nephrons of mice to determine the mechanism and molecular signaling pathway of Ang II regulation of ENaC. We found that Ang II acutely increases ENaC Po, whereas prolonged exposure to Ang II also induces translocation of α-ENaC toward the apical membrane in situ. Ang II actions on ENaC Po persist in the presence of saturated mineralocorticoid status. Moreover, aldosterone fails to stimulate ENaC acutely, suggesting that Ang II and aldosterone have different time frames of ENaC activation. AT1 but not AT2 receptors mediate Ang II actions on ENaC. Unlike its effect in vasculature, Ang II did not increase [Ca2+]i in split-opened distal nephrons as demonstrated using ratiometric Fura-2-based microscopy. However, application of Ang II to mpkCCDc14 cells resulted in generation of reactive oxygen species, as probed with fluorescent methods. Consistently, inhibiting NADPH oxidase with apocynin abolished Ang II-mediated increases in ENaC Po in murine distal nephron. Therefore, we concluded that Ang II directly regulates ENaC activity in the distal nephron, and this effect complements regulation of ENaC by aldosterone. We propose that stimulation of AT1 receptors with subsequent activation of NADPH oxidase signaling pathway mediates Ang II actions on ENaC.     

Effects of cytochrome P-450 metabolites of arachidonic acid on the epithelial sodium channel (ENaC)

Sodium reabsorption via the epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron plays a central role in the regulation of body fluid volume. Previous studies have indicated that arachidonic acid (AA) and its metabolite 11,12-EET but not other regioisomers of EETs inhibit ENaC activity in the collecting duct. The goal of this study was to investigate the endogenous metabolism of AA in cultured mpkCCD(c14) principal cells and the effects of these metabolites on ENaC activity. Liquid chromatography/mass spectrometry analysis of the mpkCCD(c14) cells indicated that these cells produce prostaglandins, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 12/8-HETE, and 15-HETE, but not 20-HETE. Single-channel patch-clamp experiments revealed that 8,9-EET, 14,15-EET, and 11,12-EET all decrease ENaC activity. Neither 5-, 12-, nor 15-HETE had any effect on ENaC activity. Diclofenac and ibuprofen, inhibitors of cyclooxygenase, decreased transepithelial Na(+) transport in the mpkCCD(c14) cells. Inhibition of cytochrome P-450 (CYP450) with MS-PPOH activated ENaC-mediated sodium transport when cells were pretreated with AA and diclofenac. Coexpression of CYP2C8, but not CYP4A10, with ENaC in Chinese hamster ovary cells significantly decreased ENaC activity in whole-cell experiments, whereas 11,12-EET mimicked this effect. Thus both endogenously formed EETs and their exogenous application decrease ENaC activity. Downregulation of ENaC activity by overexpression of CYP2C8 was PKA dependent and was prevented by myristoylated PKI treatment. Biotinylation experiments and single-channel analysis revealed that long-term treatment with 11,12-EET and overexpression of CYP2C8 decreased the number of channels in the membrane. In contrast, the acute inhibitory effects are mediated by a decrease in the open probability of the ENaC. We conclude that 11,12-EET, 8,9-EET, and 14,15-EET are endogenously formed eicosanoids that modulate ENaC activity in the collecting duct.     

Phosphatidylinositol 3,4,5-trisphosphate directs association of Src homology 2-containing signaling proteins with gelsolin

Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130(Cas), focal adhesion kinase, integrin alpha(v)beta(3), vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, c-Src, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of alpha(v)beta(3)-regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.     

Insulin activates epithelial sodium channel (ENaC) via phosphoinositide 3-kinase in mammalian taste receptor cells

Diabetes is a profound disease that results in a severe lack of regulation of systemic salt and water balance. From our earlier work on the endocrine regulation of salt taste at the level of the epithelial sodium channel (ENaC), we have begun to investigate the ability of insulin to alter ENaC function with patch-clamp recording on isolated mouse taste receptor cells (TRCs). In fungiform and vallate TRCs that exhibit functional ENaC currents (e.g., amiloride-sensitive Na(+) influx), insulin (5-20 nM) caused a significant increase in Na(+) influx at -80 mV (EC(50) = 7.53 nM). The insulin-enhanced currents were inhibited by amiloride (30 μM). Similarly, in ratiometric Na(+) imaging using SBFI, insulin treatment (20 nM) enhanced Na(+) movement in TRCs, consistent with its action in electrophysiological assays. The ability of insulin to regulate ENaC function is dependent on the enzyme phosphoinositide 3-kinase since treatment with the inhibitor LY294002 (10 μM) abolished insulin-induced changes in ENaC. To test the role of insulin in the regulation of salt taste, we have characterized behavioral responses to NaCl using a mouse model of acute hyperinsulinemia. Insulin-treated mice show significant avoidance of NaCl at lower concentrations than the control group. Interestingly, these differences between groups were abolished when amiloride (100 μM) was added into NaCl solutions, suggesting that insulin was regulating ENaC. Our results are consistent with a role for insulin in maintaining functional expression of ENaC in mouse TRCs.     

Phylogenetic characterization of the epithelial Na+ channel (ENaC) family

Summary

Twenty-one sequenced protein members of the epithelial Na⁺ channel (ENaC) family have been identified and characterized in terms of their sizes, hydropathy profiles, sequence similarities and phylogenies. These proteins derive from mammals, the frog Xenopus laevis and the worm Caenorhabditis elegans. The eleven sequenced vertebrate proteins fall into four subfamilies designated α, β, γ, and δ. The 10 C. elegans proteins do not cluster with the vertebrate proteins, and they all proved to be distantly related to each other. Nonetheless, the 21 ENaC proteins exhibit the same apparent topology, each with two transmembrane spanning segments separated by a large extracellular loop. All but two ENaC proteins possess highly conserved extracellular domains containing numerous conserved cysteine residues as well as adjacent C-terminal amphipathic transmembrane spanning segments, postulated to contribute to the formation of the hydrophilic pores of these oligomeric channel protein complexes. It is proposed that the well-conserved extracellular domains serve as receptors to control the activities of the channels. A topological model for the ENaC family proteins is presented.     

Identification of a functional phosphatidylinositol 3,4,5-trisphosphate binding site in the epithelial Na+ channel

Membrane phospholipids, such as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)), are signaling molecules that can directly modulate the activity of ion channels, including the epithelial Na(+) channel (ENaC). Whereas PI(3,4,5)P(3) directly activates ENaC, its binding site within the channel has not been identified. We identify here a region of gamma-mENaC just following the second trans-membrane domain (residues 569-583) important to PI(3,4,5)P(3) binding and regulation. Deletion of this track decreases activity of ENaC heterologously expressed in Chinese hamster ovary cells. K-Ras and its first effector phosphoinositide 3-OH kinase (PI3-K), as well as RhoA and its effector phosphatidylinositol 4-phosphate 5-kinase increase ENaC activity. Whereas the former, via generation of PI(3,4,5)P(3), increases ENaC open probability, the latter increases activity by increasing membrane levels of the channel. Deletion of the region just distal to the second trans-membrane domain disrupted regulation by K-Ras and PI3-K but not RhoA and phosphatidylinositol 4-phosphate 5-kinase. Moreover, PI(3,4,5)P(3) binds ENaC with deletion of the region following the second transmembrane domain disrupting this interaction and disrupting direct activation of the channel by PI(3,4,5)P(3). Mutation analysis revealed the importance of conserved positive and negative charged residues as well as bulky amino acids within this region to modulation of ENaC by PI3-K. The current results identify the region just distal to the second trans-membrane domain within gamma-mENaC as being part of a functional PI(3,4,5)P(3) binding site that directly impacts ENaC activity. Phospholipid binding to this site is probably mediated by the positively charged amino acids within this track, with negatively charged and bulky residues also influencing specificity of interactions.     

SPLUNC1 expression reduces surface levels of the epithelial sodium channel (ENaC) in Xenopus laevis oocytes

Throughout the body, the epithelial Na⁺ channel (ENaC) plays a critical role in salt and liquid homeostasis. In cystic fibrosis airways, for instance, improper regulation of ENaC results in hyperabsorption of sodium that causes dehydration of airway surface liquid. This dysregulation then contributes to mucus stasis and chronic lung infections. ENaC is known to undergo proteolytic cleavage, which is required for its ability to conduct Na⁺ ions. We have previously shown that the short, palate lung and nasal epithelial clone (SPLUNC1) binds to and inhibits ENaC in both airway epithelia and in Xenopus laevis oocytes. In this study, we found that SPLUNC1 was more potent at inhibiting ENaC than either SPLUNC2 or long PLUNC1 (LPLUNC1), two other PLUNC family proteins that are also expressed in airway epithelia. Furthermore, we were able to shed light on the potential mechanism of SPLUNC1''s inhibition of ENaC. While SPLUNC1 did not inhibit proteolytic activity of trypsin, it significantly reduced ENaC currents by reducing the number of ENaCs in the plasma membrane. A better understanding of ENaC''s regulation by endogenous inhibitors may aid in the development of novel therapies designed to inhibit hyperactive ENaC in cystic fibrosis epithelia.Key words: mucociliary clearance, chronic airway disease, cystic fibrosis, protease, airway surface liquid, Na⁺ absorption