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Regulated Na+ transport in the distal nephron is of fundamental importance to fluid and electrolyte homeostasis. Further upstream, Na+ is the principal driver of secondary active transport of numerous organic and inorganic solutes. In the distal nephron, Na+ continues to play a central role in controlling the body levels and concentrations of a more select group of ions, including K+, Ca++, Mg++, Cl−, and HCO3−, as well as water. Also, of paramount importance are transport mechanisms aimed at controlling the total level of Na+ itself in the body, as well as its concentrations in intracellular and extracellular compartments. Over the last several decades, the transporters involved in moving Na+ in the distal nephron, and directly or indirectly coupling its movement to that of other ions have been identified, and their interrelationships brought into focus. Just as importantly, the signaling systems and their components—kinases, ubiquitin ligases, phosphatases, transcription factors, and others—have also been identified and many of their actions elucidated. This review will touch on selected aspects of ion transport regulation, and its impact on fluid and electrolyte homeostasis. A particular focus will be on emerging evidence for site-specific regulation of the epithelial sodium channel (ENaC) and its role in both Na+ and K+ homeostasis. In this context, the critical regulatory roles of aldosterone, the mineralocorticoid receptor (MR), and the kinases SGK1 and mTORC2 will be highlighted. This includes a discussion of the newly established concept that local K+ concentrations are involved in the reciprocal regulation of Na+-Cl− cotransporter (NCC) and ENaC activity to adjust renal K+ secretion to dietary intake. [ABSTRACT FROM AUTHOR]

How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel's α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to invitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 β (GSK3β). Therefore, we investigated the effect of DYRK2 on ENaC activity in outside-out patches of Xenopus laevis oocytes heterologously expressing rat ENaC. DYRK2 included in the pipette solution significantly increased ENaC activity. In contrast, GSK3β had an inhibitory effect. Replacing S621 in αENaC with alanine (S621A) abolished the effects of both kinases. A S617A mutation reduced the inhibitory effect of GKS3β but did not prevent ENaC activation by DYRK2. Our findings suggest that phosphorylation of S621 activates ENaC and primes S617 for subsequent phosphorylation by GSK3β resulting in channel inhibition. In proof-of-concept experiments, we demonstrated that DYRK2 can also stimulate ENaC currents in microdissected mouse distal nephron, whereas GSK3β inhibits the currents. [ABSTRACT FROM AUTHOR]

Background/Aims: Apelin and its G protein-coupled receptor APLNR (also known as APJ) are widely expressed within the central nervous system and peripheral organs including heart, lung and kidney. Several studies have shown that the apelin/APJ system is involved in various important physiological processes such as energy metabolism, cardiovascular functions and fluid homeostasis. In the kidney, the apelin/APJ system performs a wide range of activities. We recently demonstrated that apelin antagonises the hydro-osmotic effect of vasopressin on aquaporin-2 water channel (AQP-2) expression by reducing its mRNA and protein levels in collecting duct principal cells. The central role of these cells in water and sodium transport is governed by AQP-2 and the epithelial sodium channel (ENaC). The coordination of these channels is essential for the control of extracellular fluid volume, sodium homeostasis and blood pressure. This study aimed at investigating the role of apelin in the regulation of sodium balance in the distal nephron, and more specifically its involvement in modulating the expression and activity of ENaC in collecting duct principal cells. Methods: mpkCCD cells were incubated in the presence of aldosterone and treated with or without apelin-13. Transepithelial Na+ current was measured and the changes in ENaC expression determined by RT-PCR and immunoblotting. Results: Our data show that apelin-13 reduces the transepithelial sodium amiloride-sensitive current in collecting duct principal cells after 8h and 24h treatment. This effect was associated with a decrease in αENaC subunit expression and mediated through the ERK pathway as well as SGK1 and Nedd4-2. Conclusion: Our findings indicate that apelin is involved in the fine regulation of sodium balance in the renal collecting duct by opposing the effects of aldosterone, likely by activation of ENaC ubiquitination. [ABSTRACT FROM AUTHOR]

Syntaxins are SNARE proteins and may play a role in epithelial sodium channel (ENaC) trafficking. The aim of the present study was to investigate the effects of syntaxin 2 (STX2), syntaxin 3 (STX3), and syntaxin 4 (STX4) on rat (rENaC) and human ENaC (hENaC). Co-expression of rENaC and STX3 or STX4 in Xenopus laevis oocytes increased amiloride-sensitive whole-cell currents (ΔIami) on average by 50% and 135%, respectively, compared to oocytes expressing rENaC alone. In contrast, STX2 had no significant effect on rENaC. Similar to its effect on rENaC, STX3 stimulated hENaC by 48%. In contrast, STX2 and STX4 inhibited hENaC by 51% and 44%, respectively. Using rENaC carrying a FLAG tag in the extracellular loop of the β-subunit, we demonstrated that the stimulatory effects of STX3 and STX4 on ΔIami were associated with an increased expression of the channel at the cell surface. Co-expression of STX3 or STX4 did not significantly alter the degree of proteolytic channel activation by chymotrypsin. STX3 had no effect on the inhibition of rENaC by brefeldin A, and the stimulatory effect of STX3 was preserved in the presence of dominant negative Rab11. This indicates that the stimulatory effect of STX3 is not mediated by inhibiting channel retrieval or by stimulating fusion of recycling endosomes. Our results suggest that the effects of syntaxins on ENaC are isoform and species dependent. Furthermore, our results demonstrate that STX3 increases ENaC expression at the cell surface, probably by enhancing insertion of vesicles carrying newly synthesized channels. [ABSTRACT FROM AUTHOR]

Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of ulcerative colitis, possibly by disrupting epithelial integrity. In the distal colon, the epithelial sodium channel (ENaC) is an important factor in the regulation of sodium absorption, and therefore plays a critical role in minimizing intestinal sodium and water losses. In the present study, we investigated whether IL-13 also acts as a potent modulator of epithelial sodium transport via ENaC, and the signalling components involved. The effect of IL-13 on ENaC was examined in HT-29/B6-GR/MR human colon cells, as well as in mouse distal colon, by measuring amiloride-sensitive short-circuit current (ISC) in Ussing chambers. The expression levels of ENaC subunits and the cellular components that contribute to ENaC activity were analysed by qRT-PCR and promoter gene assay. We show that IL-13, in both the cell model and in native intestinal tissue, impaired epithelial sodium absorption via ENaC (JNa) as a result of decreased transcription levels of β- and γ-ENaC subunits and SGK1, a post-translational regulator of ENaC activity, due to impaired promoter activity. The reduction in JNa was prevented by inhibition of JAK1/2–STAT6 signalling. This inhibition also affected the IL-13-induced decrease in p38 MAPK phosphorylation. The contribution of STAT6 to IL-13-mediated ENaC inactivation was confirmed in a STAT6−/− mouse model. In conclusion, these results indicate that IL-13, the levels of which are elevated in ulcerative colitis, contributes to impaired ENaC activity via modulation of the STAT6/p38 MAPK pathways. [ABSTRACT FROM AUTHOR]

The present study explored whether the intercalated cell Cl-/HCO3- exchanger pendrin modulates epithelial Na+ channel (ENaC) function by changing channel open probability and/or channel density. To do so, we measured ENaC subunit subcellular distribution by immunohistochemistry, single channel recordings in split open cortical collecting ducts (CCDs), as well as transepithelial voltage and Na+ absorption in CCDs from aldosterone-treated wild-type and pendrin-null mice. Because pendrin gene ablation reduced 70-kDa more than 85-kDa γ-ENaC band density, we asked if pendrin gene ablation interferes with ENaC cleavage. We observed that ENaC-cleaving protease application (trypsin) increased the lumen-negative transepithelial voltage in pendrin-null mice but not in wild-type mice, which raised the possibility that pendrin gene ablation blunts ENaC cleavage, thereby reducing open probability. In mice harboring wild-type ENaC, pendrin gene ablation reduced ENaC-mediated Na(+) absorption by reducing channel open probability as well as by reducing channel density through changes in subunit total protein abundance and subcellular distribution. Further experiments used mice with blunted ENaC endocytosis and degradation (Liddle's syndrome) to explore the significance of pendrin-dependent changes in ENaC open probability. In mouse models of Liddle's syndrome, pendrin gene ablation did not change ENaC subunit total protein abundance, subcellular distribution, or channel density, but markedly reduced channel open probability. We conclude that in mice harboring wild-type ENaC, pendrin modulates ENaC function through changes in subunit abundance, subcellular distribution, and channel open probability. In a mouse model of Liddle's syndrome, however, pendrin gene ablation reduces channel activity mainly through changes in open probability. [ABSTRACT FROM AUTHOR]

The epithelial sodium channel (ENaC) plays an important role in homeostasis of blood pressure and of the airway surface liquid, and excess function of ENaC results in refractory hypertension (in Liddle's syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsc70, is not completely understood. Our previously published data suggest that Hsc70 negatively affects ENaC activity and surface expression in Xenopus oocytes; here we investigate the mechanism by which Hsc70 acts on ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsc70, treatment with 5 μg/ml doxycycline increased total Hsc70 expression 20%. This increase in Hsc70 expression led to a decrease in ENaC activity and surface expression that corresponded to an increased rate of functional ENaC retrieval from the cell surface. In addition, Hsc70 overexpression decreased the association of newly synthesized ENaC subunits. These data support the hypothesis that Hsc70 inhibits ENaC functional expression at the apical surface of epithelia by regulating ENaC biogenesis and ENaC trafficking at the cell surface. [ABSTRACT FROM AUTHOR]

The epithelial Na(+) channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasing αENaC transcription in collecting duct principal cells. We previously reported that Af9 binds to +78/+92 of αENaC and recruits Dot1a to repress basal and aldosterone-sensitive αENaC transcription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basal αENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, an αENaC promoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild-type αENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the αENaC promoter, which was required for maximal induction of αENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229 αENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the αENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at +222/+229 represents the first identified constitutive driver of αENaC transcription, and it contributes to maximal aldosterone trans-activation of αENaC. [ABSTRACT FROM AUTHOR]

The ubiquitously expressed serum- and glucocorticoid-inducible kinase-1 (SGK1) is genomically regulated by cell stress (including cell shrinkage) and several hormones (including gluco- and mineralo- corticoids). SGK1 is activated by insulin and growth factors through PI3K and 3-phosphoinositide-depen- dent kinase PDK1. SGK1 activates a wide variety of ion channels (e.g., ENaC, SCN5A, TRPV4-6, ROMK, Kvl.3, Kvl.5, Kv4.3, KCNE1/KCNQ1, KCNQ4, ASIC1, GIuR6, CICKa/barttin, CIC2, CFTR, and Orai/STIM), which participate in the regulation of transport, hormone release, neuroexcitability, inflammation, cell proliferation, and apoptosis. SGK1-sensitive ion channels participate in the regulation of renal Na+ retention and K+ elimination, blood pressure, gastric acid secretion, cardiac action potential, hemostasis, and neuroexcit-ability. A common (~3-5% prevalence in Caucasians and ~10% in Africans) SGK1 gene variant is associated with increased blood pressure and body weight as well as increased prevalence of type II diabetes and stroke. SGK1 further contributes to the pathophysiology of allergy, peptic ulcer, fibrosing disease, ischemia, tumor growth, and neurodegeneration. The effect of SGK1 on channel activity is modest, and the channels do not require SGK1 for basic function. SGK1-dependent ion channel regulation may thus become pathophysiologically relevant primarily after excessive (pathological) expression. Therefore, SGK1 may be considered an attractive therapeutic target despite its broad range of functions. [ABSTRACT FROM AUTHOR]

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 (EC50 = 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. [ABSTRACT FROM AUTHOR]

Ion channels and transporters play a critical role in ion and fluid homeostasis and thus in normal animal physiology and pathology. Tight regulation of these transmembrane proteins is therefore essential. In recent years, many studies have focused their attention on the role of the ubiquitin system in regulating ion channels and transporters, initialed by the discoveries of the role of this system in processing of Cystic Fibrosis Transmembrane Regulator (CFTR), and in regulating endocytosis of the epithelial Na channel (ENaC) by the Nedd4 family of ubiquitin ligases (mainly Nedd4-2). In this review, we discuss the role of the ubiquitin system in ER Associated Degradation (ERAD) of ion channels, and in the regulation of endocytosis and lysosomal sorting of ion channels and transporters, focusing primarily in mammalian cells. We also briefly discuss the role of ubiquitin like molecules (such as SUMO) in such regulation, for which much less is known so far. [ABSTRACT FROM AUTHOR]

The functional expression of the epithelial sodium channel (ENaC) appears elevated in cystic fibrosis (CF) airway epithelia, but the mechanism by which this occurs is not clear. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) alters the trafficking of endogenously expressed human ENaC in the CFBE41o- model of CF bronchial epithelia. Functional expression of ENaC, as defined by amiloride-inhibited short-circuit current (Isc) in Ussing chambers, was absent under control conditions but present in CFBE41o- parental and ΔF508-CFTR-overexpressing cells after treatment with 1 μM dexamethasone (Dex) for 24 h. The effect of Dex was mimicked by incubation with the glucocorticoid hydrocortisone but not with the mineralocorticoid aldosterone. Application of trypsin to the apical surface to activate uncleaved, "near-silent" ENaC caused an additional increase in amiloride-sensitive Isc in the Dex-treated cells and was without effect in the control cells, suggesting that Dex increased ENaC cell surface expression. In contrast, Dex treatment did not stimulate amiloride-sensitive Isc in CFBE41o- cells that stably express wild-type (wt) CFTR. CFBE41o- wt cells also had reduced expression of α- and γ-ENaC compared with parental and ΔF508-CFTR-overexpressing cells. Furthermore, application of trypsin to the apical surface of Dex-treated CFBE41o- wt cells did not stimulate amiloride-sensitive Isc, suggesting that ENaC remained absent from the surface of these cells even after Dex treatment. We also tested the effect of trafficking-corrected ΔF508-CFTR on ENaC functional expression. Incubation with 1 mM 4-phenylbutyrate synergistically increased Dex-induced ENaC functional expression in ΔF508-CFTR-overexpressing cells. These data support the hypothesis that wt CFTR can regulate the whole cell, functional, and surface expression of endogenous ENaC in airway epithelial cells and that absence of this regulation may foster ENaC hyperactivity in CF airway epithelia. [ABSTRACT FROM AUTHOR]

Kinases contribute to the regulation of the epithelial sodium channel (ENaC) in a complex manner. For example, SGK1 (serum- and glucocorticoid-inducible kinase type 1) enhances ENaC surface expression by phosphorylating Nedd4-2, thereby preventing ENaC retrieval and degradation. An additional mechanism of ENaC activation by SGK1 involves an SGK consensus motif (616RSRYWS621) in the C-terminus of the channel's α-subunit. This consensus motif may also be a target for ENaC regulation by protein kinase B α (PKBα) known to be activated by insulin and growth factors. Therefore, we investigated a possible role of PKBα in the regulation of rat ENaC heterologously expressed in Xenopus laevis oocytes. We found that recombinant PKBα included in the pipette solution increased ENaC currents in outside-out patches by about 4-fold within 15-20 min. Replacing the serine residue S621 of the SGK consensus motif by an alanine (S621A) abolished this stimulatory effect. In co-expression experiments active PKBα but not catalytically inactive PKBα significantly increased ENaC whole-cell currents and surface expression by more than 50 % within 24 hours of co-expression. Interestingly, this stimulatory effect was preserved in oocytes expressing ENaC with the S621A mutation. We conclude that the acute stimulatory effect of PKBα involves a specific kinase consensus motif in the C-terminus of the channel's α-subunit. In contrast, the increase in channel surface expression caused by co-expression of PKBα does not depend on this site in the channel and is probably mediated by an effect on channel trafficking. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists used to treat type 2 diabetes. TZD treatment induces side effects such as peripheral fluid retention, often leading to discontinuation of therapy. Previous studies have shown that PPARγ activation by TZD enhances the expression or function of the epithelial sodium channel (ENaC) through different mechanisms. However, the effect of TZDs on ENaC activity is not clearly understood. Here, we show that treating Xenopus laevis oocytes expressing ENaC and PPARγ with the TZD rosiglitazone (RGZ) produced a twofold increase of amiloride-sensitive sodium current (Iam), as measured by two-electrode voltage clamp. RGZ-induced ENaC activation was PPARγ-dependent since the PPARγ antagonist GW9662 blocked the activation. The RGZ-induced Iam increase was not mediated through direct serum- and glucocorticoid-regulated kinase (SGK1)-dependent phosphorylation of serine residue 594 on the human ENaC α-subunit but by the diminution of ENaC ubiquitination through the SGK1/Nedd4-2 pathway. In accordance, RGZ increased the activity of ENaC by enhancing its cell surface expression, most probably indirectly mediated through the increase of SGK1 expression. [ABSTRACT FROM AUTHOR]

In A6 cells, a renal cell line derived from Xenopus laevis, hypotonic stress stimulates the amiloride-sensitive Na+ transport. Hypotonic action on Na+ transport consists of two phases, a nongenomic early phase and a genomic delayed phase. Although it has been reported that, during the genomic phase, hypotonic stress stimulates transcription of Na+ transport-related genes, such as serum- and glucocorticoid-inducible kinase I (SGK1) and subunits of the epithelial Na+ channel (ENaC), increasing Na+ transport, the mechanism remains unknown. We focused the present study on the role of intracellular Ca2+ in hypotonicity-induced SGK1 and ENaC subunit transcription. Since hypotonic stress raises intracellular Ca2+ concentration in A6 cells, we hypothesized that Ca2+-dependent signals participate in the genomic action. Using real-time quantitative RT-PCR and Western blot techniques and measuring short-circuit currents, we observed that 1) BAPTA-AM and W7 blunted the hypotonicity-induced expression of SGK1 mRNA and protein, 2) ionomycin dose dependently stimulated expression of SGK1 mRNA and protein under an isotonic condition and the time course of the stimulatory effect of ionomycin on SGKI mRNA was remarkably similar to that of hypotonic action on SGK1 mRNA, 3) hypotonic stress stimulated transcription of three ENaC subunits in an intracellular Ca2+-dependent manner, and 4) BAPTA-AM retarded the delayed phase of hypotonic stress-induced Na+ transport but had no effect on the early phase. These observations indicate for the first time that intracellular Ca2+ plays a role as the second messenger in hypotonic stress-induced Na+ transport by stimulating transcription of SGK1 and ENaC subunits. [ABSTRACT FROM AUTHOR]

The A663T polymorphism of the α-subunit of the human epithelial sodium channel (hENaC) increases the functional and surface expression of αβγ-hENaC in Xenopus laevis oocytes, and the context of this residue in the COOH terminus of α-hENaC is important for this effect. Query of a phosphoprotein database suggested that the α-T663 residue of hENaC might be a substrate for phosphorylation by casein kinase 1 (CK1). We tested the hypotheses that phosphorylation of α-T663-hENaC by CK1 would regulate the increased functional and surface expression of α-T663-hENaC vs. α-A663-hENaC in oocytes. General inhibition of CK1 with IC261 decreased the functional and surface expression of α-T663-hENaC, but not α-A663-hENaC. This decrease in α-T663-hENaC functional expression resulted from reduced delivery of α-T663-hENaC to the oocyte membrane. IC261 also inhibited the functional expression of α-T692-mENaC and a chimeric m(1-678)/h(650-669)α-T663, mβγ ENaC, but not α-A692-mENaC or m(1-678)/h(650-669)α-A663, mβγ ENaC. These data suggest that additional residues outside of the α-hENaC COOH terminus are important for modulation of α-T663-hENaC trafficking by CK1. Overexpression of CK1α did not alter functional expression of α-T663-hENaC. In contrast, modest overexpression of CK1δ enhanced, whereas higher levels of CK1δ overexpression inhibited α-T663-hENaC functional expression. CK1 did not phosphorylate the COOH terminus of either α-T663-hENaC or α-A663-hENaC in vitro. These data suggest that CK1, and perhaps specifically CK1δ, regulates the intracellular trafficking of the α-A663T functional polymorphism of hENaC indirectly by altering the rate of α-T663-hENaC biosynthesis and/or delivery to the plasma membrane. [ABSTRACT FROM AUTHOR]

The fine control of NaCl absorption regulated by hormones takes place in the distal nephron of the kidney. In collecting duct principal cells, the epithelial sodium channel (ENaC) mediates the apical entry of Na+, which is extruded by the basolateral Na+,K+-ATPase. Simian virus 40-transformed and “transimmortalized” collecting duct cell lines, derived from transgenic mice carrying a constitutive, conditionally, or tissue-specific promoter-regulated large T antigen, have been proven to be valuable tools for studying the mechanisms controlling the cell surface expression and trafficking of ENaC and Na+,K+-ATPase. These cell lines have made it possible to identify sets of aldosterone- and vasopressin-stimulated proteins, and have provided new insights into the concerted mechanism of action of serum- and glucocorticoid-inducible kinase 1 (Sgk1), ubiquitin ligase Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), and 14-3-3 regulatory proteins in modulating ENaC-mediated Na+ currents. Epidermal growth factor and induced leucine zipper protein have also been shown to repress and stimulate ENaC-dependent Na+ absorption, respectively, by activating or repressing the mitogen-activated protein kinase externally regulated kinase1/2. Overall, these findings have provided evidence suggesting that multiple pathways are involved in regulating NaCl absorption in the distal nephron. [ABSTRACT FROM AUTHOR]

The epithelial sodium channel (ENaC) is the major mediator of sodium transport across the apical membranes of the distal nephron, the distal colon, the respiratory tract and the ducts of exocrine glands. It is subject to feedback inhibition by increased intracellular Na+, a regulatory system wherein the ubiquitin protein ligases, Nedd4 and Nedd4-2, bind to conserved PY motifs in the C-termini of ENaC and inactivate the channel. It has been proposed recently that the kinase Sgk activates the channel as a consequence of phosphorylating Nedd4-2, thus preventing it from inhibiting the channels. This proposal predicts that Sgk should interfere with Na+ feedback regulation of ENaC. We have tested this prediction in Xenopus laevis oocytes and in mouse salivary duct cells and found that in neither system did increased activity of Sgk interrupt Na+ feedback inhibition of ENaC. We found, however, that Sgk stimulation was largely abolished in oocytes expressing ENaC channels with C-terminal truncations or mutated PY motifs. We were also unable to confirm that Sgk directly interacts with Nedd4-2 in vitro. We conclude that the stimulatory effect of Sgk on ENaC requires the presence of the channel’s PY motifs, but it is not due to the interruption of Na+ feedback regulation. [ABSTRACT FROM AUTHOR]

The role of aldosterone in the pathogenesis of heart failure (HF) is still poorly understood. Recently, aldosterone has been shown to modulate the function of cardiac Ca2+ and K+ channels, thus playing a role in the electrical remodeling process. The goal of this work was to investigate the role of aldosterone on the cardiac Na+ current (INa). We analyzed the effects of aldosterone on INa in isolated adult mouse ventricular myocytes, using the whole cell patch-clamp technique. After 24 h incubation with 1 µM aldosterone, the INa density was significantly increased (+55%), without alteration of the biophysical properties and the cell membrane capacitance. Aldosterone (10 nM) increased the INa by 23%. In 24-h coincubation experiments, with the use of actinomycin D, cycloheximide, or brefeldin A, the effect of aldosterone on INa was abolished. Spironolactone (mineralocorticoid receptor antagonist, 10 µM) prevented the 1 µM aldosterone-dependent INa increase, whereas RU-38486 (glucocorticoid receptor antagonist, 10 µM) did not. The action potential duration (APD) was longer in aldosterone-treated (APD90: +53%) than in control myocytes. In addition, the L-type Ca2+ current was also upregulated (+48%). We performed quantitative RT-PCR measurements and Western blots to quantify the mRNA and protein levels of Nav1.5 and Cav1.2 (main channels mediating cardiac INa and ICa), but no significant difference was found. In conclusion, this study shows that aldosterone upregulates the cardiac INa and suggest that this phenomenon may contribute to the HF-induced electrical remodeling process that may be reversed by spironolactone. [ABSTRACT FROM AUTHOR]

Amiloride-sensitive epithelial Na+ channels (ENaC) play a crucial role in Na+ transport and fluid reabsorption in the kidney, lung, and colon. The magnitude of ENaC-mediated Na+ transport in epithelial cells depends on the average open probability of the channels and the number of channels on the apical surface of epithelial cells. The number of channels in the apical membrane, in turn, depends on a balance between the rate of ENaC insertion and the rate of removal from the apical membrane. ENaC is made up of three homologous subunits: á, β, and γ. The COOH-terminal domain of all three subunits is intracellular and contains a proline-rich motif (PPxY). Mutations or deletion of this PPxY motif in the β- and γ-subunits prevent the binding of one isoform of a specific ubiquitin ligase, neural precursor cell-expressed, developmentally downregulated protein (Nedd4-2), to the channel in vitro and in transfected cell systems, thereby impeding ubiquitin conjugation of the channel subunits. Ubiquitin conjugation would seem to imply that ENaC turnover is determined by the ubiquitin-proteasome system, but when Madin-Darby canine kidney cells are transfected with ENaC, ubiquitin conjugation apparently leads to lysosomal degradation. However, in untransfected renal cells (A6) expressing endogenous ENaC, ENaC is indeed degraded by the ubiquitin-proteasome system. Nonetheless, in both transfected and untransfected cells, the rate of ENaC degradation is apparently controlled by Nedd4-2 activity. In this review, we discuss the role of the ubiquitin conjugation and the aitemative degradative pathways (lysosomal or proteasomal) in regulating the rate of ENaC turnover in untransfected renal cells and compare this regulation to that of transfected cell systems. [ABSTRACT FROM AUTHOR]

The epithelial Na+ channel (ENaC) is an end-effector of diverse cellular signaling cascades, including those with phosphatidylinositide second messengers. Recent evidence also suggests that in some instances, phospatidylinositides can directly interact with ENaC to increase channel activity by increasing channel open probability and/or membrane localization. We review here findings relevant to regulation of ENaC by phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3). Similar to its actions on other ion channels, PIP2 is permissive for ENaC openings having a direct effect on gating. The PIP2 binding site in ENaC involved in this regulation is most likely localized to the NH2 terminus of β-ENaC. PIP3 also affects ENaC gating but, rather than being permissive, augments open probability. The PIP3 binding site in ENaC involved in this regulation is localized to the proximal region of the COOH terminus of γ-ENaC just following the second transmembrane domain. In complementary pathways, PIP3 also impacts ENaC membrane levels through both direct actions on the channel and via a signaling cascade involving phosphoinositide 3-OH kinase (PI3-K) and the aldosterone-induced gene product serum and glucocorticoid-inducible kinase. The putative PIP3 binding site in ENaC involved in direct regulation of channel membrane levels has not yet been identified. [ABSTRACT FROM AUTHOR]

Mutations that disrupt a PY motif in epithelial Na+ channel (ENaC) subunits increase surface expression of Na+ channels in the collecting duct, resulting in greater Na+ reabsorption. Nedd4 and Nedd4-2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. We recently reported that human Nedd4-2 (hNedd4-2) is expressed as many isoforms because of alternative promoter usage and/or variable splicing. To understand the relevance of hNedd4-2 isoforms for collecting duct Na+ transport, we studied the interaction with ENaC and the intracellular localization and function of the following three naturally occurring hNedd4-2 isoforms: full-length Nedd4-2 (Nedd4-2), Nedd4-2 lacking the NH2-terminal C2 domain (Nedd4-2AC2), and Nedd4-2 lacking the C2 domain and WW domains 2 and 3 (Nedd4-2AWW2,3). Nedd4-2 and Nedd4-2AC2 associate with ENaC and robustly reduce Na+ transport in Xenopus oocytes, whereas the interaction with and functional effect of Nedd4-2ΔWW2,3 on ENaC is weak. Nedd4-2 is expressed in the mouse collecting duct, and overexpression of Nedd4-2 reduces endogenous ENaC activity in a collecting duct cell line. This reduction in ENaC activity can be reversed early with exposure to dexamethasone, an effect that is associated with an increase in sgk1 abundance. The C2 domain is required to target Nedd4-2 to the plasma membrane in response to elevation of intracellular Ca2+ concentration ([Ca2+]i) in MDCK cells, although it does not appear to mediate the inhibitory effect of [Ca2+]i on Na+ transport. Our data illustrate that naturally occurring hNedd4-2 isoforms differentially associate with ENaC to regulate its activity. [ABSTRACT FROM AUTHOR]

Reabsorption of sodium by the epithelial sodium channel (ENaC) is essential for maintaining the volume of the extracellular compartment and blood pressure. The function of ENaC is regulated primarily by aldosterone, antidiuretic hormone [arginine vasopressin (AVP)], and insulin, but the molecular mechanisms that increase channel activity are still poorly understood. It has been proposed that the related serine/threonine kinases serum- and glucocorticoid-induced kinase (Sgk1) and protein kinase B (Akt) mediate activation of ENaC. Here, we addressed the question of whether there is functional specificity of these kinases for the activation of ENaC in epithelial cells of the distal renal tubule. We demonstrate that Akt does not increase ENaC function under basal conditions or after stimulation with aldosterone, insulin, or AVP. In contrast, under the same experimental conditions, Sgkl1increases ENaC activity by 10-fold. The effect of Sgk1 is additive to that of aldosterone, whereas, in the presence of active Sgk1, cells do not further respond to insulin or AVP. We conclude that, in cells expressing both kinases, modulation of ENaC activity is mediated by Sgk1 but not by Akt1. [ABSTRACT FROM AUTHOR]

Aldosterone is a key regulator of epithelial Na+ channels (ENaC) in renal cortical collecting ducts (CCD). The goal of this study was to examine whether serum- and glucocorticoid-inducible kinase-1 (SGK1), an aldosterone-induced gene, is vital to the delayed effect of aldosterone by increasing the gene expression of ENaC subunits. To test this hypothesis, we compared the levels of ENaC mRNA in mouse CCD cells that stably express either full-length (FL)-SGK1 or a kinase-dead dominant negative (K127M)-SGK1. Our results revealed that SGK1 regulates gene expression of ENaC, whether cells are maintained in steroid-free media or in the presence of corticosteroids (CS) and/or other growth factors. Under all conditions, the loss of function of SGK1 caused a significant decrease in the expression of α- and β-ENaC, but not γ-ENaC. Compared with cells expressing FL-SGK1, K127M-SGK1 decreased the expression of α- and β-subunit mRNA by ∼45 and ∼90%, respectively. Next, to determine whether SGK1 is one of the proteins mediating the induction of α-ENaC mRNA by CS, we compared steroid induction of α-ENaC in cells expressing K127M-SGK1 vs. FL-SGK1. The maximum level of α-ENaC mRNA levels following CS was significantly (∼45%) higher in FL-SGK1-vs. K127M-SGK1-expressing cells, although the fold-induction by CS was similar in both FL-SGK1- and K127M-SGK 1-expressing cells. In summary, we report for the first time that SGK1 regulates transcription of ENaC subunits. We propose that the effect of SGK1 on ENaC transcription is mediated by the activation of unidentified transcription factors. [ABSTRACT FROM AUTHOR]

Amiloride-sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate-limiting step for Na+ absorption in kidney collecting ducts, and epidermal growth factor (EGF) inhibits Na+ transport and ENaC expression. A pathognomonic feature of polycystic kidney disease (PKD) is EGF receptor mislocalization to the apical plasma membrane and EGF/EGF receptor axis overactivity. Immunohistochemical and biochemical analysis revealed mislocalization of EGF receptor and excessive activation of the p42/44 extracellular signal-regulated protein kinase pathway (ERK1/2) in kidneys from cystic mice compared with noncystic littermates. Primary monolayer cultures of non- cystic and cystic murine collecting duct principal cells were used to identify aberrant EGF-dependent ERK1/2 activation and regulation of Na+ transport associated with autosomal recessive PKD. Addition of EGF to the basolateral bathing solution of noncystic or cystic monolayers led to p42/44 phosphorylation and inhibition of Nat transport (30–35%), whereas apical EGF was effective only in monolayers derived from cystic mice. p42/44 Phosphorylation and inhibition of Na+ transport were prevented by prior treatment of the cells with an ERK kinase inhibitor. Chronic treatment (24 h) of noncystic and cystic monolayers with basolateral EGF elicited sustained inhibition of Nat absorption (50–55%) and a reduction in steady-state ENaC mRNA levels (50–75%). In contrast, addition of EGF to the apical bathing solution (24 h) had no effect in noncystic monolayers but led to inhibition of Nat transport (50–60%) and decreased ENaC expression (45–60%) in cystic cells. Pretreatment of the monolayers with an ERK kinase inhibitor abolished the chronic effects of EGF on Na+ transport. The results of these studies reveal that the mislocalized apical EGF receptors are functionally coupled to the ERK pathway and that abnormal EGF-dependent regulation of ENaC function and expression may contribute to PKD pathophysiology. [ABSTRACT FROM AUTHOR]

The epithelial sodium channel is found in apical membranes of a variety of native epithelial tissues, where it regulates sodium and fluid balance. In vivo, a number of hormones and other endogenous factors, including polyunsaturated fatty acids (PUFAs), regulate these channels. We tested the effects of essential n-3 and n-6 PUFAs on amiloride-sensitive sodium transport in A6 epithelial cells. Eicosapentaenoic acid [EPA; C20:5(n-3)] transiently stimulated amiloride-sensitive open-circuit current (INa) from 4.0 ± 0.3 to 7.7 ± 0.3 µA/cm² within 30 min (P < 0.001). No activation was seen in the presence of 10 µM amiloride. In cell-attached but not in cell-excised patches, EPA acutely increased the open probability of sodium channels from 0.45 ± 0.08 to 0.63 ± 0.10 (P = 0.02, paired t-test), n-6 PUFAs, including linoleic acid (C18:2), eicosatetraynoic acid (C20: 4), and docosapentanoic acid (C22:5) had no effect, whereas n-3 docosahexanoic acid (C22:6) activated amiloride-sensitive INa in a manner similar to EPA. Activation of INa by EPA was prevented by H-89, a PKA inhibitor. Similarly, PKA activity was stimulated by EPA. Nonspecific stimulation of phosphodiesterase activity by COCl2 completely prevented the effect of EPA on sodium transport. We conclude that n-3 PUFAs activate epithelial sodium channels downstream of cAMP in a cAMP-dependent pathway also involving PKA. [ABSTRACT FROM AUTHOR]

The epithelial Na2 channel (ENaC) is regulated by the ubiquitin-protein ligase Nedd4-2 via interaction with ENaC PY-motifs. These PY-motifs are mutated/ deleted in Liddle's syndrome, resulting in elevated Na2 reabsorption and hypertension explained partly by impaired ENaC-Nedd4-2 interaction. We hypothesized that Nedd4-2 is a susceptibility gene for hypertension and screened 856 renal patients and healthy controls for mutations in a subset of exons of the human Nedd4-2 gene that are relevant for ENaC regulation by PCR/single-strand conformational polymorphism. Several variants were identified, and one nonsynonymous mutation (Nedd4-2-P355L) was further characterized. This mutation next to the 3' donor site of exon 15 does not affect in vitro splicing of Nedd4-2 mRNA. However, in the Xenopus oocyte expression system, Nedd4-2- P355L-dependent ENaC inhibition was weaker compared with the wild type (Nedd4-2-WT), and this difference depended on the presence of intact PY-motifs on ENaC. This could not be explained by the amount of wild type or mutant Nedd4- 2 coimmunoprecipitating with ENaC. When the phosphorylation level of human Nedd4-2 Ser448 (known to be phosphorylated by the Sgk1 kinase) was determined with a specific anti-pSer448 antibody, we observed stronger basal phosphorylation of Nedd4-2-P355L. Both the phosphorylation level and the accompanying amiloride-sensitive Na2 currents could be further enhanced to approximately the same levels by coexpressing Sgk1. In addition, the role of the two other putative Sgk1 phosphorylation sites (S342 and T367) appears also to be affected by the P355L mutation. The differential phosphorylation status between wild-type and mutant Nedd4-2 provides an explanation for the different potential to inhibit ENaC activity. [ABSTRACT FROM AUTHOR]

Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na+ channel by phosphorylating the ubiquitin ligase Nedd4-2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na+-Pi) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4-2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4-2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current (IP). Exposure to 3 mM phosphate induces an IP in NaPi IIb-expressing oocytes. Coinjection of Nedd4-2, but not the catalytically inactive mutant C938SNedd4-2, significantly downregulates IP, whereas the coinjection of S422DSGK1 markedly stimulates IP and even fully reverses the effect of Nedd4-2 on IP. The effect of S422DSGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active T308D,S473DPKB, or inactive K127NSGK1. Moreover, S422DSGK1 and SGK3 phosphorylate Nedd4-2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4-2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance. [ABSTRACT FROM AUTHOR]

Genetic analysis has demonstrated that Na absorption in the aldosterone-sensitive distal nephron (ASDN) critically determines extracellular blood volume and blood pressure variations. The epithelial sodium channel (ENaC) represents the main transport pathway for Na + absorption in the ASDN, in particular in the connecting tubule (CNT), which shows the highest capacity for ENaC-mediated Na + absorption. Gain-of-function mutations of ENaC causing hypertension target an intracellular proline-rich sequence involved in the control of ENaC activity at the cell surface. In animal models, these ENaC mutations exacerbate Na + transport in response to aldosterone, an effect that likely plays an important role in the development of volume expansion and hypertension. Recent studies of the functional consequences of mutations in genes controlling Na + absorption in the ASDN provide a new understanding of the molecular and cellular mechanisms underlying the pathogenesis of salt-sensitive hypertension. [ABSTRACT FROM AUTHOR]

Mutations that disrupt a PY motif in epithelial Na[sup +] channel (ENaC) subunits increase surface expression of Na[sup +] channels in the collecting duct, resulting in greater Na[sup +] reabsorption. Recently, Nedd4 and Nedd4-2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. To further understand the role of human Nedd4-2 (hNedd4-2), we cloned its cDNAs and determined its genomic organization using a bioinformatic approach. The gene is present as a single copy, spans at least 400 kb, and contains >40 exons. Multiple 5'-exons were identified by 5'-rapid amplification of cDNA ends, and tissuespecific expression of these transcripts was noted by RT-PCR and RNase protection assay. Alternate polyadenylation signal sequences led to varying lengths of the 3'-untranslated region. Alternate splicing events within internal exons were also noted. Open reading frame analysis indicates that hNedd4-2 encode multiple protein variants with and without a C2 domain, and with a variable number of WW domains. Coexpression, in Fischer rat thyroid epithelia, of ENaC and Nedd4-2 cDNAs leads to a significant reduction in amiloride-sensitive currents, confirming a role in Na[sup +] transport regulation. In vitro binding studies demonstrated that individual PY motifs of α-, β-, and γ-ENaC have strong affinity for WW domains 3 and 4 but not 1 and 2. These studies indicate that alternate transcripts of Nedd4-2 may interact with ENaC differently. Understanding the function of variant proteins will increase our knowledge of the role of hNedd4-2 in the regulation of ENaC and define protein domains important for Nedd4-2 function. [ABSTRACT FROM AUTHOR]

Liddle's syndrome is a monogenic form of hypertension caused by mutations in the PY motif of the COOH terminus of β- and γ-epithelial Na[sup +] channel (ENaC) subunits. These mutations lead to retention of active channels at the cell surface. Because of the critical role of this PY motif in the stability of ENaCs at the cell surface, we have investigated its contribution to the ENaC response to aldosterone and vasopressin. Mutants of the PY motif in β- and γ-ENaC subunits (βY618A, β-P616L, β-R564stop, and γ-K570stop) were stably expressed by retroviral gene transfer in a renal cortical collecting duct cell line (mpkCCD[sub c14]), and transepithelial Na[sup +] transport was assessed by measurements of the benzamil-sensitive short-circuit current (I[sub sc]). Cells that express ENaC mutants of the PY motif showed a five- to six-fold higher basal I[sub sc] compared with control cells and responded to stimulation by aldosterone (10[sup -6] M) or vasopressin (10[sup -9] M) with a further increase in I[sub sc]. The rates of the initial increases in I[sub sc] after aldosterone or vasopressin stimulation were comparable in cells transduced with wild-type and mutant ENaCs, but reversal of the effects of aldosterone and vasopressin was slower in cells that expressed the ENaC mutants. The conserved sensitivity of ENaC mutants to stimulation by aldosterone and vasopressin together with the prolonged activity at the cell surface likely contribute to the increased Na[sup +] absorption in the distal nephron of patients with Liddle's syndrome. [ABSTRACT FROM AUTHOR]

Renal A6 epithelial cells were used to determine the mechanism by which protein kinase C (PKC) decreases epithelial Na[sup +] channel (ENaC) activity. Activation of PKC reduced relative Na[sup +] reabsorption to <20% within 60 min. This decrease was sustained over the next 24-48 h. In response to PKC signaling, α-, β-, and γ-ENaC levels were 0.97, 0.36, and 0.39, respectively, after 24 h, with the levels of the latter two subunits being significantly decreased. The PKC-mediated decreases in β- and γ-ENaC were significantly reversed by simultaneous addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-½ inhibitors U-0126 and PD-98059. These inhibitors, in addition, protected Na[sup +] reabsorption from PKC, demonstrating that the MAPK½ cascade, in some instances, plays a central role in downregulation of ENaC activity. The effects of PKC on β- and γ-ENaC levels were additive with those of inhibitors of transcription (actinomycin D) and translation (emetine and cycloheximide), suggesting that PKC promotes subunit degradation and does not affect subunit synthesis. The bulk of whole cell γ-ENaC was degraded within 1 h after treatment with inhibitors of synthesis; however, a significant pool was "protected" from inhibitors for up to 12 h. PKC affected this protected pool of γ-ENaC. Moreover, proteosome inhibitors (MG-132 and lactacystin) reversed PKC effects on this protected pool of γ-ENaC. Thus PKC signaling via MAPK½ cascade activation in A6 cells promotes degradation of γ-ENaC. [ABSTRACT FROM AUTHOR]

The purpose of this study was to examine the role of the serum- and glucocorticoidinduced kinase (SGK) in the activation of the epithelial sodium channel (ENaC) by aldosterone, arginine vasopressin (AVP), and insulin. We used a tetracycline-inducible system to control the expression of wild-type (SGK[sup T, sub wt]), constitutively active (S425D mutation; SGK[sup T, sub S425D]), or inac- tive (K130M mutation ; SGK[sup T, sub K13OM]) SGK in A6 cells inde- pendently of hormonal stimulation. The effect of SGK expression on ENaC activity was monitored by measuring transepithelial amiloride-sensitive short-circuit current (I[sub SC]) of transfected A6 cell lines. Expression of SGK[sup T, sub wt] or SGK[sup T, sub S425D] and aldosterone stimulation have additive effects on I[sub SC]. Although SGK could play some role in the aldosterone response, our results suggest that other mechanisms take place. SGK[sup T, sub 425D] abrogates the responses to AVP and insulin; hence, in the signaling pathways of these hormones there is a shared step that is stimulated by SGK. Because AVP and insulin induce fusion of vesicles to the apical membrane, our results support the notion that SGK promotes incorporation of channels in the apical membrane. [ABSTRACT FROM AUTHOR]

AbstractThe serum and glucocorticoid-inducible kinase SGK1 stimulates the Na+ channels ENaC and SCN5A, the K+ channels ROMK1, Kv1.3, and KCNE1/KCNQ1, the cation conductance induced by 4F2/LAT1 and the chloride conductance induced by CFTR. The isoforms SGK2 and SGK3 have similarly been shown to regulate ENaC, SCN5A, Kv1.3 and KCNE1/KCNQ1. The kinases regulate channel abundance in the plasma membrane in part by inhibition of the ubiquitin ligase Nedd4-2 and in part by interaction with trafficking molecules such as the Na+/H+ exchanger regulating factor NHERF2. An in vivo role of SGK1 mediated ENaC channel regulation in renal salt excretion and blood pressure control is documented by the impaired ability of SGK1 knockout mice to adequately reduce renal Na+ output and maintain blood pressure during dietary salt restriction and by enhanced blood pressure in individuals carrying certain polymorphisms in the SGK1 gene. The in vivo physiological significance of SGK dependent regulation of the other channels remains to be shown even though circumstantial evidence points to involvement in the regulation of epithelial transport, cell volume, cell proliferation, cardiac action potential and neuroexcitability. There is little doubt that further channels will be identified which are modulated by the SGKs and that further in vivo physiological functions will be defined where channel regulation by the SGKs plays a critical role.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Examines the increase probability of phosphatase inhibitors of sodium channels in A6 cells. Fluctuation analysis of cell-attached patches with power spectra frequencies; Phosphorylation of regulatory molecule associated with the channel; Regulation of kinase in the changes of protein function.

Studies the concerted action of epithelial sodium channel (ENaC) and proteins Nedd4-2 and Sgk1 in transepithelial sodium transport. Suppression of ENaC cell surface expression and activity; Effect of aldosterone-induced Sgk1 on the cell surface activity and expression of ENaC; Convergence point among the ENaC, Nedd4-2 and Sgk1.

Examines the structures and various functions of epithelial sodium channel and the degenerin family of ion channels. Participation in mechanotransduction in neuronal cells; Mediation of sodium transport in epithelial and sodium homeostasis; Blockage of pharmacological ligands.