ENaC Surface Expression Research Articles

Regulation of ENaC Expression by Paraoxonase 3

The epithelial sodium channel (ENaC) mediates the rate‐limiting step of Na+ uptake across the apical membrane of specific epithelia. ENaC‐dependent Na+ absorption in the kidney has important roles in regulating extracellular fluid volume, extracellular [K+] and blood pressure. ENaC functional expression is tightly regulated by multiple intracellular and exogenous factors, including molecular chaperones that are implicated in key steps during ENaC biogenesis. We previously showed that ENaC activity was reduced by paraoxonase 2 (PON2) in Xenopus oocytes. PON2–mediated inhibition is associated with reduced surface expression of ENaC, suggesting a role of PON2 in regulating ENaC turnover and/or trafficking. In supporting this notion, PONs are the mammalian orthologues of MEC‐6, a C. elegans ER‐resident chaperone. MEC‐6 is required for the proper assembly and surface expression of the touch‐sensing MEC‐4/MEC‐10 channel in worm touch‐receptor neurons. If the chaperone function is conserved between MEC‐6 and mammalian PONs, we predict other members of this family will also regulate the functional expression of related ion channels. Indeed, we found that PON3 is expressed in principal cells of the distal nephron, where ENaC resides. The expression of PON3 inhibits ENaC activity in Xenopus oocytes. The protease chymotrypsin activates ENaC by releasing the embedded inhibitory tracts, thus increasing channel open probability (Po). We found that chymotrypsin‐mediated ENaC activation was not altered by PON3, suggesting that PON3 does not inhibit ENaC activity by reducing channel Po. In addition, ENaC expression in Fisher rat thyroid cells was reduced by PON3. Future studies will investigate the mechanism by which PON3 regulates ENaC functional expression as well as the potential roles of PON3 in regulating ENaC activity, blood pressure and extracellular [K+] in the Pon3 KO mice. Understanding mechanisms by which PON3 regulates ENaC expression will provide novel insights into both normal and altered ENaC functional states, such as hypertension.Support or Funding InformationDK103834, DK038470, DK079307This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Regulatory Mechanism of ENaC Surface Expression

A Regulatory Mechanism of ENaC Surface Expression

Inhibitors of the proteasome stimulate the epithelial sodium channel (ENaC) through SGK1 and mimic the effect of aldosterone.

The epithelial sodium channel (ENaC) marks the tightly regulated, rate-limiting step of sodium re-absorption in the aldosterone-sensitive distal nephron (ASDN). Stimulation of ENaC activity by aldosterone involves the serum and glucocorticoid-induced kinase 1 (SGK1) and is mediated via complex mechanisms including inhibition of channel retrieval. Retrieved channels may be recycled or degraded, e.g. by the proteasomal pathway. The aim of the present study was to investigate whether inhibitors of the proteasome affect ENaC activity and surface expression, and to explore a possible involvement of SGK1. Short circuit current (I SC) measurements were performed on confluent mCCDcl1 murine cortical collecting duct cells to investigate the effect of two distinct proteasomal inhibitors, MG132 and bortezomib, on amiloride-sensitive ENaC-mediated I SC. Both inhibitors robustly stimulated amiloride-sensitive I SC. The time course and magnitude of the stimulatory effect of the proteasomal inhibitors on I SC were similar to those of aldosterone. Both, MG132 and aldosterone, significantly increased the abundance of β-ENaC at the cell surface. SGK1 activity was assessed by monitoring the phosphorylation of a downstream target, NDRG1, and was found to be increased by MG132. Importantly, inhibiting SGK1 activity prevented not only the stimulatory effect of aldosterone but also that of proteasomal inhibition. In conclusion, these data suggest that ENaC stimulation following proteasomal inhibition is due to an accumulation of active SGK1 resulting in increased expression of ENaC at the cell surface. Thus, inhibition of the proteasome mimics SGK1-dependent stimulation of ENaC by aldosterone.

Expression of intersectin 1/2 is repressed by aldosterone through microRNAs in the CCD to alter ENaC‐mediated Na+ transport (893.4)

Aldosterone is a well characterized regulator of the epithelial sodium channel (ENaC), and increases transepithelial Na+ transport in the kidney CCD. Here we report on two related scaffold proteins, intersectin (Itsn) 1 and 2 that are aldosterone repressed via microRNA regulation. Itsns are involved in coordinating vesicle trafficking events and modulating several signaling pathways. In a cultured mCCD‐cl1 cell line, Itsn expression is reduced by aldosterone (50nM, from 6hrs), which in turn increases ENaC activity. Exogenous depletion of Itsn 1&2 by siRNA increases ENaC activity (175±18% over control), while overexpression of Itsns reduces ENaC activity (40±6% of control, n=8). Immunolocalization of Itsn demonstrates a vesicular staining pattern consistent with a role in mediating ENaC endocytosis. To confirm this, we performed transepithelial capacitance recordings to assess changes in membrane surface area and demonstrate an increase in membrane retrieval with Itsn overexpression. Similar to its reported role in ROMK internalization we demonstrate here that Itsn is an aldosterone‐repressed protein (via microRNAs), involved in the altering of ENaC surface expression by regulating channel endocytosis. The molecular mechanism that underlies this function is currently under investigation.Grant Funding Source: Supported by DK078917

The phosphorylation of endogenous Nedd4-2 In Na+—absorbing human airway epithelial cells

Neural precursor cell expressed, developmentally down-regulated protein 4-2 (Nedd4-2) mediates the internalisation / degradation of epithelial Na+ channel subunits (α-, β- and γ-ENaC). Serum / glucocorticoid inducible kinase 1 (SGK1) and protein kinase A (PKA) both appear to inhibit this process by phosphorylating Nedd4-2-Ser221, -Ser327 and -Thr246. This Nedd4-2 inactivation process is thought to be central to the hormonal control of Na+ absorption. The present study of H441 human airway epithelial cells therefore explores the effects of SGK1 and / or PKA upon the phosphorylation / abundance of endogenous Nedd4-2; the surface expression of ENaC subunits, and electrogenic Na+ transport. Effects on Nedd4-2 phosphorylation/abundance and the surface expression of ENaC were monitored by western analysis, whilst Na+ absorption was quantified electrometrically. Acutely (20min) activating PKA in glucocorticoid-deprived (24h) cells increased the abundance of Ser221-phosphorylated, Ser327-phosphorylated and total Nedd4-2 without altering the abundance of Thr246-phosphorylated Nedd4-2. Activating PKA under these conditions did not cause a co-ordinated increase in the surface abundance of α-, β- and γ-ENaC and had only a very small effect upon electrogenic Na+ absorption. Activating PKA (20min) in glucocorticoid-treated (0.2µM dexamethasone, 24h) cells, on the other hand, increased the abundance of Ser221-, Ser327- and Thr246-phosphorylated and total Nedd4-2; increased the surface abundance of α-, β- and γ-ENaC and evoked a clear stimulation of Na+ transport. Chronic glucocorticoid stimulation therefore appears to allow cAMP-dependent control of Na+ absorption by facilitating the effects of PKA upon the Nedd4-2 and ENaC subunits.

Hsc70 negatively regulates epithelial sodium channel trafficking at multiple sites in epithelial cells

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.

Identification of Accessory Proteins Critical for the Epithelial Sodium Channel (ENaC)

The epithelial sodium channel (ENaC) is rate‐limiting for the reabsorption of sodium in the distal nephron of the kidneys. Genetic mutations in ENaC can cause diseases such as Liddle's syndrome and pseudohypoaldosteronism type 1, causing severe hypertension or hypotension, respectively. Accessory proteins including Nedd4, ubiquitin protein‐ligase, and a serum and glucocorticoid‐regulated kinase have been shown to regulate ENaC and additional proteins are likely to play an essential role in its proper function. In an effort to identify proteins necessary for proper assembly, localization, and function of ENaC, BY4742 yeast deletion strains were transformed with αENaC, which induces a salt‐sensitive phenotype, and yeast dilution pronging assays were performed. While five deletion strains showed no effect on ENaC function in yeast, three strains lacking scj1, jem1, or lhs1 showed loss of ENaC function while deletion of ypk1 seemed to enhance ENaC function. Localization studies of αENaC in these deletion strains were used to elucidate the role of each gene on ENaC surface expression. Funded by NIH (R15GM086798) and Welch Foundation (Dept Grant A10048).

Pendrin gene ablation reduces ENaC surface expression and open probability

Epithelial sodium channels, ENaC, mediate Na+ absorption and modulate Cl− absorption in the cortical collecting duct (CCD). Since ENaC function is reduced in pendrin KO mice given aldosterone, we determined if pendrin modulates ENaC open probability (PO) and/or surface expression. Pendrin KO and wild type mice were given aldosterone by minipump. ENaC PO and density were measured in split open CCDs. ENaC subcellular distribution was determined by quantitative immunohistochemistry. Transepithelial voltage (VT) and Clabsorption (JCl) were measured in CCDs perfused in vitro. Apical membrane ENaC abundance was lower in pendrin null than in wild type mice in the CCD, although total protein abundance was similar. Single channel recordings showed that both channel number and PO were reduced in pendrin null mice. Benzamil‐sensitive JCl depends on ENaC. We examined the magnitude of benzamil‐sensitive VT and JCl. Both total and benzamil‐sensitive JCl and VT were reduced in pendrin null mice. However, increasing apical ENaC expression by introducing the Liddle's mutation into the pendrin null mouse raised JCl and VT to levels observed in wild type mice. In conclusion, pendrin regulates ENaC activity through regulating both ENaC trafficking and the channel open probability. Constitutive expression of ENaC on the apical membrane by introduction of the Liddle's mutation, restores JCl and VT in the CCD in pendrin null mice.

A mutation in the β-subunit of ENaC identified in a patient with cystic fibrosis-like symptoms has a gain-of-function effect

In some patients with atypical cystic fibrosis (CF), only one allele of the CF transmembrane conductance regulator (CFTR) gene is affected. Mutations of the epithelial sodium channel (ENaC) may contribute to the pathophysiology of the disease in these patients. To functionally characterize a mutation in the β-subunit of ENaC (βV348M) recently identified in a patient with severe CF-like symptoms (Mutesa et al. 2009), we expressed wild-type (wt) αβγENaC or mutant αβV348MγENaC in Xenopus laevis oocytes. The βV348M mutation stimulated amiloride-sensitive whole-cell current (ΔI(ami)) by ∼40% but had no effect on surface expression or single-channel conductance of ENaC. Instead the mutation increased channel open probability (P(o)). Proteolytic activation of mutant ENaC by chymotrypsin was reduced compared with that of wt ENaC (∼3.0-fold vs. ∼4.2-fold), which is consistent with the increased baseline P(o) of mutant ENaC. Similarly, the ENaC activator S3969 stimulated mutant ENaC currents to a lesser degree (by ∼2.6-fold) than wt ENaC currents (by ∼3.5-fold). The gain-of-function effect of the βV348M mutation was confirmed by whole-cell current measurements in HEK293 cells transiently transfected with wt or mutant ENaC. Computational channel modeling in combination with functional expression of different βV348 mutants in oocytes suggests that the βV348M mutation increases channel P(o) by destabilizing the closed channel state. Our findings indicate that the gain-of-function effect of the βV348M mutation may contribute to CF pathophysiology by inappropriately increasing sodium and fluid absorption in the respiratory tract.

Hsp70 Promotes Epithelial Sodium Channel Functional Expression by Increasing Its Association with Coat Complex II and Its Exit from Endoplasmic Reticulum

The epithelial sodium channel (ENaC) plays an important role in the homeostasis of blood pressure and of the airway surface liquid, and inappropriate regulation of ENaC results in refractory hypertension (in Liddle syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsp70, is not completely understood. Building on the previous suggestion by our group that Hsp70 promotes ENaC functional and surface expression in Xenopus oocytes, we investigated the mechanism by which Hsp70 acts upon ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsp70, treatment with 1 or 2 μg/ml doxycycline increased total Hsp70 expression ~2-fold and ENaC functional expression ~1.4-fold. This increase in ENaC functional expression corresponded to an increase in ENaC expression at the apical surface of the cells and was not present when an ATPase-deficient Hsp70 was similarly overexpressed. The increase in functional expression was not due to a change in the rate at which ENaC was retrieved from the apical membrane. Instead, Hsp70 overexpression increased the association of ENaC with the Sec24D cargo recognition component of coat complex II, which carries protein cargo from the endoplasmic reticulum to the Golgi. These data support the hypothesis that Hsp70 promotes ENaC biogenesis and trafficking to the apical surface of epithelial cells.

Multiple residues in the distal C terminus of the α-subunit have roles in modulating human epithelial sodium channel activity

Epithelial sodium channels (ENaC) are critically important in the regulation of ion and fluid balance in both renal and respiratory epithelia. ENaC functional polymorphisms may contribute to alterations in blood pressure in the general population. We previously reported that the A663T polymorphism in the C terminus of the α-subunit altered ENaC functional and surface expression in Xenopus laevis oocytes (Samaha FF, Rubenstein RC, Yan W, Ramkumar M, Levy DI, Ahn YJ, Sheng S, Kleyman TR. J Biol Chem 279: 23900-23907, 2004). We examined whether sites in the vicinity of 663 influenced channel activity by performing scanning Ala mutagenesis. Interestingly, only αT663/G667Aβγ channels exhibited increased currents compared with αT663βγ. This increase in channel activity reflected an increase in channel open probability and not an increase in channel surface expression. In contrast, decreases in channel activity were observed with both αT663/C664Aβγ and αT663/C664Mβγ channels. The decrease in functional expression of αT663/C664Mβγ channels correlated with decreased surface expression, suggesting that the αC664M mutation altered the intracellular trafficking of the channel. While cytoplasmic Cys residues may be modified by the addition of palmitate, we did not observe palmitoylation of αC664. Our results suggest that multiple residues in the distal part of the cytoplasmic C terminus have roles in modulating channel activity.

A physiological role of p38 on aldosterone‐dependent regulation of ENaC endocytosis in renal epithelial A6 cells

Aldosterone (ALD) is a crucial hormone to regulate ENaC‐mediated Na+ reabsorption in the kidney for controlling ECF volume and blood pressure. So far, we found ALD activated p38 and pretreatment with SB202190 (a specific p38 inhibitor: SB) abolished ALD‐stimulated benzamil‐sensitive Isc (INa ) by reducing benzamil‐sensitive conductance (GNa). In this study, we investigated a physiological role of p38 in the surface expression of ENaC through exocytic and endocytic pathways in ALD‐treated renal epithelial A6 cells by using MG132 (a proteasome inhibitor) and brefeldin A (an inhibitor of protein translocation). Pretreatment with MG132 mostly recovered the SB‐induced reduction of INa, whreas brefeldin A showed further an inhibitory effect on the SB‐decreased INa. Moreover, SB decreased both phosphorylated SGK1 and Nedd4‐2. These suggest that the p38‐ mediated signal pathway suppresses the endocytic/proteasome‐dependent ENaC degradation contributing to the stimulation of Na+ reabsorption by ALD. Supported by JSPS (19590212, 20390060), SSRF (0837, 1035) and FFPR.

Hsp70 Promotes ENaC Functional Expression and Association with COPII

Abnormal regulation of the Epithelial Sodium Channel (ENaC) causes disorders of blood pressure and of airway clearance. Our group's previous studies of ENaC regulation in Xenopus oocytes demonstrated that the highly homologous 70 kDa heat shock proteins Hsc70 and Hsp70 had opposite effects on ENaC function and surface expression (Goldfarb, et al., PNAS, 2006). Here, we probed the mechanism underlying these differential effects in MDCK epithelial cells that stably express αβγ-ENaC and have inducible overexpression of Hsp70. Hsp70 overexpression increased ENaC functional expression (amiloride-sensitive Isc) in Ussing chambers, but did not alter the rate at which functional ENaC was retrieved from the apical membrane. This correlated with increased expression of ENaC at the apical surface in biotinylation experiments. In contrast, Hsp70 overexpression increased ENaC's association with Sec24D in immunoprecipitation experiments. Sec24D is the cargo recognition component of the COPII ER exit complex. These data support the hypothesis that Hsp70 increases ENaC functional and surface expression in MDCK cells by promoting ENaC's exit from the ER and trafficking to the apical surface.

Epithelial sodium channel gating is modulated by cytoplasmic Cys‐palmitoylation

ENaC mediates amiloride-sensitive Na+ transport across the apical membrane of high resistance epithelia including kidney, lung and colon. Channel activity is modulated by changes in either membrane trafficking or gating. We previously reported that mouse ENaC gating was regulated by cytoplasmic Cys-palmitoylation of the beta subunit (Mueller et al., 2010 J Biol Chem 285, 30453–62). We also noted that the gamma subunit, but not the alpha subunit, was modified with palmitate. We now report that mutation of either of the two cytoplasmic gamma subunit Cys residues (C33A or C41A) inhibits ENaC activity and enhances Na+ self-inhibition of channels expressed in Xenopus oocytes, while ENaC surface expression and proteolytic processing are unchanged. Interestingly, single channel open probability of ENaC was significantly reduced for only one of the mutations. As protein palmitoylation is reversible and could directly regulate ENaC activity, we have begun to identify specific mouse palmitoyltransferase(s) that modulate ENaC activity when co-expressed with wild-type but not mutant ENaCs in oocytes. To date, two palmitoyltransferases that are expressed in kidney and lung enhance ENaC activity. We conclude that ENaC gating is modulated by palmitoylation of both its beta and gamma subunits. (NIH DK65161 and DK079307)

Identification of CNK3 in the Aldosterone‐regulated ENaC Regulatory Complex

Members of the Connector Enhancer of KSR (CNK) family of proteins possess multiple protein interaction domains, and have been proposed to function as scaffolds possibly assisting various interactions in a multiple signaling cascade. Mammalian CNK3 is an aldosterone‐induced protein that is essential for the activity of the epithelial sodium channel (ENaC). We have shown earlier that ENaC surface expression and activity are regulated by the ENaC‐regulatory complex, GILZ1 and SGK1 are important aldosterone‐induced components thereof. The present study examines the role of CNK3 in GILZ1/SGK1‐mediated ENaC stimulation. Co‐immunoprecipitation experiments revealed specific physical interactions of CNK3 with both GILZ1 and SGK1 in an aldosterone‐dependent manner. GILZ1 markedly enhances the interaction of CNK3 with SGK1, and also augments CNK3 multimerization, an attribute that has been shown to be critical for protein scaffolding of other larger PDZ domain‐containing proteins. The present study suggests that CNK3 is an important component of the aldosterone‐regulated ENaC regulatory complex.

Multiple Residues in the Distal C‐terminus of the Alpha Subunit Have Roles in Modulating ENaC Activity

Epithelial sodium channels (ENaC) are critically important in the regulation of ion and fluid balance in both renal and respiratory epithelia. ENaC functional polymorphisms may contribute to alterations in blood pressure in the general population. We previously showed that the A663T polymorphism in the C-terminus of the α-subunit increases ENaC functional and surface expression in Xenopus oocytes. We examined whether mutations of residues adjacent to A663 also influenced channel activity by performing scanning Ala mutagenesis of the 10 residues flanking αT663. Interestingly, only the G667A increased ENaC currents compared to wild type. This residue is not conserved across species. In contrast, C664 is conserved in the human, mouse and rat α subunit. The C664M mutation decreased the functional expression of αT663 ENaC to a level similar to that of αA663. The C664M mutation had no effect on the functional expression of αA663 ENaC. The decrease in αT663/C664M ENaC functional expression correlated with decreased expression of β ENaC at the oocyte surface, but not in whole oocyte β ENaC expression, suggesting that α T663/C664M ENaC had altered intracellular trafficking relative to αT663 ENaC. Our results suggest that multiple residues in the distal part of the cytoplasmic C-terminus have roles in modulating channel activity.

Glucocorticoid-induced Leucine Zipper 1 Stimulates the Epithelial Sodium Channel by Regulating Serum- and Glucocorticoid-induced Kinase 1 Stability and Subcellular Localization

Serum- and glucocorticoid-induced kinase 1 (SGK1) is a multifunctional protein kinase that markedly influences various cellular processes such as proliferation, apoptosis, glucose metabolism, and sodium (Na(+)) transport via the epithelial Na(+) channel, ENaC. SGK1 is a short-lived protein, which is predominantly targeted to the endoplasmic reticulum (ER) to undergo rapid proteasome-mediated degradation through the ER-associated degradation (ERAD) system. We show here that the aldosterone-induced chaperone, GILZ1 (glucocorticoid-induced leucine zipper protein-1) selectively decreases SGK1 localization to ER as well as its interaction with ER-associated E3 ubiquitin ligases, HRD1 and CHIP. GILZ1 inhibits SGK1 ubiquitinylation and subsequent proteasome-mediated degradation, thereby prolonging its half-life and increasing its steady-state expression. Furthermore, comparison of the effect of GILZ1 with that of proteasome inhibition (by MG-132) supports the idea that these effects of GILZ1 are secondary to physical interaction of GILZ1 with SGK1 and enhanced recruitment of SGK1 to targets within an "ENaC regulatory complex," thus making less SGK1 available to the ERAD machinery. Finally, effects of GILZ1 knockdown and overexpression strongly support the idea that these effects of GILZ1 are functionally important for ENaC regulation. These data provide new insight into how the manifold activities of SGK1 are selectively deployed and strengthened through modulation of its molecular interactions, subcellular localization, and stability.

A mutation of the epithelial sodium channel associated with atypical cystic fibrosis increases channel open probability and reduces Na+ self inhibition

Increased activity of the epithelial sodium channel (ENaC) in the respiratory airways contributes to the pathophysiology of cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some patients suffering from atypical CF a mutation can be identified in only one CFTR allele. We recently identified in this group of CF patients a heterozygous mutation (W493R) in the alpha-subunit of ENaC. Here, we investigate the functional effects of this mutation by expressing wild-type alpha beta gamma ENaC or mutant alpha(W493R)beta gamma ENaC in Xenopus oocytes. The alpha W493R mutation stimulated amiloride-sensitive whole-cell currents (Delta I(ami)) by approximately 4-fold without altering the single-channel conductance or surface expression of ENaC. As these data suggest that the open probability (P(o)) of the mutant channel is increased, we investigated the proteolytic activation of ENaC by chymotrypsin. Single-channel recordings revealed that chymotrypsin activated near-silent channels in outside-out membrane patches from oocytes expressing wild-type ENaC, but not in membrane patches from oocytes expressing the mutant channel. In addition, the alpha W493R mutation abolished Na(+) self inhibition of ENaC, which might also contribute to its gain-of-function effects. We conclude that the alpha W493R mutation promotes constitutive activation of ENaC by reducing the inhibitory effect of extracellular Na(+) and decreasing the pool of near-silent channels. The resulting gain-of-function phenotype of the mutant channel might contribute to the pathophysiology of CF in patients carrying this mutation.

Protein Kinase B Alpha (PKBα) Stimulates the Epithelial Sodium Channel (ENaC) Heterologously Expressed in Xenopus laevis Oocytes by Two Distinct Mechanisms

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.

USP2‐45 regulates ENaC endocytosis

The epithelial sodium channel (ENaC) is important for the maintenance of Na+ and volume homeostasis. Defects in its regulation cause hypertension and contribute to the pathogenesis of cystic fibrosis. ENaC surface expression is regulated by ubiquitination of each ENaC subunit. Previous work indicates that Nedd4‐2 catalyzes ENaC ubiquitination, which has two distinct effects on ENaC trafficking; ubiquitination increases ENaC endocytosis and increases its degradation in lysosomes, both which contribute to a reduction in ENaC surface expression. Recent work suggests that ENaC can be deubiquitinated by a ubiquitin‐specific protease, USP2‐45. Here we tested the hypothesis that USP2‐45 regulates ENaC trafficking. When coexpressed in HEK293T cells, we found that USP2‐45 produced a dose‐dependent increase in surface expression ofα, β, and γENaC, and reversed the Nedd4‐2 mediated decrease in ENaC surface expression. USP2‐45 coprecipitated with the cell surface pool of ENaC and decreased the rate of ENaC endocytosis. Moreover, USP2‐45 blunted the effect of Nedd4‐2 on ENaC endocytosis. In contrast, USP2‐45 had no effect on degradation of the surface pool of ENaC. Thus, USP2‐45 increases ENaC surface expression through a selective effect on ENaC endocytosis. This suggests a model in which USP2‐45 deubiquitinates ENaC at the cell surface but not in endosomes. Funded by the NIH.