ENaC Trafficking Research Articles

Sorting Nexins 1, 2, 3 and 17 Regulate Epithelial Sodium Channel Trafficking

The epithelial sodium channel (ENaC) facilitates sodium absorption of polarized epithelia and therefore, is required for regulation of salt and water homeostasis. ENaC’s apical membrane population is strictly controlled, with loss of this control leading to hyper‐ or hypotensive disorders such as Liddle’s Syndrome, or Pseudohypoaldosteronism type 1, respectively. Retromer and retriever are conserved endosome‐localized protein trafficking complexes that mediate recycling of membrane proteins back to the cell surface either directly, via recycling endosomes or via the trans‐Golgi network. Both retromer and retriever associate with accessory proteins including sorting nexins (SNX) proteins which play a role in the establishment of a tubulation complex leading to the formation of endosomal tubule‐shaped extensions which contain the cargo selected for recycling. We hypothesized that retromer and/or retriever are required for ENaC recycling. Using three epithelial cell lines (Fischer rat thyroid (FRT), mouse cortical collecting duct (mCCD) or human embryonic kidney cells (HEK293), we found that siRNA knockdown of retromer and retriever associated SNX proteins reduced ENaC amiloride‐sensitive short circuit current (Isc‐Amil). SNX1 and SNX2 are reported to be essential for cargo recycling through retromer. Individually, knockdown of SNX1 (n=6) or SNX2 (n=8) reduced Isc‐Amil by 38±9% and 39±8% compared with control FRT epithelia, respectively. Double‐knockdown of those SNXs reduced Isc‐Amil by 47±1% (n=6) compared with control FRT epithelia. SNX3 is known to bind directly with the retromer complex. Knockdown of SNX3 decreased Isc‐Amil by 47±5% FRT (n=9) and 35±4% in mCCD (n=9) epithelia compared with control epithelia. Additionally, it is known that SNX17 mediates cargo retrieval via the retriever complex. Knockdown of SNX17 lowered Isc‐Amil by 56±1% (n=9) in FRT and 35±6% (n=9) in mCCD epithelia compared with control epithelia. With cell surface biotinylation experiments and using FRT cells, knockdown of SNX3 and SNX17 reduced the ENaC cell surface population by 68±25% (n=3) and 77±17% (n=3), respectively, thus, attributing to the reduced Isc‐Amil. Furthermore, co‐immunoprecipitation experiments of αβHAγENaC overexpressed in HEK293 cells, demonstrated a protein‐protein interaction between retriever associated SNX17 and ENaC (n=3) suggesting SNX17 may act as a cargo binding protein between ENaC and the retriever complex. However, there was no evidence for an interaction between SNX3 and ENaC (n=3). Together, our findings suggest that SNX protein complexes play a role in ENaC recycling to the plasma membrane in polarized epithelia. Additionally, these data suggest that both the retromer and retriever recycling complexes may aid in the trafficking of ENaC to the plasma membrane.Support or Funding InformationThis work was supported by a grant from the New Zealand Lottery Health Board. Further support was provided by the School of Biomedical Sciences and the Department of Physiology of the University of Otago.

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.

Epithelial Na+ Channel: Reciprocal Control by COMMD10 and Nedd4-2.

Optimal function of the epithelial sodium channel (ENaC) in the distal nephron is key to the kidney’s long-term control of salt homeostasis and blood pressure. Multiple pathways alter ENaC cell surface populations, including correct processing and trafficking in the secretory pathway to the cell surface, and retrieval from the cell surface through ubiquitination by the ubiquitin ligase Nedd4-2, clathrin-mediated endocytosis, and sorting in the endosomal system. Members of the Copper Metabolism Murr1 Domain containing (COMMD) family of 10 proteins are known to interact with ENaC. COMMD1, 3 and 9 have been shown to down-regulate ENaC, most likely through Nedd4-2, however, the other COMMD family members remain uncharacterized. To investigate the effects of the COMMD10 protein on ENaC trafficking and function, the interaction of ENaC and COMMD10 was confirmed. Stable COMMD10 knockdown in Fischer rat thyroid epithelia decreased ENaC current and this decreased current was associated with increased Nedd4-2 protein, a known negative regulator of ENaC. However, inhibition of Nedd4-2’s ubiquitination of ENaC was only able to partially rescue the observed reduction in current. Stable COMMD10 knockdown results in defects both in endocytosis and recycling of transferrin suggesting COMMD10 likely interacts with multiple pathways to regulate ENaC and therefore could be involved in the long-term control of blood pressure.

Depletion of Cholesterol Reduces ENaC Activity by Decreasing Phosphatidylinositol-4,5-Bisphosphate in Microvilli

Background/Aims: The epithelial sodium channel (ENaC) in cortical collecting duct (CCD) principal cells plays a critical role in regulating systemic blood pressure. We have previously shown that cholesterol (Cho) in the apical cell membrane regulates ENaC; however, the underlying mechanism remains unclear. Methods: Patch-clamp technique and confocal microscopy were used to evaluate ENaC activity and density. Results: Here we show that extraction of membrane Cho with methyl-β-cyclodextrin (MβCD) significantly reduced amiloride-sensitive current and ENaC single-channel activity. The effects were reproduced by inhibition of Cho synthesis in the cells with lovastatin. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP₂), an ENaC activator, is predominantly located in the microvilli, a specialized apical membrane domain. Here, our confocal microscopy data show that α-ENaC was co-localized with PIP₂ in the microvilli and that Cho was also co-localized with PIP₂ in the microvilli. Either extraction of Cho with MβCD or inhibition of Cho synthesis with lovastatin consistently reduced the levels of Cho, PIP₂, and ENaC in the microvilli. Conclusions: Since PIP₂ can directly stimulate ENaC and also affect ENaC trafficking, these data suggest that depletion of Cho reduces ENaC apical density and activity at least in part by decreasing PIP₂ in the microvilli.

COMMD10: A Novel Regulator Of ENaC Trafficking?

The Epithelial Sodium channel (ENaC) is a heterotrimeric (consisting of one α, one β and one γ subunit) Na+ channel and is located at the apical membrane of polarised epithelia, such as the distal kidney, lung and colon, where it has an important role in Na+ reabsorption for maintenance of total body Na+, blood pressure and blood volume. ENaC's trafficking to and from the plasma membrane is a highly regulated but not completely understood process. One protein that has been identified as playing a role in ENaC trafficking is COMMD1, a member of the COMMD (Copper Metabolism Murr1 domain containing) family of proteins. The other family members however have not been as extensively studied, including COMMD10, the focus of our study. Preliminary data has suggested an interaction between COMMD10 and Arf1 (a small GTP binding protein with various functions throughout the trafficking pathway) which suggests a role for COMMD10 in protein trafficking. Our work aims to investigate whether COMMD10 is a novel regulator of the protein trafficking pathway, and thus, whether it is involved in ENaC trafficking to and/or from the plasma membrane. A stable COMMD10 knockdown reduces amiloride‐sensitive short circuit current in αβγ‐ENaC transfected FRT epithelia. A COMMD10 knockdown reduces protein expression of some proteins (Vps35 and COMMD1) involved in the retromer complex (a complex implicated in the recycling of ENaC back to the plasma membrane). These results suggest that when COMMD10 is knocked down ENaC trafficking is disrupted. We have also identified that COMMD10 and Arf1, both in its wild‐type form as well as constitutively active and constitutively inactive forms, co‐localise. However, no co‐localisation was observed between COMMD10 and other Arf family members (Arf4 and Arf5) suggesting an Arf1 specific function for COMMD10. Using a cell‐line in which COMMD10 is stabily knocked down we have identified a change in localisation of the active form of Arf1, that when taken with the other results suggests a role for COMMD10 in the protein trafficking pathway.Support or Funding InformationUniversity of Otago PhD Scholarship

Regulation of ENaC trafficking in rat kidney

Regulation of ENaC trafficking in rat kidney

Plasma membrane insertion of epithelial sodium channels occurs with dual kinetics.

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for Na(+) transport across electrically tight epithelia. Regulation of ENaC activity is critical for electrolyte and extracellular volume homeostasis, as well as for lung liquid clearance and colon Na(+) handling. ENaC activity is tightly controlled by a combination of mechanisms involving changes in open probability and plasma membrane abundance. The latter reflects a combination in channel biosynthesis and trafficking to and from the membrane. Studying ENaC trafficking with different techniques in a variety of expression systems has yielded inconsistent results, indicating either fast or slow rates of insertion and retrieval, which range from the order of minutes to several hours. Here, we use Xenopus oocytes as ENaC expression system to study channel insertion rate in the membrane using two different techniques under comparable conditions: (1) confocal microscopy coupled to fluorescence recovery after photobleaching (FRAP) measurements; and (2) fluorescent bungarotoxin (BTX) binding to ENaC subunits modified to include BTX binding sites (BBSs) in their extracellular domain, a technique that has not been previously used to study ENaC trafficking. Our confocal-FRAP data indicate a fast rate of ENaC incorporation to the membrane in a process conditioned by channel subunit composition. On the other hand, BTX binding experiments indicate much slower channel insertion rates, with matching slow ENaC retrieval rates. The data support a model that includes fast recycling of endocytosed ENaC with parallel incorporation of newly synthesized channels at a slower rate.

Epithelial Sodium Channel (ENaC) Tagged with Bungarotoxin Binding Site to Study Channel Plasma Membrane Expression and Trafficking

Transepithelial sodium reabsorption through ENaC plays a key role in electrolyte and extracellular volume homeostasis and regulation of blood pressure. ENaC is a heterotrimeric ion channel formed by α, β and γ subunits. Channel activity is tightly regulated by many local and circulating factors, which in turn control channel cell surface expression (N) and open probability (Po). ENaC upregulation resulting from decreased endocytosis or enhanced delivery to the membrane have been described. However, there are several technical challenges in the study of ENaC trafficking, including low levels of plasma membrane expression. The aim of this study was to develop a new tool to directly study the dynamics of ENaC trafficking to and from the plasma membrane. To that end we inserted an α‐bungarotoxin (BTX) binding site (BBS) in the extracellular domain of the a subunit and used fluorescently labeled BTX to study ENaC plasma membrane expression and trafficking in Xenopus laevis oocytes. Mutant αBBSβγ channels do not shown any functional abnormality in the absence or presence of the toxin. Confocal imaging of Alexa Fluor 488 (AF488)‐labeled BTX showed specific labeling in plasma membrane of oocytes expressing αBBSβγ channels. ENaC retrieval was detected by imaging AF488‐BTX labeled oocytes at different time points whereas ENaC insertion rate was detected by blocking αBBSβγ‐expressing oocytes with unlabeled BTX and then staining with AF488‐BTX at different times. Our results suggest that tagged αBBS subunits provide a useful tool to study plasma membrane ENaC abundance and trafficking to and from the membrane.

TGF-β directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury

TGF-β is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-β pathway is described, which rapidly promoted internalization of the αβγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-β applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which--together with patch-clamp and flow cytometry studies--identified ENaC as the target of TGF-β. TGF-β rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of βENaC, the subunit responsible for cell-surface stability of the αβγENaC complex. ENaC internalization was dependent on oxidation of βENaC Cys(43). Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove βENaC internalization, which was inhibited by a TGF-β neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-β signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-β-dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs.

Unexpected effect of the appetite-stimulating hormone ghrelin on ENaC: hunger for sodium?

The orexigenic hormone ghrelin acts like a hunger signal, released by the stomach in response to nutritional status. However, ghrelin and its receptor in the kidney may play other biological roles. Kemp and colleagues identify that ghrelin stimulates renal Na+ absorption through cAMP-dependent trafficking of ENaC in the cortical collecting duct. While ghrelin seems to be a physiological regulator of ENaC, future studies are necessary to clarify its physiological and pathological roles in sodium homeostasis.

Intrarenal ghrelin receptors regulate ENaC-dependent sodium reabsorption by a cAMP-dependent pathway

The main distal nephron segment sodium transporters are the distal tubule chlorothiazide-sensitive sodium chloride cotransporter (NCC) and the collecting duct amiloride-sensitive epithelial sodium channel (ENaC). The infusion of ghrelin into the renal interstitium stimulates distal nephron-dependent sodium reabsorption in normal rats, but the mechanism is unknown. Here we localize renal ghrelin receptors (GR) to the cortical collecting duct (CCD). Ghrelin significantly increased phosphorylated serum/glucocorticoid-regulated kinase-1 (pSGK1), a major upstream signaling intermediate regulating ENaC. To test whether increased apical membrane αENaC induced the antinatriuresis, ghrelin was infused in the presence of acute and chronic amiloride, a selective inhibitor of ENaC. In the presence of amiloride, renal interstitial ghrelin failed to reduce urine sodium excretion, suggesting that ghrelin-induced sodium reabsorption is dependent on intact ENaC activity. While the main sodium transporter of the CCD is ENaC, NCC is also present. In response to renal interstitial ghrelin infusion, neither total nor phosphorylated NCC levels are altered. Ghrelin-induced sodium reabsorption persisted in the presence of chlorothiazide (selective inhibitor of NCC), indicating that intact NCC activity is not necessary for ghrelin-induced antinatriuresis. Finally, renal interstitial ghrelin infusion significantly increased interstitial cAMP levels and adenylyl cyclase blockade abolished ghrelin-induced antinatriuresis. Thus, GRs expressed in the CCD regulate sodium reabsorption by cAMP-induced trafficking of ENaC to the apical membrane.

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.

The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

The epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption across tight epithelia. Cyclic-AMP (cAMP) stimulation promotes ENaC trafficking to the apical surface to increase channel number and transcellular Na+ transport. Removal of corticosteroid supplementation in a cultured cortical collecting duct cell line reduced ENaC expression. Concurrently, the number of vesicles trafficked in response to cAMP stimulation, as measured by a change in membrane capacitance, also decreased. Stimulation with aldosterone restored both the basal and cAMP-stimulated ENaC activity and increased the number of exocytosed vesicles. Knocking down ENaC directly decreased both the cAMP-stimulated short-circuit current and capacitance response in the presence of aldosterone. However, constitutive apical recycling of the Immunoglobulin A receptor was unaffected by alterations in ENaC expression or trafficking. Fischer Rat Thyroid cells, transfected with α,β,γ-mENaC had a significantly greater membrane capacitance response to cAMP stimulation compared to non-ENaC controls. Finally, immunofluorescent labeling and quantitation revealed a smaller number of vesicles in cells where ENaC expression was reduced. These findings indicate that ENaC is not a passive passenger in regulated epithelial vesicle trafficking, but plays a role in establishing and maintaining the pool of vesicles that respond to cAMP stimulation.

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.

Myosin 5 is involved in cAMP‐induced ENaC trafficking in a mCCD cell line

The molecular motor which traffics epithelial sodium channels (ENaC) to and from the apical membrane is still unknown. We hypothesized that myosins, specifically Myosin 5 (Myo5), is involved in ENaC exocytosis. To determine the role of Myo5 in ENaC trafficking, we knocked down Myo5a and Myo5c in mCCD cells using shRNAs. We then measured the short-circuit current (Isc) and membrane capacitance change (ΔCT) of cells in Ussing chambers before and following cAMP activation. The Isc and CT change following cAMP-induced trafficking in knockdowns was significantly smaller than mCCD controls. Cells with Myo5c knocked down had a ΔIsc of 2.9 μA ± 1.03 μA compared to controls ΔIsc of 4.7 μA ± 0.68μA. There was a significant reduction in ΔCT (21% for controls vs. 14% in Myo5c) following cAMP stimulation. No significant change in Isc or CT response was observed for Myo5a knockdowns. There was no significant difference in basal (unstimulated) Isc between controls and Myo5c knockdowns. These results suggest that Myo5c is specifically involved in cAMP-induced ENaC trafficking to the apical membrane in response to cAMP stimulation and not constitutive ENaC protein turnover.

上皮型ナトリウムチャネルのトラフィッキング

The epithelial sodium channel (ENaC) forms a pathway for transpepithelial Na+ transport in a variety of epithelial tissues including the kidney collecting duct, airway epithelium, and distal colon. In the kidney, ENaC plays a crucial role in body Na+ content and blood pressure control; abnormalities in ENaC function lead to several diseases such as Liddle’s syndrome and pseudohypoaldosteronism. Recent studies have revealed that ENaC function in epithelia is mainly regulated through membrane trafficking controlling the number of channels at the surface of the apical membrane. Apical membrane expression of ENaC is regulated through multiple steps, including synthesis, exocytosis, endocytosis, recycling, and degradation. Hormones such as aldosterone and vasopressin increase ENaC delivery to the apical surface by regulating those steps. This review discusses current understanding of molecular mechanisms of ENaC trafficking and introduces a newly-observed phenomenon that suggests the existence of undiscovered regulatory mechanisms of membrane trafficking: dependence of recycling rate constants on total ENaC amounts in recycle.

Protein kinase D1 modulates aldosterone‐induced ENaC trafficking and regulation in M1 renal cortical collecting duct cells

Aldosterone stimulates the phosphorylation and activation of Protein Kinase D1 in M1 cells through the transactivation of EGFR (1). PKD1 mediates rapid aldosterone‐induced subcellular trafficking of ENaC (2). ENaCα is under transcriptional control of the mineralocorticoid receptor while ENaCβ and ENaCγ are constitutively expressed in the distal nephron. Using siRNA‐mediated stable knockdown of PKD1, transepithelial current measurements (ITE) and confocal microscopy, we examined the role of PKD1 in the regulation of ENaC trafficking and activity in M1 cells. Aldosterone (10nM) increased amiloride‐sensitive ITE from 1 ± 0.21 μA/cm2 to 7 ± 0.86 μA/cm2 (n= 8, p < 0.005) in wild type M1 cells within 24h. This effect was reduced by 71.5% in the PKD1 suppressed cells. ENaCα expression was increased in wild type cells treated with aldosterone for 24h and this response was absent in PKD1 knockdown cells. Aldosterone stimulated the apical membrane insertion of ENaCβ in wild type cells, whereas no trafficking effect was observed in the PKD1 suppressed cells. PKD1 plays a central role in the aldosterone‐mediated regulation of ENaC through both transcriptional control and subcellular trafficking. Funded by Wellcome Trust, Higher Education Authority of Ireland and Science Foundation Ireland.

The Rab‐GAP, AS160, participates in the regulation of apical membrane epithelial sodium channel (ENaC) density and recycling

The Rab‐GAP, AS160 is a recently discovered Akt kinase substrate. Since phosphorylation sites for Akt are similar to those for the aldosterone (Aldo)‐induced kinase, SGK1, we postulated that phosphorylation of AS160 may promote ENaC trafficking during aldo stimulation. AS160 expression was detected in mCCD cells, in which its expression was increased by its transfection or reduced by shRNA‐mediated knockdown, followed by assays of ENaC expression and function. Aldo induced a time‐dependent increase in AS160 expression, and its phosphorylation at five sites: T568, S588, T642, S318 and S751, detected by phospho‐specific antibodies. Under non‐stimulated (basal) conditions, AS160 knockdown increased apical ENaC without changing total ENaC expression and increased Na transport. After AS160 knockdown, aldo did not further augment apical ENaC density or Na transport. Under basal conditions, AS160 over‐expression increased total ENaC expression 2.5‐fold but did not increase surface ENaC expression. However, aldo increased surface ENaC expression and Na current 2.5‐fold in epithelia expressing AS160. These findings suggest that AS160 normally retains ENaC within intracellular compartments, and that SGK1‐mediated AS160 phosphorylation blocks its Rab‐GAP activity, permitting ENaC trafficking to the apical membrane in response to aldosterone. [Supported by AHA fellowship 0725416U and NIH DK54814]

ENaC Expression Alters a Recycling Vesicle Pool Responsible for its Regulation

The epithelial sodium channel (ENaC) is the rate limiting step in sodium reabsorption across tight epithelia. In kidney cortical collecting duct (CCD) cells cAMP stimulation increases Na+ transport by trafficking ENaC from an intracellular vesicular pool to the apical surface. By reducing the activity of the serum and glucocorticoid kinase (SGK) in a cultured CCD cell line we demonstrate that both ENaC expression and the size of the vesicle‐trafficking pool responsible for ENaC trafficking is reduced. Selective stimulation of SGK with aldosterone or dexamethazone increases both the basal and cAMP‐stimulated ENaC short‐circuit currents (ISC) and the size of the ENaC‐containing vesicle pool enlarges. Fusion of ENaC‐containing vesicles with the apical membrane is measured as a change in transepithelial capacitance (CT). The cAMP‐induced ΔCT in unsupplemented, low ENaC expressing cells was 0.11±0.008 μF/cm2 which increased to 0.15±0.01 μF/cm2 in aldosterone supplemented cells. To determine if the size of the vesicle pool depends on ENaC alone or another downstream component of the SGK signaling pathway, αENaC was selectively knocked down in CCD cells by siRNA. ENaC‐dependent ISC was significantly reduced from 12.9±3.0μA/cm2 in control cells to 1.95±0.36μA/cm2 in the αENaC knockdown cells. There was also a significant reduction in the cAMP‐stimulated ΔISC and ΔCT suggesting that ENaC alone determined the size of the exocytosed vesicle pool. To verify that ENaC modulates this exocytosed vesicle pool, polarized Fischer Rat Thyroid (FRT) cells which do not endogenously express ENaC were transfected with α,β and γ‐ENaC constructs. The CT response to cAMP was significantly greater in cells transfected with ENaC. Together these results indicate the presence of a recycling vesicle pool which is regulated by ENaC expression.

Extracellular Chloride Regulates the Epithelial Sodium Channel

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl(-) concentrations in the kidney and in other epithelia. We tested whether Cl(-) alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl(-) with SO4(2-), H2PO4(-), or SCN(-) produced a large increase in ENaC current, indicating that extracellular Cl(-) inhibits ENaC. Extracellular Cl(-) also inhibited ENaC in Na+-transporting epithelia. The anion selectivity sequence was SCN(-) < SO4(2-) < H2PO4(-) < F(-) < I(-) < Cl(-) < Br(-). Crystallization of ASIC1a revealed a Cl(-) binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (alpha(H418A) and beta(R388A)) disrupted the response to Cl(-), suggesting that Cl(-) might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl(-). The response to Cl(-) was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl(-). Cl(-) regulated ENaC activity in part through enhanced Na+ self-inhibition, a process by which extracellular Na+ inhibits ENaC. Together, the data indicate that extracellular Cl(-) regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl(-) might modulate epithelial Na+ absorption.