The epithelial sodium channel and the control of sodium balance

Studies aiming at the elucidation of the genetic basis of rare monogenic forms of hypertension have identified mutations in genes coding for the epithelial sodium channel ENaC, for the mineralocorticoid receptor, or for enzymes crucial for the synthesis of aldosterone. These genetic studies clearly demonstrate the importance of the regulation of Na⁺ absorption in the aldosterone-sensitive distal nephron (ASDN), for the maintenance of the extracellular fluid volume and blood pressure.Recent studies aiming at a better understanding of the cellular and molecular basis of ENaC-mediated Na⁺ absorption in the distal part of nephron, have essentially focused on the regulation ENaC activity and on the aldosterone-signaling cascade. ENaC is a constitutively open channel, and factors controlling the number of active channels at the cell surface are likely to have profound effects on Na⁺ absorption in the ASDN, and in the amount of Na⁺ that is excreted in the final urine.A number of membrane-bound proteases, kinases, have recently been identified that increase ENaC activity at the cell surface in heterologous expressions systems. Ubiquitylation is a general process that regulates the stability of a variety of target proteins that include ENaC. Recently, deubiquitylating enzymes have been shown to increase ENaC activity in heterologous expressions systems.These regulatory mechanisms are likely to be nephron specific, since in vivo studies indicate that the adaptation of the renal excretion of Na⁺ in response to Na⁺ diet occurs predominantly in the early part (the connecting tubule) of the ASDN.An important work is presently done to determine in vivo the physiological relevance of these cellular and molecular mechanisms in regulation of ENaC activity. The contribution of the protease-dependent ENaC regulation in mediating Na⁺ absorption in the ASDN is still not clearly understood. The signaling pathway that involves ubiquitylation of ENaC does not seem to be absolutely required for the aldosterone-mediated control of ENaC. These in vivo physiological studies presently constitute a major challenge for our understanding of the regulation of ENaC to maintain the Na⁺ balance.     

Airway Surface Liquid Volume Regulation Determines Different Airway Phenotypes in Liddle Compared with βENaC-overexpressing Mice

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na⁺ channel β-subunit (βENaC-Tg) suggest that raised airway Na⁺ transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function βENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, βENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na⁺ transport measured in Ussing chambers (“flooded” conditions) was raised in both Liddle and βENaC-Tg mice. Because enhanced Na⁺ transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic “thin film” conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na⁺ absorption were intact in Liddle but defective in βENaC-Tg mice. We conclude that the capacity to regulate Na⁺ transport and ASL volume, not absolute Na⁺ transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.     

Role of Epithelial Sodium Channels and Their Regulators in Hypertension

The kidney has a central role in the regulation of blood pressure, in large part through its role in the regulated reabsorption of filtered Na⁺. Epithelial Na⁺ channels (ENaCs) are expressed in the most distal segments of the nephron and are a target of volume regulatory hormones. A variety of factors regulate ENaC activity, including several aldosterone-induced proteins that are present within an ENaC regulatory complex. Proteases also regulate ENaC by cleaving the channel and releasing intrinsic inhibitory tracts. Polymorphisms or mutations within channel subunits or regulatory pathways that enhance channel activity may contribute to an increase in blood pressure in individuals with essential hypertension.     

Acetylation Stimulates the Epithelial Sodium Channel by Reducing Its Ubiquitination and Degradation

The epithelial Na⁺ channel (ENaC) functions as a pathway for Na⁺ absorption in the kidney and lung, where it is crucial for Na⁺ homeostasis and blood pressure regulation. ENaC is regulated in part through signaling pathways that control the ubiquitination state of ENaC lysines. A defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. Here we determined that α-, β-, and γENaC are also substrates for lysine acetylation. Trichostatin A (TSA), a histone deacetylase inhibitor, enhanced ENaC acetylation and increased ENaC abundance in the total cell lysate and at the cell surface. Moreover, TSA increased ENaC current in Fischer rat thyroid and kidney collecting duct epithelia. We found that HDAC7 is expressed in the kidney collecting duct, supporting a potential role for this histone deacetylase in ENaC regulation. HDAC7 overexpression reduced ENaC abundance and ENaC current, whereas ENaC abundance and current were increased by silencing of HDAC7. ENaC and HDAC7 form a complex, as detected by coimmunoprecipitation. We observed a reciprocal relationship between acetylation and ubiquitination; TSA reduced ENaC ubiquitination, whereas HDAC7 increased ubiquitination. By reducing ENaC ubiquitination, TSA decreased the rate of ENaC degradation. Thus, acetylation increases epithelial Na⁺ absorption by antagonizing ENaC ubiquitination. This stabilizes ENaC, and hence, increases its abundance at the cell surface.     

Flow-sensitive K+-coupled ATP Secretion Modulates Activity of the Epithelial Na+ Channel in the Distal Nephron

The epithelial Na⁺ channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under tonic inhibition by a local purinergic signaling system responding to changes in dietary sodium intake. Normal BK_Ca channel function is required for flow-sensitive ATP secretion in the ASDN. We tested here whether ATP secreted through connexin channels in a coupled manner with K⁺ efflux through BK_Ca channels is required for inhibitory purinergic regulation of ENaC in response to increases in sodium intake. Inhibition of connexin channels relieves purinergic inhibition of ENaC. Deletion of the BK-β4 regulatory subunit, which is required for normal BK_Ca channel function and flow-sensitive ATP secretion in the ASDN, suppresses increases in urinary ATP in response to increases in sodium intake. As a consequence, ENaC activity, particularly in the presence of high sodium intake, is inappropriately elevated in BK-β4 null mice. ENaC in BK-β4 null mice, however, responds normally to exogenous ATP, indicating that increases in activity do not result from end-organ resistance but rather from lowered urinary ATP. Consistent with this, disruption of purinergic regulation increases ENaC activity in wild type but not BK-β4 null mice. Consequently, sodium excretion is impaired in BK-β4 null mice. These results demonstrate that the ATP secreted in the ASDN in a BK_Ca channel-dependent manner is physiologically available for purinergic inhibition of ENaC in response to changes in sodium homeostasis. Impaired sodium excretion resulting form loss of normal purinergic regulation of ENaC in BK-β4 null mice likely contributes to their elevated blood pressure.     

Control of Epithelial Ion Transport by Cl<Superscript>−</Superscript> and PDZ Proteins

Inhibition of epithelial Na⁺ channels (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated previously. Recent studies suggested a role of cytosolic Cl⁻ for the interaction of CFTR with ENaC, when studied in Xenopus oocytes. In the present study we demonstrate that the Na⁺/H⁺-exchanger regulator factor (NHERF) controls expression of CFTR in mouse collecting duct cells. Inhibition of NHERF largely attenuates CFTR expression, which is paralleled by enhanced Ca²⁺-dependent Cl⁻ secretion and augmented Na⁺ absorption by the ENaC. It is further demonstrated that epithelial Na⁺ absorption and ENaC are inhibited by cytosolic Cl⁻ and that stimulation by secretagogues enhances the intracellular Cl⁻ concentration. Thus, the data provide a clue to the question, how epithelial cells can operate as both absorptive and secretory units: Increase in intracellular Cl⁻ during activation of secretion will inhibit ENaC and switch epithelial transport from salt absorption to Cl⁻ secretion.This revised version was published online in August 2005 with a corrected cover date.     

Rapid stimulation of human renal ENaC by cAMP in Xenopus laevis oocytes

Among the compensatory mechanisms restoring circulating blood volume after severe haemorrhage, increased vasopressin secretion enhances water permeability of distal nephron segments and stimulates Na⁺ reabsorption in cortical collecting tubules via epithelial sodium channels (ENaC). The ability of vasopressin to upregulate ENaC via a cAMP-dependent mechanism in the medium to long term is well established. This study addressed the acute regulatory effect of cAMP on human ENaC (hENaC) and thus the potential role of vasopressin in the initial compensatory responses to haemorrhagic shock. The effects of raising intracellular cAMP (using 5 mmol/L isobutylmethylxanthine (IBMX) and 50 μmol/L forskolin) on wild-type and Liddle-mutated hENaC activity expressed in Xenopus oocytes and hENaC localisation in oocyte membranes were evaluated by dual-electrode voltage clamping and immunohistochemistry, respectively. After 30 min, IBMX + forskolin had stimulated amiloride-sensitive Na⁺ current by 52 % and increased the membrane density of Na⁺ channels in oocytes expressing wild-type hENaC. These responses were prevented by 5 μmol/L brefeldin A, which blocks antegrade vesicular transport. By contrast, IBMX + forskolin had no effects in oocytes expressing Liddle-mutated hENaC. cAMP stimulated rapid, exocytotic recruitment of wild-type hENaC into Xenopus oocyte membranes, but had no effect on constitutively over-expressed Liddle-mutated hENaC. Extrapolating these findings to the early cAMP-mediated effect of vasopressin on cortical collecting tubule cells, they suggest that vasopressin rapidly mobilises ENaC to the apical membrane of cortical collecting tubule cells, but does not enhance ENaC activity once inserted into the membrane. We speculate that this stimulatory effect on Na⁺ reabsorption (and hence water absorption) may contribute to the early restoration of extracellular fluid volume following severe haemorrhage.     

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

The epithelial Na⁺ channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na⁺, Cl⁻, protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na⁺ concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na⁺ binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na⁺. Mutations at selected sites altered the cation inhibitory preference to favor Li⁺ or K⁺ rather than Na⁺. Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na⁺. Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family.     

Regulation of the epithelial sodium channels (ENaC) by small G proteins and phosphatidylinositides

The epithelial Na⁺ channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. ENaC activity in these epithelia is limiting for sodium reabsorption. Abnormalities in ENaC function have been linked to disorders of total body Na⁺ homeostasis, blood volume, blood pressure, and lung fluid balance. Recently, ion channels were recognized as physiologically important effectors of small GTP-binding proteins and phosphatidylinositides. We review here recent findings relevant to regulation of ENaC by small G proteins and phosphatidylinositides.     

The Cystic Fibrosis Transmembrane Conductance Regulator Impedes Proteolytic Stimulation of the Epithelial Na+ Channel

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl⁻ secretion in CF airways causes poorly hydrated airway surfaces in CF patients, and this condition is exacerbated by excessive Na⁺ absorption. The mechanistic link between missing CFTR and increased Na⁺ absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na⁺ channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its α- and γ-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and found that ENaC associated with wild-type CFTR was protected from proteolytic cleavage and stimulation of open probability. In contrast, ΔF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full-length to total α-ENaC protein signal was consistently reduced compared with normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down-regulates Na⁺ absorption.     

Developmental Changes of ENaC Expression and Function in the Inner Ear of Pendrin Knock-Out Mice as a Perspective on the Development of Endolymphatic Hydrops

Pendrin mutations cause enlarged vestibular aqueducts and various degrees of sensorineural hearing loss. The selective abolition of pendrin causes dilation of the membranous labyrinth known as endolymphatic hydrops, loss of the endocochlear potential, and consequently loss of hearing function. Because Na⁺ transport is one of the most important driving forces for fluid transport, the epithelial Na⁺ channel (ENaC) is believed to play an important role in fluid volume regulation in the inner ear. Therefore, the dysfunction of Na⁺ transport through ENaC by the acidification of endolymph in Pendred syndrome is one of the potential causes of endolymphatic hydrops. We investigated the changes of ENaC expression and function during the development of the pendrin knock-out mouse. In the cochlea, the expression of β and γENaC was significantly increased at P56 in Pds^−/− mice compared with Pds^+/+ mice. In the vestibule, the expression of βENaC was significantly increased at P56, and γENaC expression significantly increased from P6 to P56 in Pds^−/− mice. The ENaC-dependent trans-epithelial current was not significantly different between Pds^+/+ and Pds^−/− mice in Reissner’s membrane or the saccular extramacular roof epithelium at P0, but the current was significantly increased in Pds^−/− mice at P56 compared with Pds^+/+ mice. These findings indicate that the expression and function of ENaC were enhanced in Pds^−/− mice after the development of endolymphatic hydrops as a compensatory mechanism. This result provides insight into the role of Na⁺ transport in the development and regulation of endolymphatic hydrops due to pendrin mutations.     

Inhibition of epithelial Na currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activated Cl⁻ conductance in parallel with an enhanced amiloride sensitive Na⁺ conductance (ENaC) of the respiratory epithelium. Very recently, acute downregulation of ENaC by the cystic fibrosis transmembrane conductance regulator (CFTR) was demonstrated in several studies. The mechanism, however, by which CFTR exerts its inhibitory effect on ENaC remains obscure. We demonstrate that cytosolic domains of human CFTR are sufficient to induce inhibition of rat epithelial Na⁺ currents (rENaC) when coexpressed in Xenopus oocytes and stimulated with 3-isobutyl-1-methylxanthine (IBMX). Moreover, mutations of CFTR, which occur in cystic fibrosis, abolish CFTR-dependent downregulation of rENaC. Yeast two hybrid analysis of CFTR domains and rENaC subunits suggest direct interaction between the proteins. Enhanced Na⁺ transport as found in the airways of cystic fibrosis patients is probably due to a lack of CFTR dependent downregulation of ENaC.     

Calreticulin positively regulates the expression and function of epithelial sodium channel

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

Intersubunit conformational changes mediate epithelial sodium channel gating

The epithelial Na⁺ channel (ENaC) functions as a pathway for Na⁺ absorption in the kidney and lung, where it is crucial for Na⁺ homeostasis and blood pressure regulation. However, the basic mechanisms that control ENaC gating are poorly understood. Here we define a role in gating for residues forming interfaces between the extracellular domains of the three ENaC subunits. Using cysteine substitution combined with chemical cross-linking, we determined that residues located at equivalent positions in the three subunits (α_K477, β_E446, and γ_E455) form interfaces with residues in adjacent subunits (β_V85, γ_V87, and α_L120, respectively). Cross-linking of these residues altered ENaC activity in a length-dependent manner; long cross-linkers increased ENaC current by increasing its open probability, whereas short cross-linkers reduced ENaC open probability. Cross-linking also disrupted ENaC gating responses to extracellular pH and Na⁺, signals which modulate ENaC activity during shifts in volume status. Introduction of charged side chains at the interfacing residues altered ENaC activity in a charge-dependent manner. Current increased when like charges were present at both interfacing residues, whereas opposing charges reduced current. Together, these data indicate that conformational changes at intersubunit interfaces participate in ENaC transitions between the open and closed states; movements that increase intersubunit distance favor the open state, whereas the closed state is favored when the distance is reduced. This provides a mechanism to modulate ENaC gating in response to changing extracellular conditions that threaten Na⁺ homeostasis.     

EGF and its related growth factors mediate sodium transport in mpkCCDc14 cells via ErbB2 (neu/HER‐2) receptor

Amiloride‐sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate‐limiting step for Na⁺ absorption. Epidermal growth factor (EGF) is involved in the regulation of Na⁺ transport and ENaC activity. However it is still controversial exactly how EGF regulates ENaC and Na⁺ absorption. The aim of the present study was to characterize the EGF regulation of Na⁺ transport in cultured mouse renal collecting duct principal mpkCCD_c14 cells, a highly differentiated cell line which retains many characteristics of the cortical collecting duct (CCD). EGF dose dependently regulates basal transepithelial Na⁺ transport in two phases: an acute phase (<4 h) and a chronic phase (>8 h). Similar effects were observed with TGF‐α, HB‐EGF, and amphiregulin which also belong to the EGF‐related peptide growth factor family. Inhibition of MEK1/2 by PD98059 or U0126 increased acute effects and disrupted chronic effects of EGF on Na⁺ reabsorption. Inhibition of PI3‐kinase with LY294002 abolished acute effect of EGF. As assessed by Western blotting, ErbB2 is the most predominant member of the ErbB family detected in mpkCCD_c14 cells. Immunohistochemistry analysis revealed localization of ErbB2 in the CCD in Sprague–Dawley rat kidneys. Both acute and long‐term effects of EGF were abolished when cells were treated with tyrphostin AG‐825 and ErbB2 inhibitor II, chemically dissimilar selective inhibitors of the ErbB2 receptor. Thus, we conclude that EGF and its related growth factors are important for maintaining transepithelial Na⁺ transport and that EGF biphasically modulates sodium transport in mpkCCD_c14 cells via the ErbB2 receptor. J. Cell. Physiol. 223: 252–259, 2010. © 2009 Wiley‐Liss, Inc.     

Frog atrial natriuretic peptide and cGMP activate amiloride-sensitive Na<Superscript>+</Superscript> channels in urinary bladder cells of Japanese tree frog, <Emphasis Type="Italic">Hyla japonica</Emphasis>

In our previous study, it was suggested that ANP and cGMP may increase Na⁺ absorption in the urinary bladder of the Japanese tree frog, Hyla japonica. Thus, Na⁺ transport activated by ANP was investigated electrophysiologically by using a cell-attached patch-clamp technique in freshly isolated cells from the urinary bladder. A predominant channel expressed was a low conductance Na⁺ channel in the epithelial cells. The channel exhibited conductance for inward currents of 4.9 ± 0.2 pS, long open and closed times (c.a. 190 ms), and positive reversal potential. The channel activity was decreased under the pipette solution including 10⁻⁶ M amiloride. These characteristics were similar to those of amiloride-sensitive Na⁺ channels (ENaC). Addition of 10⁻⁹ M ANP activated and significantly increased the ENaC activity from 0.58 ± 0.09 to 1.47 ± 0.34. On the other hand, mean amplitudes and conductance of single channel did not change significantly after the addition of ANP. Addition of 10⁻⁵ M 8-Br-cGMP also activated the ENaC and significantly increased the channel activity from 0.56 ± 0.10 to 2.00 ± 0.33. The addition of ANP failed to activate the ENaC in the presence of 10⁻⁶ M amiloride. These results suggested that ANP and cGMP activate Na⁺ transport via ENaC in the epithelial cells of frog urinary bladder.     

Aldosterone Reduces Crypt Colon Permeability during Low-Sodium Adaptation

Fluid and electrolyte absorption by colonic crypts depends on the transport properties of crypt cellular and paracellular routes and of the pericryptal sheath. As a low-Na⁺ diet increases aldosterone and angiotensin II secretion, either hormone could affect absorption. Control and adrenalectomized (ADX) Sprague-Dawley rats were kept at a high-NaCl (HS) diet and then switched to low-NaCl (LS) diet for 3 days. Aldosterone or angiotensin II plasma concentrations were maintained using implanted osmotic mini-pumps. The extracellular Na⁺ concentration in isolated rat distal colonic mucosa was determined by confocal microscopy using a low-affinity Na⁺-sensitive fluorescent dye (Sodium red, and Na⁺-insensitive BODIPY) bound to polystyrene beads. Crypt permeability to FITC-labelled dextran (10 kDa) was monitored by its rate of escape from the crypt lumen into the pericryptal space. Mucosal ion permeability was estimated by transepithelial electrical resistance (TER) and amiloride-sensitive short-circuit current (SCC). The epithelial Na⁺ channel, ENaC, was determined by immunolocalization. LS diet decreased crypt wall permeability to dextran by 10-fold and doubled TER. Following ADX, aldosterone decreased crypt wall dextran permeability, increased TER, increased Na⁺ accumulation in the pericryptal sheath and ENaC expression even in HS. Infusion of angiotensin II to ADX rats did not reverse the effects of aldosterone deprivation. These findings indicate that aldosterone alone is responsible for both the increase in Na⁺ absorption and the decreased paracellular and pericryptal sheath permeability.     

Larval bullfrog skin expresses ENaC despite having no amiloride-blockable transepithelial Na+ transport

Amiloride-blockable Na⁺ transport, measured as an amiloride-blockable short-circuit current (Am-SCC), is mediated by the epithelial Na⁺ channel (ENaC). Am-SCC is not normally present in bullfrog tadpole skin, but when such skin is cultured with corticoids an amiloride-blockable Na transport appears. Prolactin (PRL) inhibits its corticoid-induced development. Using specific PCR primers for adult frog ENaC and RT-PCR, we investigated whether corticoids can induce all three ENaC subunits, and whether this expression of ENaC subunit(s) can be blocked by adding PRL with the corticoids. We found that (1) the sequences of the RT-PCR products obtained using primers for α-ENaC were identical between larval and adult skins, (2) the mRNAs for all three ENaC subunits were expressed in larval skin under normal conditions despite no amiloride-blockable Na⁺ transport being detectable, (3) all three subunits were expressed in larval skins whether they were cultured with corticoids (amiloride-blockable Na transport present) or with corticoids supplemented with PRL (no amiloride-blockable Na transport present). An antibody against a peptide from the α-ENaC of adult bullfrog was localized to the apical cells of both larval and adult skins. Since no amiloride-blockable Na transport exists across larval skin under these conditions, these results suggest that ENaC protein was expressed prior to the onset of transport. ENaC may be in the plasma membrane in an inactivated form or, alternatively, within vesicles waiting to be inserted.     

Hrs Controls Sorting of the Epithelial Na+ Channel between Endosomal Degradation and Recycling Pathways

Epithelial Na⁺ absorption is regulated by Nedd4-2, an E3 ubiquitin ligase that reduces expression of the epithelial Na⁺ channel (ENaC) at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 functions through two distinct effects on trafficking, enhancing both ENaC endocytosis and ENaC degradation in lysosomes. To investigate the mechanism by which Nedd4-2 targets ENaC to lysosomes, we tested the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT)-0 complex. We found that α-, β-, and γENaC each interact with Hrs. These interactions were enhanced by Nedd4-2 and were dependent on the catalytic function of Nedd4-2 as well as its WW domains. Mutation of ENaC PY motifs, responsible for inherited hypertension (Liddle syndrome), decreased Hrs binding to ENaC. Moreover, binding of ENaC to Hrs was reduced by dexamethasone/serum- and glucocorticoid-inducible kinase and cAMP, which are signaling pathways that inhibit Nedd4-2. Nedd4-2 bound to Hrs and catalyzed Hrs ubiquitination but did not alter Hrs protein levels. Expression of a dominant negative Hrs lacking its ubiquitin-interacting motif (Hrs-ΔUIM) increased ENaC surface expression and current. This occurred through reduced degradation of the cell surface pool of proteolytically activated ENaC, which enhanced its recycling to the cell surface. In contrast, Hrs-ΔUIM had no effect on degradation of uncleaved inactive channels. The data support a model in which Nedd4-2 induces binding of ENaC to Hrs, which mediates the sorting decision between ENaC degradation and recycling.