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.     

Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains

The epithelial Na⁺ channel (ENaC) mediates the rate-limiting step in transepithelial Na⁺ transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.     

Regulation of ENaC expression at the cell surface by Rab11

The epithelial Na⁺ channel (ENaC) is an essential channel responsible for Na⁺ reabsorption. Coexpression of Rab11a and Rab3a small G proteins with ENaC results in a significant increase in channel activity. In contrast, coexpression of Rab5, Rab27a, and Arf-1 had no effect or slightly decreased ENaC activity. Inhibition of MEK with PD98059, Rho-kinase with Y27632 or PI3-kinase with LY294002 had no effect on ENaC activity in Rab11a-transfected CHO cells. Fluorescence imaging methods demonstrate that Rab11a colocalized with ENaC. Rab11a increases ENaC activity in an additive manner with dominant-negative dynamin, which is a GTPase responsible for endocytosis. Brefeldin A, an inhibitor of intracellular protein translocation, blocked the stimulatory action of Rab11a on ENaC activity. We conclude that ENaC channels, present on the apical plasma membrane, are being exchanged with channels from the intracellular pool in a Rab11-dependent manner.     

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.     

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.     

The epithelial sodium channel: from molecule to disease

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.     

Epidermal growth factor-mediated proliferation and sodium transport in normal and PKD epithelial cells

Members of the epidermal growth factor (EGF) family bind to ErbB (EGFR) family receptors which play an important role in the regulation of various fundamental cell processes including cell proliferation and differentiation. The normal rodent kidney has been shown to express at least three members of the ErbB receptor family and is a major site of EGF ligand synthesis. Polycystic kidney disease (PKD) is a group of diseases caused by mutations in single genes and is characterized by enlarged kidneys due to the formation of multiple cysts in both kidneys. Tubule cells proliferate, causing segmental dilation, in association with the abnormal deposition of several proteins. One of the first abnormalities described in cell biological studies of PKD pathogenesis was the abnormal mislocalization of the EGFR in cyst lining epithelial cells. The kidney collecting duct (CD) is predominantly an absorptive epithelium where electrogenic Na⁺ entry is mediated by the epithelial Na⁺ channel (ENaC). ENaC-mediated sodium absorption represents an important ion transport pathway in the CD that might be involved in the development of PKD. A role for EGF in the regulation of ENaC-mediated sodium absorption has been proposed. However, several investigations have reported contradictory results indicating opposite effects of EGF and its related factors on ENaC activity and sodium transport. Recent advances in understanding how proteins in the EGF family regulate the proliferation and sodium transport in normal and PKD epithelial cells are discussed here. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

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.     

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.     

Ras binding opens c-Raf to expose the docking site for mitogen-activated protein kinase kinase

A key signalling molecule, c-Raf, is situated downstream from Ras and upstream from the mitogen-activated protein kinase kinase (MEK). We studied the mechanism underlying the signal transduction from Ras to MEK by using probes based on the principle of fluorescence resonance energy transfer. In agreement with previous models, it was found that c-Raf adopted two conformations: open active and closed inactive. Ras binding induced the c-Raf transition from closed to open conformation, which enabled c-Raf to bind to MEK. In the presence of a cytosolic Ras mutant, c-Raf bound to, but failed to phosphorylate, MEK in the cytoplasm. In contrast, the cytosolic Ras mutant significantly enhanced MEK phosphorylation by a membrane-targeted c-Raf. These results demonstrated the essential role of Ras-induced conformational change in MEK activation by c-Raf.     

Associated Proteins and Renal Epithelial Na+ Channel Function

The hypothesis that amiloride-sensitive Na⁺ channel complexes immunopurified from bovine renal papillary collecting tubules contain, as their core conduction component, an ENaC subunit, was tested by functional and immunological criteria. Disulfide bond reduction with dithiothreitol (DTT) of renal Na⁺ channels incorporated into planar lipid bilayers caused a reduction of single channel conductance from 40 pS to 13 pS, and uncoupled PKA regulation of this channel. The cation permeability sequence, as assessed from bi-ionic reversal potential measurements, and apparent amiloride equilibrium dissociation constant (K ^amil _i ) of the Na⁺ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed a substantial decrease in K ^amil _i following stretch (0.44 ± 0.12 μm versus 6.9 ± 1.0 μm). Moreover, stretch activation induced a loss in the channel's ability to discriminate between monovalent cations, and even allowed Ca²⁺ to permeate. Polyclonal antibodies generated against a fusion protein of αbENaC recognized a 70 kDa polypeptide component of the renal Na⁺ channel complex. These data suggest that ENaC is present in the immunopurified renal Na⁺ channel protein complex, and that PKA sensitivity is conferred by other associated proteins. Received: 5 June 1995/Revised: 29 September 1995     

Feedback inhibition of ENaC: Acute and chronic mechanisms

Intracellular [Na⁺] ([Na⁺]_i) modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na⁺ transport, but the underlying mechanisms remain unclear. We show here that short-term (60–80 min) incubation of ENaC-expressing oocytes in high Na⁺ results in a 75% decrease in channel activity. When the β subunit was truncated, corresponding to a gain-of-function mutation found in Liddle's syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na⁺]_i. In both cases the inhibition occurred with little to no change in cell-surface expression of γENaC. Long-term incubation (18 hours) in high Na⁺ reduced activity by 92% and 75% in wild-type channels and Liddle's mutant, respectively, with concomitant 70% and 52% decreases in cell-surface γENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na⁺ incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved γENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na⁺ and 4 hrs in high Na⁺, suggesting that [Na⁺]_i increased the rate of retrieval of cleaved γ ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na⁺]_i.     

Ubiquitin-specific Peptidase 8 (USP8) Regulates Endosomal Trafficking of the Epithelial Na+ Channel

Ubiquitination plays a key role in trafficking of the epithelial Na⁺ channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na⁺ absorption.     

E Prostanoid-1 receptor regulates renal medullary αENaC in rats infused with angiotensin II

E Prostanoid (EP) receptors play an important role in urinary Na⁺ excretion. In the kidney, the epithelial sodium channel (ENaC) is the rate-limiting-step for Na⁺ reabsorption. We hypothesized that activation of EP1/EP3 regulates the expression of ENaC in the face of renin-angiotensin-aldosterone-system (RAAS) activation. In primary cultures of inner medullary collecting duct (IMCD) cells, sulprostone (EP1 > EP3 agonist, 1 μM) and 17 Phenyl trinor (17 Pt, EP1 agonist, 10 μM) prevented the up-regulation of αENaC mRNA induced by aldosterone (10 nM). In Sprague-Dawley rats infused with angiotensin II (0.4 μg/kg/min), αENaC expression was up-regulated in renal cortex and medulla coincidently with high plasma aldosterone levels. Sulprostone and/or 17 Pt prevented this effect in renal medulla but not in cortex. Immunocytochemistry demonstrated that IMCD cells express EP1. Our results suggest that specific activation of EP1 receptor during RAAS activation antagonizes the action of aldosterone on αENaC expression in the renal medulla.     

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.     

Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein

DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf.     

ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases

Epithelial Na⁺ channels facilitate the transport of Na⁺ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na⁺ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.Many ion channels are silent at rest and are activated in response to a variety of factors, including membrane potential, external ligands, and intracellular signaling processes. The ENaC² has evolved as a channel that is thought to reside primarily in an active state, facilitating the bulk movement of Na⁺ out of renal tubular or airway lumens. The regulated insertion and retrieval of channels at the plasma membrane have important roles in modulating ENaC-dependent Na⁺ transport (1). A number of factors also have a role in regulating ENaC activity via changes in channel P_o or gating. In this regard, it has become increasingly apparent that proteolysis of ENaC subunits has a key role in this process (2). This minireview addresses several questions regarding the role of ENaC subunit proteolysis in regulating channel gating. (i) Where are ENaC subunits cleaved? (ii) Which proteases mediate ENaC cleavage? (iii) Why are channels activated by proteolysis? (iv) Is proteolysis responsible, in part, for the highly variable channel P_o that has been noted for ENaC? (v) Why have ENaCs evolved as channels that require proteolysis for activation?     

Feedback inhibition of ENaC: Acute and chronic mechanisms

Intracellular [Na⁺] ([Na⁺]_i) modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na⁺ transport, but the underlying mechanisms remain unclear. We show here that short-term (60–80 min) incubation of ENaC-expressing oocytes in high Na⁺ results in a 75% decrease in channel activity. When the β subunit was truncated, corresponding to a gain-of-function mutation found in Liddle''s syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na⁺]_i. In both cases the inhibition occurred with little to no change in cell-surface expression of γENaC. Long-term incubation (18 hours) in high Na⁺ reduced activity by 92% and 75% in wild-type channels and Liddle''s mutant, respectively, with concomitant 70% and 52% decreases in cell-surface γENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na⁺ incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved γENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na⁺ and 4 hrs in high Na⁺, suggesting that [Na⁺]_i increased the rate of retrieval of cleaved γ ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na⁺]_i.     

Hypotonic Regulation of Mouse Epithelial Sodium Channel in Xenopus laevis Oocytes

The regulation of the epithelial Na⁺ channel (ENaC) during cell swelling is relevant in cellular processes in which cell volume changes occur, i.e., migration, proliferation and cell absorption. Its sensitivity to hypotonically induced swelling was investigated in the Xenopus oocyte expression system with the injection of the three subunits of mouse ENaC. We used voltage-clamp techniques to study the amiloride-sensitive Na⁺ currents (INa_(amil)) and video microscopic methodologies to assess oocyte volume changes. Under conditions of mild swelling (25 % reduced hypotonicity) inward current amplitude decreased rapidly over 1.5 min. In contrast, there was no change in current amplitude of H₂O-injected oocytes to the osmotic insult. INa_(amil) kinetics analysis revealed a decrease in the slower inactivation time constant during the hypotonic stimuli. Currents from ENaC-injected oocytes were not sensitive to external Cl^? reduction. Neither short- nor long-term cytochalasin D treatment affected the observed response. Oocytes expressing a DEG mutant β-ENaC subunit (β-S518K) with an open probability of 1 had reduced INa_(amil) hypotonic response compared to oocytes injected with wild-type ENaC subunits. Finally, during the hypotonic response ENaC-injected oocytes did not show a cell volume difference compared with water-injected oocytes. On this basis we suggest that hypotonicity-dependent ENaC inhibition is principally mediated through an effect on open probability of channels in the membrane.