Interaction of syntaxins with the amiloride-sensitive epithelial sodium channel.

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na(+) entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (alpha, beta, gamma subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.     

Expression of amiloride-sensitive epithelial sodium channels in mouse taste cells after chorda tympani nerve crush

Our previous electrophysiological study demonstrated that amiloride-sensitive (AS) and -insensitive (AI) components of NaCl responses recovered differentially after the mouse chorda tympani (CT) was crushed. AI responses reappeared earlier (at 3 weeks after the nerve crush) than did AS ones (at 4 weeks). This and other results suggested that two salt-responsive systems were differentially and independently reformed after nerve crush. To investigate the molecular mechanisms of formation of the salt responsive systems, we examined expression patterns of three subunits (alpha, beta and gamma) of the amiloride-sensitive epithelial Na(+) channel (ENaC) in mouse taste cells after CT nerve crush by using in situ hybridization (ISH) analysis. The results showed that all three ENaC subunits, as well as alpha-gustducin, a marker of differentiated taste cells, were expressed in a subset of taste bud cells from an early stage (1-2 weeks) after nerve crush, although these taste buds were smaller and fewer in number than for control mice. At 3 weeks, the mean number of each ENaC subunit and alpha-gustducin mRNA-positive cells per taste bud reached the control level. Also, the size of taste buds became similar to those of the control mice at this time. Our previous electrophysiological study demonstrated that at 2 weeks no significant response of the nerve to chemical stimuli was observed. Thus ENaC subunits appear to be expressed prior to the reappearance of AI and AS neural responses after CT nerve crush. These results support the view that differentiation of taste cells into AS or AI cells is initiated prior to synapse formation.     

The carboxyl terminus of the alpha-subunit of the amiloride-sensitive epithelial sodium channel binds to F-actin

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.     

Regulation of the amiloride-sensitive epithelial sodium channel by syntaxin 1A.

The first step in transepithelial sodium absorption lies at the apical membrane where the amiloride-sensitive, epithelial sodium channel, ENaC, facilitates sodium entry into the cell. Here we report that the vesicle traffic regulatory (SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)) protein, syntaxin 1A (S1A), inhibits ENaC mediated sodium entry. This inhibitory effect is selective for S1A and is not reproduced by syntaxin 3. The inhibition does not require the membrane anchoring domain of syntaxin 1A. It was reversed by the S1A-binding protein, Munc-18, but not by a Munc-18 mutant, which lacks syntaxin affinity. Immunostaining of epitope-tagged ENaC subunits showed that syntaxin 1A decreases ENaC current by reducing the number of ENaC channels in the plasma membrane; S1A does not interfere with ENaC protein expression. Immunoprecipitation of syntaxin 1A from the sodium-transporting epithelial cell line, A6, co-precipitates ENaC. These findings indicate that syntaxin 1A and other members of the SNARE machinery are involved in the control of plasma membrane ENaC content, and they suggest that SNARE proteins participate in the regulation of sodium absorption in relation to agonist mediated vesicle insertion-retrieval processes.     

Role of intracellular Ca2+ in the expression of the amiloride-sensitive epithelial sodium channel

The amiloride-sensitive epithelial sodium channel (ENaC), a multimeric plasma membrane protein composed of alpha-, beta-, and gamma-ENaC subunits, mediates Na(+) reabsorption in epithelial tissues, including the distal nephron, colon, lung, and secretory glands, and plays a critical role in pathophysiology of essential hypertension and cystic fibrosis (CF). The function of ENaC is tightly regulated by signals elicited by aldosterone, vasopressin, agents that increase intracellular cAMP levels, ions, ion channels, G-protein-coupled mechanisms, and cytoskeletal proteins. In this paper, the effects of Ca(2+) on the expression of the human ENaC subunits expressed in human embryonic kidney cells (HEK-293 cells) were examined. Incubation of cells with increased extracellular Ca(2+) and treatment of cells with A23187 and thapsigargin stimulated the expression of the monomeric ENaC subunits. Treatment of cells with Ca(2+)-chelating agents, EGTA and BAPTA-AM, reduced the levels of ENaC subunit expression. The pulse-chase experiments suggested that a rise in the intracellular Ca(2+) increases the ENaC subunit expression. Immunoblot analysis using the anti-ubiquitin antibody indicated that ENaC undergoes ubiquitination. A correlation between the processes that regulate ENaC function with the intracellular Ca(2+) was discussed.     

Activation of large conductance sodium channels upon expression of amiloride-sensitive sodium channel in Sf9 insect cells.

The amiloride-sensitive epithelial sodium channels (ENaC) mediate Na(+) reabsorption in epithelial tissues including distal nephron, colon, lung, and secretory glands and plays a critical role in pathophysiology of hypertension and cystic fibrosis. The ENaC is a multimeric protein composed of alpha-ENaC, beta-ENaC, and gamma-ENaC subunits. To study the biochemical properties of the channel, the subunit cDNAs of rat colon ENaC (rENaC) were subcloned into baculoviruses, and the corresponding proteins were expressed in Sf9 insect cells. The functional characteristics of the expressed rENaC were studied in planar lipid bilayers. The results show that expression of alpha-rENaC and alphabetagamma-rENaC in Sf9 insect cells results in the generation of cation-selective large conductance channels. Although the large conductance channels observed in the alpha-rENaC-containing membranes were unaffected by amiloride, the large conductance channels found in alphabetagamma-rENaC complex-containing membranes exhibited voltage-dependent flickering in the presence of micromolar amiloride. Possible implications of these observations are discussed.     

Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface

Several studies have indicated that olfactory responses are impeded byamiloride. Therefore, it was of interest to see whether, and if so which,olfactory epithelial cellular compartments have amiloride- sensitivestructures. Using ultrastructural methods that involved rapid freezing,freeze-substitution and low temperature embedding of olfactory epithelia,this study shows that, in the rat, this tissue is immunoreactive toantibodies against amiloride sensitive Na(+)- channels. However, microvilliof olfactory supporting cells, as opposed to receptor cilia, contained mostof the immunoreactive sites. Apices from which the microvilli sprout andreceptor cell dendritic knobs had much less if any of theamiloride-antibody binding sites. Using a direct ligand-bindingcytochemical method, this study also confirms earlier ones that showed thatolfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed thatsupporting cell microvilli and the receptor cilia themselves havemechanisms, different but likely complementary, that participate inregulating the salt concentration around the receptor cell cilia. In thisway, both structures help to provide the ambient mucous environment forreceptor cells to function properly. This regulation of the saltconcentration of an ambient fluid environment is a function that theolfactory epithelium shares with cells of transporting epithelia, such asthose of kidney.     

Dopamine regulation of amiloride-sensitive sodium channels in lung cells

Dopamine increases lung fluid clearance. This is partly due to activation of basolateral Na-K-ATPase. However, activation of Na-K-ATPase by itself is unlikely to produce large changes in transepithelial transport. Therefore, we examined apical and basolateral dopamine's effect on apical, highly selective sodium channels [epithelial sodium channels (ENaC)] in monolayers of an alveolar type 2 cell line (L2). Dopamine increased channel open probability (P(o)) without changing the unitary current. The D(1) receptor blocker SCH-23390 blocked the dopamine effect, but the D(2) receptor blocker sulpiride did not. The dopamine-mediated increase in ENaC activity was not a secondary effect of dopamine stimulation of Na-K-ATPase, since ouabain applied to the basolateral surface to block the activity of Na-K-ATPase did not alter dopamine-mediated ENaC activity. Protein kinase A (PKA) was not responsible for dopamine's effect since a PKA inhibitor, H89, did not reduce dopamine's effect. However, cpt-2-O-Me-cAMP, which selectively binds and activates EPAC (exchange protein activated by cAMP) but not PKA, increased ENaC P(o). An Src inhibitor, PP2, and the phosphatidylinositol-3-kinase inhibitor, LY-294002, blocked dopamine's effect on ENaC. In addition, an MEK blocker, U0126, an inhibitor of phospholipase A(2), and a protein phosphatase inhibitor also blocked the effect of dopamine on ENaC P(o). Finally, since the cAMP-EPAC-Rap1 pathway also activates DARPP32 (32-kDa dopamine response protein phosphatase), we confirmed that dopamine phosphorylates DARPP32, and okadaic acid, which blocks phosphatases (DARPP32), also blocks dopamine's effect. In summary, dopamine increases ENaC activity by a cAMP-mediated alternative signaling pathway involving EPAC and Rap1, signaling molecules usually associated with growth-factor-activated receptors.     

Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism

Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.     

FMRFamide-gated sodium channel and ASIC channels: a new class of ionotropic receptors for FMRFamide and related peptides

FMRFamide and related peptides typically exert their action through G-protein coupled receptors. However, two ionotropic receptors for these peptides have recently been identified. They are both members of the epithelial amiloride-sensitive Na+ channel and degenerin (ENaC/DEG) family of ion channels. The invertebrate FMRFamide-gated Na+ channel (FaNaC) is a neuronal Na+-selective channel which is directly gated by micromolar concentrations of FMRFamide and related tetrapeptides. Its response is fast and partially desensitizing, and FaNaC has been proposed to participate in peptidergic neurotransmission. On the other hand, mammalian acid-sensing ion channels (ASICs) are not gated but are directly modulated by FMRFamide and related mammalian peptides like NPFF and NPSF. ASICs are activated by external protons and are therefore extracellular pH sensors. They are expressed both in the central and peripheral nervous system and appear to be involved in many physiological and pathophysiological processes such as hippocampal long-term potentiation and defects in learning and memory, acquired fear-related behavior, retinal function, brain ischemia, pain sensation in ischemia and inflammation, taste perception, hearing functions, and mechanoperception. The potentiation of ASIC activity by endogenous RFamide neuropeptides probably participates in the response to noxious acidosis in sensory and central neurons. Available data also raises the possibility of the existence of still unknown FMRFamide related endogenous peptides acting as direct agonists for ASICs.     

Distribution of amiloride-sensitive sodium channels in the oral cavity of the hamster

The distribution of amiloride-sensitive sodium channels (ASSCs) in taste buds isolated from the oral cavity of hamsters was assessed by patch clamp recording. In contrast to the case for rats, taste cells from the fungiform, foliate and vallate papillae and from the soft palate all contain functional ASSCs. The differential distribution of ASSCs between the hamster and the rat may be important for understanding the physiology underlying the differing behavioral responses of these species to sodium salts.      

Oxidative stress disrupts glucocorticoid hormone-dependent transcription of the amiloride-sensitive epithelial sodium channel alpha-subunit in lung epithelial cells through ERK-dependent and thioredoxin-sensitive pathways

The amiloride-sensitive epithelial Na(+) channel (ENaC) plays a critical role in the maintenance of alveolar fluid balance. It is generally accepted that reactive oxygen and nitrogen species can inhibit ENaC activity and aggravate acute lung injury; however, the molecular mechanism for free radical-mediated ENaC inhibition is unclear. Previously, we showed that the expression of the alpha-subunit of ENaC, alpha-ENaC, which is indispensable for ENaC activity, is repressed by Ras activation in salivary epithelial cells. Here, we investigated whether exogenous H(2)O(2) modulates alpha-ENaC gene expression in lung epithelial cells through a similar molecular mechanism. Utilizing transient transfection reporter assays and site-directed mutagenesis analyses, we found that the glucocorticoid response element (GRE), located at -1334 to -1306 base pairs of the alpha-ENaC 5'-flanking region, is the major enhancer for the stimulated alpha-ENaC expression in A549 lung epithelial cells. We further demonstrate that the presence of an intact GRE is necessary and sufficient for oxidants to repress alpha-ENaC expression. Consistent with our hypothesis, exogenous H(2)O(2)-mediated repression of alpha-ENaC GRE activity is partially blocked by either a specific inhibitor for extracellular signal-regulated kinase (ERK) pathway activation, U0126, or dominant negative ERK, suggesting that, in part, activated ERK may mediate the repressive effects of H(2)O(2) on alpha-ENaC expression. In addition, overexpression of thioredoxin restored glucocorticoid receptor action on the alpha-ENaC GRE in the presence of exogenous H(2)O(2). Taken together, we hypothesize that oxidative stress impairs Na(+) transport activity by inhibiting dexamethasone-dependent alpha-ENaC GRE activation via both ERK-dependent and thioredoxin-sensitive pathways. These results suggest a putative mechanism whereby cellular redox potentials modulate the glucocorticoid receptor/dexamethasone effect on alpha-ENaC expression in lung and other tight epithelia.     

Immunocytochemical localization of amiloride-sensitive sodium channels in the lower intestine of the hen

We have used polyclonal antibodies generated against purified bovine renal amiloride-sensitive Na⁺ channels to localize amiloride-sensitive Na⁺ channels within the lower intestine (colon and coprodeum) of the hen. These antibodies cross-reacted with two polypeptides exhibiting M_r's of 235 and 150 kDa on immunoblots of detergent-solubilized apical membrane fractions from both the colon and coprodeum. The apparent molecular masses of theses polypeptides are in agreement with the M_r's of 2 of the subunits of the renal high amiloride-affintiy Na⁺ channel, namely the and the (=amiloride binding) subunits. The cellular distribution of Na⁺ channels was determined by immunoperoxidase and indirect immunofluorescence cytochemical techniques. The apical (luminal) membrane and cytoplasm of villar principal cells in both colon and coprodeum exhibited immunoreactivity, whereas goblet cells were nagative. Both principal and goblet cells of the crypts were also negative. We conclude that the amiloride-sensitive Na⁺ channels are localized to the principal cells of the intestinal villi and that these cells are responsible for intestinal Na⁺ absorption.     

Could a defective epithelial sodium channel lead to bronchiectasis

Background

Bronchiectasis is defined as a permanent dilation of the airways arising from chronic bronchial inflammation/infection. In 50% of cases, no etiology can be identified. Recently, the role of the epithelial sodium channel ENaC has been pointed out in the pathophysiology of cystic fibrosis, a disease due to mutations in the CFTR gene and causing bronchiectasis in the airways. Moreover, it was found that transgenic mice overexpressing ENaCβ present cystic fibrosis-like lung disease symptoms. Our aim was to evaluate if a defective ENaC protein could be involved in the development of bronchiectasis.

Methods

We extensively analysed ENaCβ and γ genes in 55 patients with idiopathic bronchiectasis and without two mutations in the coding regions of CFTR. Thirty-eight patients presented functional abnormalities suggesting impaired sodium transport (abnormal sweat chloride concentration or nasal potential difference measurement), and 17 had no such evidence.

Results

Sequencing of the exons and flanking introns of the ENaCβ and γ gene identified five different amino-acid changes (p.Ser82Cys, p.Pro369Thr, p.Asn288Ser in ENaCβ ; and p.Gly183Ser, p.Glu197Lys in ENaCγ) in heterozygous state in 8 patients. The p.Ser82Cys amino-acid change was found in 3 unrelated patients who were also heterozygous for a CFTR mutation or variant (1 p.F508del, 1 IVS8-5T, and 1 IVS8-5T:1716G>A (p.E528E)). The other mutations were found in patients without CFTR mutation, the p.Glu197Lys mutation in 2 patients and the other variants in single patients. Among the 8 patients bearing an ENaC mutation, 5 had functional abnormalities suggesting impaired sodium transport.

Conclusion

Our results suggest that several variants in ENaCβ and γ genes might be deleterious for ENaC function and lead to bronchiectasis, especially in patients who are trans-heterozygotes for ENaCβ/CFTR mutations or variants.     

Identification of new partners of the epithelial sodium channel alpha subunit

A fine regulation of the amiloride-sensitive Epithelial Sodium Channel (ENaC), made of alpha, beta and gamma subunits, is crucial for maintenance of Na+ balance and blood pressure. Both beta- and gamma-ENaC participate in negative regulation by interacting with Nedd4-2, an E3 ubiquitin-ligase. Disruption of this interaction results in increased ENaC activity (Liddle syndrome). By two-hybrid screenings, we identified new potential partners of alpha-ENaC: WWP1 (E3 ubiquitin-ligase protein), UBC9 and TSG101 (E2 ubiquitin/SUMO-conjugating enzymes) and confirmed these interactions in GST pull-down assays. All these partners are implicated in protein trafficking and could be involved in the regulation of ENaC activity.     

Biophysical properties and molecular characterization of amiloride-sensitive sodium channels in A549 cells

Amiloride-sensitive Na(+) channels, present in fetal and adult alveolar epithelial type II (ATII) cells, play a critical role in the reabsorption of fetal fluid shortly after birth and in limiting the extent of alveolar edema across the adult lung. Because of the difficulty in isolating and culturing ATII cells, there is considerable interest in characterizing the properties of ion channels and their response to injury of ATII cell-like cell lines such as A549 that derive from a human alveolar cell carcinoma. A549 cells were shown to contain alpha-, beta-, and gamma-epithelial Na(+) channel mRNAs. In the whole cell mode of the patch-clamp technique (bath, 145 mM Na(+); pipette, 145 mM K(+)), A549 cells exhibited inward Na(+) currents reversibly inhibited by amiloride, with an inhibition constant of 0.83 microM. Ion substitution studies showed that these channels were moderately selective for Na(+) (Na(+)-to-K(+) permeability ratio = 6:1). Inward Na(+) currents were activated by forskolin (10 microM) and inhibited by nitric oxide (300 nM) and cGMP. Recordings in cell-attached mode revealed the presence of an amiloride-sensitive Na(+) channel with a unitary conductance of 8.6 +/- 0.04 (SE) pS. Channel activity was increased by forskolin and decreased by nitric oxide and the cGMP analog 8-bromo-cGMP. These data demonstrate that A549 cells contain amiloride-sensitive Na(+) channels with biophysical properties similar to those of ATII cells.     

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.     

Subunit stoichiometry of a core conduction element in a cloned epithelial amiloride-sensitive Na+ channel.

The molecular composition of a core conduction element formed by the alpha-subunit of cloned epithelial Na+ channels (ENaC) was studied in planar lipid bilayers. Two pairs of in vitro translated proteins were employed in combinatorial experiments: 1) wild-type (WT) and an N-terminally truncated alphaDeltaN-rENaC that displays accelerated kinetics (tauo = 32 +/- 13 ms, tauc = 42 +/- 11 ms), as compared with the WT channel (tauc1 = 18 +/- 8 ms, tauc2 = 252 +/- 31 ms, and tauo = 157 +/- 43 ms); and 2) WT and an amiloride binding mutant, alphaDelta278-283-rENaC. The channels that formed in a alphaWT:alphaDeltaN mixture fell into two groups: one with tauo and tauc that corresponded to those exhibited by the alphaDeltaN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the alphaWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1alphaWT:1alphaDelta278-283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of alpha-subunits as a minimal model for the core conduction element in ENaCs.