Effect of [Cl(-)]i on ENaC activity from mouse cortical collecting duct cells

Na(+) transport via epithelial Na(+) channel (ENaC) occurs across many epithelial surfaces and plays a key role in regulating salt and water absorption. In this study, we have examined the effects of cytosolic Na(+) and Cl(-) on ENaC activity by patch clamping single channel recording method in mouse cortical collecting duct cells (M1). Cytosolic Na(+) exerts its effect in change of ENaC open probability (Po). High cytosolic Na(+) significantly reduces ENaC Po. No change in channel conductance by cytosolic Na(+) is observed. However, decrease of cytosolic Cl(-) concentration significantly increases channel conductance and ENaC Po. This effect is due to the right shift of ENaC I-V curve to positive membrane potential. The virtue of ENaC conductance remains the same. Cl(-) channels like CFTR and VRAC are unlikely to be involved in this regulation. The results suggest that cytosolic Cl(-) could serve as a mediator to regulate ENaC activity, in accordance with the activities of Cl(-) channels.     

Extracellular protons regulate human ENaC by modulating Na+ self-inhibition

The epithelial Na(+) channel, ENaC, is exposed to a wide range of proton concentrations in the kidney, lung, and sweat duct. We, therefore, tested whether pH alters ENaC activity. In Xenopus oocytes expressing human alpha-, beta-, and gammaENaC, amiloride-sensitive current was altered by protons in the physiologically relevant range (pH 8.5-6.0). Compared with pH 7.4, acidic pH increased ENaC current, whereas alkaline pH decreased current (pH(50) = 7.2). Acidic pH also increased ENaC current in H441 epithelia and in human primary airway epithelia. In contrast to human ENaC, pH did not alter rat ENaC current, indicating that there are species differences in ENaC regulation by protons. This resulted predominantly from species differences in gammaENaC. Maneuvers that lock ENaC in a high open-probability state ("DEG" mutation, proteolytic cleavage) abolished the effect of pH on human ENaC, indicating that protons alter ENaC current by modulating channel gating. Previous work showed that ENaC gating is regulated in part by extracellular Na(+) ("Na(+) self-inhibition"). Based on several observations, we conclude that protons regulate ENaC by altering Na(+) self-inhibition. First, protons reduced Na(+) self-inhibition in a dose-dependent manner. Second, ENaC regulation by pH was abolished by removing Na(+) from the extracellular bathing solution. Third, mutations that alter Na(+) self-inhibition produced corresponding changes in ENaC regulation by pH. Together, the data support a model in which protons modulate ENaC gating by relieving Na(+) self-inhibition. We speculate that this may be an important mechanism to facilitate epithelial Na(+) transport under conditions of acidosis.     

Intracellular sodium regulates proteolytic activation of the epithelial sodium channel

Na(+) transport across epithelia is mediated in part by the epithelial Na(+) channel ENaC. Previous work indicates that Na(+) is an important regulator of ENaC, providing a negative feedback mechanism to maintain Na(+) homeostasis. ENaC is synthesized as an inactive precursor, which is activated by proteolytic cleavage of the extracellular domains of the alpha and gamma subunits. Here we found that Na(+) regulates ENaC in part by altering proteolytic activation of the channel. When the Na(+) concentration was low, we found that the majority of ENaC at the cell surface was in the cleaved/active state. As Na(+) increased, there was a dose-dependent decrease in ENaC cleavage and, hence, ENaC activity. This Na(+) effect was dependent on Na(+) permeation; cleavage was increased by the ENaC blocker amiloride and by a mutation that decreases ENaC activity (alpha(H69A)) and was reduced by a mutation that activates ENaC (beta(S520K)). Moreover, the Na(+) ionophore monensin reversed the effect of the inactivating mutation (alpha(H69A)) on ENaC cleavage, suggesting that intracellular Na(+) regulates cleavage. Na(+) did not alter activity of Nedd4-2, an E3 ubiquitin ligase that modulates ENaC cleavage, but Na(+) reduced ENaC cleavage by exogenous trypsin. Our findings support a model in which intracellular Na(+) regulates cleavage by altering accessibility of ENaC cleavage sites to proteases and provide a molecular explanation for the earlier observation that intracellular Na(+) inhibits Na(+) transport via ENaC (Na(+) feedback inhibition).     

Neutrophil elastase activates near-silent epithelial Na+ channels and increases airway epithelial Na+ transport

Neutrophil elastase is a serine protease that is abundant in the airways of individuals with cystic fibrosis (CF), a genetic disease manifested by excessive airway Na(+) absorption and consequent depletion of the airway surface liquid layer. Although endogenous epithelium-derived serine proteases regulate epithelial Na(+) transport, the effects of neutrophil elastase on epithelial Na(+) transport and epithelial Na(+) channel (ENaC) activity are unknown. Low micromolar concentrations of human neutrophil elastase (hNE) applied to the apical surface of a human bronchial cell line (16HBE14o-/beta gamma) increased Na(+) transport about twofold. Similar effects were observed with trypsin, also a serine protease. Proteolytic inhibitors of hNE or trypsin selectively abolished the enzyme-induced increase of epithelial Na(+) transport. At the level of the single channel, submicromolar concentrations of hNE increased activity of near-silent ENaC approximately 108-fold in patches from NIH-3T3 cells expressing rat alpha-, beta-, and gamma-ENaC subunits. However, no enzyme effects were observed on basally active ENaCs. Trypsin exposure following hNE revealed no additional increase in amiloride-sensitive short-circuit current or in ENaC activity, suggesting these enzymes share a common mode of action for increasing Na(+) transport, likely through proteolytic activation of ENaC. The hNE-induced increase of near-silent ENaC activity in CF airways could contribute to Na(+) hyperabsorption, reduced airway surface liquid height, and dehydrated mucus culminating in inefficient mucociliary clearance.     

Nonselective cation transport in native esophageal epithelia

Rabbit esophageal epithelia actively transport Na(+) in a manner similar to that observed in classic electrically tight Na(+)-absorbing epithelia, such as frog skin. However, the nature of the apical entry step is poorly understood. To address this issue, we examined the electrophysiological and biochemical nature of this channel. Western blotting experiments with epithelial Na(+) channel (ENaC) subunit-specific antibodies revealed the presence of all three ENaC subunits in both native and immortalized esophageal epithelial cells. The amino acid sequence of the rabbit alpha-ENaC cloned from native rabbit esophageal epithelia was not significantly different from that of other published alpha-ENaC homologs. To characterize the electrophysiological properties of this native apical channel, we utilized nystatin permeabilization to eliminate the electrical contribution of the basolateral membrane in isolated native epithelia mounted in Ussing-type chambers. We find that the previously described apical Na(+) channel is nonselective for monovalent cations (Li(+), Na(+), and K(+)). Moreover, this channel was not blocked by millimolar concentrations of amiloride. These findings document the presence of a nonselective cation channel in a native Na(+) transporting epithelia, a finding that hereto has been thought to be limited to artificial culture conditions. Moreover, our data are consistent with a potential role of ENaC subunits in the formation of a native nonselective cation channel.     

Regulation of epithelial sodium channel trafficking by proprotein convertase subtilisin/kexin type 9 (PCSK9)

The epithelial Na(+) channel (ENaC) is critical for Na(+) homeostasis and blood pressure control. Defects in its regulation cause inherited forms of hypertension and hypotension. Previous work found that ENaC gating is regulated by proteases through cleavage of the extracellular domains of the α and γ subunits. Here we tested the hypothesis that ENaC is regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), a protease that modulates the risk of cardiovascular disease. PCSK9 reduced ENaC current in Xenopus oocytes and in epithelia. This occurred through a decrease in ENaC protein at the cell surface and in the total cellular pool, an effect that did not require the catalytic activity of PCSK9. PCSK9 interacted with all three ENaC subunits and decreased their trafficking to the cell surface by increasing proteasomal degradation. In contrast to its previously reported effects on the LDL receptor, PCSK9 did not alter ENaC endocytosis or degradation of the pool of ENaC at the cell surface. These results support a role for PCSK9 in the regulation of ENaC trafficking in the biosynthetic pathway, likely by increasing endoplasmic reticulum-associated degradation. By reducing ENaC channel number, PCSK9 could modulate epithelial Na(+) absorption, a major contributor to blood pressure control.     

Activation of the epithelial sodium channel by the metalloprotease meprin β subunit

The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin β (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin β or α/β in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin β, but not the α subunit. Meprin β promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin β regulates the activity of ENaC in a metalloprotease-dependent fashion.     

cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4-2

The epithelial Na(+) channel (ENaC) functions as a pathway for epithelial Na(+) transport, contributing to Na(+) homeostasis and blood pressure control. Vasopressin increases ENaC expression at the cell surface through a pathway that includes cAMP and cAMP-dependent protein kinase (PKA), but the mechanisms that link PKA to ENaC are unknown. Here we found that cAMP regulates Na(+) transport in part by inhibiting the function of Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. Consistent with this model, we found that cAMP inhibited Nedd4-2 by decreasing its binding to ENaC. Moreover, decreased Nedd4-2 expression (RNA interference) or overexpression of a dominant negative Nedd4-2 construct disrupted ENaC regulation by cAMP. Nedd4-2 was a substrate for phosphorylation by PKA in vitro and in cells; three Nedd4-2 residues were phosphorylated by PKA and were required for cAMP to inhibit Nedd4-2 (relative functional importance Ser-327 > Ser-221 > Thr-246). Previous work found that these residues are also phosphorylated by serum and glucocorticoid-inducible kinase (SGK), a downstream mediator by which aldosterone regulates epithelial Na(+) transport. Consistent with a functional interaction between these pathways, overexpression of SGK blunted ENaC stimulation by cAMP, whereas inhibition of SGK increased stimulation. Conversely, cAMP agonists decreased ENaC stimulation by SGK. The data suggest that cAMP regulates ENaC in part by phosphorylation and inhibition of Nedd4-2. Moreover, Nedd4-2 is a central convergence point for kinase regulation of Na(+) transport.     

Involvement of p38 MAPK in hypotonic stress-induced stimulation of beta- and gamma-ENaC expression in renal epithelium

We investigated a role of p38 MAPK in the regulation of transepithelial Na(+) reabsorption by chronic application (20-24h) of hypotonicity (hypotonic stress) in renal epithelial A6 cells. Pretreatment with a specific p38 MAPK inhibitor (SB202190) significantly reduced the chronic hypotonicity-stimulated transepithelial Na(+) reabsorption by diminishing the Na(+) entry through epithelial Na(+) channel (ENaC) in the apical membrane and the Na(+) extrusion via the Na(+)/K(+) ATPase (pump), although the rate limiting step was still the Na(+) entry step. We further examined whether the inhibitory effects of SB202190 on the transepithelial Na(+) reabsorption is caused through suppression of mRNA expression of ENaC participating in the transepithelial Na(+) reabsorption as the Na(+) entry pathway. The chronic hypotonicity increased the mRNA expression of alpha-, beta-, and gamma-subunits of ENaC. Moreover, we found that inhibition of p38 MAPK by SB202190 diminished the mRNA expression of beta- and gamma-ENaC but not alpha-ENaC. Based on these observations, it is suggested that the chronic hypotonicity stimulates the renal transepithelial Na(+) reabsorption by upregulating the mRNA expression of beta- and gamma-ENaC via a p38 MAPK-dependent pathway.     

Selected contribution: limiting Na(+) transport rate in airway epithelia from alpha-ENaC transgenic mice: a model for pulmonary edema.

The amiloride-sensitive epithelial Na(+) channel (ENaC) is essential for fluid clearance from the airways. An experimental animal model with a reduced expression of ENaC, the alpha-ENaC transgenic rescue mouse, is prone to develop edema under hypoxia exposure. This strongly suggests an involvement of ENaC in the pathogenesis of pulmonary edema. To investigate the pathogenesis of this type of edema, primary cultures of tracheal cells from these mice were studied in vitro. An ~60% reduction in baseline amiloride-sensitive Na(+) transport was observed, but the pharmacological characteristics and physiological regulation of the channel were similar to those observed in cells from wild-type mice. Aprotinin, an inhibitor of serine proteases, blocked 50-60% of the basal transepithelial current, hypoxia induced downregulation of Na(+) transport, and beta-adrenergic stimulation was effective to stimulate Na(+) transport after the hypoxia-induced decrease. When downregulation of ENaC activity (such as observed under hypoxia) is added to a low "constitutive" ENaC expression, the resulting reduced Na(+) transport rate may be insufficient for airway fluid clearance and favor pulmonary edema.     

Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition

Inhibition of epithelial Na(+) channel (ENaC) activity by high concentrations of extracellular Na(+) is referred to as Na(+) self-inhibition. We investigated the effects of external Zn(2+) on whole cell Na(+) currents and on the Na(+) self-inhibition response in Xenopus oocytes expressing mouse alphabetagamma ENaC. Na(+) self-inhibition was examined by analyzing inward current decay from a peak current to a steady-state current following a fast switching of a low Na(+) (1 mm) bath solution to a high Na(+) (110 mm) solution. Our results indicate that external Zn(2+) rapidly and reversibly activates ENaC in a dose-dependent manner with an estimated EC(50) of 2 microm. External Zn(2+) in the high Na(+) bath also prevents or reverses Na(+) self-inhibition with similar affinity. Zn(2+) activation is dependent on extracellular Na(+) concentration and is absent in ENaCs containing gammaH239 mutations that eliminate Na(+) self-inhibition and in alphaS580Cbetagamma following covalent modification by a sulfhydryl-reactive reagent that locks the channels in a fully open state. In contrast, external Ni(2+) inhibition of ENaC currents appears to be additive to Na(+) self-inhibition when Ni(2+) is present in the high Na(+) bath. Pretreatment of oocytes with Ni(2+) in a low Na(+) bath also prevents the current decay following a switch to a high Na(+) bath but rendered the currents below the control steady-state level measured in the absence of Ni(2+) pretreatment. Our results suggest that external Zn(2+) activates ENaC by relieving the channel from Na(+) self-inhibition, and that external Ni(2+) mimics or masks Na(+) self-inhibition.     

Gating induces a conformational change in the outer vestibule of ENaC

The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (alpha, beta, and gamma). The channel forms the pathway for Na(+) absorption in the kidney, and mutations cause disorders of Na(+) homeostasis. However, little is known about the mechanisms that control the gating of ENaC. We investigated the gating mechanism by introducing bulky side chains at a position adjacent to the extracellular end of the second membrane spanning segment (549, 520, and 529 in alpha, beta, and gammaENaC, respectively). Equivalent "DEG" mutations in related DEG/ENaC channels in Caenorhabditis elegans cause swelling neurodegeneration, presumably by increasing channel activity. We found that the Na(+) current was increased by mutagenesis or chemical modification of this residue and adjacent residues in alpha, beta, and gammaENaC. This resulted from a change in the gating of ENaC; modification of a cysteine at position 520 in betaENaC increased the open state probability from 0. 12 to 0.96. Accessibility to this side chain from the extracellular side was state-dependent; modification occurred only when the channel was in the open conformation. Single-channel conductance decreased when the side chain contained a positive, but not a negative charge. However, alterations in the side chain did not alter the selectivity of ENaC. This is consistent with a location for the DEG residue in the outer vestibule. The results suggest that channel gating involves a conformational change in the outer vestibule of ENaC. Disruption of this mechanism could be important clinically since one of the mutations that increased Na(+) current (gamma(N530K)) was identified in a patient with renal disease.     

Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric alpha gamma beta channel architecture

The extracellular domain of the epithelial Na(+) channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl(-) inhibits ENaC activity. To identify sites involved in Cl(-) inhibition, we mutated residues in the extracellular domain of α-, β-, and γENaC that are homologous to the Cl(-) binding site in acid-sensing ion channel 1a and tested the effect of Cl(-) on the activity of ENaC expressed in Xenopus oocytes. We identified two Cl(-) inhibitory sites in ENaC. One is formed by residues in the thumb domain of αENaC and the palm domain of βENaC. Mutation of residues at this interface decreased Cl(-) inhibition and decreased Na(+) self-inhibition. The second site is formed by residues at the interface of the thumb domain of βENaC and the palm domain of γENaC. Mutation of these residues also decreased Cl(-) inhibition yet had no effect on Na(+) self-inhibition. In contrast, mutations in the thumb domain of γENaC and palm of αENaC had little or no effect on Cl(-) inhibition or Na(+) self-inhibition. The data demonstrate that Cl(-) inhibits ENaC activity by two distinct Na(+)-dependent and Na(+)-independent mechanisms that correspond to the two functional Cl(-) inhibitory sites. Furthermore, based on the effects of mutagenesis on Cl(-) inhibition, the additive nature of mutations, and on differences in the mechanisms of Cl(-) inhibition, the data support a model in which ENaC subunits assemble in an αγβ orientation (listed clockwise when viewed from the top).     

The epithelial sodium channel δ-subunit: new notes for an old song

Amiloride-sensitive epithelial Na(+) channels (ENaCs) can be formed by different combinations of four homologous subunits, named α, β, γ, and δ. In addition to providing an apical entry pathway for transepithelial Na(+) reabsorption in tight epithelia such as the kidney distal tubule and collecting duct, ENaCs are also expressed in nonepithelial cells, where they may play different functional roles. The δ-subunit of ENaC was originally identified in humans and is able to form amiloride-sensitive Na(+) channels alone or in combination with β and γ, generally resembling the canonical kidney ENaC formed by α, β, and γ. However, δ differs from α in its tissue distribution and channel properties. Despite the low sequence conservation between α and δ (37% identity), their similar functional characteristics provide an excellent model for exploring structural correlates of specific ENaC biophysical and pharmacological properties. Moreover, the study of cellular mechanisms modulating the activity of different ENaC subunit combinations provides an opportunity to gain insight into the regulation of the channel. In this review, we examine the evolution of ENaC genes, channel subunit composition, the distinct functional and pharmacological features that δ confers to ENaC, and how this can be exploited to better understand this ion channel. Finally, we briefly consider possible functional roles of the ENaC δ-subunit.     

Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure

The epithelial Na(+) channel/degenerin (ENaC/DEG) protein family includes a diverse group of ion channels, including nonvoltage-gated Na(+) channels of epithelia and neurons, and the acid-sensing ion channel 1 (ASIC1). In mammalian epithelia, ENaC helps regulate Na(+) and associated water transport, making it a critical determinant of systemic blood pressure and pulmonary mucosal fluidity. In the nervous system, ENaC/DEG proteins are related to sensory transduction. While the importance and physiological function of these ion channels are established, less is known about their structure. One hallmark of the ENaC/DEG channel family is that each channel subunit has only two transmembrane domains connected by an exceedingly large extracellular loop. This subunit structure was recently confirmed when Jasti and colleagues determined the crystal structure of chicken ASIC1, a neuronal acid-sensing ENaC/DEG channel. By mapping ENaC to the structural coordinates of cASIC1, as we do here, we hope to provide insight toward ENaC structure. ENaC, like ASIC1, appears to be a trimeric channel containing 1alpha, 1beta, and 1gamma subunit. Heterotrimeric ENaC and monomeric ENaC subunits within the trimer possibly contain many of the major secondary, tertiary, and quaternary features identified in cASIC1 with a few subtle but critical differences. These differences are expected to have profound effects on channel behavior. In particular, they may contribute to ENaC insensitivity to acid and to its constitutive activity in the absence of time- and ligand-dependent inactivation. Experiments resulting from this comparison of cASIC1 and ENaC may help clarify unresolved issues related to ENaC architecture, and may help identify secondary structures and residues critical to ENaC function.     

Enac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway

Amiloride-sensitive epithelial Na(+) channels (ENaC) are responsible for trans-epithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, gamma) each containing a proline rich region (PPXY) in their carboxyl-terminal end. Mutations in this PPXY domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions with the ubiquitin ligase Neural precursor cell-expressed developmentally down-regulated protein (Nedd4). It is postulated that this results in defective endocytosis and lysosomal degradation of ENaC leading to an increase in ENaC activity. To show the pathway that degrades ENaC in epithelial cells that express functioning ENaC channels, we used inhibitors of the proteosome and measured sodium channel activity. We found that the inhibitor, MG-132, increases amiloride-sensitive trans-epithelial current in Xenopus distal nephron A6 cells. There also is an increase of total cellular as well as membrane-associated ENaC subunit molecules by Western blotting. MG-132-treated cells also have increased channel density in patch clamp experiments. Inhibitors of lysosomal function did not reproduce these findings. Our results suggest that in native renal cells the proteosomal pathway is an important regulator of ENaC function.     

Collecting duct-specific endothelin B receptor knockout increases ENaC activity

Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na(+) excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na(+) regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na(+) intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na(+) regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na(+) feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na(+) retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.     

Epithelial sodium channel inhibition by AMP-activated protein kinase in oocytes and polarized renal epithelial cells

The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.