Syntaxin 1A regulates ENaC via domain-specific interactions

The epithelial sodium channel (ENaC) is a heterotrimeric protein responsible for Na(+) absorption across the apical membranes of several absorptive epithelia. The rate of Na(+) absorption is governed in part by regulated membrane trafficking mechanisms that control the apical membrane ENaC density. Previous reports have implicated a role for the t-SNARE protein, syntaxin 1A (S1A), in the regulation of ENaC current (I(Na)). In the present study, we examine the structure-function relations influencing S1A-ENaC interactions. In vitro pull-down assays demonstrated that S1A directly interacts with the C termini of the alpha-, beta-, and gamma-ENaC subunits but not with the N terminus of any ENaC subunit. The H3 domain of S1A is the critical motif mediating S1A-ENaC binding. Functional studies in ENaC expressing Xenopus oocytes revealed that deletion of the H3 domain of co-expressed S1A eliminated its inhibition of I(Na), and acute injection of a GST-H3 fusion protein into ENaC expressing oocytes inhibited I(Na) to the same extent as S1A co-expression. In cell surface ENaC labeling experiments, reductions in plasma membrane ENaC accounted for the H3 domain inhibition of I(Na). Individually substituting C terminus-truncated alpha-, beta-, or gamma-ENaC subunits for their wild-type counterparts reversed the S1A-induced inhibition of I(Na), and oocytes expressing ENaC comprised of three C terminus-truncated subunits showed no S1A inhibition of I(Na). C terminus truncation or disruption of the C terminus beta-subunit PY motif increases I(Na) by interfering with ENaC endocytosis. In contrast to subunit truncation, a beta-ENaC PY mutation did not relieve S1A inhibition of I(Na), suggesting that S1A does not perturb Nedd4 interactions that lead to ENaC endocytosis/degradation. This study provides support for the concept that S1A inhibits ENaC-mediated Na(+) transport by decreasing cell surface channel number via direct protein-protein interactions at the ENaC C termini.     

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.     

Differential current decay profiles of epithelial sodium channel subunit combinations in polarized renal epithelial cells

In many epithelial tissues in the body, the rate of Na(+) reabsorption is governed by the activity of the epithelial sodium channel (ENaC). The assembly, trafficking, and turnover of the three ENaC subunits (alpha, beta, and gamma) is complex and not well understood. Recent experiments suggest that ENaC must be proteolytically cleaved for maximal activity and may explain the discrepancies reported in prior biochemical approaches focused on quantitating the trafficking and half-life of full-length subunits. As an alternative approach to examining the dynamics of ENaC subunits, we have generated doxycycline-repressible replication-defective recombinant adenoviruses encoding individual epitope-tagged mouse ENaC subunits and expressed these in polarized MDCK I cells. Co-infection with these viruses encoding all three subunits generates robust amiloride-sensitive currents in polarized MDCK cells. Significant current was also observed in cells expressing alpha- and gamma-mENaC in the absence of beta-mENaC. These currents did not appear to result from association with endogenous canine beta-ENaC. Treatment of alpha beta gamma-expressing cells with cycloheximide (CHX) resulted in the rapid inhibition (within 3 h) of approximately 50-80% of the initial current; however, a sizable fraction of the initial current remained even after 6 h of CHX. By contrast, CHX addition to cells expressing only alpha- and gamma-mENaC resulted in rapid decay in current with no residual fraction. Our data suggest that ENaC channels of differing stoichiometries are differentially trafficked and degraded and provide support for the possibility that noncoordinate trafficking of ENaC subunits may function in vivo as a mechanism to modulate ENaC activity.     

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.     

Regulation by vasopressin of NaCl absorption in the renal collecting duct

In the kidney, the fine control of NaCl absorption takes place in the distal nephron and is controlled by aldosterone and vasopressin. This review summarizes the effects of vasopressin on Na+ transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel in immortalized or primary cultured cortical collecting duct cells, expressing either the wild-type ENaC subunits, or mutations, or deletions of the PY domain of the beta- or gamma-ENaC subunits responsible for Liddle's syndrome, an inherited form of hypertension due to excessive salt absorption.     

Phosphatidylinositol 3,4,5-trisphosphate mediates aldosterone stimulation of epithelial sodium channel (ENaC) and interacts with gamma-ENaC

Whole cell voltage clamp experiments were performed in a mouse cortical collecting duct principal cell line using patch pipettes back-filled with a solution containing phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). PIP(3) significantly increased amiloridesensitive current in control cells but not in the cells prestimulated by aldosterone. Additionally, aldosterone stimulated amiloridesensitive current in control cells, but not in the cells that expressed a PIP(3)-binding protein (Grp1-PH), which sequestered intracellular PIP(3). 12 amino acids from the N-terminal tail (APGEKIKAKIKK) of gamma-epithelial sodium channel (gamma-ENaC) were truncated by PCRbased mutagenesis (gammaT-ENaC). Whole cell and confocal microscopy experiments were conducted in Madin-Darby canine kidney cells co-expressing alpha- and beta-ENaC only or with either gamma-ENaC or gamma(T)-ENaC. The data demonstrated that the N-terminal tail truncation significantly decreased amiloride-sensitive current and that both the N-terminal tail truncation and LY-294002 (a PI3K inhibitor) prevented ENaC translocation to the plasmamembrane. These data suggest that PIP(3) mediates aldosterone-induced ENaC activity and trafficking and that the N-terminal tail of gamma-ENaC is necessary for channel trafficking, probably channel gating as well. Additionally, we demonstrated a novel interaction between gamma-ENaC and PIP(3).     

Distribution of epithelial sodium channels and mineralocorticoid receptors in cardiovascular regulatory centers in rat brain

Epithelial sodium channels (ENaC) are important for regulating sodium transport across epithelia. Functional studies indicate that neural mechanisms acting through mineralocorticoid receptors (MR) and sodium channels (presumably ENaC) are crucial to the development of sympathoexcitation and hypertension in experimental models of salt-sensitive hypertension. However, expression and localization of the ENaC in cardiovascular regulatory centers of the brain have not yet been studied. RT-PCR and immunohistochemistry were performed to study ENaC and MR expression at the mRNA and protein levels, respectively. Both mRNA and protein for alpha-, beta-, and gamma-ENaC subunits and MR were found to be expressed in the rat brain. All three ENaC subunits and MR were present in the supraoptic nucleus, magnocellular paraventricular nucleus, hippocampus, choroid plexus, ependyma, and brain blood vessels, suggesting the presence of multimeric channels and possible regulation by mineralocorticoids. In most cortical areas, thalamus, amygdala, and suprachiasmatic nucleus, notable expression of gamma-ENaC was undetectable, whereas alpha- and beta-ENaC were abundantly expressed pointing to the possibility of a heterogeneous population of channels. The findings suggest that stoichiometrically different populations of ENaC may be present in both epithelial and neural components in the brain, which may contribute to regulation of cerebrospinal fluid and interstitial Na+ concentration as well as neuronal excitation.     

Low expression of the beta-ENaC subunit impairs lung fluid clearance in the mouse

Transepithelial alveolar sodium (Na+) transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) constitutes the driving force for removal of fluid from the alveolar space. To define the role of the beta-ENaC subunit in vivo in the mature lung, we studied a previously established mouse strain harboring a disruption of the beta-ENaC gene locus resulting in low levels of beta-ENaC mRNA expression. Real-time RT-PCR experiments confirmed that beta-ENaC mRNA levels were decreased by >90% in alveolar epithelial cells from homozygous mutant (m/m) mice. beta-ENaC protein was undetected in lung homogenates from m/m mice by Western blotting, but alpha- and gamma-ENaC proteins were increased by 83% and 45%, respectively, compared with wild-type (WT) mice. At baseline, Na+-driven alveolar fluid clearance (AFC) was significantly reduced by 32% in m/m mice. Amiloride at the concentration 1 mM inhibited AFC by 75% and 34% in WT and m/m mice, respectively, whereas a higher concentration (5 mM) induced a 75% inhibition of AFC in both groups. The beta2-agonist terbutaline significantly increased AFC in WT but not in m/m mice. These results show that despite the compensatory increase in alpha- and gamma-ENaC protein expression observed in mutant mouse lung, low expression of beta-ENaC results in a moderate impairment of baseline AFC and in decreased AFC sensitivity to amiloride, suggesting a possible change in the stoichiometry of ENaC channels. Finally, adequate beta-ENaC expression appears to be required for AFC stimulation by beta2-agonists.     

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.     

O2 can raise fetal pneumocyte Na+ conductance without affecting ENaC mRNA abundance

In fetal pneumocytes, increasing P(O(2)) can raise apical Na(+) conductance (G(Na(+))) and increase the abundance of epithelial Na(+) channel subunit (alpha-, beta-, and gamma-ENaC) mRNA, suggesting that the rise in G(Na(+)), which may be important to the perinatal maturation of the lung, reflects O(2)-evoked ENaC gene expression. However, we now show that physiologically relevant increases in P(O(2)) do not affect alpha-, beta-, and gamma-ENaC mRNA abundance in pneumocytes maintained (approximately 48 h) in hormone-free medium or in medium supplemented with dexamethasone and tri-iodothyronine, although the response does persist in cells maintained in medium containing a complex mixture of hormones/growth factors. However, parallel electrometric studies revealed clear increases in G(Na(+)) under all tested conditions and so it is now clear that O(2)-evoked increases in G(Na(+)) can occur without corresponding increases in ENaC mRNA abundance. It is therefore unlikely that this rise in G(Na(+)) is secondary to O(2)-evoked ENaC gene expression.     

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.     

Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries

Recent work from our laboratory indicates that epithelial Na(+) channel (ENaC) function plays an important role in modulating myogenic vascular reactivity. Increases in dietary sodium are known to affect vascular reactivity. Although previous studies have demonstrated that dietary salt intake regulates ENaC expression and activity in epithelial tissue, the importance of dietary salt on ENaC expression in vascular smooth muscle cells (VSMCs) and its role in myogenic constriction is unknown. Therefore, the goal of the present study was to determine whether dietary salt modulates ENaC expression and function in myogenic vasoconstriction. To accomplish this goal, we examined ENaC expression in freshly dispersed VSMCs and pressure-induced vasoconstrictor responses in isolated mesenteric resistance arteries from normotensive Sprague-Dawley rats fed a normal-salt (NS; 0.4% NaCl) or high-salt (HS; 8% NaCl for 2 wk) diet. VSMCs from the mesenteric arteries of NS-fed animals express alpha-, beta-, and gamma-ENaC. The HS diet reduced whole cell alpha- and gamma-ENaC and induced a pronounced translocation of beta-ENaC from intracellular regions toward the VSMC membrane (approximately 336 nm). Associated with this change in expression was a change in the importance of ENaC in pressure-induced constriction. Pressure-induced constriction in NS-fed animals was insensitive to ENaC inhibition with 1 microM benzamil, suggesting that ENaC proteins do not contribute to myogenic constriction in mesenteric arteries under NS intake. In contrast, ENaC inhibition blocked pressure-induced constriction in HS-fed animals. These data suggest that dietary sodium regulates ENaC expression and the quantitative importance of the vascular ENaC signaling pathway contributing to myogenic constriction.     

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.     

Roles of the C termini of alpha -, beta -, and gamma -subunits of epithelial Na+ channels (ENaC) in regulating ENaC and mediating its inhibition by cytosolic Na+

The amiloride-sensitive epithelial Na(+) channels (ENaC) in the intralobular duct cells of mouse mandibular glands are inhibited by the ubiquitin-protein ligase, Nedd4, which is activated by increased intracellular Na(+). In this study we have used whole-cell patch clamp methods in mouse mandibular duct cells to investigate the role of the C termini of the alpha-, beta-, and gamma-subunits of ENaC in mediating this inhibition. We found that peptides corresponding to the C termini of the beta- and gamma-subunits, but not the alpha-subunit, inhibited the activity of the Na(+) channels. This mechanism did not involve Nedd4 and probably resulted from the exogenous C termini interfering competitively with the protein-protein interactions that keep the channels active. In the case of the C terminus of mouse beta-ENaC, the interacting motif included betaSer(631), betaAsp(632), and betaSer(633). In the C terminus of mouse gamma-ENaC, it included gammaSer(640). Once these motifs were deleted, we were able to use the C termini of beta- and gamma-ENaC to prevent Nedd4-mediated down-regulation of Na(+) channel activity. The C terminus of the alpha-subunit, on the contrary, did not prevent Nedd4-mediated inhibition of the Na(+) channels. We conclude that mouse Nedd4 interacts with the beta- and gamma-subunits of ENaC.     

Early expression of beta- and gamma-subunits of epithelial sodium channel during human airway development

The amiloride-sensitive epithelial Na(+) channel (ENaC) is an apical membrane protein complex involved in active Na(+) absorption and in control of fluid composition in airways. There are no data reporting the distribution of its pore-forming alpha-, beta-, and gamma-subunits in the developing human lung. With use of two different rabbit polyclonal antisera raised against beta- and gamma-ENaC, immunohistochemical localization of the channel was performed in fetal (10-35 wk) and in adult human airways. Both subunits were detected after 17 wk of gestation on the apical domain of bronchial ciliated cells, in glandular ducts, and in bronchiolar ciliated and Clara cells. After 30 wk, the distribution of beta- and gamma-subunits was similar in fetal and adult airways. In large airways, the two subunits were detected in ciliated cells, in cells lining glandular ducts, and in the serous gland cells. In the distal bronchioles, beta- and gamma-subunits were identified in ciliated and Clara cells. Ultrastructural immunogold labeling confirmed the identification of beta- and gamma-ENaC proteins in submucosal serous cells and bronchiolar Clara cells. Early expression of ENaC proteins in human fetal airways suggests that Na(+) absorption might begin significantly before birth, even if secretion is still dominant.