Epithelial Na(+) Channel Subunits Research Articles

The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.

Amiloride-sensitive NaCl taste responses are associated with genetic variation of ENaC α-subunit in mice

An epithelial Na(+) channel (ENaC) is expressed in taste cells and may be involved in the salt taste transduction. ENaC activity is blocked by amiloride, which in several mammalian species also inhibits taste responses to NaCl. In mice, lingual application of amiloride inhibits NaCl responses in the chorda tympani (CT) gustatory nerve much stronger in the C57BL/6 (B6) strain than in the 129P3/J (129) strain. We examined whether this strain difference is related to gene sequence variation or mRNA expression of three ENaC subunits (alpha, beta, gamma). Real-time RT-PCR and in situ hybridization detected no significant strain differences in expression of all three ENaC subunits in fungiform papillae. Sequences of the beta- and gammaENaC subunit genes were also similar in the B6 and 129 strains, but alphaENaC gene had three single nucleotide polymorphisms (SNPs). One of these SNPs resulted in a substitution of arginine in the B6 strain to tryptophan in the 129 strain (R616W) in the alphaENaC protein. To examine association of this SNP with amiloride sensitivity of CT responses to NaCl, we produced F(2) hybrids between B6 and 129 strains. Amiloride inhibited CT responses to NaCl in F(2) hybrids with B6/129 and B6/B6 alphaENaC R616W genotypes stronger than in F(2) hybrids with 129/129 genotype. This suggests that the R616W variation in the alphaENaC subunit affects amiloride sensitivity of the ENaC channel and provides evidence that ENaC is involved in amiloride-sensitive salt taste responses in mice.

Intracellular calcium plays a role as the second messenger of hypotonic stress in gene regulation of SGK1 and ENaC in renal epithelial A6 cells

In A6 cells, a renal cell line derived from Xenopus laevis, hypotonic stress stimulates the amiloride-sensitive Na(+) transport. Hypotonic action on Na(+) transport consists of two phases, a nongenomic early phase and a genomic delayed phase. Although it has been reported that, during the genomic phase, hypotonic stress stimulates transcription of Na(+) transport-related genes, such as serum- and glucocorticoid-inducible kinase 1 (SGK1) and subunits of the epithelial Na(+) channel (ENaC), increasing Na(+) transport, the mechanism remains unknown. We focused the present study on the role of intracellular Ca(2+) in hypotonicity-induced SGK1 and ENaC subunit transcription. Since hypotonic stress raises intracellular Ca(2+) concentration in A6 cells, we hypothesized that Ca(2+)-dependent signals participate in the genomic action. Using real-time quantitative RT-PCR and Western blot techniques and measuring short-circuit currents, we observed that 1) BAPTA-AM and W7 blunted the hypotonicity-induced expression of SGK1 mRNA and protein, 2) ionomycin dose dependently stimulated expression of SGK1 mRNA and protein under an isotonic condition and the time course of the stimulatory effect of ionomycin on SGK1 mRNA was remarkably similar to that of hypotonic action on SGK1 mRNA, 3) hypotonic stress stimulated transcription of three ENaC subunits in an intracellular Ca(2+)-dependent manner, and 4) BAPTA-AM retarded the delayed phase of hypotonic stress-induced Na(+) transport but had no effect on the early phase. These observations indicate for the first time that intracellular Ca(2+) plays a role as the second messenger in hypotonic stress-induced Na(+) transport by stimulating transcription of SGK1 and ENaC subunits.

Functional cross talk between ENaC and pendrin

EPITHELIAL Na CHANNELS (ENaC) are expressed in the aldosterone-sensitive distal nephron, where they serve as the final site of renal Na reabsorption and participate in the regulation of extracellular fluid volume and blood pressure. ENaC gain-offunction mutations are associated with hypertension, whereas loss-of-function mutations are associated with hypotension. Appreciation of the features that regulate ENaC expression and activity contributes to our understanding of the role of the kidney in the regulation of blood pressure. ENaC structure, assembly, and maturation. ENaC is assembled into a presumed trimeric structure (111) from three highly homologous subunits within the endoplasmic reticulum (ER), although higher ordered stoichiometries have also been proposed (3, 12, 21). The assembly appears to be inefficient given that only a limited fraction of newly synthesized subunits

Indirect Activation of the Epithelial Na+ Channel by Trypsin

We tested the hypothesis that the serine protease trypsin can indirectly activate the epithelial Na(+) channel (ENaC). Experiments were carried out in Xenopus oocytes and examined the effects on the channel formed by all three human ENaC subunits and that formed by Xenopus epsilon and human beta and gamma subunits (epsilonbetagammaENaC). Low levels of trypsin (1-10 ng/ml) were without effects on the oocyte endogenous conductances and were specifically used to test the effects on ENaC. Addition of 1 ng/ml trypsin for 60 min stimulated the amiloride-sensitive human ENaC conductance (g(Na)) by approximately 6-fold. This effect on the g(Na) was [Na(+)]-independent, thereby ruling out an interaction with channel feedback inhibition by Na(+). The indirect nature of this activation was confirmed in cell-attached patch clamp experiments with trypsin added to the outside of the pipette. Trypsin was comparatively ineffective at activating epsilonbetagammaENaC, a channel that exhibited a high spontaneous open probability. These observations, in combination with surface binding experiments, indicated that trypsin indirectly activated membrane-resident channels. Activation by trypsin was also dependent on catalytic activity of this protease but was not accompanied by channel subunit proteolysis. Channel activation was dependent on downstream activation of G-proteins and was blocked by G-protein inhibition by injection of guanyl-5'-yl thiophosphate and by pre-stimulation of phospholipase C. These data indicate a receptor-mediated activation of ENaC by trypsin. This trypsin-activated receptor is distinct from that of protease-activated receptor-2, because the response to trypsin was unaffected by protease-activated receptor-2 overexpression or knockdown.

Small Heat Shock Protein αA-crystallin Regulates Epithelial Sodium Channel Expression

Integral membrane proteins are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). After being translocated or inserted into the ER, they fold and undergo post-translational modifications. Within the ER, proteins are also subjected to quality control checkpoints, during which misfolded proteins may be degraded by proteasomes via a process known as ER-associated degradation. Molecular chaperones, including the small heat shock protein alphaA-crystallin, have recently been shown to play a role in this process. We have now found that alphaA-crystallin is expressed in cultured mouse collecting duct cells, where apical Na(+) transport is mediated by epithelial Na(+) channels (ENaC). ENaC-mediated Na(+) currents in Xenopus oocytes were reduced by co-expression of alphaA-crystallin. This reduction in ENaC activity reflected a decrease in the number of channels expressed at the cell surface. Furthermore, we observed that the rate of ENaC delivery to the cell surface of Xenopus oocytes was significantly reduced by co-expression of alphaA-crystallin, whereas the rate of channel retrieval remained unchanged. We also observed that alphaA-crystallin and ENaC co-immunoprecipitate. These data are consistent with the hypothesis that small heat shock proteins recognize ENaC subunits at ER quality control checkpoints and can target ENaC subunits for ER-associated degradation.

Heteromeric Assembly of Acid-sensitive Ion Channel and Epithelial Sodium Channel Subunits

Amiloride-sensitive ion channels are formed from homo- or heteromeric combinations of subunits from the epithelial Na+ channel (ENaC)/degenerin superfamily, which also includes the acid-sensitive ion channel (ASIC) family. These channel subunits share sequence homology and topology. In this study, we have demonstrated, using confocal fluorescence resonance energy transfer microscopy and co-immunoprecipitation, that ASIC and ENaC subunits are capable of forming cross-clade intermolecular interactions. We have also shown that combinations of ASIC1 with ENaC subunits exhibit novel electrophysiological characteristics compared with ASIC1 alone. The results of this study suggest that heteromeric complexes of ASIC and ENaC subunits may underlie the diversity of amiloride-sensitive cation conductances observed in a wide variety of tissues and cell types where co-expression of ASIC and ENaC subunits has been observed.

Functional Role of Extracellular Loop Cysteine Residues of the Epithelial Na+ Channel in Na+ Self-inhibition

The epithelial Na(+) channel (ENaC) is typically formed by three homologous subunits (alpha, beta, and gamma) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na(+) self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na(+). All 16 Cys residues within alpha and gamma ECLs and selected beta ECL Cys residues were individually mutated to alanine or serine residues. The Na(+) self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight alpha (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one beta (Cys-7), and nine gamma (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na(+) self-inhibition. Na(+) self-inhibition was eliminated by simultaneous mutations of either the last three alpha ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both alpha and gamma ECLs. By analyzing the Na(+) self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the alphaECL (alphaCys-1/alphaCys-6, alphaCys-4/alphaCys-5, alphaCys-7/alphaCys-16, alphaCys-10/alphaCys-13, and alphaCys-11/alphaCys-12) and one pair within the gammaECL (gammaCys-7/gammaCys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the alpha and gamma ENaC subunits are required to establish the tertiary structure that ensures a proper Na(+) self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.

Determination of Epithelial Na+ Channel Subunit Stoichiometry from Single-Channel Conductances

The epithelial Na+ channel (ENaC) is a multimeric membrane protein consisting of three subunits, α, β, and γ. The total number of subunits per functional channel complex has been described variously to follow either a tetrameric arrangement of 2α:1β:1γ or a higher-ordered stoichiometry of 3α:3β:3γ. Therefore, while it is clear that all three ENaC subunits are required for full channel activity, the number of the subunits required remains controversial. We used a new approach, based on single-channel measurements in Xenopus oocytes to address this issue. Individual mutations that alter single-channel conductance were made in pore-lining residues of ENaC α, β, or γ subunits. Recordings from patches in oocytes expressing a single species, wild type or mutant, of α, β, and γ showed a well-defined current transition amplitude with a single Gaussian distribution. When cRNAs for all three wild-type subunits were mixed with an equimolar amount of a mutant α-subunit (either S589D or S592T), amplitudes corresponding to pure wild-type or mutant conductances could be observed in the same patch, along with a third intermediate amplitude most likely arising from channels with at least one wild-type and at least 1 mutant α-subunit. However, intermediate or hybrid conductances were not observed with coexpression of wild-type and mutant βG529A or γG534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2α, 1β, and 1γ come together around central pore.

GRK2 interacts with and phosphorylates Nedd4 and Nedd4-2

Epithelial Na+ channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, which bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of inhibition leads to hypertension. ENaC channels are maintained in the active state by G-protein-coupled receptor kinase 2 (GRK2), an enzyme implicated in the development of essential hypertension. Here, we report that GRK2 interacts not only with ENaC, but also with both Nedd4 and Nedd4-2. Additionally, GRK2 is capable of phosphorylating both Nedd4 and Nedd4-2 at multiple sites. Of possible significance is the phosphorylation of the threonine at position 466 in Nedd4, which is located in the area of the ww3 domain that binds ENaC. These results support and extend the role of GRK2 in sodium transport regulation.

The role of α-, β-, and γ-ENaC subunits in distal lung epithelial fluid absorption induced by pulmonary edema fluid

Edema fluid (EF) increases epithelial Na(+) transport by rat fetal distal lung epithelia (FDLE) and induces net lung fluid absorption in fetal mouse lung explants [Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM, O'Brodovich H. J Physiol (Lond) 544: 537-548, 2002]. We now show that EF increases fluid absorption across monolayers of rat FDLE in a dose-dependent manner. To study the role of subunits of the epithelial Na(+) channel (ENaC) in the phenomena, we cultured explants from the distal lungs of 16-day gestational age wild-type (WT) or alpha-, beta-, or gamma-ENaC knockout or heterozygote (HT) mice. WT explants cultured in media continuously expanded over time as a result of net fluid secretion. In contrast, when explants were exposed to EF for 24 h, net fluid absorption occurred. EF-exposed explants had normal histology, but marked changes were seen after Triton X-100 or staurosporine exposure. Transmission electron microscopy showed EF promoted lamellar body formation and abundant surfactant in the explants' lumens. EF-induced changes in explant size were similar in alpha-ENaC knockout, WT, and HT littermate fetal lung explants (P > 0.05). In contrast, EF's effect was attenuated in beta- and gamma-ENaC knockouts (P < 0.05) vs. WT and HT littermate fetal lung explants. EF exposure slightly decreased or had no effect on mRNA levels for alpha-ENaC in various mouse genotypes but decreased expression of beta- and gamma-ENaC subunit mRNAs (P < 0.01) across all genotype groups. We conclude that beta- and gamma-, but not alpha-, ENaC subunits are essential for EF to exert its maximal effect on net fluid absorption by distal lung epithelia.

The Epithelial Na+ Channel as a Determinant of Blood Pressure

The epithelial Na+ channel (ENaC) forms the rate limiting step in transepithelial Na+ absorption across aldosterone- responsive tissues such as the distal nephron. After more than a decade of investigation it is clear that the mechanisms of ENaC regulation are complex with an array of ENaC-regulatory proteins having been identified. A variety of monogenic syndromes of low renin hypertension have been identified that serve, through different mechanisms, to upregulate distal tubular ENaC activity. One of the most extensively studied, Liddles syndrome, results predominantly from mutations in the C-termini of β and γ-ENaC subunits leading to a failure of Nedd4-2 (neuronal precursor cell-expressed and developmentally down-regulated protein 4-2) mediated channel endocytosis and an increased expression of ENaC at the cell surface. The role of ENaC subunit polymorphisms as determinants of essential hypertension has been investigated in a number of studies. The T594M mutation in the β-subunit of ENaC, for example, has been screened in large study populations and there is conflicting evidence that it co-segregates with blood pressure. Reasons for such controversies are discussed. The authors conclude that ENaC is a pivotal convergence point in blood pressure regulation and that future studies must identify better ways to measure distal tubular ENaC activity and investigate the importance of combination polymorphisms of ENaC subunits and their regulatory proteins as genetic determinants of hypertension.

Na channel expression and activity in the medullary collecting duct of rat kidney

The expression and activity of epithelial Na(+) channels (ENaC) in the medullary collecting duct of the rat kidney were examined using a combination of whole cell patch-clamp measurements of amiloride-sensitive currents (I(Na)) in split-open tubules and Western blot analysis of alpha-, beta-, and gamma-ENaC proteins. In the outer medullary collecting duct, amiloride-sensitive currents were undetectable in principal cells from control animals but were robust when rats were treated with aldosterone (I(Na) = 960 +/- 160 pA/cell) or fed a low-Na diet (I(Na) = 440 +/- 120 pA/cell). In both cases, the currents were similar to those measured in principal cells of the cortical collecting duct from the same animals. In the inner medullary collecting duct, currents were much lower, averaging 120 +/- 20 pA/cell in aldosterone-treated rats. Immunoblots showed that all three ENaC subunits were expressed in the cortex, outer medulla, and inner medulla of the rat kidney. When rats were fed a low-Na diet for 1 wk, similar changes in alpha- and gamma-ENaC occurred in all three regions of the kidney; the amounts of full-length as well as putative cleaved alpha-ENaC protein increased, and the fraction of gamma-ENaC protein in the cleaved state increased at the expense of the full-length protein. The appearance of a presumably fully glycosylated form of beta-ENaC in Na-depleted animals was observed mainly in the outer and inner medulla. These findings suggest that the capability of hormone-regulated, channel-mediated Na reabsorption by the nephron extends at least into the outer medullary collecting duct.

Expression of Mineralocorticoid Receptors (MR) and Epithelial Na+ Channel (ENaC) Subunits in the Heart and Brain Nuclei of Rats Post‐MI

Blockade of central and peripheral MR in rats post MI attenuates sympathetic hyperactivity and ameliorates LV remodeling. The mechanisms involved are not yet clear. We studied mRNA expression of MR and ENaC (α, β, γ) subunits by real-time qRT-PCR and aldosterone binding densities by autoradiography in heart and brain nuclei of male Wistar rats at 2 & 4 wks post MI. Binding densities increased in LV and 2 brain nuclei, but decreased markedly in the infarct scar (table). MR mRNA decreased only at 2 wks; in SFO and OVLT (5.6±0.7 vs. 7.9±0.5, p<0.05; 2.9±0.4 vs. 5.4±0.6, p=0.05; MR/PGK [×10−2]) and showed no changes in LV and other nuclei. ENaC mRNA levels significantly increased in the scar vs. non-infarct LV & sham (41.0±6.0 vs. 3.8±1.0 & 1.5±0.4, for α; 11.3±1.0 vs. 0.3±0.1& non detectable levels for β, and 25.6±5.0 vs. 0.2±0.04 & 0.3±0.1for γ; ENaC/PGK [×10−4]). These results indicate that in rats post MI, MR expression is differentially regulated in brain nuclei. Upregulation of aldosterone binding densities and ENaC transcription in heart and brain may contribute to LV remodeling.

Functional expression and differential localization of epithelial sodium channel δ subunit isoforms

Proteins of the ENaC/DEG family of non-voltage gated Na+ channels are widely expressed in the central and peripheral nervous system. This family includes acid sensing ion channels, which are thought to be involved in certain sensory modalities in the PNS and in modulation of learning and memory in the brain. Another branch of the ENaC/DEG family includes epithelial Na+ channels (ENaCs), which mediate amiloride-sensitive Na+ transport across epithelia. ENaC subunits expression has also been documented in the nervous system, but their functional roles remain poorly studied. Others and we have recently cloned a new isoform of the human ENaC δ subunit (ENaC δ2). Expression of δ2 in Xenopus oocytes failed to induce amiloride-inhibitable Na+ currents. In contrast, when δ2 was co-expressed with ENaC accessory subunits (β and γ), an amiloride-sensitive Na+ current was detected. δ2βγ produced ten-fold less current than the classical αβγ ENaC channel, due to low expression at the plasma membrane, suggesting that the physiological partners of δ2 might be ENaC/DEG proteins other than β and γ. In situ hybridization showed that both δ isoforms are prominently expressed in pyramidal neurons of the human cortex. Double-labeling experiments demonstrated a low level of co-localization between isoforms, suggesting distinct functional roles in the human CNS. Supported by grant BFU2004-04433 (Ministerio de Educación y Ciencia, Spain)

Interregulation of Proton-gated Na+ Channel 3 and Cystic Fibrosis Transmembrane Conductance Regulator

Proton-gated Na(+) channels (ASIC) are new members of the epithelial sodium channel/degenerin gene family. ASIC3 mRNA has been detected in the homogenate of pulmonary tissues. However, whether ASIC3 is expressed in the apical membranes of lung epithelial cells and whether it regulates cystic fibrosis transmembrane conductance regulator (CFTR) function are not known at the present time. Using reverse transcription-PCR, we found that the ASIC3 mRNA was expressed in the human airway mucosal gland (Calu-3) and human airway epithelial (16HBE14o) cells. Indirect immunofluorescence microscopy revealed that ASIC3 was co-segregated with CFTR in the apical membranes of Calu-3 cells. Proton-gated, amiloride-sensitive short circuit Na(+) currents were recorded across Calu-3 monolayers mounted in an Ussing chamber. In whole-cell patch clamp studies, activation of CFTR channels with cAMP reduced proton-gated Na(+) current in Calu-3 cells from -154 +/- 28 to -33 +/- 16 pA (n = 5, p < 0.05) at -100 mV. On the other hand, cAMP-activated CFTR activity was significantly inhibited following constitutive activation of putative ASIC3 at pH 6.0. Immunoassays showed that both ASIC3 and CFTR proteins were expressed and co-immunoprecipitated mutually in Calu-3 cells. Similar results were obtained in human embryonic kidney 293T cells following transient co-transfection of ASIC3 and CFTR. Our results indicate that putative CFTR and ASIC3 channels functionally interact with each other, possibly via an intermolecular association. Because acidic luminal fluid in the cystic fibrosis airway and lung tends to stimulate ASIC3 channel expression and activity, the interaction of ASIC3 and CFTR may contribute to defective salt and fluid transepithelial transport in the cystic fibrotic pulmonary system.

Scoring of predicted GRK2 phosphorylation sites in Nedd4-2

Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4-2. These ligases bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of this inhibition leads to hypertension. We have previously reported that ENaC channels are maintained in the active state by the G protein coupled receptor kinase, GRK2. The enzyme has been implicated in the development of essential hypertension [R. D. Feldman (2002) Mol. Pharmacol., 61, 707-709]. Additional findings in our lab pointed towards a possible role for GRK2 in the phosphorylation and inactivation of Nedd4-2. We have predicted GRK2 phosphorylation sites on Nedd4-2 by combining sequence analysis, homology modeling and surface accessibility calculations. A total of 24 potential phosphorylation sites were predicted by sequence analysis. Of these, 16 could be modeled using homology modeling and 6 of these were found to have sufficient surface exposure to be accessible to the GRK2 enzyme responsible for the phosphorylation of Nedd4-2. The method provides an ordered list of the most probable GRK2 phosphorylation sites on Nedd4-2 providing invaluable guidance to future experimental studies aimed at mutating certain Nedd4-2 residues in order to prevent phosphorylation by GRK2. The method developed could be applied in a wide variety of biological applications involving the binding of one molecule to a protein. The relative effectiveness of the technique is determined mainly by the quality of the homology model built for the protein of interest. jarthur@med.usyd.edu.au

Alveolar fluid transport: a changing paradigm

UNLIKE OTHER EPITHELIAL TISSUES that transport large amounts of solutes and water, the alveolar epithelium is faced with a unique physiological challenge. To facilitate optimal gas exchange and to trap airborne particulate matter, it must maintain a thin layer of fluid on the luminal surface of its extensive alveolar network. However, the thickness of the fluid layer must be tightly regulated, since even slight increases in the fluid depth could adversely affect gas exchange, whereas dehydration of the surface would impair ciliary clearance. The alveolar flooding associated with acute respiratory distress syndrome or the airway dehydration in cystic fibrosis represents two pathological extremes of a failure to maintain proper airway fluid balance. Both of these conditions account for a significant burden of morbidity and mortality worldwide (16, 20). Extensive research done in the last two decades has shown that precise regulation of alveolar surface fluid layer is achieved through vectorial transport of solutes between the alveolar surface and interstitial spaces with active Na transport across the alveolar epithelium generating the osmotic force for movement of water (12, 17). The broadly accepted paradigm for Na transport in the alveoli involves a two-step process: movement of Na through cation channels in the apical cell membrane constitutes the first step, and extrusion of the excess Na thus acquired via basolateral Na-K-ATPase is the second step (19). However, despite extensive research using many different approaches that support this paradigm, gaps still exist in our understanding of how this fine balance is achieved in vivo (19). In particular, there are several unresolved questions that, when resolved, could significantly affect our view of alveolar fluid clearance. First, type 1 cells constitute 95% of the alveolar surface area, but their role in ion transport is not completely clear. If there are significant differences between the transport characteristics of type I and type II cells, the current paradigm describing alveolar fluid clearance would need to be modified. Second, although cation permeability pathways have been described fairly well, the precise pathways for movement of anions are unclear. The character of these channels is important since anion channels could form either co-ion pathways for the concomitant movement of Na and Cl or a secretory pathway to increase alveolar fluid. Finally, alveolar fluid clearance appears to consist of a basal and stimulated component of reabsorption. The relative contribution of different ion channels and different cell types to these two components of clearance is far from clear. These questions are all underscored by the differences between the robust increases in transport that can be produced by several different treatments in vitro and the often weak clearance response of the in vivo lung epithelium to the same treatments

Regulation of Epithelial Na+ Channels (ENaC) by Methylation: A NOVEL METHYLTRANSFERASE STIMULATES ENaC ACTIVITY

Aldosterone acts to increase apical membrane permeability by activation of epithelial Na(+) channels (ENaC). We have previously shown that aldosterone activates ENaC early in the course of its action by stimulating the methylation of the beta subunit of this heteromeric channel in A6 cells. Aldosterone also stimulates the expression and methylation of k-ras in A6 cells. To determine whether aldosterone-stimulated methylations are seen in mammalian cells, we examined the effect of aldosterone on methylation and ras activation in a continuous line of cultured epithelial cells derived from mouse cortical collecting duct (CCD) and determined that beta mENaC is a substrate for methylation by an enzyme contained in CCD cells. Aldosterone stimulated protein base labile methylation in CCD cells. Aldosterone stimulated Na(+) transport in CCD cells within 1 h of addition and without an increase in cellular amount of any ENaC subunits over the first 4 h. Inhibition of methylation, using the inhibitor 3-deaza-adenosine, blocked the stimulation of Na(+) transport induced by aldosterone at early time points (1-4 h) without affecting cellular amounts of any ENaC subunits. In contrast to 3-deaza-adenosine (3-DZA), which inhibits all methylation reactions, specific inhibitors of small G-protein methylation or prenylation had no effect on the early aldosterone-induced current. Overexpression of isoprenylcysteine carboxylmethyltransferase (PCMTase), the enzyme that methylates ras, had little effect on basal transport but enhanced aldosterone-stimulated transport in A6 cells. Overexpression of PCMTase in CCD cells had no effect on either basal or aldosterone-stimulated transport. Moreover PCMTase had no effect on ENaC activity when co-expressed in Xenopus oocytes. Aldosterone had no effect on either message or protein levels of k-ras in CCD cells. Searching a mouse kidney library, we identified a methyltransferase that stimulates ENaC activity in Xenopus oocytes without affecting surface expression of ENaC. Our results demonstrate that aldosterone stimulates protein methylation in CCD cells, and this is required for expression of the early transport response. In CCD cells this effect is not mediated via methylation of ras, which is not induced by aldosterone in these cells, and the enzyme that methylates ras has no direct effect on ENaC activity. beta ENaC is a substrate for methylation in CCD cells. A novel methyltransferase that stimulates ENaC directly has been identified in CCD cells.

Regulation of maturation and processing of ENaC subunits in the rat kidney

Antibodies directed against subunits of the epithelial Na channel (ENaC) were used together with electrophysiological measurements in the cortical collecting duct to investigate the processing of the proteins in rat kidney with changes in Na or K intake. When animals were maintained on a low-Na diet for 7-9 days, the abundance of two forms of the alpha-subunit, with apparent masses of 85 and 30 kDa, increased. Salt restriction also increased the abundance of the beta-subunit and produced an endoglycosidase H (Endo H)-resistant pool of this subunit. The abundance of the 90-kDa form of the gamma-subunit decreased, whereas that of a 70-kDa form increased and this peptide also exhibited Endo H-resistant glycosylation. These changes in alpha- and gamma-subunits were correlated with increases in Na conductance elicited by a 4-h infusion with aldosterone. Changes in all three subunits were correlated with decreases in Na conductance when Na-deprived animals drank saline for 5 h. We conclude that ENaC subunits are mainly in an immature form in salt-replete rats. With Na depletion, the subunits mature in a process that involves proteolytic cleavage and further glycosylation. Similar changes occurred in alpha- and gamma- but not beta-subunits when animals were treated with exogenous aldosterone, and in beta- and gamma- but not alpha-subunits when animals were fed a high-K diet. Changes in the processing and maturation of the channels occur rapidly enough to be involved in the daily regulation of ENaC activity and Na reabsorption by the kidney.