Assessing mucociliary transport of single particles in vivo shows variable speed and preference for the ventral trachea in newborn pigs.

Mucociliary transport (MCT) is an innate defense mechanism that removes particulates, noxious material, and microorganisms from the lung. Several airway diseases exhibit abnormal MCT, including asthma, chronic bronchitis, and cystic fibrosis. However, it remains uncertain whether MCT abnormalities contribute to the genesis of disease or whether they are secondary manifestations that may fuel disease progression. Limitations of current MCT assays and of current animal models of human disease have hindered progress in addressing these questions. Therefore, we developed an in vivo assay of MCT, and here we describe its use in newborn wild-type pigs. We studied pigs because they share many physiological, biochemical, and anatomical features with humans and can model several human diseases. We used X-ray multidetector-row-computed tomography to track movement of individual particles in the large airways of newborn pigs. Multidetector-row-computed tomography imaging provided high spatial and temporal resolution and registration of particle position to airway anatomy. We discovered that cilia orientation directs particles to the ventral tracheal surface. We also observed substantial heterogeneity in the rate of individual particle movement, and we speculate that variations in mucus properties may be responsible. The increased granularity of MCT data provided by this assay may provide an opportunity to better understand host defense mechanisms and the pathogenesis of airway disease.

Human cystic fibrosis airway epithelia have reduced Cl- conductance but not increased Na+ conductance.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF) lung disease. CFTR is expressed in airway epithelia, but how CF alters electrolyte transport across airway epithelia has remained uncertain. Recent studies of a porcine model showed that in vivo, excised, and cultured CFTR(-/-) and CFTR(ΔF508/ΔF508) airway epithelia lacked anion conductance, and they did not hyperabsorb Na(+). Therefore, we asked whether Cl(-) and Na(+) conductances were altered in human CF airway epithelia. We studied differentiated primary cultures of tracheal/bronchial epithelia and found that transepithelial conductance (Gt) under basal conditions and the cAMP-stimulated increase in Gt were markedly attenuated in CF epithelia compared with non-CF epithelia. These data reflect loss of the CFTR anion conductance. In CF and non-CF epithelia, the Na(+) channel inhibitor amiloride produced similar reductions in Gt and Na(+) absorption, indicating that Na(+) conductance in CF epithelia did not exceed that in non-CF epithelia. Consistent with previous reports, adding amiloride caused greater reductions in transepithelial voltage and short-circuit current in CF epithelia than in non-CF epithelia; these changes are attributed to loss of a Cl(-) conductance. These results indicate that Na(+) conductance was not increased in these cultured CF tracheal/bronchial epithelia and point to loss of anion transport as key to airway epithelial dysfunction in CF.

Acute regulation of tight junction ion selectivity in human airway epithelia.

Electrolyte transport through and between airway epithelial cells controls the quantity and composition of the overlying liquid. Many studies have shown acute regulation of transcellular ion transport in airway epithelia. However, whether ion transport through tight junctions can also be acutely regulated is poorly understood both in airway and other epithelia. To investigate the paracellular pathway, we used primary cultures of differentiated human airway epithelia and assessed expression of claudins, the primary determinants of paracellular permeability, and measured transepithelial electrical properties, ion fluxes, and La(3+) movement. Like many other tissues, airway epithelia expressed multiple claudins. Moreover, different cell types in the epithelium expressed the same pattern of claudins. To evaluate tight junction regulation, we examined the response to histamine, an acute regulator of airway function. Histamine stimulated a rapid and transient increase in the paracellular Na(+) conductance, with a smaller increase in Cl(-) conductance. The increase was mediated by histamine H(1) receptors and depended on an increase in intracellular Ca(2+) concentration. These results suggest that ion flow through the paracellular pathway can be acutely regulated. Such regulation could facilitate coupling of the passive flow of counter ions to active transcellular transport, thereby controlling net transepithelial salt and water transport.

Coordinate Regulation of Ribosome and tRNA Biogenesis Controls Hypoxic Injury and Translation.

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C. elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher-order-translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources.

Requirement for translocon-associated protein (TRAP) α in insulin biogenesis.

The mechanistic basis for the biogenesis of peptide hormones and growth factors is poorly understood. Here, we show that the conserved endoplasmic reticulum membrane translocon-associated protein α (TRAPα), also known as signal sequence receptor 1, plays a critical role in the biosynthesis of insulin. Genetic analysis in the nematode Caenorhabditis elegans and biochemical studies in pancreatic ß cells reveal that TRAPα deletion impairs preproinsulin translocation while unexpectedly disrupting distal steps in insulin biogenesis including proinsulin processing and secretion. The association of common intronic single-nucleotide variants in the human TRAPα gene with susceptibility to type 2 diabetes and pancreatic ß cell dysfunction suggests that impairment of preproinsulin translocation and proinsulin trafficking may contribute to the pathogenesis of type 2 diabetes.

Ageing and hypoxia cause protein aggregation in mitochondria.

Aggregation of cytosolic proteins is a pathological finding in disease states, including ageing and neurodegenerative diseases. We have previously reported that hypoxia induces protein misfolding in Caenorhabditis elegans mitochondria, and electron micrographs suggested protein aggregates. Here, we seek to determine whether mitochondrial proteins actually aggregate after hypoxia and other cellular stresses. To enrich for mitochondrial proteins that might aggregate, we performed a proteomics analysis on purified C. elegans mitochondria to identify relatively insoluble proteins under normal conditions (110 proteins identified) or after sublethal hypoxia (65 proteins). A GFP-tagged mitochondrial protein (UCR-11 - a complex III electron transport chain protein) in the normally insoluble set was found to form widespread aggregates in mitochondria after hypoxia. Five other GFP-tagged mitochondrial proteins in the normally insoluble set similarly form hypoxia-induced aggregates. Two GFP-tagged mitochondrial proteins from the soluble set as well as a mitochondrial-targeted GFP did not form aggregates. Ageing also resulted in aggregates. The number of hypoxia-induced aggregates was regulated by the mitochondrial unfolded protein response (UPRmt) master transcriptional regulator ATFS-1, which has been shown to be hypoxia protective. An atfs-1(loss-of-function) mutant and RNAi construct reduced the number of aggregates while an atfs-1(gain-of-function) mutant increased aggregates. Our work demonstrates that mitochondrial protein aggregation occurs with hypoxic injury and ageing in C. elegans. The UPRmt regulates aggregation and may protect from hypoxia by promoting aggregation of misfolded proteins.

N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3' Splice Site Function in Vivo.

Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3' splice site. In the nematode Caenorhabditis elegans, 3' splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3' splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the -5 position of the octamer motif is the most uncommon base in C. elegans 3' splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved -5 uridine residue in the C. elegans 3' splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo.

Chromoanasynthetic Genomic Rearrangement Identified in a N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Screen in Caenorhabditis elegans.

Chromoanasynthesis is a recently discovered phenomenon in humans with congenital diseases that is characterized by complex genomic rearrangements (CGRs) resulting from aberrant repair of catastrophic chromosomal damage. How these CGRs are induced is not known. Here, we describe the structure and function of dpDp667, a causative CGR that emerged from a Caenorhabditis elegans dauer suppressor screen in which animals were treated with the point mutagen N-ethyl-N-nitrosourea (ENU). dpDp667 comprises nearly 3 Mb of sequence on the right arm of the X chromosome, contains three duplications and one triplication, and is devoid of deletions. Sequences from three out of the four breakpoint junctions in dpDp667 reveal microhomologies that are hallmarks of chromoanasynthetic CGRs. Our findings suggest that environmental insults and physiological processes that cause point mutations may give rise to chromoanasynthetic rearrangements associated with congenital disease. The relatively subtle phenotype of animals harboring dpDp667 suggests that the prevalence of CGRs in the genomes of mutant and/or phenotypically unremarkable animals may be grossly underestimated.

Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans.

FoxO transcription factors promote longevity across taxa. How they do so is poorly understood. In the nematode Caenorhabditis elegans, the A- and F-isoforms of the FoxO transcription factor DAF-16 extend life span in the context of reduced DAF-2 insulin-like growth factor receptor (IGFR) signaling. To elucidate the mechanistic basis for DAF-16/FoxO-dependent life span extension, we performed an integrative analysis of isoform-specific daf-16/FoxO mutants. In contrast to previous studies suggesting that DAF-16F plays a more prominent role in life span control than DAF-16A, isoform-specific daf-16/FoxO mutant phenotypes and whole transcriptome profiling revealed a predominant role for DAF-16A over DAF-16F in life span control, stress resistance, and target gene regulation. Integration of these datasets enabled the prioritization of a subset of 92 DAF-16/FoxO target genes for functional interrogation. Among 29 genes tested, two DAF-16A-specific target genes significantly influenced longevity. A loss-of-function mutation in the conserved gene gst-20, which is induced by DAF-16A, reduced life span extension in the context of daf-2/IGFR RNAi without influencing longevity in animals subjected to control RNAi. Therefore, gst-20 promotes DAF-16/FoxO-dependent longevity. Conversely, a loss-of-function mutation in srr-4, a gene encoding a seven-transmembrane-domain receptor family member that is repressed by DAF-16A, extended life span in control animals, indicating that DAF-16/FoxO may extend life span at least in part by reducing srr-4 expression. Our discovery of new longevity genes underscores the efficacy of our integrative strategy while providing a general framework for identifying specific downstream gene regulatory events that contribute substantially to transcription factor functions. As FoxO transcription factors have conserved functions in promoting longevity and may be dysregulated in aging-related diseases, these findings promise to illuminate fundamental principles underlying aging in animals.

Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs.

Almost two decades after CFTR was identified as the gene responsible for cystic fibrosis (CF), we still lack answers to many questions about the pathogenesis of the disease, and it remains incurable. Mice with a disrupted CFTR gene have greatly facilitated CF studies, but the mutant mice do not develop the characteristic manifestations of human CF, including abnormalities of the pancreas, lung, intestine, liver, and other organs. Because pigs share many anatomical and physiological features with humans, we generated pigs with a targeted disruption of both CFTR alleles. Newborn pigs lacking CFTR exhibited defective chloride transport and developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn humans with CF. The pig model may provide opportunities to address persistent questions about CF pathogenesis and accelerate discovery of strategies for prevention and treatment.

Basolateral chloride current in human airway epithelia.

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl(-) absorption. However, the pathways for basolateral Cl(-) exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl(-) conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl(-) conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl(-) current that was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl(-) > Br(-) >> I(-). Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca(2+) and inducing cell swelling had no effect. The basolateral Cl(-) current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl(-) currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl(-) conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl(-) conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl(-) movement.

Nedd4-2 isoforms differentially associate with ENaC and regulate its activity.

Mutations that disrupt a PY motif in epithelial Na(+) channel (ENaC) subunits increase surface expression of Na(+) channels in the collecting duct, resulting in greater Na(+) reabsorption. Nedd4 and Nedd4-2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. We recently reported that human Nedd4-2 (hNedd4-2) is expressed as many isoforms because of alternative promoter usage and/or variable splicing. To understand the relevance of hNedd4-2 isoforms for collecting duct Na(+) transport, we studied the interaction with ENaC and the intracellular localization and function of the following three naturally occurring hNedd4-2 isoforms: full-length Nedd4-2 (Nedd4-2), Nedd4-2 lacking the NH(2)-terminal C2 domain (Nedd4-2DeltaC2), and Nedd4-2 lacking the C2 domain and WW domains 2 and 3 (Nedd4-2DeltaWW2,3). Nedd4-2 and Nedd4-2DeltaC2 associate with ENaC and robustly reduce Na(+) transport in Xenopus oocytes, whereas the interaction with and functional effect of Nedd4-2DeltaWW2,3 on ENaC is weak. Nedd4-2 is expressed in the mouse collecting duct, and overexpression of Nedd4-2 reduces endogenous ENaC activity in a collecting duct cell line. This reduction in ENaC activity can be reversed early with exposure to dexamethasone, an effect that is associated with an increase in sgk1 abundance. The C2 domain is required to target Nedd4-2 to the plasma membrane in response to elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in MDCK cells, although it does not appear to mediate the inhibitory effect of [Ca(2+)](i) on Na(+) transport. Our data illustrate that naturally occurring hNedd4-2 isoforms differentially associate with ENaC to regulate its activity.

New insights into epithelial sodium channel function in the kidney: site of action, regulation by ubiquitin ligases, serum- and glucocorticoid-inducible kinase and proteolysis.

PURPOSE OF REVIEW: The epithelial sodium channel (ENaC) sets the rate of Na+ reabsorption in the collecting duct. This review describes recent advances in our understanding of ENaC function. RECENT FINDINGS: First, collecting duct-specific deletion of alphaENaC does not cause Na wasting in mice, suggesting that other regions can compensate. Second, Nedd4 and Nedd4-2 are ubiquitin ligases that reduce surface expression of ENaC and inhibit Na+ transport. Nedd4-2, but not Nedd4, is negatively regulated by serum- and glucocorticoid-inducible kinase 1, an aldosterone-induced kinase, providing an attractive mechanism for the stimulatory effect of aldosterone on Na+ transport. However, mice with germline ablation of serum- and glucocorticoid-inducible kinase 1 show only modest hypotension and are able to decrease Na+ excretion rates substantially. Third, maturation of ENaC is associated with processing at consensus furin cleavage sites and this cleavage is critical for channel activity. A separate class of serine proteases, the channel-activating proteases, also stimulates ENaC activity. SUMMARY: The connecting tubule of the kidney has abundant ENaC and Na(+)- and K(+)-transport capacity and may provide much of ENaC-mediated Na+ transport in the kidney. Aldosterone may increase Na transport, in part, by serum- and glucocorticoid-inducible kinase 1-mediated inhibition of Nedd4-2 but this has not been demonstrated in the native collecting duct or connecting tubule. The mild phenotype of the serum- and glucocorticoid-inducible kinase 1-knockout mouse points to serum- and glucocorticoid-inducible kinase 1-independent mechanisms that regulate Na+ transport. Two separate classes of protease appear to regulate Na+ transport: one is furin or furin-like and cleaves ENaC subunits to stimulate transport; the other, the channel-activating proteases, may act on ENaC or a regulatory molecule.

Genomic organization of the 5' end of human beta-ENaC and preliminary characterization of its promoter.

The mRNA for the beta-subunit of the epithelial Na(+) channel (beta-ENaC) is regulated developmentally and, in some tissues, in response to corticosteroids. To understand the mechanisms of transcriptional regulation of the human beta-ENaC gene, we characterized the 5' end of the gene and its 5'-flanking regions. Adaptor-ligated human kidney and lung cDNA were amplified by 5' rapid amplification of cDNA ends, and transcription start sites of two 5' variant transcripts were determined by nuclease protection or primer extension assays. Cosmid clones that contain the 5' end of the gene were isolated, and analysis of these clones indicated that alternate first exons approximately 1.5 kb apart and approximately 45 kb upstream of a common second exon formed the basis of these transcripts. Genomic fragments that included the proximal 5'-flanking region of either transcript were able to direct expression of a reporter gene in lung epithelia and to bind Sp1 in nuclear extracts, confirming the presence of separate promoters that regulate beta-ENaC expression.

Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains.

Mutations that disrupt a PY motif in epithelial Na+ channel (ENaC) subunits increase surface expression of Na+ channels in the collecting duct, resulting in greater Na+ reabsorption. Recently, Nedd4 and Nedd4-2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. To further understand the role of human Nedd4-2 (hNedd4-2), we cloned its cDNAs and determined its genomic organization using a bioinformatic approach. The gene is present as a single copy, spans at least 400 kb, and contains >40 exons. Multiple 5'-exons were identified by 5'-rapid amplification of cDNA ends, and tissue-specific expression of these transcripts was noted by RT-PCR and RNase protection assay. Alternate polyadenylation signal sequences led to varying lengths of the 3'-untranslated region. Alternate splicing events within internal exons were also noted. Open reading frame analysis indicates that hNedd4-2 encode multiple protein variants with and without a C2 domain, and with a variable number of WW domains. Coexpression, in Fischer rat thyroid epithelia, of ENaC and Nedd4-2 cDNAs leads to a significant reduction in amiloride-sensitive currents, confirming a role in Na+ transport regulation. In vitro binding studies demonstrated that individual PY motifs of alpha-, beta-, and gamma-ENaC have strong affinity for WW domains 3 and 4 but not 1 and 2. These studies indicate that alternate transcripts of Nedd4-2 may interact with ENaC differently. Understanding the function of variant proteins will increase our knowledge of the role of hNedd4-2 in the regulation of ENaC and define protein domains important for Nedd4-2 function.

Cycloheximide increases glucocorticoid-stimulated alpha -ENaC mRNA in collecting duct cells by p38 MAPK-dependent pathway.

Aldosterone and glucocorticoids (GCs) stimulate Na(+) reabsorption in the collecting ducts by increasing the activity of the epithelial Na(+) channel (ENaC). Our laboratory has used Madin-Darby canine kidney-C7 cells to demonstrate that this effect is associated with an increase in alpha-ENaC gene transcription (Mick VE, Itani OA, Loftus RW, Husted RF, Schmidt TJ, and Thomas CP, Mol Endocrinol 15: 575-588, 2001). Cycloheximide (CHX) superinduced the GC-stimulated alpha-ENaC expression in a dose-dependent manner, but had no effect on basal or aldosterone-stimulated alpha-ENaC expression, whereas anisomycin inhibited basal and corticosteroid-stimulated alpha-ENaC expression. The superinduction of alpha-ENaC expression was also seen with hypotonicity, was blocked by RU-38486, and was independent of protein synthesis. CHX had no effect on alpha-ENaC mRNA half-life, confirming that its effect was via an increase in alpha-ENaC transcription. The effect of CHX and hypotonicity on alpha-ENaC expression was abolished by SB-202190, indicating an effect mediated via p38 MAPK. Consistent with this scheme, CHX increased pp38 and MKK6, an upstream activator of p38, stimulated alpha-ENaC promoter activity. These data confirm a model in which CHX activates p38 in Madin-Darby canine kidney-C7 cells to increase alpha-ENaC gene transcription in a GC-dependent manner.

Glucocorticoids stimulate human sgk1 gene expression by activation of a GRE in its 5'-flanking region.

In lung and collecting duct epithelia, glucocorticoid (GC)-stimulated Na+ transport is preceded by an increase in the protein kinase sgk1, which in turn regulates the activity of the epithelial Na+ channel (ENaC). We investigated the mechanism for GC-regulated human sgk1 expression in lung and renal epithelia. sgk1 mRNA was increased in these epithelia by GCs, and this was inhibited by actinomycin D and superinduced by cycloheximide, consistent with a transcriptional effect that did not require protein synthesis. To understand the basis for transcriptional regulation, the transcription initiation site was mapped and the 5'-flanking region cloned by PCR. A 3-kb fragment of the upstream region was coupled to luciferase and transfected into A549 cells. By deletion analysis, an imperfect GC response element (GRE) was identified that was necessary and sufficient for GC responsiveness. When tested with cell extracts, a specific protein recognized by an anti-GC receptor (GR) antibody bound the GRE in gel mobility shift assays. We conclude that GCs stimulate sgk1 expression in human epithelial cells via activation of a GRE in the 5'-flanking region of sgk1.

Glucocorticoid-stimulated lung epithelial Na(+) transport is associated with regulated ENaC and sgk1 expression.

H441 cells, a bronchiolar epithelial cell line, develop a glucocorticoid-regulated amiloride-sensitive Na(+) transport pathway on permeable supports (R. Sayegh, S. D. Auerbach, X. Li, R. Loftus, R. Husted, J. B. Stokes, and C. P. Thomas. J Biol Chem 274: 12431-12437, 1999). To understand its molecular basis, we examined the effect of glucocorticoids (GC) on epithelial Na(+) channel (ENaC)-alpha, -beta, and -gamma and sgk1 expression and determined the biophysical properties of Na(+) channels in these cells. GC stimulated the expression of ENac-alpha, -beta, and -gamma and sgk1 mRNA, with the first effect seen by 1 h. These effects were abolished by actinomycin D, but not by cycloheximide, indicating a direct stimulatory effect on ENaC and sgk1 mRNA synthesis. The GC effect on transcription of ENaC-alpha mRNA was accompanied by a significant increase in ENaC-alpha protein levels. GC also stimulated ENaC-alpha, -beta, and -gamma and sgk1 mRNA expression in A549 cells, an alveolar type II cell line. To determine the biophysical properties of the Na(+) channel, single-channel currents were recorded from cell-attached H441 membranes. An Na(+)-selective channel with slow kinetics and a slope conductance of 10.8 pS was noted, properties similar to ENaC-alpha, -beta, and -gamma expressed in Xenopus laevis oocytes. These experiments indicate that amiloride-sensitive Na(+) transport is mediated through classic ENaC channels in human lung epithelia and that GC-regulated Na(+) transport is accompanied by increased transcription of each of the component subunits and sgk1.