Functional interaction of CFTR and ENaC in sweat glands

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a significant role in transepithelial salt absorption as well as secretion by a number of epithelial tissues including sweat glands, airways and intestine. Early studies suggested that in absorption significant cross talk occurs between CFTR Cl(-) channels and epithelial Na(+) channels (ENaC). Studies based primarily on cultured cells of the airways and on ex vivo expression systems suggested that activating CFTR inhibits ENaC channels so that activation of CFTR and deactivation of ENaC seem reciprocal. Lack of CFTR Cl(-) conductance (g(CFTR)) in the plasma membranes was seen to enhance ENaC conductance (g(ENaC)) and Na(+) absorption from the airway surface liquid causing airway pathology in cystic fibrosis (CF). To determine if these events hold true for a purely absorptive epithelium, we investigated the role of CFTR in regulating g(ENaC) in native human sweat gland ducts. After permeabilizing the basilateral membrane of the duct with alpha-toxin, the relative activities of ENaC and CFTR in the apical membrane were characterized by correlating the effect of activating CFTR with ENaC function. We found that in contrast to reciprocal activities, activating g(CFTR) by either cAMP, cGMP or the G-proteins plus 5 mM ATP was accompanied by a concomitant activation, not inhibition, of g(ENaC). The activation of g(ENaC) appeared to be critically dependent on CFTR Cl(-) channel function because removal of Cl(-) from the medium, blockage of CFTR with inhibitor DIDS or the absence of CFTR in the DeltaF508 CF ducts prevented activation of g(ENaC) by cAMP, GMP or G-proteins. Most significantly, g(ENaC) was dramatically reduced, not increased, in CF as compared to non-CF sweat ducts. These results showed that lack of CFTR in the plasma membranes is not characteristically coupled to elevated ENaC activity or to increased Na(+) absorption in CF epithelial cells. Not only are CFTR and ENaC activated together in duct salt absorption, but ENaC activation depends on functioning CFTR. NaCl is poorly absorbed in the CF duct because CFTR activity appears to impose a loss of ENaC activity as well.     

Nitric oxide has no beneficial effects on ion transport defects in cystic fibrosis human nasal epithelium

Nitric oxide (NO) has been reported to activate Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) and inhibit epithelial Na+ absorption mediated by amiloride-sensitive epithelial Na+ channels (ENaC). These ion transport systems are defective in cystic fibrosis (CF): Cl- secretion by CFTR is impaired and Na+ absorption by ENaC is dramatically increased. By activating CFTR and depressing ENaC, NO is a potentially beneficial therapeutic agent for ion transport defects in human CF respiratory epithelia. To assess the effects of NO on human respiratory epithelial cells, the NO donors sodium nitroprusside (SNP) and spermine NONOate were applied to primary cultured nasal cells, surgically obtained from non-CF and CF patients. Measurements of transepithelial short-circuit current (ISC) showed that NO has no inhibitory potency against amiloride-sensitive nasal ENaC (nENaC) or amiloride-insensitive Na+-absorbing mechanisms in non-CF and CF epithelia. Furthermore, NO had no stimulatory effect on Cl- secretion by CFTR or any other Cl- conductance pathway in either tissue. Although NO elevated the intracellular Ca2+ concentration, we did not detect any activation of Ca2+-dependent Cl- channels. These results demonstrate that NO has no beneficial effect on CF epithelial cells of the upper airways.     

cAMP-dependent activation of CFTR inhibits the epithelial sodium channel (ENaC) without affecting its surface expression

The cystic fibrosis transmembrane conductance regulator (CFTR) is thought to modulate epithelial sodium channel (ENaC) function in various preparations. However, the molecular nature and (patho-)physiological significance of the CFTR/ENaC interaction is still unclear and may vary in different tissues. Co-expression experiments in Xenopus laevis oocytes are a popular approach to investigate a possible functional interaction of CFTR and ENaC but have revealed controversial results. We could confirm previous reports that in oocytes co-expressing ENaC and CFTR the amiloride-sensitive current was reduced during cAMP-mediated stimulation of CFTR. In contrast, co-expression of CFTR per se had no effect on baseline ENaC currents. ENaC with Liddle's syndrome mutation is also inhibited during activation of CFTR, suggesting that the C-terminus of the ENaC beta-subunit is not important for this functional interrelation. Single-channel patch-clamp recordings demonstrated that co-expression of CFTR does not alter the single-channel conductance of ENaC. Using a chemiluminescence assay we demonstrated that the inhibition of ENaC during cAMP-dependent activation of CFTR was not associated with a decrease in ENaC surface expression. We conclude that the inhibitory effect of cAMP-activated CFTR on ENaC is due to a decrease in channel open probability.     

Temporal regulation of CFTR expression during ovine lung development: implications for CF gene therapy.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a small conductance chloride ion channel that may interact directly with other channels including the epithelial sodium channel (ENaC). CFTR is known to be more abundant in the airway epithelium during the second trimester of human development than after birth. This could be a consequence of the change in function of the respiratory epithelium from chloride secretion to sodium absorption near term. Alternatively it might reflect an additional role for CFTR in the developing airway epithelium. Though the lung epithelia of CF fetuses and infants rarely show gross histological abnormalities, there is often evidence of inflammation. Our aim was to establish whether CFTR expression levels correlated with specific developmental stages or differentiated functions in the ovine fetal lung. We evaluated CFTR expression using a quantitative assay of mRNA at 14 time points through gestation and showed highest levels at the start of the second trimester followed by a gradual decline through to term. In contrast, ENaC expression increased from the start of the third trimester. These results support a role for CFTR in differentiation of the respiratory epithelium and suggest that its expression levels are not merely reflecting major changes in the sodium/chloride bulk flow close to term. These observations may have significant implications for the likely success of CF gene therapy in the postnatal lung.     

Role of the amiloride-sensitive epithelial Na+ channel in the pathogenesis and as a therapeutic target for cystic fibrosis lung disease

Increased airway Na⁺ absorption mediated by the amiloride-sensitive epithelial Na⁺ channel (ENaC) is a basic defect in cystic fibrosis (CF) lung disease. Cystic fibrosis is one of the most common lethal hereditary diseases and is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. The CFTR acts as a cAMP-dependent Cl⁻ channel and regulator of ENaC, and CFTR dysfunction causes impaired Cl⁻ secretion and increased Na⁺ absorption in the airways of CF patients. Evidence from in vitro studies suggested that increased Na⁺ absorption produces airway surface liquid (ASL) volume depletion and led to the generation of transgenic mice with airway-specific overexpression of ENaC to elucidate the role of this mechanism in the in vivo pathogenesis of lung disease. Studies of the pulmonary phenotype of βENaC-overexpressing mice demonstrated that increased airway Na⁺ absorption caused ASL depletion and reduced mucus transport, producing a CF-like lung disease with airway mucus plugging, chronic airway inflammation and pulmonary mortality. Further, recent pharmacological studies demonstrated that preventive, but not late, inhibition of increased airway Na⁺ absorption with the ENaC blocker amiloride reduced morbidity and mortality in this murine model of CF lung disease. These results support a critical role of ENaC in the in vivo pathogenesis of CF lung disease and suggest that amiloride may be an effective preventive therapy for CF patients.     

Control of epithelial Na+ conductance by the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel expressed in luminal membranes of secretory and reabsorptive epithelia. CFTR plays a predominant role in both cAMP- and Ca2+-activated secretion of electrolytes. Although Ca2+-dependent Cl- channels exist independent of CFTR in the airway epithelium, their physiological significance remains to be determined. However, CFTR seems to be the only relevant Cl- conductance in the colonic epithelium. Apart from its secretory function, CFTR also has a task in regulating the reabsorption of electrolytes by controlling the activity of the epithelial Na+ channel, ENaC. Accordingly, defects in CFTR causing the disease cystic fibrosis (CF) lead to disturbances of both the secretion and absorption of electrolytes. Therefore, it is unclear what is pathophysiologically more important for the development of CF lung disease, the impaired secretion of Cl- or the enhanced reabsorption of Na+ and consecutive hyperabsorption of electrolytes. The mechanisms of how CFTR and ENaC interact are unknown. Previous work has given rise to several interesting working hypothesis, such as direct protein interaction or interaction via cytoskeletal proteins. Recent studies demonstrate the importance of the first nucleotide binding fold of CFTR, not only for the inhibition of ENaC but also for the interaction with other ion channels. Further studies are required to demonstrate whether regulation of other ion channels and membrane transport by CFTR occur by a common mechanism.     

ENaC is inhibited by an increase in the intracellular Cl(-) concentration mediated through activation of Cl(-) channels

Activation of the CFTR Cl(-) channel inhibits epithelial Na(+) absorption, according to studies on native epithelia derived from airways, colon and kidney, and can also be demonstrated in overexpressing cells. However, Na(+) absorption is not inhibited by CFTR in the native sweat duct epithelium. The mechanism for the inhibition of epithelial sodium channels (ENaC) has been examined in most detail in Xenopus oocytes coexpressing CFTR and ENaC. It was shown that ENaC is inhibited during stimulation of CFTR in Xenopus oocytes, independent of the experimental setup and the magnitude of the whole-cell current. However, a minimal Cl(-) conductance is required for inhibition of ENaC, and inhibition is augmented at higher CFTR-to-ENaC currents ratios. Low-CFTR-to-ENaC conductance ratios may be the reason for the absence of ENaC inhibition, as described recently. Similar to CFTR, ClC-0 Cl(-) currents also inhibit ENaC, as well as high extracellular Na(+) and Cl(-) in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and could be mediated by Cl(-) flow and/or changes in the intracellular Cl(-) concentration. These results are reminiscent of the Cl(-) feedback regulation observed in mouse mandibular duct cells. Current results obtained with ENaC mutants examined in Xenopus oocytes suggest a charge interaction of Cl(-) ions with the epithelial sodium channel.     

Effects of rhinovirus infection on the expression and function of cystic fibrosis transmembrane conductance regulator and epithelial sodium channel in human nasal mucosa

BackgroundChanges in expression and function of the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) have been found to cause airway surface liquid (ASL) derangement and to impair mucociliary clearance, both of which have been linked to the pathogenesis of rhinovirus (RV) infection.ObjectivesThe effects of RV infection on the expression and function of CFTR and ENaC in nasal epithelial cells were investigated.MethodsNasal epithelial cells obtained from 14 turbinoplasty patients were infected with RV serotype 16 (RV-16) for 4 hours. Expression of CFTR, α-ENaC, β-ENaC, and γ-ENaC was determined by real-time polymerase chain reaction, Western blot analysis, and confocal immunofluorescence microscopy. Functional changes in the CFTR and ENaC proteins were assessed by measuring transepithelial resistance (TER) using a voltmeter combined with ion channel modulators.ResultsRhinovirus infection increased expression of CFTR, α-ENaC, β-ENaC, and γ-ENaC messenger RNA (mRNA) and protein compared with controls (P < .05 each) and increased the expression of all 4 proteins on confocal immunofluorescence microscopy. Treatment of cells with the ENaC blocker amiloride and the CFTR activator forskolin increased TER in RV-infected cells, whereas forskolin decreased TER in uninfected cells. The CFTR inhibitor NPPB, however, blocked CFTR more in RV-infected than in noninfected cells.ConclusionsRhinovirus increased the expression of CFTR and appeared to alter its function. In contrast, ENaC expression and function were increased by RV infection. Therefore, RV infection may impair mucociliary transport of nasal epithelium by these alterations.     

Regulation of ENaC biogenesis by the stress response protein SERP1

Cystic fibrosis lung disease is caused by reduced Cl^? secretion along with enhanced Na⁺ absorption, leading to reduced airway surface liquid and compromised mucociliary clearance. Therapeutic strategies have been developed to activate cystic fibrosis transmembrane conductance regulator (CFTR) or to overcome enhanced Na⁺ absorption by the epithelial Na⁺ channel (ENaC). In a split-ubiquitin-based two-hybrid screening, we identified stress-associated ER protein 1 (SERP1)/ribosome-associated membrane protein 4 as a novel interacting partner for the ENaC β-subunit. SERP1 is induced during cell stress and interacts with the molecular chaperone calnexin, thus controlling early biogenesis of membrane proteins. ENaC activity was measured in the human airway epithelial cell lines H441 and A549 and in voltage clamp experiments with ENaC-overexpressing Xenopus oocytes. We found that expression of SERP1 strongly inhibits amiloride-sensitive Na⁺ transport. SERP1 coimmunoprecipitated and colocalized with βENaC in the endoplasmic reticulum, together with the chaperone calnexin. In contrast to the inhibitory effects on ENaC, SERP1 appears to promote expression of CFTR. Taken together, SERP1 is a novel cochaperone and regulator of ENaC expression.     

Regulation of epithelial ion channels by the cystic fibrosis transmembrane conductance regulator

In most epithelia ion transport is tightly regulated. One major primary target of such regulation is the modulation of ion channels. The present brief review focuses on one specific example of ion channel regulation by the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a cAMP-regulated Cl^- channel. Its defect leads to the variable clinical pictures of cystic fibrosis (CF), which today is understood as a primary defect of epithelial Cl^- channels in a variety of tissues such as the respiratory tract, intestine, pancreas, skin, epididymis, fallopian tube, and others. Most recent findings suggest that CFTR also acts as a channel regulator. Three examples are discussed by which CFTR regulates other Cl^- channels, K⁺ channels, and epithelial Na⁺ channels. From this perspective it is evident that CFTR may play a major role in the integration of cellular function.Abbreviations CF Cystic fibrosis - CFTR Cystic fibrosis transmembrane conductance regulator - IBMX Isobutylmethylxanthine - ICOR Intermediate conductance outwardly rectifying - MDR Multidrug resistance protein Supported by DFG: Gr 480/11     

Electrolyte transport in the mammalian colon: mechanisms and implications for disease.

The colonic epithelium has both absorptive and secretory functions. The transport is characterized by a net absorption of NaCl, short-chain fatty acids (SCFA), and water, allowing extrusion of a feces with very little water and salt content. In addition, the epithelium does secret mucus, bicarbonate, and KCl. Polarized distribution of transport proteins in both luminal and basolateral membranes enables efficient salt transport in both directions, probably even within an individual cell. Meanwhile, most of the participating transport proteins have been identified, and their function has been studied in detail. Absorption of NaCl is a rather steady process that is controlled by steroid hormones regulating the expression of epithelial Na(+) channels (ENaC), the Na(+)-K(+)-ATPase, and additional modulating factors such as the serum- and glucocorticoid-regulated kinase SGK. Acute regulation of absorption may occur by a Na(+) feedback mechanism and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl(-) secretion in the adult colon relies on luminal CFTR, which is a cAMP-regulated Cl(-) channel and a regulator of other transport proteins. As a consequence, mutations in CFTR result in both impaired Cl(-) secretion and enhanced Na(+) absorption in the colon of cystic fibrosis (CF) patients. Ca(2+)- and cAMP-activated basolateral K(+) channels support both secretion and absorption of electrolytes and work in concert with additional regulatory proteins, which determine their functional and pharmacological profile. Knowledge of the mechanisms of electrolyte transport in the colon enables the development of new strategies for the treatment of CF and secretory diarrhea. It will also lead to a better understanding of the pathophysiological events during inflammatory bowel disease and development of colonic carcinoma.     

Morphological analysis of the trachea and pattern of breathing in βENaC-Tg mice

Cystic fibrosis (CF) is caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene which is a Cl- channel and a regulator of the epithelial Na+ channel (ENaC). We have recently shown that newborn CFTR-deficient mice exhibit abnormalities of the tracheal cartilage leading to altered ventilation (Bonvin et al., 2008). However, the mechanism by which a lack of CFTR causes tracheal cartilage defects remains unknown. The main goal of the present study was to determine whether the development of airway cartilage defects is related to ENac channel dysfunction. We thus performed macroscopic analysis of the trachea and explored ventilatory function in adult βENaC-overexpressing (βENaC-Tg) mice with airway Na+ hyperabsorption and "CF-lung" lung disease, at 2 and 5 month of age. Only minor cartilaginous abnormalities were observed in 8 out of 16 βENaC-Tg mice and in 2 out of 20 littermate controls. Breathing pattern was progressively altered in βENaC-Tg mice as evidenced by a significant decrease in respiratory frequency. Our results suggest that Na+ hyperabsorption alone is not a major contributor to the development of tracheal malformation observed in CF mice and that breathing pattern changes in βENaC-Tg mice likely reflect airflow limitation due to airway mucus obstruction.     

囊性纤维化跨膜调节因子与非囊性纤维化肺部疾病关系的研究进展

囊性纤维化跨膜调节因子(cystic fibrosis transmembrane regulatory factor,CFTR)是一种跨膜蛋白质,也是一种重要的离子通道.囊性纤维化(cystic fibrosis,CF)是一种具有家族常染色体隐性遗传的先天性疾病,CFTR基因突变使其编码的CFTR蛋白功能缺陷从而导致囊性纤维化的发生.CFTR与非囊性纤维化肺部疾病如肺水肿、急性呼吸窘迫综合征和气道上皮损伤等的发生发展密切相关.     

Mutations in the amiloride‐sensitive epithelial sodium channel in patients with cystic fibrosis‐like disease

We investigated whether mutations in the genes that code for the different subunits of the amiloride‐sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)‐like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three‐fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.–55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R‐SCNN1A polymorphism was even found to result in a four‐fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R‐SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0–10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of cystic fibrosis transmembrane conductance regulator in the human distal lung

The determination of the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the lung is essential for a full understanding of the normal lung physiology and the pathogenesis of the lung disease in cystic fibrosis (CF). However, studies on the expression of CFTR in the distal adult human lung have yielded conflicting results despite functional evidence of expression of CFTR in bronchiolar and alveolar epithelial cells. We used 2 high-affinity monoclonal anti-CFTR antibodies, MAb24-1 and MAb13-1, to determine the expression of CFTR in samples of bronchiolar and alveolar tissues obtained from the same non-CF individuals. CFTR immunostaining was detected in the epithelium of bronchiolar and alveolar tissues. The staining pattern was similar with both antibodies. In bronchioles, CFTR labeling was present mostly in ciliated cells; in alveoli, CFTR labeling was detected in both type I and type II cells. We conclude that CFTR is expressed in human bronchiolar and alveolar epithelial cells. The potential importance of CFTR expression in alveoli should be further investigated, particularly with respect to the CF lung disease and the physiology of the alveolar region.     

Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain

Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.     

Role of the cystic fibrosis transmembrane conductance channel in human airway smooth muscle

Patients with cystic fibrosis (CF) suffer from asthma-like symptoms and gastrointestinal cramps, attributed to a mutation in the CF transmembrane conductance regulator (CFTR) gene present in a variety of cells. Pulmonary manifestations of the disease include the production of thickened mucus and symptoms of asthma, such as cough and wheezing. A possible alteration in airway smooth muscle (ASM) cell function of patients with CF has not been investigated. The aim of this study was to determine whether the (CFTR) channel is present and affects function of human ASM cells. Cell cultures were obtained from the main or lobar bronchi of patients with and without CF, and the presence of the CFTR channel detected by immunofluorescence. Cytosolic Ca(2+) was measured using Fura-2 and dual-wavelength microfluorimetry. The results show that CFTR is expressed in airway bronchial tissue and in cultured ASM cells. Peak Ca(2+) release in response to histamine was significantly decreased in CF cells compared with non-CF ASM cells (357 +/- 53 nM versus 558 +/- 20 nM; P < 0.001). The CFTR pharmacological blockers, glibenclamide and N-phenyl anthranilic acid, significantly reduced histamine-induced Ca(2+) release in non-CF cells, and similar results were obtained when CFTR expression was varied using antisense oligonucleotides. In conclusion, these data show that the CFTR channel is present in ASM cells, and that it modulates the release of Ca(2+) in response to contractile agents. In patients with CF, a dysfunctional CFTR channel could contribute to the asthma diathesis and gastrointestinal problems experienced by these patients.