On the mechanism of genistein-induced activation of protein kinase A-dependent Cl– conductance in cardiac myocytes

Genistein, an inhibitor of protein tyrosine kinase (PTK), enhanced the activation of the cardiac isoform of the protein kinase A (PKA)-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– conductance in guinea-pig ventricular cells. We examined the mechanism(s) underlying this excitatory action of genistein by using patch-clamp techniques. The CFTR Cl^– conductance, activated by isoproterenol (ISO, 10 nM; [Cl^–] 153 mM extracellular, 21 mM intracellular; 36 °C), was enhanced by 20 μM genistein. Daidzein, a structural analogue of genistein with little inhibitory action on PTK, also enhanced CFTR Cl^– currents. After maximal activation of the Cl^– conductance by a cocktail of adenosine 3’,5’-cyclic monophosphate, 3-isobutyl-1-methylxanthine and okadaic acid or vanadate plus forskolin in the pipette, genistein was no longer stimulatory but was rather slightly inhibitory at 100 μM. Direct exposure of myocytes to higher concentrations of genistein (50–100 μM) elicited outwardly rectifying currents with a reversal potential of –47 mV in the absence of ISO. In the presence of 50 μM H-89, a PKA inhibitor, genistein had no effect. Vanadate in the pipette at a concentration (100 μM) inhibiting phosphotyrosine phosphatases alone did not prevent the action of genistein. In contrast, no conductance was activated by tyrphostins B42 or 51 or lavendustin A, other PTK inhibitors. Genistein’s stimulation of cardiac CFTR Cl^– conductance appears to be independent of the PTK pathway and to be due to its direct interaction with CFTR Cl^– channels. Received: 22 January 1999 / Received after revision: 9 April 1999 / Accepted: 22 April 1999     

Activation of wild-type and ΔF508-CFTR by phosphodiesterase inhibitors through cAMP-dependent and -independent mechanisms

 The cAMP-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR) and its modulation through inhibition of phosphodiesterases (PDE) were studied with the cell-attached patch-clamp technique in Calu-3 cells (expressing endogenous CFTR) and NIH3T3 cells [expressing either wild-type (Wt)-CFTR or ΔF508-CFTR]. In Calu-3 cells, CFTR current was augmented by increasing concentrations of 8-(4-chlorophenylthio)-adenosine 3′,5′-cyclic monophosphate (CPT–cAMP) and reached a saturating level at ≥60 μM. Varying the forskolin concentration also modulated CFTR activity; 10 μM was maximally effective since supplemental application of 200 μM CPT–cAMP had no additional effect. Activation of CFTR by increasing the cAMP concentration occurs through an increase of the NP _o (product of the number of functional channels and the open probability) since the single-channel amplitude remains unchanged. In Calu-3 and NIH3T3-Wt cells, PDE inhibitors, milrinone (100 μM), 8-cyclopentyl-1,3-dipropylxanthine (CPX, 25 μM), and 3-isobutyl-1-methylxanthine (IBMX, 200 μM), did not enhance CFTR current initially activated with 10 μM forskolin, but each potentiated CFTR activity elicited with a submaximal forskolin concentration (e.g., 100 nM) and prolonged the deactivation of CFTR channel current upon removal of forskolin. Millimolar IBMX increased the NP _o of both Wt- and ΔF508-CFTR even under maximal cAMP stimulation. Quantitatively, these effects of millimolar IBMX on NP _o approximate those of genistein, which potentiates the cAMP-dependent CFTR activity via a mechanism that does not involve increases in cellular cAMP. Thus, depending on the concentration, PDE inhibitors may affect CFTR through different mechanisms. Received: 27 October 1998 / Accepted: 10 November 1998     

Localisation of wild-type and DeltaF508-CFTR in nasal epithelial cells

Wild-type and the DeltaF508 mutation of the cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) were localised by confocal imaging in DeltaF508/DeltaF508 native airway epithelial cells using a well-characterised CFTR antibody. Surface nasal epithelial cells from three control and three CF individuals were obtained from nasal brushings. Cells were fixed, permeabilised and incubated with first antibody for 18 h at 4 degrees C. Following labelling with second antibody, cells were viewed with the confocal microscope. Wild-type CFTR was localised predominantly apically, whereas DeltaF508-CFTR was located mainly inside the cell in a region close to the nucleus. Incubation of cells with MPB-07 (250 microM) at 37 degrees C for 2 h resulted in pronounced movement of DeltaF508-CFTR to the cell periphery, but did not change the localisation of wild-type CFTR. The results show that DeltaF508-CFTR is mislocalised in native nasal epithelial cells and that its distribution is altered in response to the new CFTR activator, MPB-07. The findings should lead to development of a rational drug treatment for CF patients carrying the DeltaF508 mutation.     

The CFTR-mediated protein secretion defect: pharmacological correction

The cystic fibrosis transmembrane conductance regulator (CFTR) mediates secretion of mucins and serous proteins. The aim was to correct pharmacologically the CFTR defect in protein secretion in airway gland cells and so to correct the viscous mucous secretions in cystic fibrosis (CF) airways and lungs. The strategies tested included direct activation of CFTR, bypass of CFTR-mediated protein secretion and movement of the mutated form of CFTR (DeltaF(508)-CFTR) to the cell membrane. Compounds related to 3-isobutyl-1-methylxanthine (IBMX), including a selective type-IV phosphodiesterase inhibitor and the adenosine receptor antagonists 8-cyclopentyltheophylline (CPT) and 8-cyclopentyl-1,3-dipropylxanthine (CPX), corrected the defective beta-adrenergic stimulation of mucin secretion in CFTR antibody-inhibited submandibular gland cells. CPT also corrected lactoferrin secretion in DeltaF(508)/DeltaF(508)-CFTR nasal gland cells. The data suggest that correction of CFTR protein secretion activity is not mediated by excessive increase in cyclic AMP, involves direct interaction with CFTR but does not require increase in CFTR Cl(-) channel activity. Regulated glycoprotein secretion was characterised in the airway gland cell line Calu-3 to investigate whether a CFTR bypass is present. Studies of DeltaF(508)-CFTR trafficking using confocal imaging showed that some DeltaF(508)-CFTR colocalised with the apical membrane protein CD59; however a large amount was mislocalised within the cell. The results showing pharmacological correction of the defective CFTR-mediated protein secretion afford promise for the development of a rational drug therapy for CF patients.     

SERCA pump inhibitors do not correct biosynthetic arrest of deltaF508 CFTR in cystic fibrosis

Deletion of phenylalanine 508 (deltaF508) accounts for nearly 70% of all mutations that occur in the cystic fibrosis transmembrane conductance regulator (CFTR). The deltaF508 mutation is a class II processing mutation that results in very little or no mature CFTR protein reaching the apical membrane and thus no cAMP-mediated Cl- conductance. Therapeutic strategies have been developed to enhance processing of the defective deltaF508 CFTR molecule so that a functional cAMP-regulated Cl- channel targets to the apical membrane. Sarcoplasmic/endoplasmic reticulum calcium (SERCA) inhibitors, curcumin and thapsigargin, have been reported to effectively correct the CF ion transport defects observed in the deltaF508 CF mice. We investigated the effect of these compounds in human airway epithelial cells to determine if they could induce deltaF508 CFTR maturation, and Cl- secretion. We also used Baby Hamster Kidney cells, heterologously expressing deltaF508 CFTR, to determine if SERCA inhibitors could interfere with the interaction between calnexin and CFTR and thereby correct the deltaF508 CFTR misfolding defect. Finally, at the whole animal level, we tested the ability of curcumin and thapsigargin to (1) induce Cl- secretion and reduce hyperabsorption of Na+ in the nasal epithelia of the deltaF508 mouse in vivo, and (2) induce Cl- secretion in intestine (jejunum and distal colon) and the gallbladder of the deltaF508 CF mouse. We conclude that curcumin and thapsigargin failed to induce maturation of deltaF508 CFTR, or induce Cl- secretion, as measured by biochemical and electrophysiologic techniques in a variety of model systems ranging from cultured cells to in vivo studies.     

VCP/p97 AAA-ATPase does not interact with the endogenous wild-type cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis. The most common mutation, DeltaF508 CFTR, is retained in the endoplasmic reticulum, retrotranslocated into the cytosol, and degraded by the proteasome. In a proteomics screen to identify DeltaF508 CFTR interacting proteins, we found that valosin-containing protein (VCP)/p97, a Type II AAA ATPase that is a component of the retrotranslocation machinery, binds DeltaF508 CFTR, and this interaction is stabilized by proteasomal inhibition. Since wild-type (WT) CFTR has been reported to be inefficiently processed during biogenesis with as much as 75% of the newly synthesized protein degraded by the proteasome, we examined the VCP interaction in Calu-3, T-84, and 16HBE, three epithelial cell lines that endogenously express WT CFTR. The results indicate that when WT CFTR processing is efficient, as demonstrated in Calu-3 cells, VCP does not interact. Interestingly, overexpression of recombinant WT CFTR in Calu-3 cells results in inefficient processing and VCP interaction, demonstrating that CFTR processing efficiency and the VCP interaction are tightly coupled. Furthermore, induction of ER stress and activation of the unfolded protein response result in inefficient processing of WT CFTR in Calu-3 cells and promote the WT CFTR-VCP interaction. The results support the hypothesis that components of the retrotranslocation machinery such as VCP do not interact with CFTR in epithelial cells that endogenously express WT CFTR, since under normal conditions the processing of the WT protein is efficient.     

Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening

Although there have been numerous reports from around the world of mutations in the gene of chromosome 7 known as CFTR (cystic fibrosis transmembrane conductance regulator), little attention has been given to integrating these mutant alleles into a global understanding of the population molecular genetics associated with cystic fibrosis (CF). We determined the distribution of CFTR mutations in as many regions throughout the world as possible in an effort designed to: 1) increase our understanding of ancestry-genotype relationships, 2) compare mutational arrays with disease incidence, and 3) gain insight for decisions regarding screening program enhancement through CFTR multi-mutational analyses. Information on all mutations that have been published since the identification and cloning of the CFTR gene's most common allele, DeltaF508 (or F508del), was reviewed and integrated into a centralized database. The data were then sorted and regional CFTR arrays were determined using mutations that appeared in a given region with a frequency of 0.5% or greater. Final analyses were based on 72,431 CF chromosomes, using data compiled from over 100 original papers, and over 80 regions from around the world, including all nations where CF has been studied using analytical molecular genetics. Initial results confirmed wide mutational heterogeneity throughout the world; however, characterization of the most common mutations across most populations was possible. We also examined CF incidence, DeltaF508 frequency, and regional mutational heterogeneity in a subset of populations. Data for these analyses were filtered for reliability and methodological strength before being incorporated into the final analysis. Statistical assessment of these variables revealed that there is a significant positive correlation between DeltaF508 frequency and the CF incidence levels of regional populations. Regional analyses were also performed to search for trends in the distribution of CFTR mutations across migrant and related populations; this led to clarification of ancestry-genotype patterns that can be used to design CFTR multi-mutation panels for CF screening programs. From comprehensive assessment of these data, we offer recommendations that multiple CFTR alleles should eventually be included to increase the sensitivity of newborn screening programs employing two-tier testing with trypsinogen and DNA analysis.     

Interaction between calcium-activated chloride channels and the cystic fibrosis transmembrane conductance regulator

We investigated interactions between cystic fibrosis conductance regulator (CFTR) and endogenous Ca2+-activated Cl- channels (CaCC) in bovine pulmonary artery endothelium (CPAE). CPAE cells, which do not express CFTR, were transiently transfected with wild-type (WT) CFTR and the deletion mutant deltaF508 CFTR. Currents through CaCC were significantly reduced after expression of WT CFTR. This inhibition was increased by stimulation (isobutylmethylxanthine, forskolin) of CFTR in cells expressing WT CFTR. There were no such effects when deltaF508 mutant CFTR, which is retained in the endoplasmic reticulum, was expressed. It is concluded that CFTR and CaCC are functionally coupled probably through a direct channel-channel interaction.     

MPB-07 reduces the inflammatory response to Pseudomonas aeruginosa in cystic fibrosis bronchial cells

Chronic lung inflammation in cystic fibrosis (CF) is specifically characterized by predominant endobronchial neutrophil infiltrates, colonization by Pseudomonas aeruginosa, and elevated levels of cytokines and chemokines, first of all IL-8. The extensive inflammatory process in CF lungs is the basis of progressive tissue damage and is largely considered detrimental, making antiinflammatory approaches a relevant therapeutic target. This neutrophil-dominated inflammation seems to be related to an excessive proinflammatory signaling, originating from the same surface epithelial cells expressing the defective CF transmembrane conductance regulator (CFTR) protein, although the underlying mechanisms have not been completely elucidated. To investigate the relationship between defective CFTR and the inflammatory response to P. aeruginosa in CF airway cells, we studied the effect of the DeltaF508 CFTR corrector, benzo(c)quinolizinium (MPB)-07 (Dormer et al., J Cell Science 2001;114:4073-4081). CF bronchial epithelial IB3-1 and CuFi-1 cells overproduced the inflammatory molecules, IL-8 and intercellular adhesion molecule (ICAM)-1, in response to P. aeruginosa, compared with the wild-type, CFTR-expressing bronchial cells, S9, and NuLi-1 cells. In both IB3-1 and CuFi-1 cells, the corrector MPB-07 dramatically reduces the IL-8 and ICAM-1 mRNA expression elicited by P. aeruginosa infection. Correction of CFTR-dependent Cl- efflux was confirmed in MPB-07-treated IB3-1 and CuFi-1 cells. In conclusion, the DeltaF508 CFTR corrector MPB-07 produces an antiinflammatory effect in CF bronchial cells exposed to P. aeruginosa in vitro.     

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.     

Cellular localization and activity of Ad-delivered GFP-CFTR in airway epithelial and tracheal cells

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)-CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTRDeltaF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTRDeltaF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRDeltaF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl- channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl- transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.     

Prediction of cellular immune responses against CFTR in patients with cystic fibrosis after gene therapy

Different classes of mutations (class I-VI) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are responsible for lung/pancreatic disease. The most common mutation, DeltaF508, is characterized by expression of precursor forms of CFTR but no functional CFTR. Since only 5-10% of normal CFTR function is required to correct the electrophysiologic defect across the airway epithelium, gene therapy holds promise for treatment of patients with CF lung disease. However, efficient delivery and transgene expression are not the only parameters that may influence the success of gene therapy. Host-specific immune responses generated against the therapeutic CFTR protein may pose a problem, especially when the coding sequence between the normal CFTR and mutated CFTR differ. This phenomenon is more pertinent to class I mutations in which large fragments of the protein are not expressed. However, T cells directed against epitopes that span sequences containing class II-V mutations are also possible. We used MHC-binding prediction programs to predict the probability of cellular immune responses that may be generated against CFTR in DeltaF508 homozygote patients. Results obtained from running the prediction algorithms yielded a few high-scoring MHC-Class I binders within the specific sequences, suggesting that there is a possibility of the host to mount a cellular immune response against CFTR, even when the difference between therapeutic and host CFTR is a single amino acid (F) at position 508.     

Regulation of chemokine expression by NaCl occurs independently of cystic fibrosis transmembrane conductance regulator in macrophages

Chronic pulmonary inflammation and infection are the leading causes of morbidity and mortality in cystic fibrosis (CF). While the effect of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) on airways remains controversial, some groups have demonstrated increases in Na(+) and Cl(-) in CF airway surface liquid compared to normal airways. We investigated the consequences of NaCl on pro-inflammatory chemokine and cytokine production by macrophages. Stimulation of mouse macrophages with increasing amounts of NaCl induced macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-alpha (TNF-alpha) production. Further, co-incubation of macrophages with NaCl in the presence of either lipopolysaccharide (LPS) or TNF-alpha synergistically increased MIP-2 production. Both the NaCl and NaCl plus LPS responses were partially dependent on endogenous production and autocrine signaling by TNF-alpha. To investigate the role of CFTR in MIP-2 production, we compared the responses of wild-type and DeltaF508 CF mouse macrophages to NaCl and LPS. The responses of macrophages from both strains were indistinguishable. In addition, CFTR mRNA was not expressed in macrophages. Taken together, these findings suggest that NaCl stimulates MIP-2 production by macrophages through a mechanism that is partially dependent on TNF-alpha but independent of macrophage CFTR expression.     

Misprocessing of the CFTR protein leads to mild cystic fibrosis phenotype

Cystic fibrosis (CF) is mainly caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The aim of this study was to determine the mechanism of dysfunction of a disease-causing mutation associated with variable phenotypes. In order to attain these objectives, we studied the effect of the p.L206W mutation on CFTR protein production and function, and we examined the genotype-phenotype correlation of [p.L206W]+[p.F508del] patients. We showed that p.L206W is a processing (class II) mutation since the CFTR biosynthetic pathway was severely impaired, whereas single-channel measurements indicated ion conductance similar to the wild-type protein. These data raise the larger question of the phenotypic variability of class II mutants, including p.F508del. Since multiple potential partners could modify the processing of the CFTR protein during its course to the cell surface, environmental and other genetic factors might contribute to this variability.     

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.     

Regulation of gap junctional communication in CFTR-expressing pancreatic epithelial cells

Gap junction channels provide a pathway for coordinating multicellular activity. To evaluate the contribution of cell-to-cell communication in the function of epithelial cells, we studied the strength of gap junctional coupling in pancreatic acinar and duct cells exposed to agents known to elevate the intracellular concentration of Ca(2+) or cAMP. In acinar cells, we observed that maximal concentrations of acetylcholine evoked a biphasic increase in cytosolic Ca(2+) mobilization. The second sustained phase, which depends on Ca(2+) influx into the cell, was associated with the rapid closure of gap junction channels. In duct cells, stimulation of CFTR-dependent Cl(-) currents with cAMP analogs markedly increased gap junctional conductance in pairs of cells. Interestingly, cAMP had no effect on intercellular communication between cells harboring the DeltaF508 mutation of CFTR. An abnormal pattern of gap junctional coupling may contribute to the altered functions of tissues affected in cystic fibrosis.     

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.     

Cystic fibrosis transmembrane conductance regulator-mediated corneal epithelial cell ingestion of Pseudomonas aeruginosa is a key component in the pathogenesis of experimental murine keratitis

Previous findings indicate that the cystic fibrosis transmembrane conductance regulator (CFTR) is a ligand for Pseudomonas aeruginosa ingestion into respiratory epithelial cells. In experimental murine keratitis, P. aeruginosa enters corneal epithelial cells. We determined the importance of CFTR-mediated uptake of P. aeruginosa by corneal cells in experimental eye infections. Entry of noncytotoxic (exoU) P. aeruginosa into human and rabbit corneal cell cultures was inhibited with monoclonal antibodies and peptides specific to CFTR amino acids 108 to 117. Immunofluorescence microscopy and flow cytometry demonstrated CFTR in the intact murine corneal epithelium, and electron microscopy showed that CFTR binds to P. aeruginosa following corneal cell ingestion. In experimental murine eye infections, multiple additions of 5 nM CFTR peptide 103-117 to inocula of either cytotoxic (exoU+) or noncytotoxic P. aeruginosa resulted in large reductions in bacteria in the eye and markedly lessened eye pathology. Compared with wild-type C57BL/6 mice, heterozygous DeltaF508 Cftr mice infected with P. aeruginosa had an approximately 10-fold reduction in bacterial levels in the eye and consequent reductions in eye pathology. Homozygous DeltaF508 Cftr mice were nearly completely resistant to P. aeruginosa corneal infection. CFTR-mediated internalization of P. aeruginosa by buried corneal epithelial cells is critical to the pathogenesis of experimental eye infection, while in the lung, P. aeruginosa uptake by surface epithelial cells enhances P. aeruginosa clearance from this tissue.     

Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments

Recent studies have reported that mutant genomic cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) sequences can be corrected in transformed CF airway epithelial cell lines by targeted replacement with small fragments of DNA with wild-type sequence. To determine if the observed genotype modification following small fragment homologous replacement (SFHR) was limited to transformed CF cell lines, further studies were carried out in both transformed and non-transformed primary normal airway epithelial cells. The endogenous genotype of these normal cell lines was modified following liposome or dendrimer transfection using DNA fragments with DeltaF508 CFTR sequence (488 nt, complementary single strands) designed to also contain a unique restriction enzyme cleavage site (Xho I). Replacement at the appropriate genomic locus by exogenous DeltaF508 CFTR DNA and its expression as mRNA was demonstrated by PCR amplification of genomic DNA and mRNA-derived cDNA as well as Xho I digestion of the PCR products. These studies show that SFHR occurs in both transformed and non- transformed primary human airway epithelial cells and indicate that single base substitution (the silent mutation giving rise to the Xho I site) and deletion or insertion of at least three consecutive bases can be achieved in both normal and CF epithelial cells. Furthermore, these studies reiterate the potential of SFHR as a strategy for a number of gene targeting applications, such as site-specific mutagenesis, development of transgenic animals, development of isogenic cell lines and for gene therapy.