Wt CFTR Research Articles

RNA binding proteins PTBP1 and HNRNPL regulate CFTR mRNA decay

BackgroundCFTR nonsense alleles generate negligible CFTR protein due to the nonsense mutation: 1) triggering CFTR mRNA degradation by nonsense-mediated mRNA decay (NMD), and 2) terminating CFTR mRNA translation prematurely. Thus, people with cystic fibrosis (PwCF) who carry nonsense alleles cannot benefit from current modulator drugs, which target CFTR protein. In this study, we examined whether PTBP1 and HNRNPL, two RNA binding proteins that protect a subset of mRNAs with a long 3’ untranslated region (UTR) from NMD, similarly affect CFTR mRNA.Silencing RNAs were used to deplete PTBP1 or HNRNPL in 16HBE14o- human bronchial epithelial cells expressing WT, G542X, or W1282X CFTR. CFTR mRNA abundance was measured relative to controls by quantitative PCR. PTBP1 and HNRNPL were also exogenously expressed in each cell line and CFTR mRNA levels were similarly quantified. ResultsPTBP1 depletion reduced CFTR mRNA abundance in all three 16HBE14o- cell lines; HRNPL depletion reduced CFTR mRNA abundance in only the G542X and W1282X cell lines. Notably, decreased CFTR mRNA abundance correlated with increased mRNA decay. Exogenous expression of PTBP1 or HNRNPL increased CFTR mRNA abundance in all three cell lines; HNRNPL overexpression generally increased CFTR to a greater extent in G542X and W1282X 16HBE14o- cells.Our data indicate that PTBP1 and HNRNPL regulate CFTR mRNA abundance by protecting CFTR transcripts from NMD. This suggests that PTBP1 and/or HNRNPL may represent potential therapeutic targets to increase CFTR mRNA abundance and enhance responses to CFTR modulators and other therapeutic approaches in PwCF.

Differential CFTR-Interactome Proximity Labeling Procedures Identify Enrichment in Multiple SLC Transporters.

Proteins interacting with CFTR and its mutants have been intensively studied using different experimental approaches. These studies provided information on the cellular processes leading to proper protein folding, routing to the plasma membrane, recycling, activation and degradation. Recently, new approaches have been developed based on the proximity labeling of protein partners or proteins in close vicinity and their subsequent identification by mass spectrometry. In this study, we evaluated TurboID- and APEX2-based proximity labeling of WT CFTR and compared the obtained data to those reported in databases. The CFTR-WT interactome was then compared to that of two CFTR (G551D and W1282X) mutants and the structurally unrelated potassium channel KCNK3. The two proximity labeling approaches identified both known and additional CFTR protein partners, including multiple SLC transporters. Proximity labeling approaches provided a more comprehensive picture of the CFTR interactome and improved our knowledge of the CFTR environment.

CFTR‐mediated transepithelial bicarbonate conductance is affected differently than chloride conductance by CFTR correctors and potentiators

The advent of targeted CFTR therapeutics allows us to assess differences in epithelial Cl− and HCO3− conductances that might be attributed to unique combinations of CFTR variants (mutations), CFTR modulators (corrector(s) and potentiators) as well as secretory agonists. To address these questions, Fisher rat thyroid cells expressing wt or mutant CFTR were cultured on permeable supports for the measurement of transepithelial conductance (Gt) in defined ionic conditions. Initial experiments demonstrated stepwise reduction in the forskolin (Forsk)+ivacaftor (VX770)‐stimulated change in Gt as bath NaCl was partially replaced isosmotically with mannitol. Subsequent experiments were conducted in buffers containing 100 mEq/l Cl− with the addition of either 25 mM alternative anions (Na+ salts) or 50 mM mannitol. Outcomes showed forskolin‐induced Gt changes consistent with reported CFTR permselectivity: Cl− > NO3− ≥ Br− > isethionate ≥ gluconate > mannitol. All subsequent experiments were conducted to assess Gt with 100 mEq/l Cl− plus either 25 mEq/l HCO3−, 50 mM mannitol or 25 mEq/l Cl−, in a 5% CO2 atmosphere. Epithelia expressing wtCFTR respond to forskolin with a substantial increase in Gt. The response in HCO3− was ~75% of that observed with Cl−. Exposure to VX770 increased conductance, but a disproportionately lesser change was observed in HCO3− (vs. Cl−), providing an initial suggestion that VX770 promotes differential ion selectivity.Exposure of cells expressing F508del CFTR to correctors, either C17 or C18, allowed for forskolin‐induced stimulation of Gt with a modest increment of HCO3− conductance. In these conditions, VX770 induced additional Cl− conductance, but no enhancement of HCO3− conductance could be detected. Alternatively, cells exposed to the corrector combination (C17+C18) showed a substantial forskolin‐stimulated increase in anion conductance and a modest increase in CFTR‐associated HCO3− conductance. Thus, the combination of C17 and C18 gives a different outcome regarding F508del‐CFTR HCO3− conductance than either corrector alone.Cells expressing G551D CFTR exhibited little response to forskolin, but substantial responses to VX770. Responses to VX770 in HCO3− approached the magnitude of the responses observed in Cl−.Taken together, the results suggest that CFTR correctors and potentiators can enhance CFTR‐mediated anion conductance, but that significant differences in the conductances of HCO3− relative to Cl− are observed between CFTR variants and that responses can be influenced by the correctors employed. Ultimately, these results suggest that CFTR‐associated pH regulation will be impacted by both the patient’s mutation and the CFTR modulator treatment. Furthermore, additional studies are required to evaluate the CFTR modulators for their effects on Cl− and HCO3− conductances on the many CF causing CFTR variants.Support or Funding InformationCFF BRIDGE18XX0; K‐State CVM SMILE

Slowing ribosome velocity restores folding and function of mutant CFTR.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.

Sweat rate analysis of ivacaftor potentiation of CFTR in non-CF adults

To determine if ivacaftor (Kalydeco) influences non-CF human CFTR function in vivo, we measured CFTR-dependent (C-sweat) and CFTR-independent (M-sweat) rates from multiple identified sweat glands in 8 non-CF adults. The two types of sweating were stimulated sequentially with intradermal injections of appropriate reagents; each gland served as its own control via alternating off-on drug tests on both arms, given at weekly intervals with 3 off and 3 on tests per subject. We compared drug effects on C-sweating stimulated by either high or low concentrations of β-adrenergic cocktail, and on methacholine-stimulated M-sweating. For each subject we measured ~700 sweat volumes from ~75 glands per arm (maximum 12 readings per gland), and sweat volumes were log-transformed for statistical analysis. T-tests derived from linear mixed models (LMMs) were more conservative than the familiar paired sample t-tests, and show that ivacaftor significantly increased C-sweating stimulated by both levels of agonist, with a larger effect in the low cocktail condition; ivacaftor did not increase M-sweat. Concurrent sweat chloride tests detected no effect of ivacaftor. We conclude that ivacaftor in vivo increases the open channel probability (PO) of WT CFTR, provided it is not already maximally stimulated.

The CFTR-Associated Ligand Arrests the Trafficking of the Mutant ΔF508 CFTR Channel in the ER Contributing to Cystic Fibrosis

Background/Aims: The CFTR-Associated Ligand (CAL), a PDZ domain containing protein with two coiled-coil domains, reduces cell surface WT CFTR through degradation in the lysosome by a well-characterized mechanism. However, CAL’s regulatory effect on ΔF508 CFTR has remained almost entirely uninvestigated. Methods: In this study, we describe a previously unknown pathway for CAL by which it regulates the membrane expression of ΔF508 CFTR through arrest of ΔF508 CFTR trafficking in the endoplasmic reticulum (ER) using a combination of cell biology, biochemistry and electrophysiology. Results: We demonstrate that CAL is an ER localized protein that binds to ΔF508 CFTR and is degraded in the 26S proteasome. When CAL is inhibited, ΔF508 CFTR retention in the ER decreases and cell surface expression of mature functional ΔF508 CFTR is observed alongside of enhanced expression of plasma membrane scaffolding protein NHERF1. Chaperone proteins regulate this novel process, and ΔF508 CFTR binding to HSP40, HSP90, HSP70, VCP, and Aha1 changes to improve ΔF508 CFTR cell surface trafficking. Conclusion: Our results reveal a pathway in which CAL regulates the cell surface availability and intracellular retention of ΔF508 CFTR.

The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9.

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.

CFTR Controls the Activity of NF-κB by Enhancing the Degradation of TRADD

Background/Aims: Chronic lung infection in cystic fibrosis leads to an inflammatory response that persists because of the chronic presence of bacteria and ultimately leads to a catastrophic failure of lung function. Methods: We use a combination of biochemistry, cell and molecular biology to study the interaction of TRADD, a key adaptor molecule in TNFα signaling, with CFTR in the regulation of NFκB. Results: We show that Wt CFTR binds to and colocalizes with TRADD. TRADD is a key signaling intermediate connecting TNFα with activation of NFκB. By contrast, ΔF508 CFTR does not bind to TRADD. NF-κB activation is higher in CFBE expressing ΔF508 CFTR than in cells expressing Wt CFTR. However, this differential effect is abolished when TRADD levels are knocked down. Transfecting Wt CFTR into CFBE cells reduces NF-κB activity. However the reduction is abolished by the CFTR chloride transport inhibitor-172. Consistently, transfecting in the correctly trafficked CFTR conduction mutants G551D or S341A also fail to reduce NFκB activity. Thus CFTR must be functional if it is to regulate NF-κB activity. We also found that TNFα produced a greater increase in NF-κB activity in CFBE cells than in the same cell when Wt CFTR-corrected. Consistently, the effect is also abolished when TRADD is knocked down by shRNA. Thus, Wt CFTR control of TRADD modulates the physiological activation of NF-κB by TNFα. Based on studies with proteosomal and lysosomal inhibitors, the mechanism by which Wt CFTR, but not ΔF508 CFTR, suppresses TRADD is by lysosomal degradation. Conclusion: We have uncovered a novel mechanism whereby Wt CFTR regulates TNFα signaling by enhancing TRADD degradation. Thus by reducing the levels of TRADD, Wt CFTR suppresses downstream proinflammatory NFκB signaling. By contrast, suppression of NF-κB activation fails in CF cells expressing ΔF508 CFTR.

Evidence against resveratrol as a viable therapy for the rescue of defective ΔF508 CFTR

BackgroundResveratrol, a natural phenolic compound, has been reported to rescue mutant ΔF508 CFTR in expression systems and primary epithelial cells. Although this implies a therapeutic benefit to patients with CF, investigations were performed using resveratrol concentrations greatly in excess of those achievable in plasma. We evaluated the efficacy of resveratrol as a CFTR corrector in relevant primary airway cells, using physiologically achievable resveratrol concentrations. MethodsCells expressing wt or ΔF508 CFTR were exposed to chronic or acute resveratrol. CFTR mRNA and protein expression were monitored. The effects of resveratrol on primary ΔF508 human airway cells were evaluated by equivalent current analysis using modified Ussing chambers. ResultsConsistent with previously published data in heterologous expression systems, high doses of resveratrol increased CFTR expression; however physiologically relevant concentrations were without effect. In contrast to heterologous expression systems, resveratrol was unable to increase mutant CFTR channel activity in primary airway cells. Elevated amiloride-sensitive currents, indicative of sodium transport and characteristically elevated in CF airway cells, were also unaffected by resveratrol. ConclusionsHigh concentrations of resveratrol can increase CFTR mRNA and protein in some cell types. In addition, acute resveratrol exposure can stimulate CFTR mediated chloride secretion, probably by increasing cellular cAMP levels. Resveratrol at physiologically achievable levels yielded no benefit in primary ΔF508 airway cells, either in terms of amiloride-sensitive currents of CFTR currents. General significanceTaken together, our results do not support the use of resveratrol supplements as a therapy for patients with cystic fibrosis. It is possible that further modifications of the resveratrol backbone would yield a more efficacious compound.

Prevention of ubiquitination at lysines in the N tail, R, ICL4, and NBD2 domains disrupts CFTR trafficking and immune response

RationalePost‐translational modification of lysine residues in CFTR is predicted to regulate CFTR trafficking and recycling. We hypothesized that disruption of ubiquitination in lysine residues will accelerate destruction of wt CFTR and increase inflammatory responses.MethodsSite‐directed mutagenesis, Immunoblot, confocal analysis, ELISA assay, etc.ResultsWe identify seven key lysines in CFTR protein that regulate expression, maturation, poly‐ubiquitination, and trafficking of CFTR and subsequently, its role in inflammatory responses. More specifically, K14/K68/K1218 residues are important for lysosomal degradation, while the K710/K716/K1080 residues are necessary for stabilization/poly‐ubiquitination of CFTR, and the K1041 residue is critical for overall expression of CFTR. Moreover, the K710R, K716R and K710/716R mutants are inflammatory however, inhibition of lysosomal function dampens inflammatory signaling by these mutations. K14R, K68R, K1041R, K1080R and K1218R mutants are not inflammatory. Inhibition of proteasomal degradation enhances non‐inflammatory effect of K14R and K1080R mutants.ConclusionsPost‐translational modifications in CFTR lysine residues regulate maturation and trafficking of CFTR and the inflammatory responses.Supported by R01 HL 59410; Eudowood Division of Pediatric Respiratory Sciences

Role of SLC6A14 as a Modifier of Cystic Fibrosis Phenotype

Cystic Fibrosis (CF) is considered to be a single gene disorder, most commonly the result of a F508del mutation in the CFTR gene. However, patients homozygous for F508del mutation show a considerable variation in disease severity. Consistent with this observation, recent genome wide association studies identified secondary genes that modify disease. A SNP in the putative promoter region of SLC6A14 gene showed the highest significance (p=1.28x10−12). SLC6A14 is a cationic/neutral amino acid transporter, which transports 2Na+, 1 Cl− and a basic/neutral amino-acid into the cell from the apical surface of intestinal/bronchial epithelium. We hypothesize that expression of SLC6A14 is protective against meconium ileus, a CF phenotype detected at birth. To test this hypothesis we over-expressed SLC6A14 in BHK cells stably expressing either WT or F508del-CFTR. We measured CFTR channel activity using the halide sensitive fluorophore, SPQ. We found that CFTR function is enhanced with over-expression of SLC6A14 in cells expressing WT CFTR or F508del CFTR (after rescue of its trafficking defect), (p<0.05). This suggests a possible functional interaction between SLC6A14 and CFTR. We also monitored the functional interaction of SLC6A14 and CFTR in Caco-2 cells, an intestinal cell line that endogenously expresses both CFTR and SLC6A14. Caco-2 cells form three-dimensional, cyst-like cultures wherein CFTR mediated secretion determines the volume of its internal cavity. We found that the area of the cyst cavity increases upon cAMP stimulation of CFTR stimulation by 5.5 fold and that this expansion was decreased by 44% in the presence of the SLC6A14 inhibitor (α-methyltryptophan). These results suggest that SLC6A14 exerts a positive effect on regulated CFTR function and future studies will explore the possibility that agonists of SLC6A14 could constitute a means for therapeutic intervention.(Studies supported by grants to C.E.B and J.M.R. from CFC, CIHR)

Contribution of Casein Kinase 2 and Spleen Tyrosine Kinase to CFTR Trafficking and Protein Kinase A-Induced Activity

Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

BackgroundPrevious observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.MethodsElectrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.ResultsMembrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.ConclusionsThe data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.

The expression of the mitochondrial gene MT-ND4 is downregulated in cystic fibrosis

Cystic fibrosis (CF) is a disease produced by mutations in the CFTR channel. We have previously reported that the CFTR chloride transport activity indirectly regulates the differential expression of several genes, including SRC and MUC1. Here we report that MT-ND4, a mitochondrial gene encoding a subunit of the mitochondrial Complex I (mtCx-I), is also a CFTR-dependent gene. A reduced expression of MT-ND4 was observed in CFDE cells (derived from a CF patient) when compared to CFDE cells ectopically expressing wild-type CFTR. The differential expression of MT-ND4 in CF was confirmed by RT-PCR. In situ hybridizations of deparaffinized human lung tissue slices derived from wt-CFTR or CF patients also showed downregulation of ND4 in CF. In addition, the CFTR chloride transport inhibitors glibenclamide and CFTR(inh)-172 also reduced MT-ND4 expression in CFDE cells ectopically expressing wt CFTR. These results suggest that the CFTR chloride transport activity indirectly up-regulates MT-ND4 expression.

PBA increases CFTR expression but at high doses inhibits Cl(-) secretion in Calu-3 airway epithelial cells.

Sodium 4-phenylbutyrate (PBA), a short-chain fatty acid, has been approved to treat patients with urea cycle enzyme deficiencies and is being evaluated in the management of sickle cell disease, thalassemia, cancer, and cystic fibrosis (CF). Because relatively little is known about the effects of PBA on the expression and function of the wild-type CF transmembrane conductance regulator (wt CFTR), the goal of this study was to examine the effects of PBA and related compounds on wt CFTR-mediated Cl(-) secretion. To this end, we studied Calu-3 cells, a human airway cell line that expresses endogenous wt CFTR and has a serous cell phenotype. We report that chronic treatment of Calu-3 cells with a high concentration (5 mM) of PBA, sodium butyrate, or sodium valproate but not of sodium acetate reduced basal and 8-(4-chlorophenylthio)-cAMP-stimulated Cl(-) secretion. Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and increased the intensity of CFTR staining in the apical plasma membrane. PBA also increased protein expression of Na(+)-K(+)-ATPase. PBA reduced CFTR Cl(-) currents across the apical membrane but had no effect on Na(+)-K(+)-ATPase activity in the basolateral membrane. Thus a high concentration of PBA (5 mM) reduces Cl(-) secretion by inhibiting CFTR Cl(-) currents across the apical membrane. In contrast, lower therapeutic concentrations of PBA (0.05-2 mM) had no effect on cAMP-stimulated Cl(-) secretion across Calu-3 cells. We conclude that PBA concentrations in the therapeutic range are unlikely to have a negative effect on Cl(-) secretion. However, concentrations >5 mM might reduce transepithelial Cl(-) secretion by serous cells in submucosal glands in individuals expressing wt CFTR.

Genistein potentiates wild-type and delta F508-CFTR channel activity.

Effects of genistein on wild-type (wt) and delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) were studied in NIH/3T3 cells stably transfected with wt or mutant CFTR cDNA. As measured by I- efflux, half-maximal concentration of agonist (K1/2) for forskolin-dependent activation was greater for delta F508-CFTR than wt-CFTR. Genistein decreased the K1/2 for both forms of the channel and increased the maximal activity of delta F508-CFTR by 3.7-fold. In cell-attached patches, 10 microM forskolin induced minimal delta F508-CFTR activity with characteristic prolonged closed times (estimated time constant, > 30 s). Genistein increased the forskolin-induced macroscopic currents of wt-CFTR and delta F508-CFTR by 3- and 19-fold, respectively. Variance analysis suggested that in the presence of forskolin and genistein the open probabilities (Po) of wt- and delta F508-CFTR were identical. In single-channel studies, at maximal adenosine 3',5'-cyclic monophosphate (cAMP) stimulation, genistein increased the Po of wt-CFTR by prolonging the open time, but, at submaximal cAMP stimulation, the Po was increased by prolonging the open time and shortening the closed time. In excised patches with CFTR channels preactivated in the cell-attached mode, genistein increased ATP-dependent wt- and delta F508-CFTR current about twofold by prolonging the open time. Our results thus suggest that phosphorylation-dependent activation of delta F508-CFTR is defective and that genistein corrects this defect at least in part by binding to the CFTR protein.