Wt Cystic Fibrosis Transmembrane Conductance Regulator Research Articles

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator correctors VX-445 and VX-121.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large-conductance calcium-activated potassium channel (BKCa, KCa1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated BKCa activity from both primary HBEs and HEK cells stably expressing the α subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of BKCa in the lung yet may lead to adverse events through BKCa activation elsewhere.

Structural Comparative Modeling of Multi-Domain F508del CFTR.

Cystic fibrosis (CF) is a rare genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs-which increases the risk of infection and eventually causes death. CFTR is an ATP-binding cassette (ABC) transporter family protein composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 (F508del), making F508del CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain F508del CFTR structure has been determined and few studies have modeled F508del using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a F508del model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and F508del CFTR structural models. These models demonstrate the destabilizing effects of F508del on NBD1 and the NBD1/TMD interface in both the inactive and active conformation of CFTR. Furthermore, we modeled F508del/R1070W and F508del bound to the CFTR corrector VX-809. Our models reveal the stabilizing effects of VX-809 on multi-domain models of F508del CFTR and pave the way for rational design of additional drugs that target F508del CFTR for treatment of CF.

Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most common cause of cystic fibrosis. The F508 residue is located on nucleotide-binding domain 1 (NBD1) in contact with the cytosolic extensions of the transmembrane helices, in particular intracellular loop 4 (ICL4). To investigate how absence of F508 at this interface impacts the CFTR protein, we carried out a mutagenesis scan of ICL4 by introducing second-site mutations at 11 positions in cis with F508del. Using an image-based fluorescence assay, we measured how each mutation affected membrane proximity and ion-channel function. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains (F1068, R1070W, and F1074, mimicking F1068, F508, and F1074 in WT CFTR). F508del-CFTR displayed a distorted aromatic stack, with F1068 displaced toward the space vacated by F508, while in F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR included F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. These studies highlight how both hydrophobic interactions and conformational flexibility might be important at the ICL4/NBD1 interface, suggesting possible structural underpinnings of F508del-induced dysfunction.

NBD2 Is Required for the Rescue of Mutant F508del CFTR by a Thiazole-Based Molecule: A Class II Corrector for the Multi-Drug Therapy of Cystic Fibrosis.

Cystic fibrosis (CF) is caused by loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) protein, an anion channel that regulates epithelial surface fluid secretion. The deletion of phenylalanine at position 508 (F508del) is the most common CFTR mutation. F508del CFTR is characterized by folding and trafficking defects, resulting in decreased functional expression of the protein on the plasma membrane. Several classes of small molecules, named correctors, have been developed to rescue defective F508del CFTR. Although individual correctors failed to improve the clinical status of CF patients carrying the F508del mutation, better results were obtained using correctors combinations. These results were obtained according to the premise that the administration of correctors having different sites of action should enhance F508del CFTR rescue. We investigated the putative site of action of an aminoarylthiazole 4-(3-chlorophenyl)-N-(3-(methylthio)phenyl)thiazol-2-amine, named FCG, with proven CFTR corrector activity, and its synergistic effect with the corrector VX809. We found that neither the total expression nor the maturation of WT CFTR transiently expressed in human embryonic kidney 293 cells was influenced by FCG, administrated alone or in combination with VX809. On the contrary, FCG was able to enhance F508del CFTR total expression, and its combination with VX809 provided a further effect, being able to increase not only the total expression but also the maturation of the mutant protein. Analyses on different CFTR domains and groups of domains, heterologously expressed in HEK293 cells, show that NBD2 is necessary for FCG corrector activity. Molecular modelling analyses suggest that FCG interacts with a putative region located into the NBD2, ascribing this molecule to class II correctors. Our study indicates that the continuous development and testing of combinations of correctors targeting different structural and functional defects of mutant CFTR is the best strategy to ensure a valuable therapeutic perspective to a larger cohort of CF patients.

Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation

BackgroundW1282X-CFTR variant (c.3846G>A) is the second most common nonsense cystic fibrosis (CF)-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Even though remarkable breakthroughs have been done towards CF treatment with the approval of four CFTR protein modulators, none of these are approved for patients with nonsense mutations. CRISPR gene editing tools can be of great value to permanently correct the genetic defects caused by these mutations. MethodsWe compared the capacity of homology-directed repair (HDR) mediated by Cas9 or Cas12a to correct W1282X CFTR mutation in the CFF-16HBEge W1282X CFTR cell line (obtained from CFF), using Cas9/gRNA and Cas12a/gRNA ribonucleoproteins (RNPs) and single strand DNA (ssODN) oligonucleotide donors. ResultsCas9 shows higher levels of correction than Cas12a as, by electroporating cells with Cas9 RNPs and ssODN donor, nearly 18% of precise editing was achieved compared to just 8% for Cas12a. Such levels of correction increase the abundance of CFTR mRNA and protein, and partially restore CFTR function in the pool of edited cells to 18% of WT CFTR function. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT CFTR. ConclusionAltogether, this work demonstrates the potential of gene editing as a therapeutic strategy for CF directly correcting the root cause of the disease.

Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination.

Based on its clinical benefits, Trikafta — the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) — was FDA approved for treatment of patients with cystic fibrosis (CF) carrying deletion of phenylalanine at position 508 (F508del) of the CF transmembrane conductance regulator (CFTR) on at least 1 allele. Neither the mechanism of action of VX-445 nor the susceptibility of rare CF folding mutants to Trikafta are known. Here, we show that, in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1) membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661 plus VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to approximately 62% of WT CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K, and N1303K), confined to MSD1, MSD2, NBD1, and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.

Identification and Characterization of Novel CFTR Potentiators.

There is still a high unmet need for the treatment of most patients with cystic fibrosis (CF). The identification and development of new Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators is necessary to achieve higher clinical benefit in patients. In this report we describe the characterization of novel potentiators. From a small screening campaign on F508del CFTR, hits were developed leading to the identification of pre-clinical candidates GLPG1837 and GLPG2451, each derived from a distinct chemical series. Both drug candidates enhance WT CFTR activity as well as low temperature or corrector rescued F508del CFTR, and are able to improve channel activity on a series of Class III, IV CFTR mutants. The observed activities in YFP halide assays translated well to primary cells derived from CF lungs when measured using Trans-epithelial clamp circuit (TECC). Both potentiators improve F508del CFTR channel opening in a similar manner, increasing the open time and reducing the closed time of the channel. When evaluating the potentiators in a chronic setting on corrected F508del CFTR, no reduction of channel activity in presence of potentiator was observed. The current work identifies and characterizes novel CFTR potentiators GLPG1837 and GLPG2451, which may offer new therapeutic options for CF patients.

Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene.

Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR, supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease.

CFTR supports cell death through ROS-dependent activation of TMEM16F (anoctamin 6).

Cystic fibrosis transmembrane conductance regulator (CFTR) is the essential chloride and bicarbonate channel in the apical membrane of epithelial cells. CFTR was also proposed earlier to conduct glutathione (GSH) out of airway epithelial cells to be enriched in the apical airway surface liquid to neutralize reactive oxygen species (ROS). Although earlier studies suggested that release of GSH by wild type (wt) CFTR may lead to an increase in cytosolic ROS, we did not detect different ROS levels in cells expressing wt-CFTR and mutant F508del-CFTR, independent of CFTR-activation or exposure to the ROS donor tert-butyl hydroperoxide. The Ca2+-activated phospholipid scramblase and ion channel TMEM16F (anoctamin 6, ANO6) is also expressed in airway cells. ANO6 produced outwardly rectifying Cl- currents (ORCC) and scrambled plasma membrane phospholipids when activated by increase in cytosolic ROS and consecutive peroxidation of plasma membrane lipids. ANO6 activity is enhanced by CFTR, probably through translocation of signaling proteins to the plasma membrane. The present data suggest that enhanced cell death in CFTR-expressing cells is due to upregulation of ANO6-activity. In ANO6 knockout mice, the number of apoptotic cells in the intestinal epithelium was strongly reduced, supporting the role of ANO6 for cell death. Thus, ANO6 and CFTR act cooperatively on ROS-mediated cell death, which is not further augmented by cAMP-dependent stimulation. We propose that ANO6 supports cell death correlated with expression of CFTR, possibly by inducing ferroptosis.

Phosphorylation Modifies Coupling of the Membrane Domains and NBD1 of Full Length CFTR

Cystic fibrosis is a common genetic recessive disease caused by mutations of the Cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-Binding Cassette (ABC) anion channel. CFTR consists of two membrane spanning domains (MSDs), two nucleotide binding domains (NBDs), intracellular loops (ICLs) that “couple” MSDs and NBDs, and a regulatory (R) domain. CFTR activity is predominantly regulated by protein kinase A phosphorylation at the R domain. However, little is known of the effect of phosphorylation on intramolecular interactions of full length CFTR. Intrinsic tryptophan fluorescence studies of a similar ABC transporter, BtuCD, have shown that urea dissociates the ICL:NBD interface which we were interested to apply on purified full length CFTR. Our results were similar to BtuCD studies, suggesting the dissociation of ICL:NBD interface of full length CFTR. Phosphorylation modified urea denaturation like VX-661, a suggested modulator of ICL:NBD1 interface. Phosphorylation and VX-661 similarly modified quenching of tryptophan residues suggesting that monitored tryptophan residues were located within an electrostatic environment. Our biophysical studies led us to hypothesize that phosphorylation may modify the intramolecular interactions at the ICL:NBD1 interface of full length CFTR. A phosphorylation site on the regulatory insertion, which has been suggested to open up upon phosphorylation to allow interaction of ICLs to NBD1 is located near many tryptophan residues from the CFTR homology model. To specifically study the interactions, cysteine crosslinking studies were conducted on a Wt CFTR lacking native cysteines with V510C (NBD1) and A1067C (ICL4) engineered and transfected in HEK cells. Phosphorylation significantly enhanced the crosslinking and interaction at the ICL4:NBD1 more than the ICL:NBD1 modulator VX-661. This study provides insight into the effect of phosphorylation on the intramolecular interactions of full length CFTR.

Simple image-based no-wash method for quantitative detection of surface expressed CFTR

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, compromising its trafficking to the epithelial cell apical membrane. The absence of functional CFTR leads to depletion of airway surface liquid, impaired clearance of mucus and bacteria from the lung, and predisposes to recurrent infections. Ultimately, respiratory failure results from inflammation and bronchiectasis.Although high throughput screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, most primary CF drug discovery assays have employed measurements of CFTR’s anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Screening efforts have also included assays for detection of extracellularly HA-tagged or HRP-tagged CFTR, which require multiple washing steps. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules, and have extended this assay to the 96-well format. This new and simple no-wash assay of F508del CFTR at the cell surface may permit the discovery of more efficacious drugs, and hopefully thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform should make it useful for other diseases where loss-of-function results from folding and/or trafficking defects in membrane proteins.

Deleterious impact of Pseudomonas aeruginosa on cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells.

The epithelial response to bacterial airway infection, a common feature of lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis, has been extensively studied. However, its impact on cystic fibrosis transmembrane conductance regulator (CFTR) channel function is not clearly defined. Our aims were, therefore, to evaluate the effect of Pseudomonas aeruginosa on CFTR function and expression in non-cystic fibrosis airway epithelial cells, and to investigate its impact on ΔF508-CFTR rescue by the VRT-325 corrector in cystic fibrosis cells. CFTR expression/maturation was evaluated by immunoblotting and its function by short-circuit current measurements. A 24-h exposure to P. aeruginosa diffusible material (PsaDM) reduced CFTR currents as well as total and membrane protein expression of the wildtype (wt) CFTR protein in CFBE-wt cells. In CFBE-ΔF508 cells, PsaDM severely reduced CFTR maturation and current rescue induced by VRT-325. We also confirmed a deleterious impact of PsaDM on wt-CFTR currents in non-cystic fibrosis primary airway cells as well as on the rescue of ΔF508-CFTR function induced by VRT-325 in primary cystic fibrosis cells. These findings show that CFTR function could be impaired in non-cystic fibrosis patients infected by P. aeruginosa. Our data also suggest that CFTR corrector efficiency may be affected by infectious components, which should be taken into account in screening assays of correctors.

Investigating the Effect of PKA Phosphorylation on Intramolecular Interactions in Purified Full Length Wildtype CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is an unique anion channel of the ATP-Binding Cassette (ABC) superfamily. CFTR consists of two nucleotide binding domains (NBDs), two membrane spanning domains (MSDs) and a regulatory (R) domain. There are also alpha-helical extensions and intracellular loops (ICLs) which couple the NBDs and MSDs via “coupling helices”. The regulation CFTR channel activity involves protein kinase A (PKA) phosphorylation at the R domain. However, not much is known about the effect of phosphorylation on the intramolecular interactions of full length CFTR. We propose that phosphorylation modifies the interactions of full length CFTR, especially at the ICL4:NBD1 interface. Previous studies of a similar ABC transporter, BtuCD, have shown that intrinsic tryptophan fluorescence can detect urea sensitive changes in the coupling between the MSDs and NBDs of full length BtuCD. Thus, we decided monitor intrinsic tryptophan fluorescence to study the effect of phosphorylation on the purified full length Wt CFTR. Interestingly, we found that the urea sensitive changes in the intrinsic tryptophan fluorescence of full length CFTR were modified upon PKA phosphorylation. We were interested to determine whether this result was due to phosphorylation modifying the ICL4:NBD1 interface. In order to specifically study that interface, cysteine crosslinking studies were employed using a short cell permeable cysteine crosslinker on a cys-less CFTR mutant with two cysteines, V510C (NBD1) and A106C7C (ICL4), transfected in HEK293 cells. Phosphorylation induced by cAMP agonists in cells resulted in a significantly increased the crosslinking of cysteines at the ICL4:NBD1 interface compared to the inhibition of phosphorylation with adenyl cyclase inhibitors. This suggests that phosphorylation modifies the ICL4 and NBD1 interface. These studies further our understanding of the molecular mechanisms underlying phosphorylation dependent gating of CFTR.

Interaction of the Isolated Nucleotide Binding Domains of CFTR Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. Defective function of CFTR is responsible for cystic fibrosis, a lethal genetic disease manifested in defective chloride transport across the epithelial cells in various tissues. The structure of CFTR comprises two transmembrane domains (TMDs) that form the channel pore, two intracellular nucleotide-binding domains (NBDs) and a unique regulatory domain. The opening of CFTR channels is coupled to ATP binding to the NBDs and subsequent NBD dimerization. ATP hydrolysis leads to dimer separation and channel closure. In the past few years tremendous progress has been made in the characterization of CFTR gating, but the conformational changes behind the gating transitions observed in these functional studies can only be inferred based mostly on the crystal structures available from a few ABC transporters. Dynamic structural information governing the mechanisms behind CFTR function at the molecular level is still lacking. Recent biochemical breakthroughs in purifying CFTR now make it possible to address some of the outstanding questions in the CFTR field. Our goal is to investigate the NBD dimer formation and separation (dimerization dynamics) using the state-of-the-art single-molecule Forster resonance energy transfer (smFRET) technique for WT and mutant CFTR channels. Here we show our progress so far in this work, namely the characterization of the purified NBDs: the apparent biding affinity, hydrolysis competence and FRET data demonstrating the association of the two isolated domains in the presence of ATP. The purpose of this study is to address fundamental questions about the molecular mechanisms behind the function of CFTR channels, and when completed, will no doubt enhance our understanding of the relationship between structure, dynamics and function.

Respiratory Syncytial Virus Infection Disrupts Monolayer Integrity and Function in Cystic Fibrosis Airway Cells

Background: Respiratory Syncytial Virus (RSV) infection is a common contributor to pulmonary symptoms in children with cystic fibrosis (CF). Here we examined RSV infection in immortalized bronchial epithelial cells (CFBE41o-) expressing wild-type (wt) or F508del cystic fibrosis transmembrane conductance regulator (CFTR), for monolayer integrity and RSV replication. Methods: CFBE41o- monolayers expressing wt or F508del CFTR were grown on permeable supports and inoculated with RSV A2 strain. Control experiments utilized UV-inactivated RSV and heat-killed RSV. Monolayer resistance and RSV production was monitored for up to six days post-infection. Results: Within 24 h, a progressive decrease in monolayer resistance was observed in RSV infected F508del CFBE41o- cells, while the monolayer integrity of RSV infected wt CFTR CFBE41o- cells remained stable. RSV replication was necessary to disrupt F508del CFBE41o- monolayers as UV-irradiated and heat killed RSV had no effect on monolayer integrity, with an earlier and much more pronounced peak in RSV titer noted in F508del relative to wt CFTR-expressing cells. RSV infection of wt CFBE41o- monolayers also resulted in blunting of CFTR response. Conclusions: These findings identify an enhanced sensitivity of CFBE41o- cells expressing F508del CFTR to RSV infection, replication and monolayer disruption independent of the cellular immune response, and provide a novel mechanism by which cystic fibrosis airway epithelia are susceptible to RSV-dependent injury.

Transcomplementation by a Truncation Mutant of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Enhances ΔF508 Processing through a Biomolecular Interaction

We previously showed that a truncation mutant of CFTR missing the first four transmembrane segments of TMD1, Δ264 CFTR, binds to key elements in the ER quality control mechanism to increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Here, we created a new construct, Δ27-264 CFTR. Even though Δ27-264 CFTR is rapidly degraded in the proteasome, steady state protein can be detected by Western blot. Δ27-264 CFTR can also increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Electrophysiology experiments show that Δ27-264 CFTR can restore chloride channel currents. Further experiments with the conduction mutant S341A show conclusively that currents are indeed generated by rescued channel function of ΔF508 CFTR. Immunoprecipitation studies show that Δ27-264 binds to ΔF508-CFTR, suggesting a bimolecular interaction. Thus the adeno-associated viral vector, rAAV-Δ27-264 CFTR, is a highly promising CF gene therapy vector, because it increases the amount of mature band C protein both from wt and ΔF508 CFTR, and rescues channel activity of ΔF508 CFTR.

Thermal Instability of ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function: Protection by Single Suppressor Mutations and Inhibiting Channel Activity

Deletion of Phe508 from cystic fibrosis transmembrane conductance regulator (CFTR) results in a temperature-sensitive folding defect that impairs protein maturation and chloride channel function. Both of these adverse effects, however, can be mitigated to varying extents by second-site suppressor mutations. To better understand the impact of second-site mutations on channel function, we compared the thermal sensitivity of CFTR channels in Xenopus oocytes. CFTR-mediated conductance of oocytes expressing wt or ΔF508 CFTR was stable at 22 °C and increased at 28 °C, a temperature permissive for ΔF508 CFTR expression in mammalian cells. At 37 °C, however, CFTR-mediated conductance was further enhanced, whereas that due to ΔF508 CFTR channels decreased rapidly toward background, a phenomenon referred to here as "thermal inactivation." Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Another mutation, K1250A, known to increase open probability (P(o)) of ΔF508 CFTR channels, exacerbated thermal inactivation. Application of potentiators known to increase P(o) of ΔF508 CFTR channels at room temperature failed to protect channels from inactivation at 37 °C and one, PG-01, actually exacerbated thermal inactivation. Unstimulated ΔF508CFTR channels or those inhibited by CFTR(inh)-172 were partially protected from thermal inactivation, suggesting a possible inverse relationship between thermal stability and gating transitions. Thermal stability of channel function and temperature-sensitive maturation of the mutant protein appear to reflect related, but distinct facets of the ΔF508 CFTR conformational defect, both of which must be addressed by effective therapeutic modalities.

CFTR regulation in human airway epithelial cells requires integrity of the actin cytoskeleton and compartmentalized cAMP and PKA activity

The cystic fibrosis transmembrane conductance regulator (CFTR) mutation ΔF508CFTR still causes regulatory defects when rescued to the apical membrane, suggesting that the intracellular milieu might affect its ability to respond to cAMP regulation. We recently reported that overexpression of the Na(+)/H(+) exchanger regulatory factor NHERF1 in the cystic fibrosis (CF) airway cell line CFBE41o-rescues the functional expression of ΔF508CFTR by promoting F-actin organization and formation of the NHERF1-ezrin-actin complex. Here, using real-time FRET reporters of both PKA activity and cAMP levels, we find that lack of an organized subcortical cytoskeleton in CFBE41o-cells causes both defective accumulation of cAMP in the subcortical compartment and excessive cytosolic accumulation of cAMP. This results in reduced subcortical levels and increased cytosolic levels of PKA activity. NHERF1 overexpression in CFBE41o-cells restores chloride secretion, subcortical cAMP compartmentalization and local PKA activity, indicating that regulation of ΔF508CFTR function requires not only stable expression of the mutant CFTR at the cell surface but also depends on both generation of local cAMP signals of adequate amplitude and activation of PKA in proximity of its target. Moreover, we found that the knockdown of wild-type CFTR in the non-CF 16HBE14o-cells results in both altered cytoskeletal organization and loss of cAMP compartmentalization, whereas stable overexpression of wt CFTR in CF cells restores cytoskeleton organization and re-establishes the compartmentalization of cAMP at the plasma membrane. This suggests that the presence of CFTR on the plasma membrane influences the cytoskeletal organizational state and, consequently, cAMP distribution. Our data show that a sufficiently high concentration of cAMP in the subcortical compartment is required to achieve PKA-mediated regulation of CFTR activity.

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.

Regulation of endogenous ENaC functional expression by CFTR and ΔF508-CFTR in airway epithelial cells

The functional expression of the epithelial sodium channel (ENaC) appears elevated in cystic fibrosis (CF) airway epithelia, but the mechanism by which this occurs is not clear. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) alters the trafficking of endogenously expressed human ENaC in the CFBE41o⁻ model of CF bronchial epithelia. Functional expression of ENaC, as defined by amiloride-inhibited short-circuit current (I(sc)) in Ussing chambers, was absent under control conditions but present in CFBE41o⁻ parental and ΔF508-CFTR-overexpressing cells after treatment with 1 μM dexamethasone (Dex) for 24 h. The effect of Dex was mimicked by incubation with the glucocorticoid hydrocortisone but not with the mineralocorticoid aldosterone. Application of trypsin to the apical surface to activate uncleaved, "near-silent" ENaC caused an additional increase in amiloride-sensitive I(sc) in the Dex-treated cells and was without effect in the control cells, suggesting that Dex increased ENaC cell surface expression. In contrast, Dex treatment did not stimulate amiloride-sensitive I(sc) in CFBE41o⁻ cells that stably express wild-type (wt) CFTR. CFBE41o⁻ wt cells also had reduced expression of α- and γ-ENaC compared with parental and ΔF508-CFTR-overexpressing cells. Furthermore, application of trypsin to the apical surface of Dex-treated CFBE41o⁻ wt cells did not stimulate amiloride-sensitive I(sc), suggesting that ENaC remained absent from the surface of these cells even after Dex treatment. We also tested the effect of trafficking-corrected ΔF508-CFTR on ENaC functional expression. Incubation with 1 mM 4-phenylbutyrate synergistically increased Dex-induced ENaC functional expression in ΔF508-CFTR-overexpressing cells. These data support the hypothesis that wt CFTR can regulate the whole cell, functional, and surface expression of endogenous ENaC in airway epithelial cells and that absence of this regulation may foster ENaC hyperactivity in CF airway epithelia.