Cystic Fibrosis Transmembrane Conductance Regulator Regulation Research Articles

Female mice display sex-specific differences in cerebrovascular function and subarachnoid haemorrhage-induced injury

In male mice, a circadian rhythm in myogenic reactivity influences the extent of brain injury following subarachnoid haemorrhage (SAH). We hypothesized that female mice have a different cerebrovascular phenotype and consequently, a distinct SAH-induced injury phenotype. SAH was modelled by pre-chiasmatic blood injection. Olfactory cerebral resistance arteries were functionally assessed by pressure myography; these functional assessments were related to brain histology and neurobehavioral assessments. Cystic fibrosis transmembrane conductance regulator (CFTR) expression was assessed by PCR and Western blot. We compared non-ovariectomized and ovariectomized mice. Cerebrovascular myogenic reactivity is not rhythmic in females and no diurnal differences in SAH-induced injury are observed; ovariectomy does not unmask a rhythmic phenotype for any endpoint. CFTR expression is rhythmic, with similar expression levels compared to male mice. CFTR inhibition studies, however, indicate that CFTR activity is lower in female arteries. Pharmacologically increasing CFTR expression invivo (3mg/kg lumacaftor for 2 days) reduces myogenic tone at Zeitgeber time 11, but not Zeitgeber time 23. Myogenic tone is not markedly augmented following SAH in female mice and lumacaftor loses its ability to reduce myogenic tone; nevertheless, lumacaftor confers at least some injury benefit in females with SAH. Female mice possess a distinct cerebrovascular phenotype compared to males, putatively due to functional differences in CFTR regulation. This sex difference eliminates the CFTR-dependent cerebrovascular effects of SAH and may alter the therapeutic efficacy of lumacaftor compared to males. Brain Aneurysm Foundation, Heart and Stroke Foundation and Ted Rogers Centre for Heart Research.

SUMOylation Inhibition Enhances Protein Transcription under CMV Promoter: A Lesson from a Study with the F508del-CFTR Mutant.

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a selective anion channel expressed in the epithelium of various organs. The most frequent mutation is F508del. This mutation leads to a misfolded CFTR protein quickly degraded via ubiquitination in the endoplasmic reticulum. Although preventing ubiquitination stabilizes the protein, functionality is not restored due to impaired plasma membrane transport. However, inhibiting the ubiquitination process can improve the effectiveness of correctors which act as chemical chaperones, facilitating F508del CFTR trafficking to the plasma membrane. Previous studies indicate a crosstalk between SUMOylation and ubiquitination in the regulation of CFTR. In this study, we investigated the potential of inhibiting SUMOylation to increase the effects of correctors and enhance the rescue of the F508del mutant across various cell models. In the widely used CFBE41o-cell line expressing F508del-CFTR, inhibiting SUMOylation substantially boosted F508del expression, thereby increasing the efficacy of correctors. Interestingly, this outcome did not result from enhanced stability of the mutant channel, but rather from augmented cytomegalovirus (CMV) promoter-mediated gene expression of F508del-CFTR. Notably, CFTR regulated by endogenous promoters in multiple cell lines or patient cells was not influenced by SUMOylation inhibitors.

Activation of farnesoid X receptor retards expansion of renal collecting duct cell-derived cysts via inhibition of CFTR-mediated Cl- secretion.

The transcription factor farnesoid X receptor (FXR) regulates energy metabolism. Specifically, FXR functions to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion in intestinal epithelial cells. Therefore, this study aimed to investigate the role of FXR in CFTR-mediated Cl- secretion in renal tubular cells and to further elucidate its effects on renal cyst formation and growth. CFTR-mediated Cl- transport was evaluated via short-circuit current (ISC) measurements in Madin-Darby canine kidney (MDCK) cell monolayers and primary rat inner medullary collecting duct cells. The role of FXR in renal cyst formation and growth was determined by the MDCK cell-derived cyst model. Incubation with synthesized (GW4064) and endogenous (CDCA) FXR ligands reduced CFTR-mediated Cl- secretion in a concentration- and time-dependent manner. The inhibitory effect of FXR ligands was not due to the result of reduced cell viability and was attenuated by cotreatment with an FXR antagonist. FXR activation significantly decreased CFTR protein but not its mRNA. In addition, FXR activation inhibited CFTR-mediated Cl- secretion in primary renal collecting duct cells. FXR activation decreased ouabain-sensitive ISC without altering Na+-K+-ATPase mRNA and protein levels. Furthermore, FXR activation significantly reduced the number of cysts and renal cyst expansion. These inhibitory effects were correlated with a decrease in the expression of protein synthesis regulators mammalian target of rapamycin/S6 kinase. This study shows that FXR activation inhibits Cl- secretion in renal cells via inhibition of CFTR expression and retards renal cyst formation and growth. The discoveries point to a physiological role of FXR in the regulation of CFTR and a potential therapeutic application in polycystic kidney disease treatment.NEW & NOTEWORTHY The present study reveals that farnesoid X receptor (FXR) activation reduces microcyst formation and enlargement. This inhibitory effect of FXR activation is involved with decreased cell proliferation and cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion in renal collecting duct cells. FXR might represent a novel target for the treatment of autosomal dominant polycystic kidney disease.

Laboratory Tools to Predict CFTR Modulator Therapy Effectiveness and to Monitor Disease Severity in Cystic Fibrosis.

The implementation of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulator drugs into clinical practice has been attaining remarkable therapeutic outcomes for CF, a life-threatening autosomal recessive genetic disease. However, there is elevated CFTR allelic heterogeneity, and various individuals carrying (ultra)rare CF genotypes remain without any approved modulator therapy. Novel translational model systems based on individuals' own cells/tissue are now available and can be used to interrogate in vitro CFTR modulator responses and establish correlations of these assessments with clinical features, aiming to provide prediction of therapeutic effectiveness. Furthermore, because CF is a progressive disease, assessment of biomarkers in routine care is fundamental in monitoring treatment effectiveness and disease severity. In the first part of this review, we aimed to focus on the utility of individual-derived in vitro models (such as bronchial/nasal epithelial cells and airway/intestinal organoids) to identify potential responders and expand personalized CF care. Thereafter, we discussed the usage of CF inflammatory biomarkers derived from blood, bronchoalveolar lavage fluid, and sputum to routinely monitor treatment effectiveness and disease progression. Finally, we summarized the progress in investigating extracellular vesicles as a robust and reliable source of biomarkers and the identification of microRNAs related to CFTR regulation and CF inflammation as novel biomarkers, which may provide valuable information for disease prognosis.

It Takes Two to Tango! Protein-Protein Interactions behind cAMP-Mediated CFTR Regulation.

Over the last fifteen years, with the approval of the first molecular treatments, a breakthrough era has begun for patients with cystic fibrosis (CF), the rare genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). These molecules, known as CFTR modulators, have led to unprecedented improvements in the lung function and quality of life of most CF patients. However, the efficacy of these drugs is still suboptimal, and the clinical response is highly variable even among individuals bearing the same mutation. Furthermore, not all patients carrying rare CFTR mutations are eligible for CFTR modulator therapies, indicating the need for alternative and/or add-on therapeutic approaches. Because the second messenger 3',5'-cyclic adenosine monophosphate (cAMP) represents the primary trigger for CFTR activation and a major regulator of different steps of the life cycle of the channel, there is growing interest in devising ways to fine-tune the cAMP signaling pathway for therapeutic purposes. This review article summarizes current knowledge regarding the role of cAMP signalosomes, i.e., multiprotein complexes bringing together key enzymes of the cAMP pathway, in the regulation of CFTR function, and discusses how modulating this signaling cascade could be leveraged for therapeutic intervention in CF.

Ionocyte-Specific Regulation of Cystic Fibrosis Transmembrane Conductance Regulator.

CFTR (cystic fibrosis transmembrane conductance regulator) is a tightly regulated anion channel that mediates chloride and bicarbonate conductance in many epithelia and in other tissues, but whether its regulation varies depending on the cell type has not been investigated. Epithelial CFTR expression is highest in rare cells called ionocytes. We studied CFTR regulation in control and ionocyte-enriched cultures by transducing bronchial basal cells with adenoviruses that encode only eGFP or FOXI1 (forkhead box I1) + eGFP as separate polypeptides. FOXI1 dramatically increased the number of transcripts for ionocyte markers ASCL3 (Achaete-Scute Family BHLH Transcription Factor 3), BSND, ATP6V1G3, ATP6V0D2, KCNMA1, and CFTR without altering those for secretory (SCGB1A1), basal (KRT5, KRT6, TP63), goblet (MUC5AC), or ciliated (FOXJ1) cells. The number of cells displaying strong FOXI1 expression was increased 7-fold, and there was no evidence for a broad increase in background immunofluorescence. Total CFTR mRNA and protein levels increased 10-fold and 2.5-fold, respectively. Ionocyte-enriched cultures displayed elevated basal current, increased adenylyl cyclase 5 expression, and tonic suppression of CFTR activity by the phosphodiesterase PDE1C, which has not been shown previously to regulate CFTR activity. The results indicate that CFTR regulation depends on cell type and identifies PDE1C as a potential target for therapeutics that aim to increase CFTR function specifically in ionocytes.

Cell signaling and regulation of CFTR expression in cystic fibrosis cells in the era of high efficiency modulator therapy

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and protein kinase A (PKA)-regulated channel, expressed on the luminal surface of secretory and absorptive epithelial cells. CFTR has a complex, cell-specific regulatory network playing a major role in cAMP- and Ca2+-activated secretion of electrolytes. It secretes intracellular Cl– and bicarbonate and regulates absorption of electrolytes by differentially controlling the activity of the epithelial Na+ channel (ENaC) in colon, airways, and sweat ducts. The CFTR gene expression is regulated by cell-specific, time-dependent mechanisms reviewed elsewhere [1]. This review will focus on the transcriptional, post-transcriptional, and translational regulation of CFTR by cAMP-PKA, non-coding (nc)RNAs, and TGF-β signaling pathways in cystic fibrosis (CF) cells.

Combined Treatment of Bronchial Epithelial Calu-3 Cells with Peptide Nucleic Acids Targeting miR-145-5p and miR-101-3p: Synergistic Enhancement of the Expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene encodes for a chloride channel defective in Cystic Fibrosis (CF). Accordingly, upregulation of its expression might be relevant for the development of therapeutic protocols for CF. MicroRNAs are deeply involved in the CFTR regulation and their targeting with miRNA inhibitors (including those based on Peptide Nucleic Acids, PNAs)is associated with CFTR upregulation. Targeting of miR-145-5p, miR-101-3p, and miR-335-5p with antisense PNAs was found to be associated with CFTR upregulation. The main objective of this study was to verify whether combined treatments with the most active PNAs are associated with increased CFTR gene expression. The data obtained demonstrate that synergism of upregulation of CFTR production can be obtained by combined treatments of Calu-3 cells with antisense PNAs targeting CFTR-regulating microRNAs. In particular, highly effective combinations were found with PNAs targeting miR-145-5p and miR-101-3p. Content of mRNAs was analyzed by RT-qPCR, the CFTR production by Western blotting. Combined treatment with antagomiRNAs might lead to maximized upregulation of CFTR and should be considered in the development of protocols for CFTR activation in pathological conditions in which CFTR gene expression is lacking, such as Cystic Fibrosis.

Krüppel-Like Factor 5 Regulates CFTR Expression Through Repression by Maintaining Chromatin Architecture Coupled with Direct Enhancer Activation

Single cell RNA-sequencing has accurately identified cell types within the human airway that express the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene. Low abundance CFTR transcripts are seen in many secretory cells, while high levels are restricted to rare pulmonary ionocytes. Here we focus on the mechanisms coordinating basal CFTR expression in the secretory compartment. Cell-selective regulation of CFTR is achieved within its invariant topologically associating domain by the recruitment of cis-regulatory elements (CREs). CRE activity is coordinated by cell-type-selective transcription factors. One such factor, Krüppel-Like Factor 5 (KLF5), profoundly represses CFTR transcript and protein in primary human airway epithelial cells and airway cell lines. Here we reveal the mechanism of action of KLF5 upon the CFTR gene. We find that depletion or ablation of KLF5 from airway epithelial cells changes higher order chromatin structure at the CFTR locus. Critical looping interactions that are required for normal gene expression are altered, the H3K27ac active chromatin mark is redistributed, and CTCF occupancy is modified. However, mutation of a single KLF5 binding site within a pivotal airway cell CRE abolishes CFTR expression. Hence, KLF5 has both direct activating and indirect repressive effects, which together coordinate CFTR expression in the airway.

Role of Protein Kinase A-Mediated Phosphorylation in CFTR Channel Activity Regulation.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel expressed on the apical membrane of epithelial cells, where it plays a pivotal role in chloride transport and overall tissue homeostasis. CFTR constitutes a unique member of the ATP-binding cassette transporter superfamily, due to its distinctive cytosolic regulatory (R) domain carrying multiple phosphorylation sites that allow the tight regulation of channel activity and gating. Mutations in the CFTR gene cause cystic fibrosis, the most common lethal autosomal genetic disease in the Caucasian population. In recent years, major efforts have led to the development of CFTR modulators, small molecules targeting the underlying genetic defect of CF and ultimately rescuing the function of the mutant channel. Recent evidence has highlighted that this class of drugs could also impact on the phosphorylation of the R domain of the channel by protein kinase A (PKA), a key regulatory mechanism that is altered in various CFTR mutants. Therefore, the aim of this review is to summarize the current knowledge on the regulation of the CFTR by PKA-mediated phosphorylation and to provide insights into the different factors that modulate this essential CFTR modification. Finally, the discussion will focus on the impact of CF mutations on PKA-mediated CFTR regulation, as well as on how small molecule CFTR regulators and PKA interact to rescue dysfunctional channels.

A Peptide-Nucleic Acid Targeting miR-335-5p Enhances Expression of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene with the Possible Involvement of the CFTR Scaffolding Protein NHERF1.

(1) Background: Up-regulation of the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) might be of great relevance for the development of therapeutic protocols for cystic fibrosis (CF). MicroRNAs are deeply involved in the regulation of CFTR and scaffolding proteins (such as NHERF1, NHERF2 and Ezrin). (2) Methods: Content of miRNAs and mRNAs was analyzed by RT-qPCR, while the CFTR and NHERF1 production was analyzed by Western blotting. (3) Results: The results here described show that the CFTR scaffolding protein NHERF1 can be up-regulated in bronchial epithelial Calu-3 cells by a peptide-nucleic acid (PNA) targeting miR-335-5p, predicted to bind to the 3′-UTR sequence of the NHERF1 mRNA. Treatment of Calu-3 cells with this PNA (R8-PNA-a335) causes also up-regulation of CFTR. (4) Conclusions: We propose miR-335-5p targeting as a strategy to increase CFTR. While the efficiency of PNA-based targeting of miR-335-5p should be verified as a therapeutic strategy in CF caused by stop-codon mutation of the CFTR gene, this approach might give appreciable results in CF cells carrying other mutations impairing the processing or stability of CFTR protein, supporting its application in personalized therapy for precision medicine.

Dynamic Regulation of Bicarbonate Permeability through CFTR Channel by WNK1

Increase in relative HCO3− permeability (PHCO3/PCl) of cystic fibrosis transmembrane conductance regulator (CFTR) channel by[Cl−]i-sensitive kinases is essential for pancreatic HCO3− secretion. Defects in pancreatic HCO3− secretion caused by CFTR mutations are associated with cystic fibrosis and pancreatitis. However, the molecular mechanisms by which [Cl−]i-sensitive kinases regulate CFTR anion selectivity are unknown. We examined the mechanisms of CFTR PHCO3/PCl regulation by [Cl−]i-sensitive kinases using the electrophysiological and molecular approach, including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. Moreover, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR. Although SPAK and/or OSR, which constitute a [Cl−]i-sensitive kinase cascade, wasn't expressed, the expression of WNK1 alone was sufficient to increase the CFTR PHCO3/PCl in patch clamp recordings. In molecular dissection of the WNK1 protein, the WNK1 kinase domain contributes CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions regulate the [Cl−]i-sensitivity of WNK1. Therefore, the inclusion of WNK1 N-terminal recombinant proteins in excised patch clamp recordings increased the CFTR PHCO3/PCl in a dose-dependent manner. The pancreatitis-causing R74Q and R75Q mutations in elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation. The CFTR PHCO3/PCl is regulated by [Cl−]i and a WNK1-dependent mechanism. In low [Cl−]i, physical associations between the N-terminal region of WNK1 and CFTR near p.R75 increase PHCO3/PCl. Defects in this process underlie the pathogenesis of some CFTR-related disorders.

Regulation of CFTR Bicarbonate Channel Activity by WNK1: Implications for Pancreatitis and CFTR-Related Disorders.

Backgraoud & AimsAberrant epithelial bicarbonate (HCO3−) secretion caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with several diseases including cystic fibrosis and pancreatitis. Dynamically regulated ion channel activity and anion selectivity of CFTR by kinases sensitive to intracellular chloride concentration ([Cl−]i) play an important role in epithelial HCO3− secretion. However, the molecular mechanisms of how [Cl−]i-dependent mechanisms regulate CFTR are unknown.MethodsWe examined the mechanisms of the CFTR HCO3− channel regulation by [Cl−]i-sensitive kinases using an integrated electrophysiological, molecular, and computational approach including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. In addition, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR.ResultsAmong the WNK1, SPAK, and OSR1 kinases that constitute a [Cl−]i-sensitive kinase cascade, the expression of WNK1 alone was sufficient to increase the CFTR bicarbonate permeability (PHCO3/PCl) and conductance (GHCO3) in patch clamp recordings. Molecular dissection of the WNK1 domains revealed that the WNK1 kinase domain is responsible for CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions mediate the [Cl−]i-sensitivity of WNK1. Furthermore, the pancreatitis-causing R74Q and R75Q mutations in the elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation.ConclusionThe CFTR HCO3− channel activity is regulated by [Cl−]i and a WNK1-dependent mechanism. Our results provide new insights into the regulation of the ion selectivity of CFTR and the pathogenesis of CFTR-related disorders.

CFTR regulation of aquaporin-mediated water transport.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel responsible for the direct transport of bicarbonate and chloride. CFTR-dependent ionic transport is crucial for pH regulation and fluid homeodynamics among epithelial surfaces. Particularly, CFTR performs an essential role in the male reproductive tract, which requires a tight regulation of water and electrolytes in order to produce healthy spermatozoa. The absence or malfunction of CFTR results in cystic fibrosis, the most common lethal disease among Caucasians, that is characterized by an impaired fluid and ionic homeostasis in the whole organism. Due to the wide expression and importance of CFTR, the male reproductive tract is highly affected by cystic fibrosis, resulting in male infertility. Although CFTR is not permeable to water, this protein acts as a regulator of other protein channels, such as aquaporins. In fact, CFTR acts as a molecular partner of aquaporins in epithelial cells, regulating fluid homeodynamics. Herein, up-to-date data concerning the regulation of aquaporin-mediated water transport by CFTR will be discussed, highlighting the role of both channels in the male reproductive tract.

Thickness of the airway surface liquid layer in the lung is affected in cystic fibrosis by compromised synergistic regulation of the ENaC ion channel.

The lung epithelium is lined with a layer of airway surface liquid (ASL) that is crucial for healthy lung function. ASL thickness is controlled by two ion channels: epithelium sodium channel (ENaC) and cystic fibrosis (CF) transmembrane conductance regulator (CFTR). Here, we present a minimal mathematical model of ENaC, CFTR and ASL regulation that sheds light on the control of ENaC by the short palate lung and nasal epithelial clone 1 (SPLUNC1) protein and by phosphatidylinositol 4,5-biphosphate (PI(4,5)P2). The model, despite its simplicity, yields a good fit to experimental observations and is an effective tool for exploring the interplay between ENaC, CFTR and ASL. Steady-state data and dynamic information constrain the model's parameters without ambiguities. Testing the hypothesis that PI(4,5)P2 protects ENaC from ubiquitination suggests that this protection does not improve the model results and that the control of the ENaC opening probability by PI(4,5)P2 is sufficient to explain all available data. The model analysis further demonstrates that ASL at the steady state is sensitive to small changes in PI(4,5)P2 abundance, particularly in CF conditions, which suggests that manipulation of phosphoinositide metabolism may promote therapeutic benefits for CF patients.

Cystic Fibrosis Transmembrane Conductance Regulator Is Heterologously Expressed in Human Heart

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP‐binding cassette transporter family and is a chloride channel protein. CFTR dysfunction is considered the major factor in development of cystic fibrosis. While CFTR research has focused primarily on its role in the lung and intestinal epithelia, CFTR is also present in a variety of tissues including heart. Information regarding CFTR's role in heart function is limited but possible roles for CFTR in myocardial protection and progression of heart disease have been implicated.Using biomaterials in the Duke Human Heart Repository (DHHR), an extensive and well‐curated source of human cardiac samples, we have initiated molecular and genetic studies focused on the CFTR locus in human hearts. Using a next generation sequence panel customized for CFTR gene (Ion Torrent) we obtained CFTR sequences from 48 genomic DNA samples in the DHHR (3 groups, n=16, i) no heart failure (NF), ii) non‐ischemic cardiomyopathy (NICM), and iii) ischemic cardiomyopathy (ICM)). Sequences mapping to a putative CFTR enhancer element emerged as differential between DNA from hearts +/− end‐stage heart failure. Since these changes map to a region which may control CFTR gene expression, we hypothesized that there may be differential protein expression between failing and non‐failing human hearts. To test this hypothesis we evaluated CFTR protein (using antibody 570, John Riordan, UNC) in human heart samples via western blot analysis. CFTR protein was present in all heart regions evaluated. In tissues from right ventricle (RV) and left ventricle (LV), CFTR protein expression was consistently higher in NF samples than in corresponding samples with heart failure. The highest level of CFTR expression was observed in the right atrium (RA) from ICM patient samples. The levels of CFTR may diminish early in the transition of healthy myocardium to early heart disease as indicated by the analysis of NF samples with and without left ventricular hypertrophy (LVH) an early indication of heart disease, where CFTR expression was dramatically less in LVH patients. Although preliminary, this may indicate loss of CFTR may occur early in the progression and initiation of heart disease and could potentially be part of early diagnostic testing if this effect proves to be a valid indicator of early heart disease. The mechanism of loss of CFTR in failed tissue is an ongoing line of investigation to assess whether CFTR affects progression of heart disease. As potential targets for intervention in the CFTR regulation pathway we are investigating CFTR turnover and phosphorylation in cardiac tissues as well as functional aspects of low expression or over‐expression especially as it relates to mitochondrial function and calcium handling in the intact, functioning myocardium.Support or Funding InformationThis research was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Numbers UL1TR001117 and UL1TR001111. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recent Progress in CFTR Interactome Mapping and Its Importance for Cystic Fibrosis.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride channel found in secretory epithelia with a plethora of known interacting proteins. Mutations in the CFTR gene cause cystic fibrosis (CF), a disease that leads to progressive respiratory illness and other complications of phenotypic variance resulting from perturbations of this protein interaction network. Studying the collection of CFTR interacting proteins and the differences between the interactomes of mutant and wild type CFTR provides insight into the molecular machinery of the disease and highlights possible therapeutic targets. This mini review focuses on functional genomics and proteomics approaches used for systematic, high-throughput identification of CFTR-interacting proteins to provide comprehensive insight into CFTR regulation and function.

The Mechanistic Links between Insulin and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- Channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel belongs to the ATP-binding cassette (ABC) transporter superfamily and regulates Cl− secretion in epithelial cells for water secretion. Loss-of-function mutations to the CFTR gene cause dehydrated mucus on the apical side of epithelial cells and increase the susceptibility of bacterial infection, especially in the airway and pulmonary tissues. Therefore, research on the molecular properties of CFTR, such as its gating mechanism and subcellular trafficking, have been intensively pursued. Dysregulated CFTR trafficking is one of the major pathological hallmarks in cystic fibrosis (CF) patients bearing missense mutations in the CFTR gene. Hormones that activate cAMP signaling, such as catecholamine, have been found to regulate the intracellular trafficking of CFTR. Insulin is one of the hormones that regulate cAMP production and promote trafficking of transmembrane proteins to the plasma membrane. The functional interactions between insulin and CFTR have not yet been clearly defined. In this review article, I review the roles of CFTR in epithelial cells, its regulatory role in insulin secretion, and a mechanism of CFTR regulation by insulin.

Hydrogen sulfide stimulates CFTR in Xenopus oocytes by activation of the cAMP/PKA signalling axis

Hydrogen sulfide (H2S) has been recognized as a signalling molecule which affects the activity of ion channels and transporters in epithelial cells. The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial anion channel and a key regulator of electrolyte and fluid homeostasis. In this study, we investigated the regulation of CFTR by H2S. Human CFTR was heterologously expressed in Xenopus oocytes and its activity was electrophysiologically measured by microelectrode recordings. The H2S-forming sulphur salt Na2S as well as the slow-releasing H2S-liberating compound GYY4137 increased transmembrane currents of CFTR-expressing oocytes. Na2S had no effect on native, non-injected oocytes. The effect of Na2S was blocked by the CFTR inhibitor CFTR_inh172, the adenylyl cyclase inhibitor MDL 12330A, and the protein kinase A antagonist cAMPS-Rp. Na2S potentiated CFTR stimulation by forskolin, but not that by IBMX. Na2S enhanced CFTR stimulation by membrane-permeable 8Br-cAMP under inhibition of adenylyl cyclase-mediated cAMP production by MDL 12330A. These data indicate that H2S activates CFTR in Xenopus oocytes by inhibiting phosphodiesterase activity and subsequent stimulation of CFTR by cAMP-dependent protein kinase A. In epithelia, an increased CFTR activity may correspond to a pro-secretory response to H2S which may be endogenously produced by the epithelium or H2S-generating microflora.

Synergy of cAMP and calcium signaling pathways in CFTR regulation

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport.