Transmembrane Conductance Regulator Chloride Channel Research Articles

General trends in the effects of VX-661 and VX-445 on the plasma membrane expression of clinical CFTR variants

Cystic fibrosis (CF) is caused by mutations that compromise the expression and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Most people with CF harbor a common misfolded variant (ΔF508) that can be partially rescued by therapeutic "correctors" that restore its expression. Nevertheless, many other CF variants are insensitive to correctors. Using deep mutational scanning, we quantitatively compare the effects of two correctors on the plasma membrane expression of 129 CF variants. Though structural calculations suggest corrector binding provides similar stabilization to most variants, it's those with intermediate expression and mutations near corrector binding pockets that exhibit the greatest response. Deviations in sensitivity appear to depend on the degree of variant destabilization and the timing of misassembly. Combining correctors appears to rescue more variants by doubling the binding energy and stabilizing distinct cotranslational folding transitions. These results provide an overview of rare CF variant expression and establish new tools for precision pharmacology.

Easy-to-Build and Reusable Microfluidic Device for the Dynamic Culture of Human Bronchial Cystic Fibrosis Epithelia.

Cystic fibrosis (CF) is one of the most frequent genetic diseases, caused by dysfunction of the CF transmembrane conductance regulator (CFTR) chloride channel. CF particularly affects the epithelium of the respiratory system. Therapies aim at rescuing CFTR defects in the epithelium, but CF genetic heterogeneity hinders the finding of a single and generally effective treatment. Therefore, in vitro models have been developed to study CF and guide patient therapy. Here, we show a CF model on-chip by coupling the feasibility of the human bronchial epithelium differentiated in vitro at the air-liquid interface and the innovation of microfluidics. We demonstrate that the dynamic flow enhanced cilia distribution and increased mucus quantity, thus promoting tissue differentiation in a short time. The microfluidic devices highlighted differences between CF and non-CF epithelia, as shown by electrophysiological measures, mucus quantity, viscosity, and the analysis of ciliary beat frequency. The described model on-chip may be a handy instrument for studying CF and setting up therapies. As a proof of principle, we administrated the corrector VX-809 on-chip and observed a decrease in mucus thickness and viscosity.

Rescue by elexacaftor-tezacaftor-ivacaftor of the G1244E cystic fibrosis mutation's stability and gating defects are dependent on cell background

BackgroundCystic fibrosis is caused by mutations impairing expression, trafficking, stability and/or activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The G1244E mutation causes a severe gating defect that it is not completely rescued by ivacaftor but requires the use of a second compound (a co-potentiator). Recently, it has been proposed that the corrector elexacaftor may act also as a co-potentiator. MethodsBy using molecular, biochemical and functional analyses we performed an in-depth characterization of the G1244E-CFTR mutant in heterologous and native cell models. ResultsOur studies demonstrate that processing and function of the mutant protein, as well as its pharmacological sensitivity, are markedly dependent on cell background. In heterologous expression systems, elexacaftor mainly acted on G1244E-CFTR as a co-potentiator, thus ameliorating the gating defect. On the contrary, in the native nasal epithelial cell model, elexacaftor did not act as a co-potentiator, but it increased mature CFTR expression possibly by improving mutant's defective stability at the plasma membrane. ConclusionsOur study highlights the importance of the cell background in the evaluation of CFTR modulator effects. Further, our results draw attention to the need for the development of novel potentiators having different mechanisms with respect to ivacaftor to improve channel activity for mutants with severe gating defect.

SUCCESSFUL MANAGEMENT OF REFRACTORY CONSTIPATION USING KAMPO MEDICINE MASHININGAN IN A PATIENT WITH WILD-TYPE ATTR CARDIAC AMYLOIDOSIS

TYPE: Case Report TOPIC: Cardiovascular Disease INTRODUCTION: A wild-type ATTR amyloidosis is a systemic disease with multi-organ dysfunction, involving heart, kidney, skin, and gastrointestinal tract, due to deposition of wild-type transthyretin in each organ. CASE PRESENTATION: We had a 76-year-old man diagnosed with wild-type ATTR cardiac amyloidosis, whose heart failure symptom improved by anti-heart failure medications but constipation refractory to multiple conventional medications persisted. Following the conversion from lubiprostone to Kampo medicine mashiningan, his average days per one evacuation decreased from around 7.0 days down to 1.6 days. DISCUSSION: Amyloid-related gastrointestinal comorbidities are often refractory to the conventional medications. Mashiningan might increase the proportion of soft feces via increasing intestinal fluid secretion through cystic fibrosis transmembrane conductance regulator chloride channel activation. CONCLUSIONS: Mashininigan might be alternative option to improve refractory constipation in patients with cardiac amyloidosis. DISCLOSURE: Nothing to declare. KEYWORD: heart failure

A Phase 2a randomized, single-center, double-blind, placebo-controlled study to evaluate the safety and preliminary efficacy of oral iOWH032 against cholera diarrhea in a controlled human infection model

Cholera remains a major cause of infectious diarrhea globally. Despite the increased availability of cholera vaccines, there is still an urgent need for other effective interventions to reduce morbidity and mortality. Furthermore, increased prevalence of antibiotic-resistant Vibrio cholerae threatens the use of many drugs commonly used to treat cholera. We developed iOWH032, a synthetic small molecule inhibitor of the cystic fibrosis transmembrane conductance regulator chloride channel, as an antisecretory, host-directed therapeutic for cholera. In the study reported here, we tested iOWH032 in a Phase 2a cholera controlled human infection model. Forty-seven subjects were experimentally infected with V. cholerae El Tor Inaba strain N16961 in an inpatient setting and randomized to receive 500 mg iOWH032 or placebo by mouth every 8 hours for 3 days to determine the safety and efficacy of the compound as a potential treatment for cholera. We found that iOWH032 was generally safe and achieved a mean (± standard deviation) plasma level of 4,270 ng/mL (±2,170) after 3 days of oral dosing. However, the median (95% confidence interval) diarrheal stool output rate for the iOWH032 group was 25.4 mL/hour (8.9, 58.3), compared to 32.6 mL/hour (15.8, 48.2) for the placebo group, a reduction of 23%, which was not statistically significant. There was also no significant decrease in diarrhea severity and number or frequency of stools associated with iOWH032 treatment. We conclude that iOWH032 does not merit future development for treatment of cholera and offer lessons learned for others developing antisecretory therapeutic candidates that seek to demonstrate proof of principle in a cholera controlled human infection model study.Trial registration: This study is registered with ClinicalTrials.gov as NCT04150250.

Chloride transport modulators as drug candidates

Chloride transport across cell membranes is broadly involved in epithelial fluid transport, cell volume and pH regulation, muscle contraction, membrane excitability, and organellar acidification. The human genome encodes at least 53 chloride-transporting proteins with expression in cell plasma or intracellular membranes, which include chloride channels, exchangers, and cotransporters, some having broad anion specificity. Loss-of-function mutations in chloride transporters cause a wide variety of human diseases, including cystic fibrosis, secretory diarrhea, kidney stones, salt-wasting nephropathy, myotonia, osteopetrosis, hearing loss, and goiter. Although impactful advances have been made in the past decade in drug treatment of cystic fibrosis using small molecule modulators of the defective cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, other chloride channels and solute carrier proteins (SLCs) represent relatively underexplored target classes for drug discovery. New opportunities have emerged for the development of chloride transport modulators as potential therapeutics for secretory diarrheas, constipation, dry eye disorders, kidney stones, polycystic kidney disease, hypertension, and osteoporosis. Approaches to chloride transport-targeted drug discovery are reviewed herein, with focus on chloride channel and exchanger classes in which recent preclinical advances have been made in the identification of small molecule modulators and in proof of concept testing in experimental animal models.

Functional stability of CFTR depends on tight binding of ATP at its degenerate ATP-binding site.

Opening of the cystic fibrosis transmembrane conductance regulator (CFTR) channel is coupled to the motion of its two nucleotide-binding domains: they form a heterodimer sandwiching two functionally distinct ATP-binding sites (sites 1 and 2). While active ATP hydrolysis in site 2 triggers rapid channel closure, the functional role of stable ATP binding in the catalysis-incompetent (or degenerate) site 1, a feature conserved in many other ATP-binding cassette (ABC) transporter proteins, remains elusive. Here, we found that CFTR loses its prompt responsiveness to ATP after the channel is devoid of ATP for tens to hundreds of seconds. Mutants with weakened ATP binding in site 1 and the most prevalent disease-causing mutation, F508del, are more vulnerable to ATP depletion. In contrast, strengthening ligand binding in site 1 with N6 -(2-phenylethyl)-ATP, a high-affinity ATP analogue, or abolishing ATP hydrolysis in site 2 by the mutation D1370N, helps sustain a durable function of the otherwise unstable mutant channels. Thus, tight binding of ATP in the degenerate ATP-binding site is crucial to the functional stability of CFTR. Small molecules targeting site 1 may bear therapeutic potential to overcome the membrane instability of F508del-CFTR. KEY POINTS: During evolution, many ATP-binding cassette transporters - including the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, whose dysfunction causes cystic fibrosis (CF) - lose the ability to hydrolyse ATP in one of the two ATP-binding sites. Here we show that tight ATP binding at this degenerate site in CFTR is central for maintaining the stable, robust function of normal CFTR. We also demonstrate that membrane instability of the most common CF-causing mutant, F508del-CFTR, can be rescued by strengthening ATP binding at CFTR's degenerate site. Our data thus explain an evolutionary puzzle and offer a potential therapeutic strategy for CF.

β-eudesmol but not atractylodin exerts an inhibitory effect on CFTR-mediated chloride transport in human intestinal epithelial cells

Oriental herbal medicine with the two bioactive constituents, β-eudesmol (BE) and atractylodin (AT), has been used as a remedy for gastrointestinal disorders. There was no scientific evidence reporting their antidiarrheal effect and underpinning mechanisms. Therefore, we aimed to investigate the anti-secretory activity of these two compounds in vitro. The inhibitory effect of BE and AT on cAMP-induced Cl- secretion was evaluated by Ussing chamber in human intestinal epithelial (T84) cells. Short-circuit current (ISC) and apical Cl- current (ICl-) were measured after adding indirect and direct cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activator. MTT assay was used to determine cellular cytotoxicity. Protein-ligand interaction was investigated by in silico molecular docking analysis. BE, but not AT concentration-dependently (IC50 of ~1.05 µM) reduced cAMP-mediated, CFTRinh-172 inhibitable Cl− secretion as determined by transepithelial ISC across a monolayer of T84 cells. Potency of CFTR-mediated ICl- inhibition by BE did not change with the use of different CFTR activators suggesting a direct blockage of the channel active site(s). Pretreatment with BE completely prevented cAMP-induced ICl-. Furthermore, BE at concentrations up to 200 µM (24 h) had no effect on T84 cell viability. In silico studies indicated that BE could best dock onto dephosphorylated structure of CFTR at ATP-binding pockets in nucleotide-binding domain (NBD) 2 region. These findings provide the first evidence for the anti-secretory effect of BE involving inhibition of CFTR function. BE represents a promising candidate for the therapeutic or prophylactic intervention of diarrhea resulted from intestinal hypersecretion of Cl-

Stability Prediction for Mutations in the Cytosolic Domains of Cystic Fibrosis Transmembrane Conductance Regulator.

Cystic Fibrosis (CF) is caused by mutations to the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. CFTR is composed of two membrane spanning domains, two cytosolic nucleotide-binding domains (NBD1 and NBD2) and a largely unstructured R-domain. Multiple CF-causing mutations reside in the NBDs and some are known to compromise the stability of these domains. The ability to predict the effect of mutations on the stability of the cytosolic domains of CFTR and to shed light on the mechanisms by which they exert their effect is therefore important in CF research. With this in mind, we have predicted the effect on domain stability of 59 mutations in NBD1 and NBD2 using 15 different algorithms and evaluated their performances via comparison to experimental data using several metrics including the correct classification rate (CCR), and the squared Pearson correlation (R2) and Spearman's correlation (ρ) calculated between the experimental ΔTm values and the computationally predicted ΔΔG values. Overall, the best results were obtained with FoldX and Rosetta. For NBD1 (35 mutations), FoldX provided R2 and ρ values of 0.64 and -0.71, respectively, with an 86% correct classification rate (CCR). For NBD2 (24 mutations), FoldX R2, ρ, and CCR were 0.51, -0.73, and 75%, respectively. Application of the Rosetta high-resolution protocol (Rosetta_hrp) to NBD1 yielded R2, ρ, and CCR of 0.64, -0.75, and 69%, respectively, and for NBD2 yielded R2, ρ, and CCR of 0.29, -0.27, and 50%, respectively. The corresponding numbers for the Rosetta's low-resolution protocol (Rosetta_lrp) were R2 = 0.47, ρ = -0.69, and CCR = 69% for NBD1 and R2 = 0.27, ρ = -0.24, and CCR = 63% for NBD2. For NBD1, both algorithms suggest that destabilizing mutations suffer from destabilizing vdW clashes, whereas stabilizing mutations benefit from favorable H-bond interactions. Two triple consensus approaches based on FoldX, Rosetta_lpr, and Rosetta_hpr were attempted using either "majority-voting" or "all-voting". The all-voting consensus outperformed the individual predictors, albeit on a smaller data set. In summary, our results suggest that the effect of mutations on the stability of CFTR's NBDs could be largely predicted. Since NBDs are common to all ABC transporters, these results may find use in predicting the effect and mechanism of the action of multiple disease-causing mutations in other proteins.

On the relationship between anion binding and chloride conductance in the CFTR anion channel

Mutations at many sites within the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore region result in changes in chloride conductance. Although chloride binding in the pore – as well as interactions between concurrently bound chloride ions – are thought to be important facets of the chloride permeation mechanism, little is known about the relationship between anion binding and chloride conductance. The present work presents a comprehensive investigation of a number of anion binding properties in different pore mutants with differential effects on chloride conductance. When multiple pore mutants are compared, conductance appears best correlated with the ability of anions to bind to the pore when it is already occupied by chloride ions. In contrast, conductance was not correlated with biophysical measures of anion:anion interactions inside the pore. Although these findings suggest anion binding is required for high conductance, mutations that strengthened anion binding had very little effect on conductance, especially at high chloride concentrations, suggesting that the wild-type CFTR pore is already close to saturated with chloride ions. These results are used to support a revised model of chloride permeation in CFTR in which the overall chloride occupancy of multiple loosely-defined chloride binding sites results in high chloride conductance through the pore.

A SNARE protein Syntaxin 17 captures CFTR to potentiate autophagosomal clearance under stress.

Autophagy, a cellular stress response to starvation and bacterial infection, is executed by double-membrane-bound organelles called autophagosomes. Autophagosomes transfer cytosolic material to acidified lysosomes for degradation following soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-dependent fusion processes. Many of the autophagy-related disorders stem from defective end-step proteolysis inside lysosomes. The role of epithelial cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel has been argued to be critical for efficient lysosomal clearance; however, its context to autophagic clearance and the underlying mechanism is poorly defined. Here, we report that syntaxin17 (Stx17), an autophagic SNARE protein interacts with CFTR under nutritional stress and bacterial infection and incorporates it into mature autophagosomes to mediate an efficient lysosomal clearance. Lack of CFTR function and Stx17 and loss of CFTR-Stx17 interaction impairs bacterial clearance. We discover a specialized role of the Stx17-CFTR protein complex that is critical to prevent defective autophagy as has been the reported scenario in CF airway epithelial cells, infectious diseases, and lysosomal clearance disorders.

Epithelial barrier function properties of the 16HBE14o- human bronchial epithelial cell culture model.

The human bronchial epithelial cell line, 16HBE14o- (16HBE), is widely used as a model for respiratory epithelial diseases and barrier function. During differentiation, transepithelial electrical resistance (TER) increased to approximately 800 Ohms × cm2, while 14C-d-mannitol flux rates (Jm) simultaneously decreased. Tight junctions (TJs) were shown by diffusion potential studies to be anion-selective with PC1/PNa = 1.9. Transepithelial leakiness could be induced by the phorbol ester, protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the proinflammatory cytokine, tumor necrosis factor-α (TNF-α). Basal barrier function could not be improved by the micronutrients, zinc, or quercetin. Of methodological significance, TER was observed to be more variable and to spontaneously, significantly decrease after initial barrier formation, whereas Jm did not significantly fluctuate or increase. Unlike the strong inverse relationship between TER and Jm during differentiation, differentiated cell layers manifested no relationship between TER and Jm. There was also much greater variability for TER values compared with Jm. Investigating the dependence of 16HBE TER on transcellular ion conductance, inhibition of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel with GlyH-101 produced a large decrease in short-circuit current (Isc) and a slight increase in TER, but no significant change in Jm. A strong temperature dependence was observed not only for Isc, but also for TER. In summary, research utilizing 16HBE as a model in airway barrier function studies needs to be aware of the complexity of TER as a parameter of barrier function given the influence of CFTR-dependent transcellular conductance on TER.

Serum-Based Proteomics Profiling in Adult Patients with Cystic Fibrosis

Cystic fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, is caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel gene. Despite significant advances in the management of CF patients, novel disease-related biomarkers and therapies must be identified. We performed serum proteomics profiling in CF patients (n = 28) and healthy subjects (n = 10) using the 2D-DIGE MALDI-TOF proteomic approach. Out of a total of 198 proteins identified, 134 showed a statistically significant difference in abundance and a 1.5-fold change (ANOVA, p < 0.05), including 80 proteins with increased abundance and 54 proteins with decreased abundance in CF patients. A multiple reaction monitoring-mass spectrometry analysis of six differentially expressed proteins identified by a proteomic approach (DIGE-MALD-MS) showed a significant increase in C3 and CP proteins and a decrease in APOA1, Complement C1, Hp, and RBP4proteins compared with healthy controls. Fifteen proteins were identified as potential biomarkers for CF diagnosis. An ingenuity pathway analysis of the differentially regulated proteins indicates that the central nodes dysregulated in CF subjects involve pro-inflammatory cytokines, ERK1/2, and P38 MAPK, which are primarily involved in catalytic activities and metabolic processes. The involved canonical pathways include those related to FXR/RXR, LXR/RXR, acute phase response, IL12, nitric oxide, and reactive oxygen species in macrophages. Our data support the current efforts toward augmenting protease inhibitors in patients with CF. Perturbations in lipid and vitamin metabolism frequently observed in CF patients may be partly due to abnormalities in their transport mechanism.

Identification, Structure-Activity Relationship, and Biological Characterization of 2,3,4,5-Tetrahydro-1H-pyrido[4,3-b]indoles as a Novel Class of CFTR Potentiators.

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole core as a novel chemotype of potentiators. In-depth structure–activity relationship studies led to the discovery of enantiomerically pure 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.

Novel ANO1 Inhibitor from Mallotus apelta Extract Exerts Anticancer Activity through Downregulation of ANO1.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including human prostate cancer and oral squamous cell carcinomas. ANO1 plays a critical role in tumor growth and maintenance of these cancers. In this study, we have isolated two new compounds (1 and 2) and four known compounds (3–6) from Mallotus apelta. These compounds were evaluated for their inhibitory effects on ANO1 channel activity and their cytotoxic effects on PC-3 prostate cancer cells. Interestingly, compounds 1 and 2 significantly reduced both ANO1 channel activity and cell viability. Electrophysiological study revealed that compound 2 (Ani-D2) is a potent and selective ANO1 inhibitor, with an IC50 value of 2.64 μM. Ani-D2 had minimal effect on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity and intracellular calcium signaling. Notably, Ani-D2 significantly reduced ANO1 protein expression levels and cell viability in an ANO1-dependent manner in PC-3 and oral squamous cell carcinoma CAL-27 cells. In addition, Ani-D2 strongly reduced cell migration and induced activation of caspase-3 and cleavage of PARP in PC-3 and CAL-27 cells. This study revealed that a novel ANO1 inhibitor, Ani-D2, has therapeutic potential for the treatment of several cancers that overexpress ANO1, such as prostate cancer and oral squamous cell carcinoma.

Pathways in the Pathophysiology of Coronavirus 19 Lung Disease Accessible to Prevention and Treatment.

Background: In COVID 19 related lung disease, which is a leading cause of death from this disease, cytokines like tumor necrosis factor-alpha (TNF alpha) may be pivotal in the pathogenesis. TNF alpha reduces fluid absorption due to impairment of sodium and chloride transport required for building an osmotic gradient across epithelial cells, which in the airways maintains airway surface liquid helping to keep airways open and enabling bacterial clearance and aids water absorption from the alveolar spaces. TNF alpha can, through Rho-kinase, disintegrate the endothelial and epithelial cytoskeleton, and thus break up intercellular tight junctional proteins, breaching the intercellular barrier, which prevents flooding of the interstitial and alveolar spaces with fluid.Hypotheses: (1) Preservation and restoration of airway and alveolar epithelial sodium and chloride transport and the cytoskeleton dependent integrity of the cell barriers within the lung can prevent and treat COVID 19 lung disease. (2) TNF alpha is the key mediator of pulmonary edema in COVID 19 lung disease.Confirmation of hypothesis and implications: The role of a reduction in the function of epithelial sodium and chloride transport could with regards to chloride transport be tested by analysis of chloride levels in exhaled breath condensate and levels correlated with TNF alpha concentrations. Reduced levels would indicate a reduction of the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and a correlation with TNF alpha levels indicative of its involvement. Anti-TNF alpha treatment with antibodies is already available and needs to be tested in randomized controlled trials of COVID 19 lung disease. TNF alpha levels could also be reduced by statins, aspirin, and curcumin. Chloride transport could be facilitated by CFTR activators, including curcumin and phosphodiesterase-5 inhibitors. Sodium and chloride transport could be further regulated to prevent accumulation of alveolar fluid by use of Na(+)/K(+)/2Cl(−) cotransporter type 1 inhibitors, which have been associated with improved outcome in adults ventilated for acute respiratory distress syndrome (ARDS) in randomized controlled trials. Primary prevention of coronavirus infection and TNF alpha release in response to it could be improved by induction of antimicrobial peptides LL-37 and human beta defensin-2 and reduction of TNF alpha production by vitamin D prophylaxis for the population as a whole.

Novel Anti-Inflammatory Approaches for Cystic Fibrosis Lung Disease: Identification of Molecular Targets and Design of Innovative Therapies.

Cystic fibrosis (CF) is the most common genetic disorder among Caucasians, estimated to affect more than 70,000 people in the world. Severe and persistent bronchial inflammation and chronic bacterial infection, along with airway mucus obstruction, are hallmarks of CF lung disease and participate in its progression. Anti-inflammatory therapies are, therefore, of particular interest for CF lung disease. Furthermore, a better understanding of the molecular mechanisms involved in airway infection and inflammation in CF has led to the development of new therapeutic approaches that are currently under evaluation by clinical trials. These new strategies dedicated to CF inflammation are designed to treat different dysregulated aspects such as oxidative stress, cytokine secretion, and the targeting of dysregulated pathways. In this review, we summarize the current understanding of the cellular and molecular mechanisms that contribute to abnormal lung inflammation in CF, as well as the new anti-inflammatory strategies proposed to CF patients by exploring novel molecular targets and novel drug approaches.

Disrupted local innervation results in less VIP expression in CF mice tissues

Vasoactive Intestinal Peptide (VIP) is the major physiological agonist of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride channel activity. VIP functions as a neuromodulator and neurotransmitter secreted by neurons innervating all exocrine glands. VIP is also a potent vasodilator and bronchodilator that regulates exocrine gland secretions, contributing to local innate defense by stimulating the movement of water and chloride transport across intestinal and tracheobronchial epithelia. Previous human studies have shown that the rich intrinsic neuronal networks for VIP secretion around exocrine glands could be lost in tissues from patients with cystic fibrosis. Our research has since confirmed, in vitro and in vivo, the need for chronic VIP exposure to maintain functional CFTR chloride channels at the cell surface of airways and intestinal epithelium, as well as normal exocrine tissues morphology [1]. The goal of the present study was to examine changes in VIP in the lung, duodenum and sweat glands of 8- and 17-weeks old F508del/F508del mice and to investigate VIPergic innervation in the small intestine of CF mice, before important signs of the disease development. Our data show that a low amount of VIP is found in CF tissues prior to tissue damage. Moreover, we found a specific reduction in VIPergic and cholinergic innervation of the small intestine. The general innervation of the primary and secondary myenteric plexus was lost in CF tissues, with the presence of enlarged ganglionic cells in the tertiary layer. We propose that low amount of VIP in CF tissues is due to a reduction in VIPergic and cholinergic innervation and represents an early defect that constitutes an aggravating factor for CF disease progression.

Cystic Fibrosis Presenting as Pseudo-Bartter Syndrome: An Important Diagnosis that is Missed!

Cystic fibrosis (CF), an autosomal recessive disorder, occurs due to mutations in CFTR gene resulting in impaired cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel function in various epithelia. In addition to the well-known pulmonary and pancreatic morbidities, CF is characterized by electrolyte and acid-base abnormalities- hypochloremia, hyponatremia, hypokalemia and metabolic alkalosis. These are collectively known as Pseudo-Bartter syndrome, as similar abnormalities are seen in Bartter syndrome- an inherited tubulopathy affecting thick ascending limb of loop of Henle. There may be a significant clinical overlap between the Classic Bartter syndrome, Gitelman syndrome and CF presenting as Pseudo-Bartter syndrome, especially in early childhood. This review focuses on Pseudo-Bartter syndrome in CF, its pathogenesis and differentiation from Bartter/Gitelman syndrome. Other causes of metabolic abnormalities resembling Bartter syndrome are also highlighted.

Changes in the R-region interactions depend on phosphorylation and contribute to PKA and PKC regulation of the cystic fibrosis transmembrane conductance regulator chloride channel.

The CFTR chloride channel is regulated by phosphorylation at PKA and PKC consensus sites within its regulatory region (R‐region) through a mechanism, which is still not completely understood. We used a split‐CFTR construct expressing the N‐term‐TMD1‐NBD1 (Front Half; FH), TMD2‐NBD2‐C‐Term (Back Half; BH), and the R‐region as separate polypeptides (Split‐R) in BHK cells, to investigate in situ how different phosphorylation conditions affect the R‐region interactions with other parts of the protein. In proximity ligation assays, we studied the formation of complexes between the R‐region and each half of the Split‐CFTR. We found that at basal conditions, the density of complexes formed between the R‐region and both halves of the split channel were equal. PKC stimulation alone had no effect, whereas PKA stimulation induced the formation of more complexes between the R‐region and both halves compared to basal conditions. Moreover, PKC + PKA stimulation further enhanced the formation of FH‐R complexes by 40% from PKA level. In cells expressing the Split‐R with the two inhibitory PKC sites on the R‐region inactivated (SR‐S641A/T682A), density of FH‐R complexes was much higher than in Split‐R WT expressing cells after PKC or PKC + PKA stimulation. No differences were observed for BH‐R complexes measured at all phosphorylation conditions. Since full‐length CFTR channels display large functional responses to PKC + PKA in WT and S641A/T682A mutant, we conclude that FH‐R interactions are important for CFTR function. Inactivation of consensus PKC site serine 686 (S686A) significantly reduced the basal BH‐R interaction and prevented the PKC enhancing effect on CFTR function and FH‐R interaction. The phospho‐mimetic mutation (S686D) restored basal BH‐R interaction and the PKC enhancing effect on CFTR function with enhanced FH‐R interaction. As the channel function is mainly stimulated by PKA phosphorylation of the R‐region, and this response is known to be enhanced by PKC phosphorylation, our data support a model in which the regulation of CFTR activation results from increased interactions of the R‐region with the N‐term‐TMD1‐NBD1. Also, serine S686 was found to be critical for the PKC enhancing effect which requires a permissive BH‐R interaction at basal level and increased FH‐R interaction after PKC + PKA phosphorylation.