Fibrosis Transmembrane Conductance Regulator Channel Research Articles

Cystic fibrosis in childhood and adulthood

Cystic fibrosis (CF, or mucoviscidosis) is one of the rare diseases with a fatal course and with the highest prevalence. Formerly known as apurely childhood disease, this multisystemic disease follows an autosomal recessive inheritance pattern and results in amalfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, leading to the production of viscous secretions. The prognosis and outcome of CF are determined by the severity of the involvement of the lungs. Other typically affected organs include the pancreas, liver and intestines. This article reviews the clinical presentation and evolution of CF with afocus on the new era of the highly effective CFTR modulator treatment. An overview of the current state of knowledge on the care for CF patients is presented. The introduction of the CF newborn screening, the increased understanding of the disease and the development of novel treatment options have substantially increased the quality of life and life expectancy of people with CF. As aresult, more than half of CF patients in Germany are now older than 18years of age and the complications of a chronic disease as well as organ damage due to the intensive treatment are gaining in importance. The highly effective CFTR modulator treatment results in a significant improvement in CFTR function, lung function, body mass index and quality of life and is available to approximately 90% of patients in Germany, based on the genotype. Nevertheless, further research including the development of causal treatment, e.g., gene therapy, targeting the underlying defect in the remaining 10% of CF patients, is urgently needed. Even in adult patients, CF with a mild course or aCFTR-related disease should be considered, e.g., in cases of bronchiectasis and/or recurrent abdominal complaints.

Apical pH regulates calcium-activated chloride secretion across human airway epithelia

Anion secretion across airway epithelia contributes to host defense by increasing airway surface liquid (ASL) volume and pH. Anion channels in the apical membrane of airway epithelia include Ca2+-activated Cl− channels (CaCCs) and cystic fibrosis transmembrane conductance regulator (CFTR) channels. Mutations in the CFTR gene that encode nonfunctional CFTR channels cause cystic fibrosis (CF); thus, activating CaCCs may restore anion secretion in people with CF. ATP secreted into the ASL activates purinergic receptors, leading to an increase in intracellular Ca2+ (Cai2+) and the opening of CaCCs. Data from docking simulations suggest that the protonation state of ATP influences its affnity for purinergic receptors, therefore we tested the hypothesis that ASL pH influences the effcacy of ATP to activate CaCCs in airway epithelia. In saline solutions buffered with 5 mM ATP, titration experiments revealed a pKa of ~6.9 at 37°C. Thus, ATP exists in its protonated ATP3− and deprotonated ATP4− in ASL that can range from pH 6 to pH 7.5. To determine the effect of pH on ATP-stimulated Cl− secretion through CaCCs, we measured Cl− secretion across CF epithelia generated by several ATP concentrations with apical pH-6.00, -6.70, and -7.40 solutions. As pH became alkaline, the EC50 for the relationship between [ATP] and Cl− secretion decreased. Using the Henderson-Hasselbalch equation and the pKa for ATP, we determined the relationship between ATP4− and Cl− secretion. This analysis revealed that regardless of apical pH values, the relationship between [ATP4−] and Cl− secretion aligned to a single experimental fit. These data suggest that pH regulates the CaCC response by deprotonating ATP. To examine the pH dependence of purinergic receptor activation, we measured ATP-induced Cai2+ responses in acutely dissociated human airway epithelial cells loaded with FURA-2 and perfused with pH-7.40 or -6.50 solutions. Separate experiments measuring intracellular pH in pH-7.40 or 6.50 solutions informed Kd values for FURA-2 experiments. With an [ATP] that induces similar levels of Cl− secretion from pH 6.00 to pH 7.40 (100 μM ATP), we observed similar Cai2+ responses for pH-7.40 and -6.50 solutions. However, when we repeated the experiment with an [ATP] that induces pH-sensitive Cl− secretion (10 μM ATP), we observed larger Ca2+ responses with pH-7.40 solutions than with pH-6.50 solutions. These data indicate that pH influences purinergic receptor activation near the pKa of ATP, a finding consistent with ATP4− activating purinergic receptors. These data have implications in CF and airway inflammation. In CF, decreased CFTR-mediated HCO−3 secretion acidifies the ASL, thus a large portion of ATP will exist in the protonated ATP3− species requiring a higher total [ATP] to open CaCCs. Inflammatory cytokines that cause airway inflammation increase the ASL pH, therefore a larger portion of ATP will exist in the deprotonated ATP4− species requiring less total [ATP] to open CaCCs. Cystic Fibrosis Foundation Research Development Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Airway basal cells from human-induced pluripotent stem cells: a new frontier in cystic fibrosis research.

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases, regenerative medicine, and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs, which involves step-by-step differentiation of hiPSCs into definitive endoderm, anterior foregut endoderm, NKX2.1+ lung progenitors, and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs, expressing basal cell markers (NGFR, KRT5, and TP63), could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs, potentially applicable for gene therapy research.

The Impact of Antimicrobial Resistance in Cystic Fibrosis.

The phenomenon of antimicrobial resistance (AMR) is a critical global health challenge, with prospects indicating its potential to become the leading cause of death worldwide in the coming years. Individuals with pre-existing conditions, such as neoplastic disease undergoing chemotherapy, those on immunosuppressive therapy, and individuals with rare diseases like cystic fibrosis (CF), face heightened challenges due to AMR. CF is a rare disease caused by a deficiency in the synthesis of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel protein, resulting in multi-organ clinical symptoms, particularly in the respiratory system. PwCF experience recurrent pulmonary exacerbations triggered by bacterial or viral infections, making them particularly vulnerable to the impact of AMR. This review delves into the complex relationship between AMR and climate dynamics, focusing on the unique challenges faced by individuals with CF. It discusses the methods employed to measure AMR, its global impact on antibiotic resistance, and the specific microbial communities present in the CF airway. The review also explores the intricacies of antimicrobial resistance within the context of cystic fibrosis, emphasizing the urgent need for research in this field.

Recommended Tool Compounds for Modifying the Cystic Fibrosis Transmembrane Conductance Regulator Channel Variants.

Cystic fibrosis (CF) is a genetic disorder arising from variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to multiple organ system defects. CFTR tool compounds are molecules that can modify the activity of the CFTR channel. Especially, patients that are currently not able to benefit from approved CFTR modulators, such as patients with rare CFTR variants, benefit from further research in discovering novel tools to modulate CFTR. This Review explores the development and classification of CFTR tool compounds, including CFTR blockers (CFTRinh-172, GlyH-101), potentiators (VRT-532, Genistein), correctors (VRT-325, Corr-4a), and other approved and unapproved modulators, with detailed descriptions and discussions for each compound. The challenges and future directions in targeting rare variants and optimizing drug delivery, and the potential synergistic effects in combination therapies are outlined. CFTR modulation holds promise not only for CF treatment but also for generating CF models that contribute to CF research and potentially treating other diseases such as secretory diarrhea. Therefore, continued research on CFTR tool compounds is critical.

Physiological and Pathological Significance of Chloride Channels.

Cl- influx and efflux through Cl- channels play a role in regulating the homeostasis of biological functions. Therefore, the hyperfunction or dysfunction of Cl- channels elicits pathological mechanisms. The Cl- channel superfamily includes voltage-gated Cl- (ClC) channels, Ca2+-activated Cl- channels (ClCa; TMEM16A/TMEM16B), cystic fibrosis transmembrane conductance regulator channels, and ligand-gated Cl- channels. These channels are ubiquitously expressed to regulate ion homeostasis, muscle tonus, membrane excitability, cell volume, survival, neurotransmission, and transepithelial transport. The activation or inhibition of Cl- channels changes the membrane potential, thereby affecting cytosolic Ca2+ signals. An elevation in cytosolic [Ca2+] triggers physiological and pathological responses in most cells. However, the roles of Cl- channels have not yet been examined as extensively as cation (Na+, Ca2+, and K+) channels. We recently reported the functional expression of: (i) TMEM16A/ClCa channels in portal vein and pulmonary arterial smooth muscle cells (PASMC), pinealocytes, and brain capillary endothelial cells; (ii) TMEM16B/ClCa channels in pinealocytes; (iii) ClC-3 channels in PASMC and chondrocytes; and (iv) ClC-7 channels in chondrocytes. We also showed that the down-regulation of TMEM16A and ClC-7 channel expression was associated with cirrhotic portal hypertension and osteoarthritis, respectively, whereas the enhanced expression of TMEM16A and ClC-3 channels was involved in the pathogenesis of cerebral ischemia and pulmonary arterial hypertension, respectively. Further investigations on the physiological/pathological functions of Cl- channels will provide insights into biological functions and contribute to the screening of novel target(s) of drug discovery for associated diseases.

Two rare variants that affect the same amino acid in CFTR have distinct responses to ivacaftor.

Some residues in the cystic fibrosis transmembrane conductance regulator (CFTR) channel are the site of more than one CFTR variant that cause cystic fibrosis. Here, we investigated the function of S1159F and S1159P, two variants associated with different clinical phenotypes, which affect the same pore-lining residue in transmembrane segment 12 that are both strongly potentiated by ivacaftor when expressed in CFBE41o- bronchial epithelial cells. To study the single-channel behaviour of CFTR, we applied the patch-clamp technique to Chinese hamster ovary cells heterologously expressing CFTR variants incubated at 27°C to enhance channel residence at the plasma membrane. S1159F- and S1159P-CFTR formed Cl- channels activated by cAMP-dependent phosphorylation and gated by ATP that exhibited thermostability at 37°C. Both variants modestly reduced the single-channel conductance of CFTR. By severely attenuating channel gating, S1159F- and S1159P-CFTR reduced the open probability (Po ) of wild-type CFTR by ≥75% at ATP (1mM); S1159F-CFTR caused the greater decrease in Po consistent with its more severe clinical phenotype. Ivacaftor (10-100nM) doubled the Po of both CFTR variants without restoring Po values to wild-type levels, but concomitantly, ivacaftor decreased current flow through open channels. For S1159F-CFTR, the reduction of current flow was marked at high (supersaturated) ivacaftor concentrations (0.5-1μM) and voltage-independent, identifying an additional detrimental action of elevated ivacaftor concentrations. In conclusion, S1159F and S1159P are gating variants, which also affect CFTR processing and conduction, but not stability, necessitating the use of combinations of CFTR modulators to optimally restore their channel activity. KEY POINTS: Dysfunction of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes the genetic disease cystic fibrosis (CF). This study investigated two rare pathogenic CFTR variants, S1159F and S1159P, which affect the same amino acid in CFTR, to understand the molecular basis of disease and response to the CFTR-targeted therapy ivacaftor. Both rare variants diminished CFTR function by modestly reducing current flow through the channel and severely inhibiting ATP-dependent channel gating with S1159F exerting the stronger adverse effect, which correlates with its association with more severe disease. Ivacaftor potentiated channel gating by both rare variants without restoring their activity to wild-type levels, but concurrently reduced current flow through open channels, particularly those of S1159F-CFTR. Our data demonstrate that S1159F and S1159P cause CFTR dysfunction by multiple mechanisms that require combinations of CFTR-targeted therapies to fully restore channel function.

Delivery Characterization of SPL84 Inhaled Antisense Oligonucleotide Drug for 3849 + 10 kb C- > T Cystic Fibrosis Patients.

Recent advances in the therapeutic potential of RNA-related treatments, specifically for antisense oligonucleotide (ASO)-based drugs, have led to increased numbers of ASO regulatory approvals. In this study, we focus on SPL84, an inhaled ASO-based drug, developed for the treatment of the pulmonary disease cystic fibrosis (CF). Pulmonary drug delivery is challenging, due to a variety of biological, physical, chemical, and structural barriers, especially when targeting the cell nucleus. The distribution of SPL84 throughout the lungs, penetration into the epithelial cells and nucleus, and structural stability are critical parameters that will impact drug efficacy in a clinical setting. In this study, we demonstrate broad distribution, as well as cell and nucleus penetration of SPL84 in mouse and monkey lungs. In vivo and in vitro studies confirmed the stability of our inhaled drug in CF patient-derived mucus and in lung lysosomal extracts. The mobility of SPL84 through hyperconcentrated mucus was also demonstrated. Our results, supported by a promising preclinical pharmacological effect of full restoration of cystic fibrosis transmembrane conductance regulator channel activity, emphasize the high potential of SPL84 as an effective drug for the treatment of CF patients. In addition, successfully tackling the lung distribution of SPL84 offers immense opportunities for further development of SpliSense's inhaled ASO-based drugs for unmet needs in pulmonary diseases.

Airway and Lung Organoids from Human-Induced Pluripotent Stem Cells Can Be Used to Assess CFTR Conductance

Airway and lung organoids derived from human-induced pluripotent stem cells (hiPSCs) are current models for personalized drug screening, cell-cell interaction studies, and lung disease research. We analyzed the existing differentiation protocols and identified the optimal conditions for obtaining organoids. In this article, we describe a step-by-step protocol for differentiating hiPSCs into airway and lung organoids. We obtained airway and lung organoids from a healthy donor and from five donors with cystic fibrosis. Analysis of the cellular composition of airway and lung organoids showed that airway organoids contain proximal lung epithelial cells, while lung organoids contain both proximal and distal lung epithelial cells. Forskolin-induced swelling of organoids derived from a healthy donor showed that lung organoids, as well as airway organoids, contain functional epithelial cells and swell after 24 h exposure to forskolin, which makes it a suitable model for analyzing the cystic fibrosis transmembrane conductance regulator (CFTR) channel conductance in vitro. Thus, our results demonstrate the feasibility of generating and characterizing airway and lung organoids from hiPSCs, which can be used for a variety of future applications.

Rescue of Rare CFTR Trafficking Mutants Highlights a Structural Location-Dependent Pattern for Correction.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Currently, more than 2100 variants have been identified in the gene, with a large number being very rare. The approval of modulators that act on mutant CFTR protein, correcting its molecular defect and thus alleviating the burden of the disease, revolutionized the field of CF. However, these drugs do not apply to all patients with CF, especially those with rare mutations-for which there is a lack of knowledge on the molecular mechanisms of the disease and the response to modulators. In this work, we evaluated the impact of several rare putative class II mutations on the expression, processing, and response of CFTR to modulators. Novel cell models consisting of bronchial epithelial cell lines expressing CFTR with 14 rare variants were created. The variants studied are localized at Transmembrane Domain 1 (TMD1) or very close to the signature motif of Nucleotide Binding Domain 1 (NBD1). Our data show that all mutations analyzed significantly decrease CFTR processing and while TMD1 mutations respond to modulators, those localized in NBD1 do not. Molecular modeling calculations confirm that the mutations in NBD1 induce greater destabilization of CFTR structure than those in TMD1. Furthermore, the structural proximity of TMD1 mutants to the reported binding site of CFTR modulators such as VX-809 and VX-661, make them more efficient in stabilizing the CFTR mutants analyzed. Overall, our data suggest a pattern for mutation location and impact in response to modulators that correlates with the global effect of the mutations on CFTR structure.

SARS-CoV-2 viral entry and replication is impaired in Cystic Fibrosis airways due to ACE2 downregulation

As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis could be considered a comorbidity for coronavirus disease 2019. Instead, current clinical evidence seems to be heading in the opposite direction. To clarify whether host factors expressed by the Cystic Fibrosis epithelia may influence coronavirus disease 2019 progression, here we describe the expression of SARS-CoV-2 receptors in primary airway epithelial cells. We show that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral entry and replication in Cystic Fibrosis cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin 6 in healthy donor-derived primary airway epithelial cells, but a very weak response in primary Cystic Fibrosis cells. Collectively, these data support that Cystic Fibrosis condition may be at least partially protecting from SARS-CoV-2 infection.

Patient-derived cell models for personalized medicine approaches in cystic fibrosis

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel that perturb anion transport across the epithelia of the airways and other organs. To treat cystic fibrosis, strategies that target mutant CFTR have been developed such as correctors that rescue folding and enhance transfer of CFTR to the apical membrane, and potentiators that increase CFTR channel activity. While there has been tremendous progress in development and approval of CFTR therapeutics for the most common (F508del) and several other CFTR mutations, around 10-20% of people with cystic fibrosis have rare mutations that are still without an effective treatment. In the current decade, there was an impressive evolution of patient-derived cell models for precision medicine. In cystic fibrosis, these models have played a crucial role in characterizing the molecular defects in CFTR mutants and identifying compounds that target these defects. Cells from nasal, bronchial, and rectal epithelia are most suitable to evaluate treatments that target CFTR. In vitro assays using cultures grown at an air-liquid interface or as organoids and spheroids allow the diagnosis of the CFTR defect and assessment of potential treatment strategies. An overview of currently established cell culture models and assays for personalized medicine approaches in cystic fibrosis will be provided in this review. These models allow theratyping of rare CFTR mutations with available modulator compounds to predict clinical efficacy. Besides evaluation of individual personalized responses to CFTR therapeutics, patient-derived culture models are valuable for testing responses to developmental treatments such as novel RNA- and DNA-based therapies.

Mucins and CFTR: Their Close Relationship.

Mucociliary clearance is a critical defense mechanism for the lungs governed by regionally coordinated epithelial cellular activities, including mucin secretion, cilia beating, and transepithelial ion transport. Cystic fibrosis (CF), an autosomal genetic disorder caused by the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, is characterized by failed mucociliary clearance due to abnormal mucus biophysical properties. In recent years, with the development of highly effective modulator therapies, the quality of life of a significant number of people living with CF has greatly improved; however, further understanding the cellular biology relevant to CFTR and airway mucus biochemical interactions are necessary to develop novel therapies aimed at restoring CFTR gene expression in the lungs. In this article, we discuss recent advances of transcriptome analysis at single-cell levels that revealed a heretofore unanticipated close relationship between secretory MUC5AC and MUC5B mucins and CFTR in the lungs. In addition, we review recent findings on airway mucus biochemical and biophysical properties, focusing on how mucin secretion and CFTR-mediated ion transport are integrated to maintain airway mucus homeostasis in health and how CFTR dysfunction and restoration of function affect mucus properties.

Anticancer effect of verteporfin on non-small cell lung cancer via downregulation of ANO1

Anoctamin 1 (ANO1) is a calcium-activated chloride channel found in various cell types and is overexpressed in non-small cell lung cancer (NSCLC), a major cause of cancer-related mortality. With the rising interest in development of druggable compounds for NSCLC, there has been a corresponding rise in interest in ANO1, a novel drug target for NSCLC. However, as ANO1 inhibitors that have been discovered simultaneously exhibit both the functions of an inhibition of ANO1 channel as well as a reduction of ANO1 protein levels, it is unclear which of the two functions directly causes the anticancer effect. In this study, verteporfin, a chemical compound that reduces ANO1 protein levels was identified through high-throughput screening. Verteporfin did not inhibit ANO1-induced chloride secretion but reduced ANO1 protein levels in a dose-dependent manner with an IC50 value of ~300 nM. Moreover, verteporfin inhibited neither P2Y receptor-induced intracellular Ca2+ mobilization nor cystic fibrosis transmembrane conductance regulator (CFTR) channel activity, and molecular docking studies revealed that verteporfin bound to specific sites of ANO1 protein. Confirming that verteporfin reduces ANO1 protein levels, we then investigated the molecular mechanisms involved in its effect on NSCLC cells. Interestingly, verteporfin decreased ANO1 protein levels, the EGFR-STAT3 pathway as well as ANO1 mRNA expression. Verteporfin reduced the viability of ANO1-expressing cells (PC9, and gefitinib-resistant PC9) and induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage. However, it did not affect hERG channel activity. These results show that the anticancer mechanism of verteporfin is caused via the down-regulation of ANO1.

Downregulation of ANO1 activity in PC‐9 cells modulated by a new novel candidate TMEMinh001

Relevant to multiple studies, calcium activated chloride channel called Anoctamin1 (ANO1) is identified as an excellent biomarker for tumor growth reported in different cancer cells such as prostate, breast, and lung cancer. Growing evidence suggests, ANO1 promotes tumor growth via regulating EGFR signaling and modulating MAPK and protein kinase B pathway. In non‐small cell lung cancer (NSCLC), the ANO1 expression level is high, leading to high cancer‐related mortality. To date, several natural and synthetic compounds have shown their modulatory effect on ANO1 activity but are out of the limelight due to selectivity. Our present study is carried out based on finding a selective and potent modulator of ANO1 activity by in silico and in vitro assay. According to the hypothesis, inhibitors of ANO1 are screened out, where an inhibitor reduced ANO1 expression without modulating intracellular Ca+ level and cystic fibrosis transmembrane conductance regulator (CFTR) channel activity. The molecular docking study revealed negative high binding free energy than known inhibitor Ani9 and other candidate compounds. Furthermore, our potential compound suppressed cell viability and migration process in ANO1 expressing PC9 cells, while not in H1975 cells not expressing ANO1. Besides, it initiated the apoptosis process by up‐regulating caspase‐3 activity. Thus, the above results provided significant evidence of the anticancer effect of the final compound against ANO1, reducing its protein level in NSCLC.

Functional interplay between CFTR and pendrin: physiological and pathophysiological relevance.

The transport of chloride and bicarbonate across epithelia controls the pH and volume of the intracellular and luminal fluids, as well as the systemic pH and vascular volume. The anion exchanger pendrin (SLC26A4) and the cystic fibrosis transmembrane conductance regulator (CFTR) channel are expressed in the apical membrane of epithelial cells of various organs and tissues, including the airways, kidney, thyroid, and inner ear. While pendrin drives chloride reabsorption and bicarbonate, thiocyanate or iodide secretion within the apical compartment, CFTR represents a pathway for the apical efflux of chloride, bicarbonate, and possibly iodide. In the airways, pendrin and CFTR seems to be involved in alkalinization of the apical fluid via bicarbonate secretion, especially during inflammation, while CFTR also controls the volume of the apical fluid via a cAMP-dependent chloride secretion, which is stimulated by pendrin. In the kidney, pendrin is expressed in the cortical collecting duct and connecting tubule and co-localizes with CFTR in the apical membrane of β intercalated cells. Bicarbonate secretion occurs via pendrin, which also drives chloride reabsorption. A functional CFTR is required for pendrin activity. Whether CFTR stimulates pendrin via a direct molecular interaction or other mechanisms, or simply provides a pathway for chloride recycling across the apical membrane remains to be established. In the thyroid, CFTR and pendrin might have overlapping functions in driving the apical flux of iodide within the follicular lumen. In other organs, including the inner ear, the possible functional interplay between pendrin and CFTR needs to be explored.

Подбор CFTR-модуляторов для детей – носителей генетического варианта W1282R

Objective. To study the clinical manifestations of W1282R variant, functional activity of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, and effectiveness of CFTR modulator therapy in patients with the W1282R variant. Materials and methods included medical records, intestinal current measurement method (ICM) and method of intestinal organoids (IO). Results. The c.3844T>C (p.Trp1282Arg, W1282R) variant refers to missense mutations. The Cystic Fibrosis (CF) Patient Registry of the Russian Federation (2019) provides the data on 34 patients with the W1282R variant in the genotype. Clinical manifestations in two patients with the W1282R variant corresponds to the severe course of CF. ICM method revealed the loss of CFTR function. The effect of VX-809 corrector, VX-770 potentiator and their combined use were assessed in each patient using the method of IO. Conclusion. The results indicated a “severe” W1282R variant. The application of VX-809 corrector had no effect on the restoration of chloride channel function, unlike the F508del homozygous control sample (class II mutations). The VX-770 potentiator had no effect on the kinetic properties of CFTR protein. But in both patients, a significant restoration of lost channel function (~45%) was recorded with the combined use of VX-809 corrector and VX-770 potentiator, which is typical for class III and class IV variants. This challenges the conclusions that the W1282R variant has class II characteristics only. Combination therapy with VX-809 and VX-770 may be recommended for patients with the W1282R/CFTRdel2,3 and W1282R/F508del genotypes. Key words: cystic fibrosis, CFTR gene, W1282R pathogenic variant, intestinal current measurement method, intestinal organoids, forskolin-induced swelling assay, correctors, potentiators, target therapy, ivacaftor, lumacaftor

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.

Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

Alteration of Membrane Cholesterol Content Plays a Key Role in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Channel Activity.

Altered cholesterol homeostasis in cystic fibrosis patients has been reported, although controversy remains. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms. However, whether cholesterol directly modulates cystic fibrosis transmembrane conductance regulator (CFTR) channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized fischer rat thyroid (FRT) cells and primary human bronchial epithelial (HBE) cells. Treatment with methyl-β-cyclodextrin (MβCD) significantly reduced total cholesterol content in FRT cells, which significantly decreased forskolin (FSK)-mediated activation of both wildtype (WT-) and P67L-CFTR. This effect was also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of CFTR by FSK. In addition, alteration of cholesterol increased the potency of VX-770, a clinically used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations; this likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the plasma membrane cholesterol level significantly modulate CFTR channel function and consequently may affect sensitivity to clinical therapeutics in CF patients.