Ivacaftor attenuates gentamicin-induced ototoxicity through the CFTR-Nrf2-HO1/NQO1 pathway.

OBJECTIVES: Gentamicin is one of the most common ototoxic drugs that can lower patients' quality of life. Oxidative stress is a key factors inducing sensory hair cell death during gentamicin administration. So far, there are no effective drugs to prevent or treat gentamicin- induced hearing loss. A recent study found cystic fibrosis transmembrane conductance regulator (CFTR) as a new target to modulate cellular oxidative balance. The objective of this study was to estimate the effect of the CFTR activator ivacaftor on gentamicin-induced ototoxicity and determine its mechanism. METHODS: The hair cell count was analyzed by Myosin 7a staining. Apoptosis was analyzed by TUNEL Apoptosis Kit. Cellular reactive oxygen species (ROS) level was detected by DCFH-DA probes. The Nrf2 related proteins expression levels were analyzed by western blot. RESULTS: An in vitro cochlear explant model showed that gentamicin caused ROS accumulation in sensory hair cells and induced apoptosis, and this effect was alleviated by pretreatment with ivacaftor. Western blotting showed that ivacaftor administration markedly increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO1), and NAD(P)H:quinone oxidoreductase 1 (NQO1). The protective effect of ivacaftor was abolished by the Nrf2 inhibitor ML385. DISCUSSION: Our results indicate the protective role of the CFTR-Nrf2-HO1/NQO1 pathway in gentamicin-induced ototoxicity. Ivacaftor may be repositioned or repurposed towards aminoglycosides-induced hearing loss.

Functional Consequences of CFTR Interactions in Cystic Fibrosis.

Cystic fibrosis (CF) is a fatal autosomal recessive disorder caused by the loss of function mutations within a single gene for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CFTR is a chloride channel that regulates ion and fluid transport across various epithelia. The discovery of CFTR as the CF gene and its cloning in 1989, coupled with extensive research that went into the understanding of the underlying biological mechanisms of CF, have led to the development of revolutionary therapies in CF that we see today. The highly effective modulator therapies have increased the survival rates of CF patients and shifted the epidemiological landscape and disease prognosis. However, the differential effect of modulators among CF patients and the presence of non-responders and ineligible patients underscore the need to develop specialized and customized therapies for a significant number of patients. Recent advances in the understanding of the CFTR structure, its expression, and defined cellular compositions will aid in developing more precise therapies. As the lifespan of CF patients continues to increase, it is becoming critical to clinically address the extra-pulmonary manifestations of CF disease to improve the quality of life of the patients. In-depth analysis of the molecular signature of different CF organs at the transcriptional and post-transcriptional levels is rapidly advancing and will help address the etiological causes and variability of CF among patients and develop precision medicine in CF. In this review, we will provide an overview of CF disease, leading to the discovery and characterization of CFTR and the development of CFTR modulators. The later sections of the review will delve into the key findings derived from single-molecule and single-cell-level analyses of CFTR, followed by an exploration of disease-relevant protein complexes of CFTR that may ultimately define the etiological course of CF disease.

Discovery of GLPG2737, a Potent Type 2 Corrector of CFTR for the Treatment of Cystic Fibrosis in Combination with a Potentiator and a Type 1 Co-corrector.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. This epithelial anion channel regulates the active transport of chloride and bicarbonate ions across membranes. Mutations result in reduced surface expression of CFTR channels with impaired functionality. Correctors are small molecules that support the trafficking of CFTR to increase its membrane expression. Such correctors can have different mechanisms of action. Combinations may result in a further improved therapeutic benefit. We describe the identification and optimization of a new pyrazolol3,4-bl pyridine-6-carboxylic acid series with high potency and efficacy in rescuing CFTR from the cell surface. Investigations showed that carboxylic acid group replacement with acylsulfonamides and acylsulfonylureas improved ADMET and PK properties, leading to the discovery of the structurally novel co-corrector GLPG2737. The addition of GLPG2737 to the combination of the potentiator GLPG1837 and C1 corrector 4 led to an 8-fold increase in the F508del CFTR activity.

Functional rescue of CFTR in rectal organoids from patients carrying R334W variant by CFTR modulators and PDE4 inhibitor Roflumilast.

BACKGROUND: Many disease-causing variants in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene remain uncharacterized and untreated. Restoring the function of the impaired CFTR protein is the goal of personalized medicine, particularly in patients carrying rare CFTR variants. In this study, functional defects related to the rare R334W variant were evaluated after treatment with CFTR modulators or Roflumilast, a phosphodiesterase-4 inhibitor (PDE4i). METHODS: Rectal organoids from subjects with R334W/2184insA and R334W/2183AA > G genotypes were used to perform the Forskolin-induced swelling (FIS) assay. Organoids were left drug-untreated or treated with modulators VX-770 (I), VX-445 (E), and VX-661 (T) mixed, and their combination (ETI). Roflumilast (R) was used alone or as a combination of I + R. RESULTS: Our data show a significant increase in FIS rate following treatment with I alone. The combined use of modulators, such as ETI, did not increase further swelling than I alone, nor in protein maturation. Treatment with R shows an increase in FIS response similar to those of I, and the combination R + I significantly increases the rescue of CFTR activity. CONCLUSIONS: Equivalent I and ETI treatment efficacy was observed for both genotypes. Furthermore, significant organoid swelling was observed with combined I + R used that supports the recently published data describing a potentiating effect of only I in patients carrying the variant R334W and, at the same time, corroborating the role of strategies that include PDE4 inhibitors further to potentiate the effect of I for this variant.

Use of CFTR Modulators for Cystic Fibrosis in a Patient with Liver Transplant and ESRD on Hemodialysis.

Cystic fibrosis is an autosomal recessive disorder caused by mutations of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most recent therapeutic approach to cystic fibrosis aims to correct structural and functional abnormalities of CFTR protein. CFTR modulators including ivacaftor-tezacaftor-elexacaftor are used in patients with F508del mutation, with clinical improvement. To date, there are no experiences of CFTR modulator therapy in cystic fibrosis patients with organ transplantation and severe renal impairment. We report the case of a patient diagnosed with cystic fibrosis with F508del mutation, who underwent liver transplantation at the age of 19 and started hemodialysis at the age of 24 due to end-stage renal disease secondary to membranous glomerulonephritis. She was treated with Kaftrio (ivacaftor-tezacaftor-elexacaftor) with clinical benefits on appetite, improvement of body mass index, and reduction of pulmonary exacerbations. A reduction of dosage to 75% of the standard dose was required due to alterations of the liver function. Conclusions. Use of CFTR modulators in patient with cystic fibrosis, liver transplant and end-stage renal disease could be considered safe but a clinical and laboratoristic monitoring of hepatic function is needed.

Organic Synthesis and Current Understanding of the Mechanisms of CFTR Modulator Drugs Ivacaftor, Tezacaftor, and Elexacaftor.

The monogenic rare disease Cystic Fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance (CFTR) protein, an anion channel expressed at the apical plasma membrane of epithelial cells. The discovery and subsequent development of CFTR modulators-small molecules acting on the basic molecular defect in CF-have revolutionized the standard of care for people with CF (PwCF), thus drastically improving their clinical features, prognosis, and quality of life. Currently, four of these drugs are approved for clinical use: potentiator ivacaftor (VX-770) alone or in combination with correctors lumacaftor, (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445). Noteworthily, the triple combinatorial therapy composed of ivacaftor, tezacaftor, and elexacaftor constitutes the most effective modulator therapy nowadays for the majority of PwCF. In this review, we exploit the organic synthesis of ivacaftor, tezacaftor, and elexacaftor by providing a retrosynthetic drug analysis for these CFTR modulators. Furthermore, we describe the current understanding of the mechanisms of action (MoA's) of these compounds by discussing several studies that report the key findings on the molecular mechanisms underlying their action on the CFTR protein.

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.

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

A mathematical model of ENaC and Slc26a6 regulation by CFTR in salivary gland ducts.

Cystic fibrosis (CF) is a genetic disease caused by the mutations of cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis transmembrane conductance regulator gene. Cftr is a critical ion channel expressed in the apical membrane of mouse salivary gland striated duct cells. Although Cftr is primarily a Cl- channel, its knockout leads to higher salivary Cl- and Na+ concentrations and lower pH. Mouse experiments show that the activation of Cftr upregulates epithelial Na+ channel (ENaC) protein expression level and Slc26a6 (a 1Cl-:2[Formula: see text] exchanger of the solute carrier family) activity. Experimentally, it is difficult to predict how much the coregulation effects of CFTR contribute to the abnormal Na+, Cl-, and [Formula: see text] concentrations and pH in CF saliva. To address this question, we construct a wild-type mouse salivary gland model and simulate CFTR knockout by altering the expression levels of CFTR, ENaC, and Slc26a6. By reproducing the in vivo and ex vivo final saliva measurements from wild-type and CFTR knockout animals, we obtain computational evidence that ENaC and Slc26a6 activities are downregulated in CFTR knockout in salivary glands.NEW & NOTEWORTHY This paper describes a salivary gland mathematical model simulating the ion exchange between saliva and the salivary gland duct epithelium. The novelty lies in the implementation of CFTR regulating ENaC and Slc26a6 in a CFTR knockout gland. By reproducing the experimental saliva measurements in wild-type and CFTR knockout glands, the model shows that CFTR regulates ENaC and Slc26a6 anion exchanger in salivary glands. The method could be used to understand the various cystic fibrosis phenotypes.

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.

Role of CFTR in diabetes-induced pancreatic ductal fluid and HCO3 - secretion.

Type 1 diabetes is a disease of the endocrine pancreas; however, it also affects exocrine function. Although most studies have examined the effects of diabetes on acinar cells, much less is known regarding ductal cells, despite their important protective function in the pancreas. Therefore, we investigated the effect of diabetes on ductal function. Diabetes was induced in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice following an i.p. administration of streptozotocin. Pancreatic ductal fluid and HCO3 - secretion were determined using fluid secretion measurements and fluorescence microscopy, respectively. The expression of ion transporters was measured by real-time PCR and immunohistochemistry. Transmission electron microscopy was used for the morphological characterization of the pancreas. Serum secretin and cholecystokinin levels were measured by an enzyme-linked immunosorbent assay. Ductal fluid and HCO3 - secretion, CFTR activity, and the expression of CFTR, Na+ /H+ exchanger-1, anoctamine-1 and aquaporin-1 were significantly elevated in diabetic mice. Acute or chronic glucose treatment did not affect HCO3 - secretion, but increased alkalizing transporter activity. Inhibition of CFTR significantly reduced HCO3 - secretion in both normal and diabetic mice. Serum levels of secretin and cholecystokinin were unchanged, but the expression of secretin receptors significantly increased in diabetic mice. Diabetes increases fluid and HCO3 - secretion in pancreatic ductal cells, which is associated with the increased function of ion and water transporters, particularly CFTR. KEY POINTS: There is a lively interaction between the exocrine and endocrine pancreas not only under physiological conditions, but also under pathophysiological conditions The most common disease affecting the endocrine part is type-1 diabetes mellitus (T1DM), which is often associated with pancreatic exocrine insufficiency Compared with acinar cells, there is considerably less information regarding the effect of diabetes on pancreatic ductal epithelial cells, despite the fact that the large amount of fluid and HCO3 - produced by ductal cells is essential for maintaining normal pancreatic functions Ductal fluid and HCO3 - secretion increase in T1DM, in which increased cystic fibrosis transmembrane conductance regulator activation plays a central role. We have identified a novel interaction between T1DM and ductal cells. Presumably, the increased ductal secretion represents a defence mechanism in the prevention of diabetes, but further studies are needed to clarify this issue.

Bicarbonate secretion and acid/base sensing by the intestine.

The transport of bicarbonate across the enterocyte cell membrane regulates the intracellular as well as the luminal pH and is an essential part of directional fluid movement in the gut. Since the first description of "active" transport of HCO3- ions against a concentration gradient in the 1970s, the fundamental role of HCO3- transport for multiple intestinal functions has been recognized. The ion transport proteins have been identified and molecularly characterized, and knockout mouse models have given insight into their individual role in a variety of functions. This review describes the progress made in the last decade regarding novel techniques and new findings in the molecular regulation of intestinal HCO3- transport in the different segments of the gut. We discuss human diseases with defects in intestinal HCO3- secretion and potential treatment strategies to increase luminal alkalinity. In the last part of the review, the cellular and organismal mechanisms for acid/base sensing in the intestinal tract are highlighted.

Highly effective cystic fibrosis transmembrane conductance (regulator) modulator therapy: shifting the curve for most while leaving some further behind.

PURPOSE OF REVIEW: Traditional cystic fibrosis (CF) care had been focused on early intervention and symptom mitigation. With the advent of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy (HEMT), in particular, the approval of elexacaftor/tezacaftor/ivacaftor in 2019, there has been a dramatic improvement in outcomes in CF. The purpose of this article is to review the benefits, limitations, and impact of HEMT as well as discuss the new implications, challenges, and hope that modulators bring to people with CF (pwCF). RECENT FINDINGS: HEMT has demonstrated sustained improvement in lung function, nutrition, quality of life, and survival for over 90% of pwCF. As HEMT has delivered such promise, there is a small but significant portion of pwCF who do not benefit from HEMT due to ineligible mutations, intolerance, or lack of accessibility to modulators. SUMMARY: HEMT has significantly improved outcomes, but continued research is needed to understand the new challenges and implications the era of HEMT will bring, as well as how to provide equitable care to those who are unable to benefit from HEMT.

CFTR is required for zinc-mediated antibacterial defense in human macrophages.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion transporter required for epithelial homeostasis in the lung and other organs, with CFTR mutations leading to the autosomal recessive genetic disease CF. Apart from excessive mucus accumulation and dysregulated inflammation in the airways, people with CF (pwCF) exhibit defective innate immune responses and are susceptible to bacterial respiratory pathogens such as Pseudomonas aeruginosa. Here, we investigated the role of CFTR in macrophage antimicrobial responses, including the zinc toxicity response that is used by these innate immune cells against intracellular bacteria. Using both pharmacological approaches, as well as cells derived from pwCF, we show that CFTR is required for uptake and clearance of pathogenic Escherichia coli by CSF-1-derived primary human macrophages. CFTR was also required for E. coli-induced zinc accumulation and zinc vesicle formation in these cells, and E. coli residing in macrophages exhibited reduced zinc stress in the absence of CFTR function. Accordingly, CFTR was essential for reducing the intramacrophage survival of a zinc-sensitive E. coli mutant compared to wild-type E. coli. Ectopic expression of the zinc transporter SLC30A1 or treatment with exogenous zinc was sufficient to restore antimicrobial responses against E. coli in human macrophages. Zinc supplementation also restored bacterial killing in GM-CSF-derived primary human macrophages responding to P. aeruginosa, used as an in vitro macrophage model relevant to CF. Thus, restoration of the zinc toxicity response could be pursued as a therapeutic strategy to restore innate immune function and effective host defense in pwCF.

Structural identification of a selectivity filter in CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that regulates transepithelial salt and fluid homeostasis. CFTR dysfunction leads to reduced chloride secretion into the mucosal lining of epithelial tissues, thereby causing the inherited disease cystic fibrosis. Although several structures of CFTR are available, our understanding of the ion-conduction pathway is incomplete. In particular, the route that connects the cytosolic vestibule with the extracellular space has not been clearly defined, and the structure of the open pore remains elusive. Furthermore, although many residues have been implicated in altering the selectivity of CFTR, the structure of the "selectivity filter" has yet to be determined. In this study, we identify a chloride-binding site at the extracellular ends of transmembrane helices 1, 6, and 8, where a dehydrated chloride is coordinated by residues G103, R334, F337, T338, and Y914. Alterations to this site, consistent with its function as a selectivity filter, affect ion selectivity, conductance, and open channel block. This selectivity filter is accessible from the cytosol through a large inner vestibule and opens to the extracellular solvent through a narrow portal. The identification of a chloride-binding site at the intra- and extracellular bridging point leads us to propose a complete conductance path that permits dehydrated chloride ions to traverse the lipid bilayer.

Stress stimulation promotes the injury repair process of airway epithelial cells through the [Cl-]i-FAK signaling axis.

The airway epithelium serves as a critical interface with the external environment, making it vulnerable to various external stimuli. Airway epithelial stress acts as a catalyst for the onset of numerous pulmonary and systemic diseases. Our previous studies have highlighted the impact of acute stress stimuli, especially bacterial lipopolysaccharide (LPS) and hydrogen peroxide (H2O2), on the continuous elevation of intracellular chloride concentration ([Cl-]i). However, the precise mechanism behind this [Cl-]i elevation and the consequential effects of such stress on the injury repair function of airway epithelial cells remain unclear. Our findings indicate that H2O2 induces an elevation in [Cl-]i by modulating the expression of CF transmembrane conductance regulator (CFTR) and Ca-activated transmembrane protein 16 A (TMEM16A) in airway epithelial cells (BEAS-2B), whereas LPS achieves this solely through CFTR. Subsequently, the elevated [Cl-]i level facilitated the injury repair process of airway epithelial cells by activating focal adhesion kinase (FAK). In summary, the [Cl-]i-FAK axis appears to play a promoting effect on the injury repair process triggered by stress stimulation. Furthermore, our findings suggest that abnormalities in the [Cl-]i-FAK signaling axis may play a crucial role in the pathogenesis of chronic airway diseases. Therefore, controlling the structure and function of airway epithelial barriers through the modulation of [Cl-]i holds promising prospects for future applications in managing and treating such conditions.

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 (1 mM); S1159F-CFTR caused the greater decrease in Po consistent with its more severe clinical phenotype. Ivacaftor (10-100 nM) 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 (0.5-1 µM) 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.

CFTR expression in human salivary gland acinar cells.

The key role of CFTR in secretory epithelia has been extensively documented. Additionally, CFTR plays a significant role in ion absorption in exocrine glands, including salivary and sweat glands. Most of the knowledge about CFTR expression comes from animal models such as the mouse or the rat, but there is limited information about CFTR expression in human tissues. In the present study, we assessed the expression of CFTR in human submandibular and parotid glands. Consistent with findings in rodent salivary glands, our immunolocalization studies show that CFTR is expressed in duct cells. However, CFTR expression in human salivary glands differs from that in rodents, as immunolocalization and single-cell RNA sequencing analysis from a previous study performed in the human parotid gland revealed the presence of CFTR protein and transcripts within a distinct cell cluster. Based on cell marker expression, this cluster corresponds to acinar cells. To obtain functional evidence supporting CFTR expression, we isolated human parotid acinar cells through collagenase digestion. Acinar cells displayed an anion conductance that was activated in response to cAMP-increasing agents and was effectively blocked by CFTRInh172, a known CFTR blocker. This study provides novel evidence of CFTR expression within acinar cells of human salivary glands. This finding challenges the established model positioning CFTR exclusively in duct cells from exocrine glands.NEW & NOTEWORTHY This study addresses the uncertainty about the impact of CFTR on human salivary gland function. We found CFTR transcripts in a subset of duct cells known as ionocytes, as well as in acinar cells. Isolated human parotid acinar cells exhibited Cl- conductance consistent with CFTR activity. This marks the first documented evidence of functional CFTR expression in human salivary gland acinar cells.

Genetic analysis and functional study of novel CFTR variants in Chinese children with cystic fibrosis.

OBJECTIVES: To describe the clinical characteristics of Chinese cystic fibrosis (CF) patients and to investigate the variants of CFTR and their potential pathogenicity. STUDY DESIGN: Chinese patients with potential CF diagnosis were studied. Clinical data were reviewed retrospectively from medical records. Whole exome sequencing and genetic evaluation were conducted to explore potential gene variants. The disruption of the variants to protein structure and function was explored and validated using in vitro experiments and in silico analysis. RESULTS: Four patients were recruited to the study, three of them were diagnosed as CF, and one was diagnosed as CFTR-related disorder. The age at symptom onset for the patients in this study ranged from newborn to 6 years, while the age at diagnosis varied from 3 to 11 years. All four patients exhibited bilateral diffuse bronchiectasis with Pseudomonas aeruginosa infections, and three of them had malnutrition. Finger clubbing was observed in three patients, two of whom displayed mixed ventilatory dysfunction. The CFTR variants spectrum of Chinese children with CF differs from that of Caucasian. A total of six variants were identified, two of which were first reported (c.1219G > T [p.Glu407*] and c.1367delT [p.Ala457Leufs*12]). The nonsense variants c.1219G > T, c.1657C > T and c.2551C > T and the frameshift variant c.1367delT were predicted to introduce premature stop codon and produce shorten CFTR protein, which was also first validated by in vitro truncation assay in this study. The missense variant c.1810A > C was predicted to disrupt the function of the nucleotide-binding domain 1 (NBD1) in the CFTR protein. The splicing variant c.1766 + 5G > T caused skipping of exon 13 and damaged the integrity of CFTR protein. CONCLUSIONS: Our study expands the spectrum of phenotypes and genotypes for CF of Chinese origin, which differs significantly from that of Caucasian. Genetic analysis and counseling are crucial and deserve extensive popularization for the diagnosis ofCF in patients of Chinese origin.

Benchmarking AlphaMissense pathogenicity predictions against cystic fibrosis variants.

Variants in the cystic fibrosis transmembrane conductance regulator gene (CFTR) result in cystic fibrosis-a lethal autosomal recessive disorder. Missense variants that alter a single amino acid in the CFTR protein are among the most common cystic fibrosis variants, yet tools for accurately predicting molecular consequences of missense variants have been limited to date. AlphaMissense (AM) is a new technology that predicts the pathogenicity of missense variants based on dual learned protein structure and evolutionary features. Here, we evaluated the ability of AM to predict the pathogenicity of CFTR missense variants. AM predicted a high pathogenicity for CFTR residues overall, resulting in a high false positive rate and fair classification performance on CF variants from the CFTR2.org database. AM pathogenicity score correlated modestly with pathogenicity metrics from persons with CF including sweat chloride level, pancreatic insufficiency rate, and Pseudomonas aeruginosa infection rate. Correlation was also modest with CFTR trafficking and folding competency in vitro. By contrast, the AM score correlated well with CFTR channel function in vitro-demonstrating the dual structure and evolutionary training approach learns important functional information despite lacking such data during training. Different performance across metrics indicated AM may determine if polymorphisms in CFTR are recessive CF variants yet cannot differentiate mechanistic effects or the nature of pathophysiology. Finally, AM predictions offered limited utility to inform on the pharmacological response of CF variants i.e., theratype. Development of new approaches to differentiate the biochemical and pharmacological properties of CFTR variants is therefore still needed to refine the targeting of emerging precision CF therapeutics.