OrgaSegment: deep-learning based organoid segmentation to quantify CFTR dependent fluid secretion.

Epithelial ion and fluid transport studies in patient-derived organoids (PDOs) are increasingly being used for preclinical studies, drug development and precision medicine applications. Epithelial fluid transport properties in PDOs can be measured through visual changes in organoid (lumen) size. Such organoid phenotypes have been highly instrumental for the studying of diseases, including cystic fibrosis (CF), which is characterized by genetic mutations of the CF transmembrane conductance regulator (CFTR) ion channel. Here we present OrgaSegment, a MASK-RCNN based deep-learning segmentation model allowing for the segmentation of individual intestinal PDO structures from bright-field images. OrgaSegment recognizes spherical structures in addition to the oddly-shaped organoids that are a hallmark of CF organoids and can be used in organoid swelling assays, including the new drug-induced swelling assay that we show here. OrgaSegment enabled easy quantification of organoid swelling and could discriminate between organoids with different CFTR mutations, as well as measure responses to CFTR modulating drugs. The easy-to-apply label-free segmentation tool can help to study CFTR-based fluid secretion and possibly other epithelial ion transport mechanisms in organoids.

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.

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.

Abnormal functional lymphoid tolerance and enhanced myeloid exocytosis are characteristics of resting and stimulated PBMCs in cystic fibrosis patients.

Introduction: Cystic Fibrosis (CF) is the commonest genetically inherited disease (1 in 4,500 newborns) and 70% of people with CF (pwCF) harbour the F508Del mutation, resulting in misfolding and incorrect addressing of the channel CFTR to the epithelial membrane and subsequent dysregulation of fluid homeostasis. Although studies have underscored the importance and over-activation of myeloid cells, and in particular neutrophils in the lungs of people with CF (pwCF), relatively less emphasis has been put on the potential immunological bias in CF blood cells, at homeostasis or following stimulation/infection. Methods: Here, we revisited, in an exhaustive fashion, in pwCF with mild disease (median age of 15, median % FEV1 predicted = 87), whether their PBMCs, unprimed or primed with a 'non specific' stimulus (PMA+ionomycin mix) and a 'specific' one (live P.a =PAO1 strain), were differentially activated, compared to healthy controls (HC) PBMCs. Results: 1) we analysed the lymphocytic and myeloid populations present in CF and Control PBMCs (T cells, NKT, Tgd, ILCs) and their production of the signature cytokines IFN-g, IL-13, IL-17, IL-22. 2) By q-PCR, ELISA and Luminex analysis we showed that CF PBMCs have increased background cytokines and mediators production and a partial functional tolerance phenotype, when restimulated. 3) we showed that CF PBMCs low-density neutrophils release higher levels of granule components (S100A8/A9, lactoferrin, MMP-3, MMP-7, MMP-8, MMP-9, NE), demonstrating enhanced exocytosis of potentially harmful mediators. Discussion: In conclusion, we demonstrated that functional lymphoid tolerance and enhanced myeloid protease activity are key features of cystic fibrosis PBMCs.

Prevalence of common autosomal recessive mutation carriers in women in the Southern Vietnam following the application of expanded carrier screening.

The common autosomal recessive (AR) mutation carrier is still unknown in Vietnam. This study aims to identify the most common AR gene mutation carriers in women of reproductive age to build a Vietnamese-specific carrier screening panel for AR and X-linked disorders in the preconception and prenatal healthcare program. A cross-sectional study was conducted at University Medical Center-Branch 2 in Ho Chi Minh City from December 1st, 2020, to June 30th, 2023. 338 women have consented to take a 5 mL blood test to identify 540 recessive genes. The carrier screening panel was designed based on the American College of Medical Genetics and Genomics (ACMG)-recommended genes and suggestions from 104 clinical experts in Vietnam. Obstetricians and genetic experts counseled all positive testing results to discuss the possibility of recessive diseases in their offspring. The most common recessive disorders were defined at a prevalence of 1 in 60 or greater, and those were added to a Vietnamese-specific carrier screening panel. 338 non-pregnant and pregnant women underwent the expanded carrier screening (ECS). The carrier frequency was 63.6%, in which 215 women carried at least one AR gene mutation. GJB2 hearing impairment was identified as the most common chronic condition (1 in 5). The second most common AR disorder was beta-thalassemia (1 in 16), followed by cystic fibrosis (1 in 23), G6PD deficiency (1 in 28), Wilson's disease (1 in 31), Usher's syndrome (1 in 31), and glycogen storage disease (1 in 56). Seven common recessive genes were added in ethnic-based carrier screening. Women in the South of Vietnam have been carried for many recessive conditions at high frequency, such as hearing impairment, genetic anemia, and cystic fibrosis. It is necessary to implement a preconception and prenatal screening program by using seven widely popular AR genes in a Vietnamese-specific carrier screening panel to reduce the burden related to AR and X-linked disorders.

Diversity of CFTR variants across ancestries characterized using 454,727 UK biobank whole exome sequences.

BACKGROUND: Limited understanding of the diversity of variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene across ancestries hampers efforts to advance molecular diagnosis of cystic fibrosis (CF). The consequences pose a risk of delayed diagnoses and subsequently worsened health outcomes for patients. Therefore, characterizing the spectrum of CFTR variants across ancestries is critical for revolutionizing molecular diagnoses of CF. METHODS: We analyzed 454,727 UK Biobank (UKBB) whole-exome sequences to characterize the diversity of CFTR variants across ancestries. Using the PanUKBB classification, the participants were assigned into six major groups: African (AFR), American/American Admixed (AMR), Central South Asia (CSA), East Asian (EAS), European (EUR), and Middle East (MID). We segregated ancestry-specific CFTR variants, including those that are CF-causing or clinically relevant. The ages of certain CF-causing variants were determined and analyzed for selective pressure effects, and curated phenotype analysis was performed for participants with clinically relevant CFTR genotypes. RESULTS: We detected over 4000 CFTR variants, including novel ancestry-specific variants, across six ancestries. Europeans had the most unique CFTR variants [n = 2212], while the American group had the least unique variants [n = 23]. F508del was the most prevalent CF-causing variant found in all ancestries, except in EAS, where V520F was the most prevalent. Common EAS variants such as 3600G > A, V456A, and V520, which appeared approximately 270, 215, and 338 generations ago, respectively, did not show evidence of selective pressure. Sixteen participants had two CF-causing variants, with two being diagnosed with CF. We found 154 participants harboring a CF-causing and varying clinical consequences (VCC) variant. Phenotype analysis performed for participants with multiple clinically relevant variants returned significant associations with CF and its pulmonary phenotypes [Bonferroni-adjusted p < 0.05]. CONCLUSIONS: We leveraged the UKBB database to comprehensively characterize the broad spectrum of CFTR variants across ancestries. The detection of over 4000 CFTR variants, including several ancestry-specific and uncharacterized CFTR variants, warrants the need for further characterization of their functional and clinical relevance. Overall, the presentation of classical CF phenotypes seen in non-CF diagnosed participants with more than one CF-causing variant indicates that they may benefit from current CFTR modulator therapies.

Influence of the BsmI polymorphism of the vitamin D receptor gene on the levels of vitamin D, inflammatory and oxidative stress profile in patients with cystic fibrosis supplemented with Colecalciferol megadose.

OBJECTIVE: Evaluate the influence of the BsmI polymorphism of the vitamin D receptor gene on vitamin D levels, and inflammatory and oxidative stress markers in patients with Cystic Fibrosis supplemented with cholecalciferol megadose. METHODS: We performed a single-arm, non-randomized pre- and post-study of 17 patients aged 5 to 20 years with cystic fibrosis diagnosed with vitamin D insufficiency/deficiency 25-hydroxy vitamin< 30 ng/mL. Individuals were genotyped for the BsmI polymorphism of the vitamin D receptor gene and all received cholecalciferol supplementation of 4,000 IU daily for children aged 5 to 10 years and 10,000 IU for children over 10 years of age for 8 weeks. Interviews were conducted with personal data, sun exposure, anthropometric and blood samples of 25-hydroxy vitamin parathormone, serum calcium, ultrasensitive C-reactive protein, alpha 1 acid glycoprotein, total antioxidant capacity, malondialdehyde and kidney and liver function. Inter- and intra-group assessment was assessed by paired t-test Anova test or its non-parametric counterparts. RESULTS: The individuals were mostly male and reported no adverse effects from the use of supplementation, 64 % had 25-hydroxy vitamin levels >30 ng/mL. Patients with BB and Bb genotypes showed increased serum levels of 25-hydroxy vitamin. The group with BB genotype showed a reduction in alpha 1 acid glycoprotein. And individuals with the bb genotype had high levels of malondialdehyde compared to the pre-intervention time. CONCLUSION: It is concluded that variations of the BsmI polymorphism of the vitamin D receptor gene have different responses in vitamin D levels and markers of inflammation and oxidative stress.

Comprehensive Assessment of CFTR Modulators' Therapeutic Efficiency for N1303K Variant.

p.Asn1303Lys (N1303K) is a common missense variant of the CFTR gene, causing cystic fibrosis (CF). In this study, we initially evaluated the influence of CFTR modulators on the restoration of N1303K-CFTR function using intestinal organoids derived from four CF patients expressing the N1303K variant. The forskolin-induced swelling assay in organoids offered valuable insights about the beneficial effects of VX-770 + VX-661 + VX-445 (Elexacaftor + Tezacaftor + Ivacaftor, ETI) on N1303K-CFTR function restoration and about discouraging the prescription of VX-770 + VX-809 (Ivacaftor + Lumacaftor) or VX-770 + VX-661 (Ivacaftor + Tezacaftor) therapy for N1303K/class I patients. Then, a comprehensive assessment was conducted on an example of one patient with the N1303K/class I genotype to examine the ETI effect on the restoration of N1303K-CFTR function using in vitro the patient's intestinal organoids, ex vivo the intestinal current measurements (ICM) method and assessment of the clinical status before and after targeted therapy. All obtained results are consistent with each other and have proven the effectiveness of ETI for the N1303K variant. ETI produced a significant positive effect on forskolin-induced swelling in N1303K/class I organoids indicating functional improvement of the CFTR protein; ICM demonstrated that ETI therapy restored CFTR function in the intestinal epithelium after three months of treatment, and the patient improved his clinical status and lung function, increased his body mass index (BMI) and reduced the lung pathogenic flora diversity, surprisingly without improving the sweat test results.

[Therapeutics in cystic fibrosis: Clinical revolution and new challenges]. / Traitements de la mucoviscidose - Révolution clinique et nouveaux défis.

Over time, cystic fibrosis has become a model of synergy between research in pathophysiology and cell biology, and clinical advances. Therapies targeting the CFTR protein, in particular CFTR modulators, have transformed the prognosis of patients, bringing the hope of a normal life with the possibility of starting a family and growing old, challenging established statistics. However, patients are not yet cured, and side effects remain insufficiently documented. Epidemiological changes create new challenges for the management of cystic fibrosis. Approximately 10 % of patients still lack a therapeutic option. The community of researchers, pharmaceutical industries, patient associations, and health authorities remains committed to monitor the long-term effects of these still poorly characterised treatments, and to explore new pharmacological approaches, such as gene therapies.

Title: Traitements de la mucoviscidose - Révolution clinique et nouveaux défis. Abstract: Avec le temps, la mucoviscidose est devenue un exemple de synergie entre la recherche en biologie cellulaire et les progrès cliniques. Les thérapies protéiques ont enfin apporté l'espoir d'une vie normale aux patients, bouleversant ainsi les statistiques épidémiologiques établies. Néanmoins, les patients ne guérissent pas, et l'évolution épidémiologique de la maladie ouvre de nouveaux défis pour la prise en charge des malades. Par ailleurs, environ 10 % des patients demeurent sans solution thérapeutique. De nouvelles stratégies sont ainsi envisagées et la communauté des chercheurs, industriels, patients et autorités de santé reste mobilisée pour suivre les effets à long terme de ces nouveaux traitements et explorer de nouvelles approches pharmacologiques.

A uniquely efficacious type of CFTR corrector with complementary mode of action.

Three distinct pharmacological corrector types (I, II, III) with different binding sites and additive behavior only partially rescue the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding and trafficking defect observed in cystic fibrosis. We describe uniquely effective, macrocyclic CFTR correctors that were additive to the known corrector types, exerting a complementary "type IV" corrector mechanism. Macrocycles achieved wild-type-like folding efficiency of F508del-CFTR at the endoplasmic reticulum and normalized CFTR currents in reconstituted patient-derived bronchial epithelium. Using photo-activatable macrocycles, docking studies and site-directed mutagenesis a highly probable binding site and pose for type IV correctors was identified in a cavity between lasso helix-1 (Lh1) and transmembrane helix-1 of membrane spanning domain (MSD)-1, distinct from the known corrector binding sites. Since only F508del-CFTR fragments spanning from Lh1 until MSD2 responded to type IV correctors, these likely promote cotranslational assembly of Lh1, MSD1, and MSD2. Previously corrector-resistant CFTR folding mutants were also robustly rescued, suggesting substantial therapeutic potential for type IV correctors.

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.

Lumacaftor/Ivacaftor Population Pharmacokinetics in Pediatric Patients with Cystic Fibrosis: A First Step Toward Personalized Therapy.

BACKGROUND: A major breakthrough in cystic fibrosis (CF) therapy was achievedAQ1 with CFTR modulators. The lumacaftor/ivacaftor combination is indicated for the treatment of CF in pediatric patients above 6 years old. Pharmacokinetic (PK) studies of lumacaftor/ivacaftor in these vulnerable pediatric populations are AQ2crucial to optimize treatment protocols. OBJECTIVES AND METHODS: The objectives of this study were to describe the population PK (PPK) of lumacaftor and ivacaftor in children with CF, and to identify factors associated with interindividual variability. The association between drug exposure and clinical response was also investigated. RESULTS: A total of 75 children were included in this PPK study, with 191 concentrations available for each compound and known metabolites (lumacaftor, ivacaftor, ivacaftor-M1, and ivacaftor-M6). PPK analysis was performed using Monolix software. A large interindividual variability was observed. The main sources of interpatient variability identified were patient bodyweight and hepatic function (aspartate aminotransferase). Forced expiratory volume in the first second (FEV1) was statistically associated with the level of exposure to ivacaftor after 48 weeks of treatment. CONCLUSIONS: This study is the first analysis of lumacaftor/ivacaftor PPK in children with CF. These data suggest that dose adjustment is required after identifying variability factors to optimize efficacy. The use of therapeutic drug monitoring as a basis for dose adjustment in children with CF may be useful.

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.

Sotagliflozin attenuates liver-associated disorders in cystic fibrosis rabbits.

Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene lead to CF, a life-threating autosomal recessive genetic disease. While recently approved Trikafta dramatically ameliorates CF lung diseases, there is still a lack of effective medicine to treat CF-associated liver disease (CFLD). To address this medical need, we used a recently established CF rabbit model to test whether sotagliflozin, a sodium-glucose cotransporter 1 and 2 (SGLT1/2) inhibitor drug that is approved to treat diabetes, can be repurposed to treat CFLD. Sotagliflozin treatment led to systemic benefits to CF rabbits, evidenced by increased appetite and weight gain as well as prolonged lifespan. For CF liver-related phenotypes, the animals benefited from normalized blood chemistry and bile acid parameters. Furthermore, sotagliflozin alleviated nonalcoholic steatohepatitis-like phenotypes, including liver fibrosis. Intriguingly, sotagliflozin treatment markedly reduced the otherwise elevated endoplasmic reticulum stress responses in the liver and other affected organs of CF rabbits. In summary, our work demonstrates that sotagliflozin attenuates liver disorders in CF rabbits and suggests sotagliflozin as a potential drug to treat CFLD.

Comparative genomics reveals distinct diversification patterns among LysR-type transcriptional regulators in the ESKAPE pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a harmful nosocomial pathogen associated with cystic fibrosis and burn wounds, encodes for a large number of LysR-type transcriptional regulator proteins. To understand how and why LTTR proteins evolved with such frequency and to establish whether any relationships exist within the distribution we set out to identify the patterns underpinning LTTR distribution in P. aeruginosa and to uncover cluster-based relationships within the pangenome. Comparative genomic studies revealed that in the JGI IMG database alone ~86 000 LTTRs are present across the sequenced genomes (n=699). They are widely distributed across the species, with core LTTRs present in >93 % of the genomes and accessory LTTRs present in <7 %. Analysis showed that subsets of core LTTRs can be classified as either variable (typically specific to P. aeruginosa) or conserved (and found to be distributed in other Pseudomonas species). Extending the analysis to the more extensive Pseudomonas database, PA14 rooted analysis confirmed the diversification patterns and revealed PqsR, the receptor for the Pseudomonas quinolone signal (PQS) and 2-heptyl-4-quinolone (HHQ) quorum-sensing signals, to be amongst the most variable in the dataset. Successful complementation of the PAO1 pqsR - mutant using representative variant pqsR sequences suggests a degree of structural promiscuity within the most variable of LTTRs, several of which play a prominent role in signalling and communication. These findings provide a new insight into the diversification of LTTR proteins within the P. aeruginosa species and suggests a functional significance to the cluster, conservation and distribution patterns identified.

Cystic fibrosis in Europe: improved lung function and longevity - reasons for cautious optimism, but challenges remain.

BACKGROUND: Prognosis and disease severity in cystic fibrosis (CF) are linked to declining lung function. To characterise lung function by the number of adults in countries with different levels of Gross National Income (GNI), data from the European Cystic Fibrosis Society Patient Registry were utilised. METHODS: Annual data including age, forced expiratory volume in 1 s (FEV1), anthropometry, genotype, respiratory cultures and CF-related diabetes (CFRD) were retrieved between 2011 and 2021. All countries were stratified into GNI per capita to reflect differences within Europe. RESULTS: A consistent improvement in FEV1 % pred and survival was observed among the 47 621 people with CF (pwCF), including subjects with chronic Pseudomonas aeruginosa infection, CFRD and/or undernutrition. Mean values of FEV1 % pred changed from 85% to 94.2% for children and from 63.6% to 74.7% for adults. FEV1 % pred further increased among those carrying the F508del mutation in 2021, when elexacaftor/tezacaftor/ivacaftor was available. The number of adult pwCF increased from 13 312 in 2011 to 21 168 in 2021, showing a 60% increase. PwCF living in European lower income countries did not demonstrate a significant annual increase in FEV1 % pred or in the number of adults. CONCLUSION: This pan-European analysis demonstrates a consistent improvement in FEV1 % pred, number of adult pwCF and survival over the last decade only in European higher and middle income countries. Urgent action is needed in the lower income countries where such improvement was not observed. The notable improvement observed in pwCF carrying the F508del mutation emphasises the need to develop treatments for all CF mutations.

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.