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In the common genetic disorder autosomal dominant polycystic kidney disease (ADPKD), kidney function is disrupted by multiple fluid-filled epithelial cysts. Cyst growth in ADPKD involves fluid accumulation within the cyst lumen driven by cystic fibrosis transmembrane conductance regulator (CFTR)- mediated transepithelial Cl- secretion. This suggests that inhibitors of the CFTR Cl- channel might retard cyst growth. This review considers how knowledge of CFTR structure and function and its role in transepithelial salt and water movements provides insight into the mechanism of action of CFTR inhibitors. Some small molecules, termed open-channel blockers, inhibit directly the CFTR Cl- channel by physically obstructing the CFTR pore and preventing Cl- flow. By contrast, other small molecules, termed allosteric inhibitors, bind to CFTR at a site remote from the channel pore and interfere with conformational changes that open the pore. The application of high-throughput screening to CFTR drug discovery has led to the identification of new inhibitors of the CFTR Cl- channel including the thiazolidinone CFTRinh-172 and the glycine hydrazide GlyH-101. The demonstration that CFTR inhibitors retard cyst expansion and kidney enlargement in mouse models of ADPKD provides proof of concept for the use of small-molecule CFTR inhibitors in the treatment of ADPKD. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl− channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating. [ABSTRACT FROM AUTHOR]

Comments on the findings of a study appearing in the August 2004 issue of the "Journal of General Physiology" regarding cystic fibrosis transmembrane conductance regulator (CFTR) pharmacology. Absence of potent, specific inhibitors of anion channels and implications for CFTR pharmacology studies; Discovery of a glycine hydrazides and its impact on CFTR inhibitors; Relationship between the chemical structure of glycine hydrazides and CFTR inhibition.

Discusses the structure and function of the cystic fibrosis transmembrane regulator (CFTR) chloride channel. Topology; Domain structure; Biophysical properties of channel pore; Identification of residues that influence conduction and that line the pore; Ion selectivity; Contribution of the intracellular and extracellular loops; Regulating effect of adenosine 5'-triphosphate hydrolysis.

Presents a study on the pharmacological agents that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) chlorine channel. Agents which stimulate CFTR by interacting with the nucleotide-binding domains that control channel gating; CFTR inhibitors characterized by binding within the channel pore and preventing chlorine permeation; Use of molecular pharmacology of CFTR in the treatment of diseases caused by CFTR dysfunction.

Describes studies designed to develop an expression system for cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane of Fischer rat thyroid epithelia. CFTR expression systems; Measurements of apical membrane chloride current; Immunoprecipitation; Immunofluorescence studies.

Reports that, when expressed in heterologous epithelial cells, missense mutations associated with cystic fibrosis in which arginine is replaced by histidine, tryptophan or proline were correctly processed and generated cyclic AMP-regulated apical Cl- currents. Cystic fibrosis transmembrane conductance regulator (CFTR); Fischer Rat Thyroid (FRT) epithelia; Whole-cell properties of R117H; Further details.

Tests the hypothesis that one membrane-spanning domain--nucleotide binding domain (MSD-NBD) motif could form a chlorine channel at the amino terminal of the cystic fibrosis transmembrane conductance regulator (CFTR). Expression of D836X generates cAMP-regulated chlorine currents; Regulation of D836X chlorine channels by intracellular nucleotides; Implications for cystic fibrosis.

Cystic fibrosis transmembrane conductance regulator (CFTR) correctors are small molecules that target the most common cause of cystic fibrosis: misfolded F508del-CFTR. Using differential scanning fluorimetry, Sampson et al. (2010) identify a CFTR corrector that interacts directly with the CFTR domain affected by the F508del mutation. [Copyright &y& Elsevier]

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most common cause of cystic fibrosis. The F508 residue is located on nucleotide-binding domain 1 (NBD1) in contact with the cytosolic extensions of the transmembrane helices, in particular intracellular loop 4 (ICL4). To investigate how absence of F508 at this interface impacts the CFTR protein, we carried out a mutagenesis scan of ICL4 by introducing secondsite mutations at 11 positions in cis with F508del. Using an image-based fluorescence assay, we measured how each mutation affected membrane proximity and ion-channel function. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains (F1068, R1070W, and F1074, mimicking F1068, F508, and F1074 in WT CFTR). F508del-CFTR displayed a distorted aromatic stack, with F1068 displaced toward the space vacated by F508, while in F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR included F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. These studies highlight how both hydrophobic interactions and conformational flexibility might be important at the ICL4/NBD1 interface, suggesting possible structural underpinnings of F508del-induced dysfunction. [ABSTRACT FROM AUTHOR]

Background: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. Methods: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. Results: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. Conclusion: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This document provides an overview of experimental pharmacology in precision medicine, focusing on recent advancements and challenges in the field. It discusses the application of precision medicine in areas such as drug metabolism, drug-drug interactions, therapeutic drug dose monitoring, and interventions for cystic fibrosis. The article also mentions the 19th World Congress of Basic & Clinical Pharmacology (WCP2023), which explored precision medicine approaches for improving human health. The importance of molecular information and quantitative proteomics in enhancing drug safety and efficacy, as well as predicting individual variations in drug absorption, distribution, metabolism, and elimination, is emphasized. The document highlights the success and challenges of precision medicine in specific cases, including drug-drug interactions, targeted cancer therapies, and cystic fibrosis. It also underscores the significance of collaboration between patient advocacy groups, academia, industry, and regulatory authorities in advancing precision medicine. [Extracted from the article]

The authors comment on a study published in this issue of the journal which suggested that individual airway surface epithelial cells oscillate between absorptive and secretory states. Included is a list of principal observations underlying the fluid recirculation model. The authors believe that the study's bioelectric measurements of the airway epithelium are a major technical achievement. They contend that it highlights the flaw in bidirectional models of ion transport by airway epithelia.

The article introduces a series of studies on the physiology of anion transport.

One therapeutic strategy for cystic fibrosis (CF) seeks to restore anion transport to affected epithelia by targeting other apical membrane Cl − channels to bypass dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel. The properties and regulation of the Ca 2+ -activated Cl − channel TMEM16A argue that long-acting small molecules which target directly TMEM16A are required to overcome CFTR loss. Through genetic studies of lung diseases, SLC26A9, a member of the solute carrier 26 family of anion transporters, has emerged as a promising target to bypass CFTR dysfunction. An alternative strategy to circumvent CFTR dysfunction is to deliver to CF epithelia artificial anion transporters that shuttle Cl − across the apical membrane. Recently, powerful, non-toxic, biologically-active artificial anion transporters have emerged. [ABSTRACT FROM AUTHOR]

Key points The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP., To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH., We found that short-lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP-binding site 1 formed by the nucleotide-binding domains., Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects., Abstract Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl− channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP-binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single-channel activity of human CFTR at different intracellular pH (pHi) values. When compared with the control (pHi 7.3), acidifying pHi to 6.3 or alkalinizing pHi to 8.3 and 8.8 caused small reductions in the open-time constant (τo) of wild-type CFTR. By contrast, the fast closed-time constant (τcf), which describes the short-lived closures that interrupt open bursts, was greatly increased at pHi 5.8 and 6.3. To analyse intraburst kinetics, we used linear three-state gating schemes. All data were satisfactorily modelled by the C1 ↔ O ↔ C2 kinetic scheme. Changing the intracellular ATP concentration was without effect on τo, τcf and their responses to pHi changes. However, mutations that disrupt the interaction of ATP with ATP-binding site 1, including K464A, D572N and the CF-associated mutation G1349D all abolished the prolongation of τcf at pHi 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP-dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP-binding site 1 modulates intraburst gating. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Defective CFTR leads to accumulation of dehydrated viscous mucus within the small intestine, luminal acidification and altered intestinal motility, resulting in blockage. These changes promote gut microbial dysbiosis, adversely influencing the normal proliferation and differentiation of intestinal epithelial cells. Using Illumina 16S rRNA gene sequencing and immunohistochemistry, we assessed changes in mucosa-attached microbiome and epithelial cell profile in the small intestine of CF mice and a CF patient compared to wild-type mice and non-CF humans. We found increased abundance of pro-inflammatory Escherichia and depletion of beneficial secondary bile-acid producing bacteria in the ileal mucosa-attached microbiome of CFTR-null mice. The ileal mucosa in a CF patient was dominated by a non-aeruginosa Pseudomonas species and lacked numerous beneficial anti-inflammatory and short-chain fatty acid-producing bacteria. In the ileum of both CF mice and a CF patient, the number of absorptive enterocytes, Paneth and glucagon-like peptide 1 and 2 secreting L-type enteroendocrine cells were decreased, whereas stem and goblet cell numbers were increased. These changes in mucosa-attached microbiome and epithelial cell profile suggest that microbiota-host interactions may contribute to intestinal CF disease development with implications for therapy. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Defective CFTR leads to accumulation of dehydrated viscous mucus within the small intestine, luminal acidification and altered intestinal motility, resulting in blockage. These changes promote gut microbial dysbiosis, adversely influencing the normal proliferation and differentiation of intestinal epithelial cells. Using Illumina 16S rRNA gene sequencing and immunohistochemistry, we assessed changes in mucosa-attached microbiome and epithelial cell profile in the small intestine of CF mice and a CF patient compared to wild-type mice and non-CF humans. We found increased abundance of pro-inflammatory Escherichia and depletion of beneficial secondary bile-acid producing bacteria in the ileal mucosa-attached microbiome of CFTR-null mice. The ileal mucosa in a CF patient was dominated by a non-aeruginosa Pseudomonas species and lacked numerous beneficial anti-inflammatory and short-chain fatty acid-producing bacteria. In the ileum of both CF mice and a CF patient, the number of absorptive enterocytes, Paneth and glucagon-like peptide 1 and 2 secreting L-type enteroendocrine cells were decreased, whereas stem and goblet cell numbers were increased. These changes in mucosa-attached microbiome and epithelial cell profile suggest that microbiota-host interactions may contribute to intestinal CF disease development with implications for therapy. [ABSTRACT FROM AUTHOR]

Background and Purpose: Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators are small molecules developed to treat the genetic disease cystic fibrosis (CF). They interact directly with CFTR Cl− channels at the plasma membrane to enhance channel gating. Here, we investigate the action of a new CFTR potentiator, CP‐628006 with a distinct chemical structure. Experimental Approach Using electrophysiological assays with CFTR‐expressing heterologous cells and CF patient‐derived human bronchial epithelial (hBE) cells, we compared the effects of CP‐628006 with the marketed CFTR potentiator ivacaftor. Key Results: CP‐628006 efficaciously potentiated CFTR function in epithelia from cultured hBE cells. Its effects on the predominant CFTR variant F508del‐CFTR were larger than those with the gating variant G551D‐CFTR. In excised inside‐out membrane patches, CP‐628006 potentiated wild‐type, F508del‐CFTR, and G551D‐CFTR by increasing the frequency and duration of channel openings. CP‐628006 increased the affinity and efficacy of F508del‐CFTR gating by ATP. In these respects, CP‐628006 behaved like ivacaftor. CP‐628006 also demonstrated notable differences with ivacaftor. Its potency and efficacy were lower than those of ivacaftor. CP‐628006 conferred ATP‐dependent gating on G551D‐CFTR, whereas the action of ivacaftor was ATP‐independent. For G551D‐CFTR, but not F508del‐CFTR, the action of CP‐628006 plus ivacaftor was greater than ivacaftor alone. CP‐628006 delayed, but did not prevent, the deactivation of F508del‐CFTR at the plasma membrane, whereas ivacaftor accentuated F508del‐CFTR deactivation. Conclusions and Implications: CP‐628006 has distinct effects compared to ivacaftor, suggesting a different mechanism of CFTR potentiation. The emergence of CFTR potentiators with diverse modes of action makes therapy with combinations of potentiators a possibility. [ABSTRACT FROM AUTHOR]

Dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes a wide spectrum of disease, including cystic fibrosis (CF) and CFTR‐related diseases (CFTR‐RDs). Here, we investigate genotype–phenotype–CFTR function relationships using human nasal epithelial (hNE) cells from a small cohort of non‐CF subjects and individuals with CF and CFTR‐RDs and genotypes associated with either residual or minimal CFTR function using electrophysiological techniques. Collected hNE cells were either studied directly with the whole‐cell patch‐clamp technique or grown as primary cultures at an air–liquid interface after conditional reprogramming. The properties of cAMP‐activated whole‐cell Cl− currents in freshly isolated hNE cells identified them as CFTR‐mediated. Their magnitude varied between hNE cells from individuals within the same genotype and decreased in the rank order: non‐CF > CFTR residual function > CFTR minimal function. CFTR‐mediated whole‐cell Cl− currents in hNE cells isolated from fully differentiated primary cultures were identical to those in freshly isolated hNE cells in both magnitude and behaviour, demonstrating that conditional reprogramming culture is without effect on CFTR expression and function. For the cohort of subjects studied, CFTR‐mediated whole‐cell Cl− currents in hNE cells correlated well with CFTR‐mediated transepithelial Cl− currents measured in vitro with the Ussing chamber technique, but not with those determined in vivo with the nasal potential difference assay. Nevertheless, they did correlate with the sweat Cl− concentration of study subjects. Thus, this study highlights the complexity of genotype–phenotype–CFTR function relationships, but emphasises the value of conditionally reprogrammed hNE cells in CFTR research and therapeutic testing. Key points: The genetic disease cystic fibrosis is caused by pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel, which controls anion flow across epithelia lining ducts and tubes in the body.This study investigated CFTR function in nasal epithelial cells from people with cystic fibrosis and CFTR variants with a range of disease severity.CFTR function varied widely in nasal epithelial cells depending on the identity of CFTR variants, but was unaffected by conditional reprogramming culture, a cell culture technique used to grow large numbers of patient‐derived cells.Assessment of CFTR function in vitro in nasal epithelial cells and epithelia, and in vivo in the nasal epithelium and sweat gland highlights the complexity of genotype–phenotype–CFTR function relationships. [ABSTRACT FROM AUTHOR]

In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pHi). CFTR modulates pHi through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pHi. Here, we investigate the direct effects of pHi on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pHi increased the open probability (Po) of wild-type CFTR, whereas alkaline pH, decreased Po and inhibited Cl- flow through the channel. Acidic pHi potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pHi inhibited the MgATP dependence of wildtype CFTR by decreasing channel activity. Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR), Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pH, and inhibition by alkaline pHi, suggesting that site 2 is a critical determinant of the pHi sensitivity of CFTR. The effects of pHi also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl- channel senses directly pHi. The direct regulation of CFTR by pH, has important implications for the regulation of epithelial ion transport. [ABSTRACT FROM AUTHOR]

The chemical solvent tetrahydrofuran (THF) increases short-circuit current (Isc) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases Isc, we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased Isc in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (KD) of 134 mM. This THF-induced increase in Isc was enhanced by forskolin (10 µM), inhibited by the PKA inhibitor H-89 (10 µM) and the thiazolidinone CFTRinh-172 (10 µM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100 mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase Isc in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100 mM) potentiated CFTR Cl- channels open in the presence of ATP (1 mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75 nM), THF (100 mM) did not potentiate channel activity. Similar results were obtained with the ▵R-S660A-CFTR Cl- channel that is not regulated by PKA-dependent phosphorylation and using 2'deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR. [ABSTRACT FROM AUTHOR]

The chemical solvent tetrahydrofuran (THF) increases short-circuit current (Isc) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases Isc, we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased Isc in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (KD) of 134 mM. This THF-induced increase in Isc was enhanced by forskolin (10 µM), inhibited by the PKA inhibitor H-89 (10 µM) and the thiazolidinone CFTRinh-172 (10 µM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100 mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase Isc in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100 mM) potentiated CFTR Cl- channels open in the presence of ATP (1 mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75 nM), THF (100 mM) did not potentiate channel activity. Similar results were obtained with the ▵R-S660A-CFTR Cl- channel that is not regulated by PKA-dependent phosphorylation and using 2'deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR. [ABSTRACT FROM AUTHOR]

Keywords: cystic fibrosis; CFTR; CFTR correctors and potentiators; premature termination codons (PTCs) Because single-channel studies revealed that G542X-CFTR had wild-type CFTR-like conductance properties, these tiny currents represent full-length CFTR protein with the PTC endogenously suppressed. By contrast, E60Q-CFTR possessed the same single-channel conductance and gating pattern as wild-type CFTR and E60X-CFTR treated with G418. [Extracted from the article]

The genetic disease cystic fibrosis (CF) is caused by loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Two CF mutants, G551D and G1349D, affect equivalent residues in the highly conserved LSGGQ motifs that are essential components of the ATP-binding sites of CFTR. Both mutants severely disrupt CFTR channel gating by decreasing mean burst duration (MBD) and prolonging greatly the interburst interval (IBI). To identify small molecules that rescue the gating defects of G551D- and G1349D-CFTR and understand better how these agents work, we used the patch clamp technique to study the effects on G551D- and G1349D-CFTR of phloxine B, pyrophosphate (PPi), and 2′-deoxy ATP (2′-dATP), three agents that strongly enhance CFTR channel gating. Phloxine B (5 μM) potentiated robustly G551D-CFTR Cl- channels by altering both MBD and IBI. In contrast, phloxine B (5 μM) decreased the IBI of G1349D-CFTR, but this effect was insufficient to rescue G1349D-CFTR channel gating. PPi (5 μM) potentiated weakly G551D-CFTR and was without effect on the G1349D-CFTR Cl- channel. However, by altering both MBD and IBI, albeit with different efficacies, 2′-dATP ( 1 mM) potentiated both G551D- and G1349D-CFTR Cl- channels. Using the ATP-driven nucleotide-binding domain dimerization model of CFTR channel gating, we suggest that phloxine B, PPi and 2′-dATP alter channel gating by distinct mechanisms. We conclude that G551D- and G1349D-CFTR have distinct pharmacological profiles and speculate that drug therapy for CF is likely to be mutation-specific. [ABSTRACT FROM AUTHOR]

The relationship between cell proliferation, Cl− secretion, and renal cyst growth: A study using CFTR inhibitors.Background.In autosomal-dominant polycystic kidney disease (ADPKD), cAMP-stimulated cell proliferation and Cl− secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel drive the enlargement of fluid-filled epithelial cysts. To investigate how CFTR blockers inhibit cyst growth, we studied cAMP-dependent Cl− secretion, cell proliferation, and cyst growth using type I Madin Darby canine kidney (MDCK) cells as a model of renal cyst development and growth.Methods.We grew MDCK cysts in collagen gels in the presence of the cAMP agonist forskolin, measured Cl− secretion with the Ussing chamber technique, and assayed cell proliferation using nonpolarized and polarized MDCK cells. To inhibit CFTR, we used glibenclamide, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), genistein, and the specific CFTR inhibitor CFTRinh-172. As controls, we tested the effects of blockers of other types of apical membrane Cl− channels and inhibitors of basolateral membrane ion channels and transporters.Results.In the absence of inhibitors of transepithelial ion transport, forskolin stimulated dramatic cyst growth. CFTR blockers and inhibitors of basolateral membrane ion channels and transporters retarded cyst growth. In contrast, blockers of other types of apical membrane Cl− channels, which were without effect on CFTR, failed to inhibit cyst growth. Inhibition of cyst growth by CFTR blockers was correlated with inhibition of cAMP-stimulated Cl− current (correlation coefficient= 0.81;P<0.05), but not cell proliferation (correlation coefficient= 0.50;P>0.05).Conclusion.Our data suggest that CFTR blockers might retard cyst growth predominantly by inhibiting fluid accumulation within the cyst lumen. [ABSTRACT FROM AUTHOR]

When excised inside-out membrane patches are bathed in symmetrical Cl[sup -]-rich solutions, the current-voltage (I-V) relationship of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl[sup -] currents inwardly rectifies at large positive voltages. To investigate the mechanism of inward rectification, we studied CFTR Cl[sup -] channels in excised inside-out membrane patches from cells expressing wild-type human and murine CFTR using voltage-ramp and -step protocols. Using a voltage-ramp protocol, the magnitude of human CFTR Cl[sup -] current at +100 mV was 74 ± 2% (n = 10) of that at -100 mV. This rectification of macroscopic CFTR Cl[sup -] current was reproduced in full by ensemble currents generated by averaging single-channel currents elicited by an identical voltage-ramp protocol. However, using a voltage-step protocol the single-channel current amplitude (i) of human CFTR at +100 mV was 88 ± 2% (n = 10) of that at -100 mV. Based on these data, we hypothesized that voltage might alter the gating behavior of human CFTR. Using linear three-state kinetic schemes, we demonstrated that voltage has marked effects on channel gating. Membrane depolarization decreased both the duration of bursts and the interburst interval, but increased the duration of gaps within bursts. However, because the voltage dependencies of the different rate constants were in opposite directions, voltage was without large effect on the open probability (P[sub o]) of human CFTR. In contrast, the P[sub o] of murine CFTR was decreased markedly at positive voltages, suggesting that the rectification of murine CFTR is stronger than that of human CFTR. We conclude that inward rectification of CFTR is caused by a reduction in i and changes in gating kinetics. We suggest that inward rectification is an intrinsic property of the CFTR Cl[sup -] channel and not the result of pore block. [ABSTRACT FROM AUTHOR]

• CFTR correctors rescue the plasma membrane expression of F508del-CFTR. • Extracellular phosphate enhances F508del-CFTR function rescued by CFTR correctors. • Cystic fibrosis airway epithelia express the phosphate transporter SLC34A2. • Extracellular phosphate levels might contribute to variable drug responses. Background : The clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulators varies between people with cystic fibrosis (CF) of the same genotype, in part through the action of solute carriers encoded by modifier genes. Here, we investigate whether phosphate transport by SLC34A2 modulates the function of F508del-CFTR after its rescue by CFTR correctors. Methods : With Fischer rat thyroid (FRT) cells heterologously expressing wild-type and F508del-CFTR and fully-differentiated CF and non-CF human airway epithelial cells, we studied SLC34A2 expression and the effects of phosphate on CFTR-mediated transepithelial ion transport. F508del-CFTR was trafficked to the plasma membrane by incubation with different CFTR correctors (alone or in combination) or by low temperature. Results : Quantitative RT-PCR demonstrated that both FRT and primary airway epithelial cells express SLC34A2 mRNA and no differences were found between cells expressing wild-type and F508del-CFTR. For both heterologously expressed and native F508del-CFTR rescued by either VX-809 or C18, the magnitude of CFTR-mediated Cl− currents was dependent on the presence of extracellular phosphate. However, this effect of phosphate was not detected with wild-type and low temperature-rescued F508del-CFTR Cl− currents. Importantly, the modulatory effect of phosphate was observed in native CF airway cells exposed to VX-445, VX-661 and VX-770 (Trikafta) and was dependent on the presence of both sodium and phosphate. Conclusions : Extracellular phosphate modulates the magnitude of CFTR-mediated Cl− currents after F508del-CFTR rescue by clinically-approved CFTR correctors. This effect likely involves electrogenic phosphate transport by SLC34A2. It might contribute to inter-individual variability in the clinical response to CFTR correctors. [ABSTRACT FROM AUTHOR]

The gasotransmitter carbon monoxide (CO) regulates fluid and electrolyte movements across epithelial tissues. However, its action on anion channels is incompletely understood. Here, we investigate the direct action of CO on the cystic fibrosis transmembrane conductance regulator (CFTR) by applying CO-releasing molecules (CO-RMs) to the intracellular side of excised inside-out membrane patches from cells heterologously expressing wild-type human CFTR. Addition of increasing concentrations of tricarbonyldichlororuthenium(II) dimer (CORM-2) (1–300 μM) inhibited CFTR channel activity, whereas the control RuCl3 (100 μM) was without effect. CORM-2 predominantly inhibited CFTR by decreasing the frequency of channel openings and, hence, open probability (Po). But, it also reduced current flow through open channels with very fast kinetics, particularly at elevated concentrations. By contrast, the chemically distinct CO-releasing molecule CORM-3 inhibited CFTR by decreasing Po without altering current flow through open channels. Neither depolarizing the membrane voltage nor raising the ATP concentration on the intracellular side of the membrane affected CFTR inhibition by CORM-2. Interestingly, CFTR inhibition by CORM-2, but not by CFTRinh-172, was prevented by prior enhancement of channel activity by the clinically approved CFTR potentiator ivacaftor. Similarly, when added after CORM-2, ivacaftor completely relieved CFTR inhibition. In conclusion, CORM-2 has complex effects on wild-type human CFTR consistent with allosteric inhibition and open-channel blockade. Inhibition of CFTR by CO-releasing molecules suggests that CO regulates CFTR activity and that the gasotransmitter has tissue-specific effects on epithelial ion transport. The action of ivacaftor on CFTR Cl− channels inhibited by CO potentially expands the drug’s clinical utility. [ABSTRACT FROM AUTHOR]

Parathyroid hormone (PTH) enhances cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion secretion by the human intestinal epithelial cell line Caco-2. With the patch-clamp and Ussing chamber techniques, we investigated how PTH stimulates CFTR activity in Caco-2 cells. Cell-attached recordings revealed that PTH stimulated the opening of CFTR-like channels, while impedance analysis demonstrated that PTH increased apical membrane capacitance, a measure of membrane surface area. Using ion substitution experiments, the PTH-stimulated increase in short-circuit current (I sc), a measure of transepithelial ion transport, was demonstrated to be Cl−- and HCO 3 −-dependent. However, the PTH-stimulated increase in I sc was unaffected by the carbonic anhydrase inhibitor acetazolamide, but partially blocked by the intermediate-conductance Ca2+-activated K+ channel (IKCa) inhibitor clotrimazole. TRAM-34, a related IKCa inhibitor, failed to directly inhibit CFTR Cl− channels in cell-free membrane patches, excluding its action on CFTR. In conclusion, PTH enhances CFTR-mediated anion secretion by Caco-2 monolayers by increasing the expression and function of CFTR in the apical membrane and IKCa activity in the basolateral membrane. • The action of PTH on CFTR-mediated anion secretion by Caco-2 epithelia was studied. • PTH opened CFTR-like channels and increased CFTR expression in the apical membrane. • PTH increased the activity of IKCa in the basolateral membrane. [ABSTRACT FROM AUTHOR]

• Co-translational folding of NBD1 is disrupted by CF mutations. • FRET analysis of transmembrane constructs reveals misfolding by CF mutations. • The impact of the F508del mutation on CFTR is species-dependent. • Two potentiators with distinct actions restore CFTR activity to rare CF mutations. The treatment of cystic fibrosis (CF) has been transformed by orally-bioavailable small molecule modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), which restore function to CF mutants. However, CFTR modulators are not available to all people with CF and better modulators are required to prevent disease progression. Here, we review selectively recent advances in CFTR folding, function and pharmacology. We highlight ensemble and single-molecule studies of CFTR folding, which provide new insight into CFTR assembly, its perturbation by CF mutations and rescue by CFTR modulators. We discuss species-dependent differences in the action of the F508del-CFTR mutation on CFTR expression, stability and function, which might influence pharmacological studies of CFTR modulators in CF animal models. Finally, we illuminate the identification of combinations of two CFTR potentiators (termed co-potentiators), which restore therapeutically-relevant levels of CFTR activity to rare CF mutations. Thus, mechanistic studies of CFTR folding, function and pharmacology inform the development of highly effective CFTR modulators. [ABSTRACT FROM AUTHOR]

• Understanding CFTR's structure-function relationship is key to learning how CF patient mutations and modulators affect the protein. • New structural insights on CFTR gating support the identified potentiator binding site. • Modulation of CFTR has clear clinical benefit, but more work is needed to secure life-long treatments. Structural biology and functional studies are a powerful combination to elucidate fundamental knowledge about the cystic fibrosis transmembrane conductance regulator (CFTR). Here, we discuss the latest findings, including how clinically-approved drugs restore function to mutant CFTR, leading to better clinical outcomes for people with cystic fibrosis (CF). Despite the prospect of regulatory approval of a CFTR-targeting therapy for most CF mutations, strenuous efforts are still needed to fully comprehend CFTR structure-and-function for the development of better drugs to enable people with CF to live full and active lives. [ABSTRACT FROM AUTHOR]

Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl- channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl- channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl- channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl- channels. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) mutations have complex effects on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. They disrupt its processing to and stability at the plasma membrane and function as an ATP-gated Cl − channel. Here, we review therapeutic strategies to overcome defective CFTR processing and stability. Because CF mutations have multiple impacts on the assembly of CFTR protein, combination therapy with several pharmacological chaperones is likely to be required to rescue mutant CFTR expression at the plasma membrane. Alternatively, proteostasis regulators, proteins which regulate the synthesis, intracellular transport and membrane stability of CFTR might be targeted to enhance the plasma membrane expression of mutant CFTR. Finally, we consider an innovative approach to bypass CFTR dysfunction in CF, the delivery of artificial anion transporters to CF epithelia to shuttle Cl − across the apical membrane. The identification of therapies or combinations of therapies, which rescue all CF mutations, is now a priority. [ABSTRACT FROM AUTHOR]

Ivacaftor is the first drug to target directly defects in the cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis (CF). To understand better how ivacaftor potentiates CFTR channel gating, here we investigated the effects of temperature on its action. As a control, we studied the benzimidazolone UCCF-853, which potentiates CFTR by a different mechanism. Using the patch-clamp technique and cells expressing recombinant CFTR, we studied the single-channel behavior of wild-type and F508del-CFTR, the most common CF mutation. Raising the temperature of the intracellular solution from 23 to 37°C increased the frequency but reduced the duration of wild-type and F508del-CFTR channel openings. Although the open probability (Po) of wild-type CFTR increased progressively as temperature was elevated, the relationship between Po and temperature for F508del-CFTR was bell-shaped with a maximum Po at ~30°C. For wild-type CFTR and to a greatly reduced extent F508del-CFTR, the temperature dependence of channel gating was asymmetric with the opening rate demonstrating greater temperature sensitivity than the closing rate. At all temperatures tested, ivacaftor and UCCF-853 potentiated wild-type and F508del-CFTR. Strikingly, ivacaftor but not UCCF-853 abolished the asymmetric temperature dependence of CFTR channel gating. At all temperatures tested, Po values of wild-type CFTR in the presence of ivacaftor were approximately double those of F508del-CFTR, which were equivalent to or greater than those of wild-type CFTR at 37°C in the absence of the drug. We conclude that the principal effect of ivacaftor is to promote channel opening to abolish the temperature dependence of CFTR channel gating. [ABSTRACT FROM AUTHOR]

Background and Purpose: Rescue of F508del-cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the most common CF mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004, a small molecule designed to independently correct protein processing and channel gating defects.Experimental Approach: Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques.Key Results: Chronic treatment with CFFT-004 improved modestly F508del-CFTR processing, but not its plasma membrane stability. By contrast, CFFT-004 rescued F508del-CFTR channel gating better than C18, an analogue of the clinically used CFTR corrector lumacaftor. Subsequent acute addition of CFFT-004, but not C18, potentiated F508del-CFTR channel gating. However, CFFT-004 was without effect on A561E-CFTR, a CF mutation with a comparable mechanism of CFTR dysfunction as F508del-CFTR. To investigate the mechanism of action of CFFT-004, we used F508del-CFTR revertant mutations. Potentiation by CFFT-004 was unaffected by revertant mutations, but correction was abolished by the revertant mutation G550E. These data suggest that correction, but not potentiation, by CFFT-004 might involve nucleotide-binding domain 1 of CFTR.Conclusions and Implications: CFFT-004 is a dual-acting small molecule with independent corrector and potentiator activities that partially rescues F508del-CFTR in recombinant cells and native airway epithelia. The limited efficacy and potency of CFFT-004 suggests that combinations of small molecules targeting different defects in F508del-CFTR might be a more effective therapeutic strategy than a single agent. [ABSTRACT FROM AUTHOR]

Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by mutations that disrupt the plasma membrane expression, stability, and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Two small molecules, the CFTR corrector lumacaftor and the potentiator ivacaftor, are now used clinically to treat CF, although some studies suggest that they have counteracting effects on CFTR stability. Here, we investigated the impact of these compounds on the instability of F508del-CFTR, the most common CF mutation. To study individual CFTR Cl- channels, we performed single-channel recording, whereas to assess entire CFTR populations, we used purified CFTR proteins and macroscopicCFTRCl-currents. At 37 °C, low temperature-rescued F508del-CFTR more rapidly lost function in cell-free membrane patches and showed altered channel gating and current flow through open channels. Compared with purified wildtype CFTR, the full-length F508del-CFTR was about 10 °C less thermostable. Lumacaftor partially stabilized purified fulllength F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl- channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl- currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl- channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development. [ABSTRACT FROM AUTHOR]

The anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) transporter. CFTR plays a pivotal role in transepithelial ion transport as its dysfunction in the genetic disease cystic fibrosis (CF) dramatically demonstrates. Phylogenetic analysis suggests that CFTR first appeared in aquatic vertebrates fulfilling important roles in osmosensing and organ development. Here, we review selectively, knowledge of CFTR structure, function and pharmacology, gleaned from cross-species comparative studies of recombinant CFTR proteins, including CFTR chimeras. The data argue that subtle changes in CFTR structure can affect strongly channel function and the action of CF mutations. [ABSTRACT FROM AUTHOR]

Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF)., Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR., We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function., Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del., Abstract Cross-species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. [ABSTRACT FROM AUTHOR]

Background and Purpose Dysfunction of the cystic fibrosis transmembrane conductance regulator ( CFTR) Cl− channel causes the genetic disease cystic fibrosis ( CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561 E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561 E causes a temperature-sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease. Experimental Approach Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch-clamp techniques. Key Results Low temperature incubation delivered a small proportion of A561 E- CFTR protein to the cell surface. Like F508del- CFTR, low temperature-rescued A561 E- CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561 E- CFTR also exhibited thermoinstability, losing function more quickly than F508del- CFTR in cell-free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small-molecule ivacaftor, partially restored function to A561 E- CFTR. Interestingly, ivacaftor restored wild-type levels of channel activity (as measured by open probability) to single A561 E- and F508del- CFTR Cl− channels. However, it accentuated the thermoinstability of both mutants in cell-free membrane patches. Conclusions and Implications Like F508del- CFTR, A561 E- CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature-rescued A561 E- CFTR. Transformational drug therapy for A561 E- CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability. [ABSTRACT FROM AUTHOR]

Background and Purpose: Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel causes the genetic disease cystic fibrosis (CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561E causes a temperature-sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease.Experimental Approach: Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch-clamp techniques.Key Results: Low temperature incubation delivered a small proportion of A561E-CFTR protein to the cell surface. Like F508del-CFTR, low temperature-rescued A561E-CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561E-CFTR also exhibited thermoinstability, losing function more quickly than F508del-CFTR in cell-free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small-molecule ivacaftor, partially restored function to A561E-CFTR. Interestingly, ivacaftor restored wild-type levels of channel activity (as measured by open probability) to single A561E- and F508del-CFTR Cl(-) channels. However, it accentuated the thermoinstability of both mutants in cell-free membrane patches.Conclusions and Implications: Like F508del-CFTR, A561E-CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature-rescued A561E-CFTR. Transformational drug therapy for A561E-CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability. [ABSTRACT FROM AUTHOR]

Transmembrane anion carriers (anionophores) have potential in biological research and medicine, provided high activities can be obtained. There is particular interest in treating cystic fibrosis (CF), a genetic illness caused by deficient anion transport. Previous work has found that anionophore designs featuring axial ureas on steroid and trans-decalin scaffolds can be especially effective. Here we show that replacement of ureas by thioureas yields substantial further enhancements. Six new bis-thioureas have been prepared and tested for Cl-/NO3- exchange in 1-palmitoyl- 2-oleoylphosphatidylcholine/cholesterol large unilamellar vesicles (LUVs). The bis-thioureas are typically >10 times more effective than the corresponding ureas and are sufficiently active that transport by molecules acting singly in LUVs is readily detected. The highest activity is shown by decalin 9, which features N-(3,5-bis(trifluoromethyI)phenyl)thioureido and octyl ester substituents. A single molecule of transporter 9 in a 200 nm vesicle promotes Cl-/NO3- exchange with a half-life of 45 s and an absolute rate of 850 chloride anions per second Weight-for-weight, this carrier is only slightly less effective than CFTR, the natural anion channel associated with CF. [ABSTRACT FROM AUTHOR]