118 results on '"Sheppard DN"'
Search Results
2. Inhibition of protein kinase CK2 closes the CFTR Cl channel, but has no effect on the cystic fibrosis mutant Δf508-CFTR
- Author
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Chen, JH, Best, OG, Xu, Z, Treharne, KJ, Sheppard, DN, Kunzelmann, K, Mehta, A, Hegyi, P, Gruenert, DC, Gray, MA, and Cassidy, DM
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,animal structures ,ATP-binding cassette transporter ,Chloride ion channel ,Protein kinase CK2 ,respiratory system ,CFTR ,Channel regulation ,digestive system diseases ,Cystic fibrosis ,respiratory tract diseases - Abstract
Background: Deletion of phenylalanine-508 (ΔF508) from the first nucleotide-binding domain (NBD1) in the wild-type cystic fibrosis (CF) transmembrane-conductance regulator (wtCFTR) causes CF. However, the mechanistic relationship between ΔF508-CFTR and the diversity of CF disease is unexplained. The surface location of F508 on NBD1 creates the potential for protein-protein interactions and nearby, lies a consensus sequence (SYDE) reported to control the pleiotropic protein kinase CK2. Methods: Electrophysiology, immunofluorescence and biochemistry applied to CFTR-expressing cells, Xenopus oocytes, pancreatic ducts and patient biopsies. Results: Irrespective of PKA activation, CK2 inhibition (ducts, oocytes, cells) attenuates CFTR-dependent Cl- transport, closing wtCFTR in cell-attached membrane patches. CK2 and wtCFTR co-precipitate and CK2 co-localized with wtCFTR (but not ΔF508-CFTR) in apical membranes of human airway biopsies. Comparing wild-type and ΔF508CFTR expressing oocytes, only ΔF508-CFTR Cl- currents were insensitive to two CK2 inhibitors. Furthermore, wtCFTR was inhibited by injecting a peptide mimicking the F508 region, whereas the ΔF508-equivalent peptide had no effect. Conclusions: CK2 controls wtCFTR, but not ΔF508-CFTR. Others find that peptides from the F508 region of NBD1 allosterically control CK2, acting through F508. Hence, disruption of CK2-CFTR interaction by ΔF508-CFTR might disrupt multiple, membrane-associated, CK2-dependent pathways, creating a new molecular disease paradigm for deleted F508 in CFTR. Copyright © 2009 S. Karger AG, Basel., link_to_subscribed_fulltext
- Published
- 2009
3. Treatment of cystic fibrosis lung disease: The development of drugs targeting cystic fibrosis transmembrane conductance regulator dysfunction
- Author
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Sheppard, DN, Liu, Jia, Zhou, Z, and Mall, MA
- Subjects
epithelial Na + channel (ENaC) ,enac blockers ,CFTR correctors ,osmotic agents ,CFTR potentiators ,cystic fibrosis transmembrane conductance regulator (CFTR) ,respiratory system ,F508del ,airway surface liquid (ASL) ,respiratory tract diseases - Abstract
Chronic lung disease is the major cause of morbidity and mortality in the common, life-shortening genetic disease cystic fibrosis (CF). Since the identification of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), detailed understanding of how CF mutations cause chronic lung disease has emerged. In turn, this knowledge has stimulated the development of drug therapies that target the root cause of CF lung disease rather than the consequences of CFTR dysfunction. This article reviews recent progress towards the development of drug therapies for CF lung disease based on a rational understanding of how CF mutations cause CFTR dysfunction Refereed/Peer-reviewed
- Published
- 2011
4. In vitro platform to model the function of ionocytes in the human airway epithelium.
- Author
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Vilà-González M, Pinte L, Fradique R, Causa E, Kool H, Rodrat M, Morell CM, Al-Thani M, Porter L, Guo W, Maeshima R, Hart SL, McCaughan F, Granata A, Sheppard DN, Floto RA, Rawlins EL, Cicuta P, and Vallier L
- Subjects
- Humans, Cell Differentiation physiology, Cells, Cultured, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Epithelial Cells metabolism, Organoids metabolism, Induced Pluripotent Stem Cells metabolism, Respiratory Mucosa metabolism, Respiratory Mucosa cytology
- Abstract
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., (© 2024. The Author(s).)
- Published
- 2024
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5. Two rare variants that affect the same amino acid in CFTR have distinct responses to ivacaftor.
- Author
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Li H, Rodrat M, Al-Salmani MK, Veselu DF, Han ST, Raraigh KS, Cutting GR, and Sheppard DN
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- Cricetinae, Animals, Humans, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, CHO Cells, Cricetulus, Amino Acids, Ion Channel Gating, Aminophenols pharmacology, Adenosine Triphosphate metabolism, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Quinolones
- Abstract
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., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)- Published
- 2024
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6. CFTR Modulators: From Mechanism to Targeted Therapeutics.
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Yeh HI, Sutcliffe KJ, Sheppard DN, and Hwang TC
- Subjects
- Humans, Mutation, Signal Transduction, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics
- Abstract
People with cystic fibrosis (CF) suffer from a multi-organ disorder caused by loss-of-function variants in the gene encoding the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Tremendous progress has been made in both basic and clinical sciences over the past three decades since the identification of the CFTR gene. Over 90% of people with CF now have access to therapies targeting dysfunctional CFTR. This success was made possible by numerous studies in the field that incrementally paved the way for the development of small molecules known as CFTR modulators. The advent of CFTR modulators transformed this life-threatening illness into a treatable disease by directly binding to the CFTR protein and correcting defects induced by pathogenic variants. In this chapter, we trace the trajectory of structural and functional studies that brought CF therapies from bench to bedside, with an emphasis on mechanistic understanding of CFTR modulators., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)
- Published
- 2024
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7. Experimental pharmacology in precision medicine.
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Urbaniak A, Thummel KE, Alade AN, Rettie AE, Prasad B, De Nicolò A, Martin JH, Sheppard DN, and Jarvis MF
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- Drug Discovery, Precision Medicine, Pharmacogenetics
- Published
- 2023
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8. Alterations of mucosa-attached microbiome and epithelial cell numbers in the cystic fibrosis small intestine with implications for intestinal disease.
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Kelly J, Al-Rammahi M, Daly K, Flanagan PK, Urs A, Cohen MC, di Stefano G, Bijvelds MJC, Sheppard DN, de Jonge HR, Seidler UE, and Shirazi-Beechey SP
- Subjects
- Animals, Bacteria genetics, Cell Count, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Goblet Cells, Humans, Intestinal Mucosa microbiology, Intestine, Small microbiology, Mice, RNA, Ribosomal, 16S genetics, Cystic Fibrosis microbiology, Intestinal Diseases complications, Microbiota
- Abstract
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., (© 2022. The Author(s).)
- Published
- 2022
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9. A small molecule CFTR potentiator restores ATP-dependent channel gating to the cystic fibrosis mutant G551D-CFTR.
- Author
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Liu J, Berg AP, Wang Y, Jantarajit W, Sutcliffe KJ, Stevens EB, Cao L, Pregel MJ, and Sheppard DN
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- Adenosine Triphosphate, Aminophenols pharmacology, Cell Line, Cells, Cultured, Humans, Mutation, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator metabolism
- Abstract
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., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)- Published
- 2022
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10. CFTR bearing variant p.Phe312del exhibits function inconsistent with phenotype and negligible response to ivacaftor.
- Author
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Raraigh KS, Paul KC, Goralski JL, Worthington EN, Faino AV, Sciortino S, Wang Y, Aksit MA, Ling H, Osorio DL, Onchiri FM, Patel SU, Merlo CA, Montemayor K, Gibson RL, West NE, Thakerar A, Bridges RJ, Sheppard DN, Sharma N, and Cutting GR
- Subjects
- Aminophenols pharmacology, Aminophenols therapeutic use, Chlorides metabolism, Humans, Phenotype, Quinolones, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism
- Abstract
The chloride channel dysfunction caused by deleterious cystic fibrosis transmembrane conductance regulator (CFTR) variants generally correlates with severity of cystic fibrosis (CF). However, 3 adults bearing the common severe variant p.Phe508del (legacy: F508del) and a deletion variant in an ivacaftor binding region of CFTR (p.Phe312del; legacy: F312del) manifested only elevated sweat chloride concentration (sw[Cl-]; 87-105 mEq/L). A database review of 25 individuals with F312del and a CF-causing variant revealed elevated sw[Cl-] (75-123 mEq/L) and variable CF features. F312del occurs at a higher-than-expected frequency in the general population, confirming that individuals with F312del and a CF-causing variant do not consistently develop overt CF features. In primary nasal cells, CFTR bearing F312del and F508del generated substantial chloride transport (66.0% ± 4.5% of WT-CFTR) but did not respond to ivacaftor. Single-channel analysis demonstrated that F312del did not affect current flow through CFTR, minimally altered gating, and ablated the ivacaftor response. When expressed stably in CF bronchial epithelial (CFBE41o-) cells, F312del-CFTR demonstrated residual function (50.9% ± 3.3% WT-CFTR) and a subtle decrease in forskolin response compared with WT-CFTR. F312del provides an exception to the established correlation between CFTR chloride transport and CF phenotype and informs our molecular understanding of ivacaftor response.
- Published
- 2022
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11. Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains.
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Prins S, Corradi V, Sheppard DN, Tieleman DP, and Vergani P
- Subjects
- Humans, Protein Domains, Protein Structure, Secondary, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator, Mutation
- Abstract
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 second-site 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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12. Correlating genotype with phenotype using CFTR-mediated whole-cell Cl - currents in human nasal epithelial cells.
- Author
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Noel S, Servel N, Hatton A, Golec A, Rodrat M, Ng DRS, Li H, Pranke I, Hinzpeter A, Edelman A, Sheppard DN, and Sermet-Gaudelus I
- Subjects
- Chlorides metabolism, Epithelial Cells metabolism, Genotype, Humans, Nasal Mucosa metabolism, Nasal Mucosa pathology, Phenotype, Cystic Fibrosis genetics, Cystic Fibrosis pathology, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism
- Abstract
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., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)- Published
- 2022
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13. Extracellular phosphate enhances the function of F508del-CFTR rescued by CFTR correctors.
- Author
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Saint-Criq V, Wang Y, Delpiano L, Lin J, Sheppard DN, and Gray MA
- Subjects
- Aminophenols pharmacology, Animals, Benzodioxoles pharmacology, Cell Membrane metabolism, Cystic Fibrosis genetics, Drug Therapy, Combination, Humans, Indoles pharmacology, Ion Transport, Pyrazoles pharmacology, Pyridines pharmacology, Pyrrolidines pharmacology, Quinolones pharmacology, Rats, Rats, Inbred F344, Cystic Fibrosis drug therapy, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator drug effects, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Phosphates metabolism, Sodium-Phosphate Cotransporter Proteins, Type IIb metabolism
- Abstract
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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with the contents of this manuscript., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)- Published
- 2021
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14. A topological switch in CFTR modulates channel activity and sensitivity to unfolding.
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Scholl D, Sigoillot M, Overtus M, Martinez RC, Martens C, Wang Y, Pardon E, Laeremans T, Garcia-Pino A, Steyaert J, Sheppard DN, Hendrix J, and Govaerts C
- Subjects
- Cystic Fibrosis Transmembrane Conductance Regulator chemistry, Cystic Fibrosis Transmembrane Conductance Regulator isolation & purification, Fluorescence Resonance Energy Transfer, HEK293 Cells, Humans, Models, Molecular, Protein Conformation, Protein Unfolding, Cystic Fibrosis Transmembrane Conductance Regulator metabolism
- Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2021
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15. Carbon monoxide-releasing molecules inhibit the cystic fibrosis transmembrane conductance regulator Cl - channel.
- Author
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Rodrat M, Jantarajit W, Ng DRS, Harvey BSJ, Liu J, Wilkinson WJ, Charoenphandhu N, and Sheppard DN
- Subjects
- Adenosine Triphosphate metabolism, Animals, Carbon Monoxide metabolism, Humans, Carbon Monoxide pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Ion Channel Gating drug effects, Ion Transport drug effects
- Abstract
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 RuCl
3 (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.- Published
- 2020
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16. Parathyroid hormone increases CFTR expression and function in Caco-2 intestinal epithelial cells.
- Author
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Jantarajit W, Wongdee K, Lertsuwan K, Teerapornpuntakit J, Aeimlapa R, Thongbunchoo J, Harvey BSJ, Sheppard DN, and Charoenphandhu N
- Subjects
- Anions metabolism, Caco-2 Cells, Cystic Fibrosis Transmembrane Conductance Regulator analysis, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Humans, Intestinal Mucosa cytology, Ion Transport, Up-Regulation, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Intestinal Mucosa metabolism, Parathyroid Hormone metabolism
- Abstract
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 HCO3 - -dependent. However, the PTH-stimulated increase in Isc 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., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2020
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17. CFTR: New insights into structure and function and implications for modulation by small molecules.
- Author
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Kleizen B, Hunt JF, Callebaut I, Hwang TC, Sermet-Gaudelus I, Hafkemeyer S, and Sheppard DN
- Subjects
- Humans, Ion Channel Gating drug effects, Ion Channel Gating genetics, Mutation, Treatment Outcome, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis physiopathology, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Molecular Targeted Therapy methods
- Abstract
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., Competing Interests: Declaration of Competing Interest DNS is the recipient of a Vertex Innovation Award, IS-G is the recipient of two Vertex Innovation Awards and a member of the scientific boards of Eloxx, PTC Therapeutics and Vertex Therapeutics. T-CH has an ongoing service agreement with Abbvie and a sponsored research grant from Nanova., (Copyright © 2019. Published by Elsevier B.V.)
- Published
- 2020
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18. Towards next generation therapies for cystic fibrosis: Folding, function and pharmacology of CFTR.
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Bose SJ, Krainer G, Ng DRS, Schenkel M, Shishido H, Yoon JS, Haggie PM, Schlierf M, Sheppard DN, and Skach WR
- Subjects
- Animals, Humans, Molecular Medicine methods, Molecular Medicine trends, Mutation, Pharmacogenomic Testing, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Membrane Transport Modulators pharmacology, Molecular Targeted Therapy methods, Molecular Targeted Therapy trends
- Abstract
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., Competing Interests: Declaration of Competing Interest HS, JSY and WRS are employees of the Cystic Fibrosis Foundation. DNS is the recipient of a Vertex Innovation Award from Vertex Pharmaceuticals (Europe) Ltd. All the other authors have no conflicts of interest to declare., (Copyright © 2019. Published by Elsevier B.V.)
- Published
- 2020
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19. Suppressing 'nonsense' in cystic fibrosis.
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Hinzpeter A, Sermet-Gaudelus I, and Sheppard DN
- Subjects
- Aminophenols, Codon, Nonsense, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Humans, Cystic Fibrosis, Quinolones
- Published
- 2020
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20. Anion carriers as potential treatments for cystic fibrosis: transport in cystic fibrosis cells, and additivity to channel-targeting drugs.
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Li H, Valkenier H, Thorne AG, Dias CM, Cooper JA, Kieffer M, Busschaert N, Gale PA, Sheppard DN, and Davis AP
- Abstract
Defective anion transport is a hallmark of the genetic disease cystic fibrosis (CF). One approach to restore anion transport to CF cells utilises alternative pathways for transmembrane anion transport, including artificial anion carriers (anionophores). Here, we screened 22 anionophores for biological activity using fluorescence emission from the halide-sensitive yellow fluorescent protein. Three compounds possessed anion transport activity similar to or greater than that of a bis-( p -nitrophenyl)ureidodecalin previously shown to have promising biological activity. Anion transport by these anionophores was concentration-dependent and persistent. All four anionophores mediated anion transport in CF cells, and their activity was additive to rescue of the predominant disease-causing variant F508del-CFTR using the clinically-licensed drugs lumacaftor and ivacaftor. Toxicity was variable but minimal at the lower end. The results provide further evidence that anionophores, by themselves or together with other treatments that restore anion transport, offer a potential therapeutic strategy for CF., (This journal is © The Royal Society of Chemistry 2019.)
- Published
- 2019
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21. Differential thermostability and response to cystic fibrosis transmembrane conductance regulator potentiators of human and mouse F508del-CFTR.
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Bose SJ, Bijvelds MJC, Wang Y, Liu J, Cai Z, Bot AGM, de Jonge HR, and Sheppard DN
- Subjects
- Adenosine Triphosphate metabolism, Aminophenols pharmacology, Aminopyridines pharmacology, Animals, Benzodioxoles pharmacology, CHO Cells, Colforsin pharmacology, Cricetulus, Cyclic AMP-Dependent Protein Kinases metabolism, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Gene Expression, Genistein pharmacology, Ion Transport drug effects, Mice, NIH 3T3 Cells, Patch-Clamp Techniques, Protein Stability, Quinolones pharmacology, Species Specificity, Temperature, Transgenes, Base Sequence, Chloride Channel Agonists pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Sequence Deletion
- Abstract
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.- Published
- 2019
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22. Preferred Formation of Heteromeric Channels between Coexpressed SK1 and IKCa Channel Subunits Provides a Unique Pharmacological Profile of Ca 2+ -Activated Potassium Channels.
- Author
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Higham J, Sahu G, Wazen RM, Colarusso P, Gregorie A, Harvey BSJ, Goudswaard L, Varley G, Sheppard DN, Turner RW, and Marrion NV
- Subjects
- Cell Line, Fluorescence Resonance Energy Transfer, HEK293 Cells, Humans, Microscopy, Stochastic Processes, Intermediate-Conductance Calcium-Activated Potassium Channels metabolism, Multiprotein Complexes metabolism, Small-Conductance Calcium-Activated Potassium Channels metabolism
- Abstract
Three small conductance calcium-activated potassium channel (SK) subunits have been cloned and found to preferentially form heteromeric channels when expressed in a heterologous expression system. The original cloning of the gene encoding the intermediate conductance calcium-activated potassium channel (IKCa) was termed SK4 because of the high homology between channel subtypes. Recent immunovisualization suggests that IKCa is expressed in the same subcellular compartments of some neurons as SK channel subunits. Stochastic optical reconstruction microscopy super-resolution microscopy revealed that coexpressed IKCa and SK1 channel subunits were closely associated, a finding substantiated by measurement of fluorescence resonance energy transfer between coexpressed fluorophore-tagged subunits. Expression of homomeric SK1 channels produced current that displayed typical sensitivity to SK channel inhibitors, while expressed IKCa channel current was inhibited by known IKCa channel blockers. Expression of both SK1 and IKCa subunits gave a current that displayed no sensitivity to SK channel inhibitors and a decreased sensitivity to IKCa current inhibitors. Single channel recording indicated that coexpression of SK1 and IKCa subunits produced channels with properties intermediate between those observed for homomeric channels. These data indicate that SK1 and IKCa channel subunits preferentially combine to form heteromeric channels that display pharmacological and biophysical properties distinct from those seen with homomeric channels., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)
- Published
- 2019
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23. Pore-forming small molecules offer a promising way to tackle cystic fibrosis.
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Sheppard DN and Davis AP
- Subjects
- Cystic Fibrosis Transmembrane Conductance Regulator, Humans, Ion Channels, Cystic Fibrosis
- Published
- 2019
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24. Fluorinated synthetic anion carriers: experimental and computational insights into transmembrane chloride transport.
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Spooner MJ, Li H, Marques I, Costa PMR, Wu X, Howe ENW, Busschaert N, Moore SJ, Light ME, Sheppard DN, Félix V, and Gale PA
- Abstract
A series of fluorinated tripodal tris-thioureas function as highly active anion transporters across lipid bilayers and cell membranes. Here, we investigate their mechanism of action using anion transport assays in cells and synthetic vesicles and molecular modelling of transporter-lipid interactions. When compared with non-fluorinated analogues, fluorinated compounds demonstrate a different mechanism of membrane transport because the free transporter cannot effectively diffuse through the membrane. As a result, in H
+ /Cl- cotransport assays, fluorinated transporters require the presence of oleic acid to form anionic oleate complexes for recycling of the transporter, whereas non-fluorinated analogues readily diffuse through the membrane as free transporters and show synergistic transport with the proton transporter gramicidin. Molecular dynamics simulations revealed markedly stronger transporter-lipid interactions for fluorinated compounds compared with non-fluorinated analogues and hence, higher energy barriers for fluorinated compounds to cross the membrane as free transporters. With use of appropriate proton transporters to ensure measurement of the correct rate-limiting steps, the transport rates determined in synthetic vesicle assays show excellent agreement with the anion transport rates determined in cell-based assays. We conclude that integration of computational and experimental methods provides a strategy to optimise transmembrane anion transporter design for biomedical applications.- Published
- 2018
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25. Potentiation of the cystic fibrosis transmembrane conductance regulator Cl - channel by ivacaftor is temperature independent.
- Author
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Wang Y, Cai Z, Gosling M, and Sheppard DN
- Subjects
- Animals, Benzodioxoles pharmacology, Cell Line, Cricetinae, Cystic Fibrosis metabolism, Humans, Ion Transport drug effects, Mice, Mutation drug effects, Temperature, Aminophenols pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Ion Channel Gating drug effects, Mice, Inbred CFTR metabolism, Quinolones pharmacology
- Abstract
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 UC
CF -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.- Published
- 2018
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26. N1303K: Leaving no stone unturned in the search for transformational therapeutics.
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Noel S, Sermet-Gaudelus I, and Sheppard DN
- Subjects
- Humans, Cystic Fibrosis, Cystic Fibrosis Transmembrane Conductance Regulator
- Published
- 2018
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27. Residual function of cystic fibrosis mutants predicts response to small molecule CFTR modulators.
- Author
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Han ST, Rab A, Pellicore MJ, Davis EF, McCague AF, Evans TA, Joynt AT, Lu Z, Cai Z, Raraigh KS, Hong JS, Sheppard DN, Sorscher EJ, and Cutting GR
- Subjects
- Aminophenols therapeutic use, Aminopyridines therapeutic use, Benzodioxoles therapeutic use, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Drug Combinations, Drug Therapy, Combination, HEK293 Cells, Humans, Mutation, Quinolones therapeutic use, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator drug effects, Cystic Fibrosis Transmembrane Conductance Regulator metabolism
- Abstract
Treatment of individuals with cystic fibrosis (CF) has been transformed by small molecule therapies that target select pathogenic variants in the CF transmembrane conductance regulator (CFTR). To expand treatment eligibility, we stably expressed 43 rare missense CFTR variants associated with moderate CF from a single site in the genome of human CF bronchial epithelial (CFBE41o-) cells. The magnitude of drug response was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants expressed stably in Fischer rat thyroid (FRT) cells showed nearly identical correlations. Subsets of variants were identified that demonstrated statistically significantly higher responses to specific treatments. Furthermore, nearly all variants studied in CFBE cells (40 of 43) and FRT cells (13 of 16) demonstrated greater response to ivacaftor-lumacaftor combination therapy than either modulator alone. Together, these variants represent 87% of individuals in the CFTR2 database with at least 1 missense variant. Thus, our results indicate that most individuals with CF carrying missense variants are (a) likely to respond modestly to currently available modulator therapy, while a small fraction will have pronounced responses, and (b) likely to derive the greatest benefit from combination therapy.
- Published
- 2018
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28. Partial rescue of F508del-cystic fibrosis transmembrane conductance regulator channel gating with modest improvement of protein processing, but not stability, by a dual-acting small molecule.
- Author
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Liu J, Bihler H, Farinha CM, Awatade NT, Romão AM, Mercadante D, Cheng Y, Musisi I, Jantarajit W, Wang Y, Cai Z, Amaral MD, Mense M, and Sheppard DN
- Subjects
- Animals, Cell Line, Cell Membrane physiology, Cells, Cultured, Cricetinae, Epithelial Cells physiology, Humans, Ion Channel Gating, Mice, Protein Processing, Post-Translational, Protein Stability, Cystic Fibrosis Transmembrane Conductance Regulator physiology
- Abstract
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., (© 2018 The British Pharmacological Society.)
- Published
- 2018
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29. Therapeutic approaches to CFTR dysfunction: From discovery to drug development.
- Author
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Li H, Pesce E, Sheppard DN, Singh AK, and Pedemonte N
- Subjects
- Humans, Ion Transport drug effects, Ion Transport physiology, Mutation, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Drug Development
- Abstract
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., (Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)- Published
- 2018
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30. Anion transport by ortho-phenylene bis-ureas across cell and vesicle membranes.
- Author
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Dias CM, Li H, Valkenier H, Karagiannidis LE, Gale PA, Sheppard DN, and Davis AP
- Subjects
- Anions metabolism, Bacterial Proteins, Cell Membrane metabolism, Halogens pharmacology, Intracellular Membranes metabolism, Luminescent Proteins, Urea chemistry, Urea metabolism, Ion Transport, Phenylenediamines chemistry, Urea analogs & derivatives
- Abstract
Ortho-Phenylene bis-ureas serve as anionophores in cells expressing halide-sensitive yellow fluorescent protein, as well as in synthetic vesicles. Activities can reach high levels, and are strongly dependent on the deliverability of the transporters.
- Published
- 2018
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31. Editorial overview: Respiratory: Transformational therapies for cystic fibrosis.
- Author
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Sheppard DN, Bear CE, and de Jonge HR
- Subjects
- Animals, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Humans, Mutation genetics, Cystic Fibrosis therapy
- Published
- 2017
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32. Bypassing CFTR dysfunction in cystic fibrosis with alternative pathways for anion transport.
- Author
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Li H, Salomon JJ, Sheppard DN, Mall MA, and Galietta LJ
- Subjects
- Animals, Anoctamin-1 metabolism, Antiporters metabolism, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Humans, Ion Transport, Respiratory Mucosa metabolism, Sulfate Transporters, Cystic Fibrosis metabolism
- Abstract
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 Ca2+ -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., (Copyright © 2017. Published by Elsevier Ltd.)- Published
- 2017
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33. Alteration of protein function by a silent polymorphism linked to tRNA abundance.
- Author
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Kirchner S, Cai Z, Rauscher R, Kastelic N, Anding M, Czech A, Kleizen B, Ostedgaard LS, Braakman I, Sheppard DN, and Ignatova Z
- Subjects
- Cystic Fibrosis Transmembrane Conductance Regulator metabolism, HEK293 Cells, HeLa Cells, Humans, Polymorphism, Single Nucleotide, Protein Stability, Structure-Activity Relationship, Cystic Fibrosis Transmembrane Conductance Regulator genetics, RNA, Transfer metabolism, Silent Mutation
- Abstract
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.
- Published
- 2017
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34. Two Small Molecules Restore Stability to a Subpopulation of the Cystic Fibrosis Transmembrane Conductance Regulator with the Predominant Disease-causing Mutation.
- Author
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Meng X, Wang Y, Wang X, Wrennall JA, Rimington TL, Li H, Cai Z, Ford RC, and Sheppard DN
- Subjects
- Animals, Cell Line, Cell Membrane metabolism, Cell-Free System, Chromatography, Cricetinae, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Hot Temperature, Humans, Mutation, Patch-Clamp Techniques, Protein Denaturation, Aminophenols pharmacology, Aminopyridines pharmacology, Benzodioxoles pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Quinolones pharmacology
- Abstract
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 macroscopic CFTR Cl- 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 wild-type CFTR, the full-length F508del-CFTR was about 10 °C less thermostable. Lumacaftor partially stabilized purified full-length 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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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35. Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl - channel.
- Author
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Chen JH, Xu W, and Sheppard DN
- Subjects
- 3T3 Cells, Action Potentials, Adenosine Triphosphate metabolism, Animals, Binding Sites, Cystic Fibrosis Transmembrane Conductance Regulator chemistry, Extracellular Space metabolism, Humans, Hydrogen-Ion Concentration, Mice, Protein Binding, Rats, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Ion Channel Gating
- Abstract
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., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)- Published
- 2017
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36. Targeted anion transporter delivery by coiled-coil driven membrane fusion.
- Author
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Mora NL, Bahreman A, Valkenier H, Li H, Sharp TH, Sheppard DN, Davis AP, and Kros A
- Abstract
Synthetic anion transporters (anionophores) have potential as biomedical research tools and therapeutics. However, the efficient and specific delivery of these highly lipophilic molecules to a target cell membrane is non-trivial. Here, we investigate the delivery of a powerful anionophore to artificial and cell membranes using a coiled-coil-based delivery system inspired by SNARE membrane fusion proteins. Incorporation of complementary lipopeptides into the lipid membranes of liposomes and cell-sized giant unilamellar vesicles (GUVs) facilitated the delivery of a powerful anionophore into GUVs, where its anion transport activity was monitored in real time by fluorescence microscopy. Similar results were achieved using live cells engineered to express a halide-sensitive fluorophore. We conclude that coiled-coil driven membrane fusion is a highly efficient system to deliver anionophores to target cell membranes.
- Published
- 2016
- Full Text
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37. From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations.
- Author
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Veit G, Avramescu RG, Chiang AN, Houck SA, Cai Z, Peters KW, Hong JS, Pollard HB, Guggino WB, Balch WE, Skach WR, Cutting GR, Frizzell RA, Sheppard DN, Cyr DM, Sorscher EJ, Brodsky JL, and Lukacs GL
- Subjects
- Animals, Chloride Channel Agonists pharmacology, Chloride Channel Agonists therapeutic use, Cystic Fibrosis classification, Cystic Fibrosis drug therapy, Cystic Fibrosis Transmembrane Conductance Regulator agonists, Genetic Predisposition to Disease, Humans, Ion Channel Gating, Mutation, Missense, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics
- Abstract
More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients., (© 2016 Veit et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)
- Published
- 2016
- Full Text
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38. Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia.
- Author
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Li H, Valkenier H, Judd LW, Brotherhood PR, Hussain S, Cooper JA, Jurček O, Sparkes HA, Sheppard DN, and Davis AP
- Subjects
- Animals, Anions metabolism, Bacterial Proteins genetics, Cell Membrane drug effects, Cell Membrane metabolism, Cell Proliferation drug effects, Cell Survival drug effects, Chlorine metabolism, Cyclohexanes pharmacokinetics, Cyclohexanes toxicity, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Dogs, Drug Carriers chemistry, Drug Design, Electrophysiological Phenomena, Epithelial Cells drug effects, HeLa Cells, Humans, Hydrogen Bonding, Ion Transport, Luminescent Proteins genetics, Madin Darby Canine Kidney Cells, Microscopy, Fluorescence, Molecular Structure, Naphthalenes pharmacokinetics, Naphthalenes toxicity, Phosphatidylcholines chemistry, Rats, Inbred F344, Steroids pharmacokinetics, Steroids toxicity, Cyclohexanes chemistry, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Epithelial Cells metabolism, Naphthalenes chemistry, Steroids chemistry
- Abstract
Transmembrane anion transporters (anionophores) have potential for new modes of biological activity, including therapeutic applications. In particular they might replace the activity of defective anion channels in conditions such as cystic fibrosis. However, data on the biological effects of anionophores are scarce, and it remains uncertain whether such molecules are fundamentally toxic. Here, we report a biological study of an extensive series of powerful anion carriers. Fifteen anionophores were assayed in single cells by monitoring anion transport in real time through fluorescence emission from halide-sensitive yellow fluorescent protein. A bis-(p-nitrophenyl)ureidodecalin shows especially promising activity, including deliverability, potency and persistence. Electrophysiological tests show strong effects in epithelia, close to those of natural anion channels. Toxicity assays yield negative results in three cell lines, suggesting that promotion of anion transport may not be deleterious to cells. We therefore conclude that synthetic anion carriers are realistic candidates for further investigation as treatments for cystic fibrosis.
- Published
- 2016
- Full Text
- View/download PDF
39. Exploiting species differences to understand the CFTR Cl- channel.
- Author
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Bose SJ, Scott-Ward TS, Cai Z, and Sheppard DN
- Subjects
- Adenosine Monophosphate metabolism, Animals, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator classification, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Humans, Ion Channel Gating genetics, Mutation, Phylogeny, Species Specificity, Adenosine Triphosphate metabolism, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Ion Channel Gating physiology
- Abstract
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., (© 2015 Authors; published by Portland Press Limited.)
- Published
- 2015
- Full Text
- View/download PDF
40. Impact of the F508del mutation on ovine CFTR, a Cl- channel with enhanced conductance and ATP-dependent gating.
- Author
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Cai Z, Palmai-Pallag T, Khuituan P, Mutolo MJ, Boinot C, Liu B, Scott-Ward TS, Callebaut I, Harris A, and Sheppard DN
- Subjects
- Adenosine Triphosphate physiology, Animals, CHO Cells, Cricetulus, HEK293 Cells, Humans, Ion Channel Gating, Models, Molecular, Mutation, Sheep, Cystic Fibrosis Transmembrane Conductance Regulator chemistry, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator physiology
- Abstract
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., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)
- Published
- 2015
- Full Text
- View/download PDF
41. Chronic ivacaftor treatment: getting F508del-CFTR into more trouble?
- Author
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Mall MA and Sheppard DN
- Subjects
- Humans, Aminophenols pharmacology, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator drug effects, Quinolones pharmacology
- Published
- 2014
- Full Text
- View/download PDF
42. CFTR potentiators partially restore channel function to A561E-CFTR, a cystic fibrosis mutant with a similar mechanism of dysfunction as F508del-CFTR.
- Author
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Wang Y, Liu J, Loizidou A, Bugeja LA, Warner R, Hawley BR, Cai Z, Toye AM, Sheppard DN, and Li H
- Subjects
- Aminophenols pharmacology, Animals, Biotinylation, Cell Line, Cricetinae, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Genistein pharmacology, Iodides metabolism, Ion Channel Gating drug effects, Mutation, Quinolones pharmacology, Temperature, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator physiology
- Abstract
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., (© 2014 The British Pharmacological Society.)
- Published
- 2014
- Full Text
- View/download PDF
43. Preorganized bis-thioureas as powerful anion carriers: chloride transport by single molecules in large unilamellar vesicles.
- Author
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Valkenier H, Judd LW, Li H, Hussain S, Sheppard DN, and Davis AP
- Subjects
- Anions chemistry, Chlorides administration & dosage, Chlorides chemistry, Humans, Ion Transport, Models, Molecular, Nitrates administration & dosage, Nitrates chemistry, Anions administration & dosage, Cholesterol chemistry, Phosphatidylcholines chemistry, Thiourea chemistry, Unilamellar Liposomes chemistry
- Abstract
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(trifluoromethyl)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.
- Published
- 2014
- Full Text
- View/download PDF
44. Understanding how cystic fibrosis mutations disrupt CFTR function: from single molecules to animal models.
- Author
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Wang Y, Wrennall JA, Cai Z, Li H, and Sheppard DN
- Subjects
- Animals, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Humans, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Disease Models, Animal
- Abstract
Defective epithelial ion transport is the hallmark of the life-limiting genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the ATP-binding cassette transporter that functions as a ligand-gated anion channel. Since the identification of the CFTR gene, almost 2000 disease-causing mutations associated with a spectrum of clinical phenotypes have been reported, but the majority remain poorly characterised. Studies of a small number of mutations including the most common, F508del-CFTR, have identified six general mechanisms of CFTR dysfunction. Here, we review selectively progress to understand how CF mutations disrupt CFTR processing, stability and function. We explore CFTR structure and function to explain the molecular mechanisms of CFTR dysfunction and highlight new knowledge of disease pathophysiology emerging from large animal models of CF. Understanding CFTR dysfunction is crucial to the development of transformational therapies for CF patients., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
45. Revertant mutants modify, but do not rescue, the gating defect of the cystic fibrosis mutant G551D-CFTR.
- Author
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Xu Z, Pissarra LS, Farinha CM, Liu J, Cai Z, Thibodeau PH, Amaral MD, and Sheppard DN
- Subjects
- Animals, Cattle, Cell Line, Cricetinae, Protein Structure, Secondary, Protein Structure, Tertiary, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator chemistry, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Ion Channel Gating genetics, Mutation genetics
- Abstract
Cystic fibrosis (CF) is caused by dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). One strategy to restore function to CF mutants is to suppress defects in CFTR processing and function using revertant mutations. Here, we investigate the effects of the revertant mutations G550E and 4RK (the simultaneous disruption of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K and R766K) on the CF mutant G551D, which impairs severely channel gating without altering protein processing and which affects a residue in the same α-helix as G550 and R555. Both G550E and 4RK augmented strongly CFTR-mediated iodide efflux from BHK cells expressing G551D-CFTR. To learn how revertant mutations influence G551D-CFTR function, we studied protein processing and single-channel behaviour. Neither G550E nor 4RK altered the expression and maturation of G551D-CFTR protein. By contrast, both revertants had marked effects on G551D-CFTR channel gating, increasing strongly opening frequency, while 4RK also diminished noticeably the duration of channel openings. Because G551D-CFTR channel gating is ATP independent, we investigated whether revertant mutations restore ATP dependence to G551D-CFTR. Like wild-type CFTR, the activity of 4RK-G551D-CFTR varied with ATP concentration, suggesting that 4RK confers some ATP dependence on the G551D-CFTR channel. Thus, the revertant mutations G550E and 4RK alter the gating pattern and ATP dependence of G551D-CFTR without restoring single-channel activity to wild-type levels. Based on their impact on the CF mutants F508del and G551D, we conclude that G550E and 4RK have direct effects on CFTR structure, but that their action on CFTR processing and channel function is CF mutation specific.
- Published
- 2014
- Full Text
- View/download PDF
46. Loop diuretics are open-channel blockers of the cystic fibrosis transmembrane conductance regulator with distinct kinetics.
- Author
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Ju M, Scott-Ward TS, Liu J, Khuituan P, Li H, Cai Z, Husbands SM, and Sheppard DN
- Subjects
- Animals, Bumetanide pharmacology, Cricetinae, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Dose-Response Relationship, Drug, Furosemide pharmacology, Humans, Kinetics, Membrane Potentials, Mice, Molecular Structure, Rats, Sodium Potassium Chloride Symporter Inhibitors chemistry, Structure-Activity Relationship, Sulfonamides pharmacology, Xipamide pharmacology, Chlorides metabolism, Cystic Fibrosis Transmembrane Conductance Regulator antagonists & inhibitors, Sodium Potassium Chloride Symporter Inhibitors pharmacology
- Abstract
Background and Purpose: Loop diuretics are widely used to inhibit the Na(+), K(+), 2Cl(-) co-transporter, but they also inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Here, we investigated the mechanism of CFTR inhibition by loop diuretics and explored the effects of chemical structure on channel blockade., Experimental Approach: Using the patch-clamp technique, we tested the effects of bumetanide, furosemide, piretanide and xipamide on recombinant wild-type human CFTR., Key Results: When added to the intracellular solution, loop diuretics inhibited CFTR Cl(-) currents with potency approaching that of glibenclamide, a widely used CFTR blocker with some structural similarity to loop diuretics. To begin to study the kinetics of channel blockade, we examined the time dependence of macroscopic current inhibition following a hyperpolarizing voltage step. Like glibenclamide, piretanide blockade of CFTR was time and voltage dependent. By contrast, furosemide blockade was voltage dependent, but time independent. Consistent with these data, furosemide blocked individual CFTR Cl(-) channels with 'very fast' speed and drug-induced blocking events overlapped brief channel closures, whereas piretanide inhibited individual channels with 'intermediate' speed and drug-induced blocking events were distinct from channel closures., Conclusions and Implications: Structure-activity analysis of the loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that loop diuretics are open-channel blockers of CFTR with distinct kinetics, affected by molecular dimensions and lipophilicity., (© 2013 The British Pharmacological Society.)
- Published
- 2014
- Full Text
- View/download PDF
47. Acute inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by thyroid hormones involves multiple mechanisms.
- Author
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Cai Z, Li H, Chen JH, and Sheppard DN
- Subjects
- Adenosine Triphosphate chemistry, Adenosine Triphosphate metabolism, Animals, Cell Line, Cricetinae, Genistein pharmacology, Glyburide pharmacology, Humans, Ion Channel Gating drug effects, Membrane Potentials drug effects, Mice, Patch-Clamp Techniques, Cystic Fibrosis Transmembrane Conductance Regulator antagonists & inhibitors, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Thyroxine metabolism, Triiodothyronine metabolism
- Abstract
The chemical structures of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) resemble those of small-molecules that inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We therefore tested the acute effects of T3, T4 and reverse T3 (rT3) on recombinant wild-type human CFTR using the patch-clamp technique. When added directly to the intracellular solution bathing excised membrane patches, T3, T4, and rT3 (all tested at 50 μM) inhibited CFTR in several ways: they strongly reduced CFTR open probability by impeding channel opening; they moderately decreased single-channel current amplitude, and they promoted transitions to subconductance states. To investigate the mechanism of CFTR inhibition, we studied T3. T3 (50 μM) had multiple effects on CFTR gating kinetics, suggestive of both allosteric inhibition and open-channel blockade. Channel inhibition by T3 was weakly voltage dependent and stronger than the allosteric inhibitor genistein, but weaker than the open-channel blocker glibenclamide. Raising the intracellular ATP concentration abrogated T3 inhibition of CFTR gating, but not the reduction in single-channel current amplitude nor the transitions to subconductance states. The decrease in single-channel current amplitude was relieved by membrane depolarization, but not the transitions to subconductance states. We conclude that T3 has complex effects on CFTR consistent with both allosteric inhibition and open-channel blockade. Our results suggest that there are multiple allosteric mechanisms of CFTR inhibition, including interference with ATP-dependent channel gating and obstruction of conformational changes that gate the CFTR pore. CFTR inhibition by thyroid hormones has implications for the development of innovative small-molecule CFTR inhibitors.
- Published
- 2013
- Full Text
- View/download PDF
48. Impact of the cystic fibrosis mutation F508del-CFTR on renal cyst formation and growth.
- Author
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Li H, Yang W, Mendes F, Amaral MD, and Sheppard DN
- Subjects
- Animals, Cell Proliferation, Cells, Cultured, Cystic Fibrosis metabolism, Cystic Fibrosis pathology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Dogs, Kidney metabolism, Kidney pathology, Kidney Diseases, Cystic metabolism, Kidney Diseases, Cystic pathology, Mutation, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Epithelial Cells metabolism, Kidney Diseases, Cystic genetics
- Abstract
In autosomal dominant polycystic kidney disease (ADPKD), cystic fibrosis transmembrane conductance regulator (CFTR), the protein product of the gene defective in cystic fibrosis (CF), plays a crucial role in fluid accumulation, which promotes cyst swelling. Several studies have identified individuals afflicted by both ADPKD and CF. Two studies suggested that CF mutations might attenuate the severity of ADPKD, whereas a third found no evidence of a protective effect. In this study, we investigated the impact of the commonest CF mutation F508del-CFTR on the formation and growth of renal cysts. As a model system, we used Madin-Darby canine kidney (MDCK) epithelial cells engineered to express wild-type and F508del human CFTR. We grew MDCK cysts in collagen gels in the presence of the cAMP agonist forskolin and measured transepithelial resistance and Cl(-) secretion with the Ussing chamber technique and assayed cell proliferation using nonpolarized MDCK cells. When compared with untransfected MDCK cells, cells expressing wild-type CFTR generated substantial numbers of large cysts, which grew markedly over time. By contrast, MDCK cells expressing F508del-CFTR formed very few tiny cysts that failed to enlarge. Interestingly, treatment of F508del-CFTR cysts with the CFTR corrector VRT-325 and the CFTR corrector-potentiator VRT-532 increased the number, but not size, of F508del-CFTR cysts, possibly because VRT-325 inhibited strongly cell proliferation. Based on its effects on transepithelial resistance, Cl(-) secretion, and cell proliferation, we conclude that the F508del-CFTR mutation disrupts cyst formation and growth by perturbing strongly fluid accumulation within the cyst lumen without compromising epithelial integrity.
- Published
- 2012
- Full Text
- View/download PDF
49. The small airways accordion: concurrent or alternating fluid absorption and secretion?
- Author
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de Jonge HR and Sheppard DN
- Subjects
- Animals, Female, Male, Body Fluids physiology, Lung physiology, Respiratory Mucosa physiology
- Published
- 2012
- Full Text
- View/download PDF
50. CFTR channel pharmacology: insight from a flock of clones. Focus on "Divergent CFTR orthologs respond differently to the channel inhibitors CFTRinh-172, glibenclamide, and GlyH-101".
- Author
-
Sheppard DN
- Subjects
- Animals, Female, Glycine pharmacology, Humans, Male, Benzoates pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator antagonists & inhibitors, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Glyburide pharmacology, Glycine analogs & derivatives, Hydrazines pharmacology, Thiazolidines pharmacology
- Published
- 2012
- Full Text
- View/download PDF
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