Two Small Molecules Restore Stability to a Sub-population of the Cystic Fibrosis Transmembrane conductance Regulator with the Predominant Disease-causing Mutation (original) (raw)
Related papers
2017
Edited by Norma Allewell 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 wildtype CFTR, the full-length F508del-CFTR was about 10°C less thermostable. Lumacaftor partially stabilized purified fulllength F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl ؊ channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl ؊ currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl ؊ channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development. Cystic fibrosis (CF) 8 is a common life-shortening inherited disease, mostly affecting people of European origin (1). The disease affects multiple organ systems throughout the body, especially the respiratory airways and intestine, leading to the blockage of ducts and tubes by thick tenacious mucus and a failure of host defense systems (1, 2). CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) (3), a unique ATP-binding cassette (ABC) transporter that functions as a Cl Ϫ channel with complex regulation (4). Located in the apical membrane of epithelia, CFTR plays a pivotal role in transepithelial ion transport, regulating the quantity and composition of epithelial secretions (5). To date, Ͼ2,000 mutations have been identified in the CFTR gene (see the Hospital for Sick Children in Toronto Cystic Fibrosis Mutation Database), although the vast majority are very rare and not all lead to CF (1). By far the most common disease-causing mutation, with a prevalence as high as 90%, is F508del, the deletion of the phenylalanine residue at position 508 of the CFTR amino acid sequence (1). F508del affects a residue located in a crucial position on the surface of the first nucleotide-binding domain (NBD1) (6). Structural studies reveal that the absence of Phe-508 causes local structural changes to the ABC ␣-subdomain of NBD1, mainly affecting the loop spanning residues 509-511 (7). However, loss of Phe-508 disrupts domain-domain interactions critical for correct assembly and function of CFTR (8-11). F508del-CFTR is rec
Journal of Pharmacology and Experimental Therapeutics, 2009
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl Ϫ channel. The mutations G551D and G1349D, which affect the nucleotidebinding domains (NBDs) of CFTR protein, reduce channel activity. This defect can be corrected pharmacologically by small molecules called potentiators. CF mutations residing in the intracellular loops (ICLs), connecting the transmembrane segments of CFTR, may also reduce channel activity. We have investigated the extent of loss of function caused by ICL mutations and the sensitivity to pharmacological stimulation. We found that E193K and G970R (in ICL1 and ICL3, respectively) cause a severe loss of CFTR channel activity that can be rescued by the same potentiators that are effective on NBD mutations. We compared potency and efficacy of three different potentiators for E193K, G970R, and G551D. The 1,4dihydropyridine felodipine and the phenylglycine PG-01 [2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2phenylacetamide] were strongly effective on the three CFTR mutants. The efficacy of sulfonamide SF-01 [6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide], another CFTR potentiator, was instead significantly lower than felodipine and PG-01 for the E193K and G970R mutations, and almost abolished for G551D. Furthermore, SF-01 modified the response of G551D and G970R to the other two potentiators, an effect that may be explained by an allosteric antagonistic effect. Our results indicate that CFTR potentiators correct the basic defect caused by CF mutations residing in different CFTR domains. However, there are differences among potentiators, with felodipine and PG-01 having a wider pharmacological activity, and SF-01 being more mutation specific. Our observations are useful in the prioritization and development of drugs targeting the CF basic defect.
Journal of Pharmacology and Experimental Therapeutics, 2014
The mutated protein F508del-cystic fibrosis transmembrane conductance regulator (CFTR) failed to traffic properly as a result of its retention in the endoplasmic reticulum and functions as a chloride (Cl 2) channel with abnormal gating and endocytosis. Small chemicals (called correctors) individually restore F508del-CFTR trafficking and Cl 2 transport function, but recent findings indicate that synergistic pharmacology should be considered to address CFTR defects more clearly. We studied the function and maturation of F508del-CFTR expressed in HeLa cells using a combination of five correctors [miglustat, IsoLAB (1,4-dideoxy-2-hydroxymethyl-1,4-imino-L-threitol), Corr4a (N-[2-(5-chloro-2methoxy-phenylamino)-49-methyl-[4,59]bithiazolyl-29-yl]-benzamide), VX-809 [3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid], and suberoylamilide hydroxamic acid (SAHA)]. Using the whole-cell patch-clamp technique, the current density recorded in response to CFTR activators (forskolin 1 genistein) was significantly increased in the presence of the following combinations: VX-809 1
Clinical pharmacology, 2016
Mutations of the CFTR gene cause cystic fibrosis (CF), the most common recessive monogenic disease worldwide. These mutations alter the synthesis, processing, function, or half-life of CFTR, the main chloride channel expressed in the apical membrane of epithelial cells in the airway, intestine, pancreas, and reproductive tract. Lung disease is the most critical manifestation of CF. It is characterized by airway obstruction, infection, and inflammation that lead to fatal tissue destruction. In spite of great advances in early and multidisciplinary medical care, and in our understanding of the pathophysiology, CF is still considerably reducing the life expectancy of patients. This review highlights the current development in pharmacological modulators of CFTR, which aim at rescuing the expression and/or function of mutated CFTR. While only Kalydeco® and Orkambi® are currently available to patients, many other families of CFTR modulators are undergoing preclinical and clinical investigations. Drug repositioning and personalized medicine are particularly detailed in this review as they represent the most promising strategies for restoring CFTR function in CF.
Proceedings of the …, 1998
CFTR is a cyclic AMP (cAMP)-activated chloride (Cl ؊) channel and a regulator of outwardly rectifying Cl ؊ channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl ؊ channel function and͞or ORCC regulator function, mutant CFTRs with N-and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36 Cl ؊ eff lux assays, and whole cell patch-clamp recordings were used to assay for the Cl ؊ channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted ␣-helices 5 and 6, forms an essential part of the Cl ؊ channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1͞R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl ؊ channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved. CFTR is a transmembrane protein involved in the regulation of several processes, including the activation of outwardly rectifying Cl Ϫ channels (1, 2) and the inhibition of Na ϩ channels by cAMP-dependent protein kinase A (PKA) (3-5). Mutations in CFTR cause cystic fibrosis (CF). Both channels lose this pattern of PKA sensitivity when CFTR is absent or its function is severely compromised in mutant forms. Other members of the ATP-binding cassette (ABC) transporter superfamily also regulate other processes. For example, the multidrug transporter, MDR, may regulate volume-activated chloride channels (6-9). The sulfonylurea receptor (SUR) binds sulfonylurea compounds such as glybenclamide and confers sulfonylurea inhibition upon a separate ATP-gated K ϩ channel protein in pancreatic  cells (10-15). More recent results suggest that CFTR can act as a SUR for ATP-gated K ϩ channels in kidney (16). We have shown previously that CFTR regulates outwardly rectifying Cl Ϫ channels (ORCCs) by an autocrine mechanism involving ATP release that is CFTR dependent. The ATP released binds to purinergic receptors to stimulate ORCCs (17, 18). The mechanism of how ATP is released, either through CFTR itself or by a separate mechanism, remains highly controversial (19-21). Two possibilities are that CFTR either transports ATP directly or activates an alternate ATP-release pathway. A key question in CF research is: How does CFTR allow protein kinase A to activate separate populations of ORCCs and inhibit a distinct family of Na ϩ-conductive channels? In this study, we tested the hypothesis that the complex, multidomain structure of CFTR supports its multifunctional behavior and that separate domains within the CFTR protein perform Cl Ϫ channel function independent of its regulatory functions. We show that the ability of CFTR to regulate ORCCs is not dependent upon CFTR's Cl Ϫ channel function and that conductance regulation is separate from CFTR's ability to conduct Cl Ϫ. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Activating Cystic Fibrosis Transmembrane Conductance Regulator Channels with Pore Blocker Analogs
Journal of Biological Chemistry, 2005
Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is ⌬F508-CFTR, which inefficiently traffics to the surfaces of most cells. The ⌬F508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and ⌬F508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment ⌬F508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and ⌬F508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of ⌬F508-CFTR channels in excised membrane patches by 10-15-fold under conditions in which wildtype channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the ⌬F508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface. Cystic fibrosis (CF) 1 is caused by inadequate CF transmembrane conductance regulator (CFTR) channel activity in the lung and intestines (1). CFTR channels are normally activated by MgATP binding to the nucleotide-binding domains (NBDs) and phosphorylation of the regulatory domain by protein kinase A (PKA) (2, 3). Many different mutations in these domains
The Journal of biological chemistry, 2015
In this study, we present data indicating a robust and specific domain interaction between the CFTR first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed binding kinetics were not altered by the common clinical mutation, F508del. Both intrinsic ATPase activity and CFTR channel gating were severely inhibited by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR disregulation has been implicated in pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease (COPD), acquired rhinosinusitis, pancreatitis and lethal secretory diarrhea (e.g. cholera). Based on clinical relevance of CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508...
Molecular biology of the cell, 2016
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 mutatio...