Epithelial Cell Chloride Channel Activity Correlates with Improved Airway Function in Cystic Fibrosis Patients with the Major Mutant: Delta F508

2002 ◽  
Vol 52 (5) ◽  
pp. 625-627
Author(s):  
J. F. KIDD
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cell ◽  
Chloride Channel ◽  
Channel Activity ◽  
Airway Function

The cystic fibrosis mutation (ΔF508) does not influence the chloride channel activity of CFTR

1993 ◽  
Vol 3 (4) ◽  
pp. 311-316 ◽  
Author(s):  
Canhui Li ◽  
Mohabir Ramjeesingh ◽  
Evangelica Reyes ◽  
Tim Jensen ◽  
Xiubao Chang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Channel Activity ◽  
Cystic Fibrosis Mutation

scholarly journals Ablation of Internalization Signals in the Carboxyl-terminal Tail of the Cystic Fibrosis Transmembrane Conductance Regulator Enhances Cell Surface Expression

2002 ◽  
Vol 277 (51) ◽  
pp. 49952-49957 ◽  
Author(s):  
Krisztina Peter ◽  
Karoly Varga ◽  
Zsuzsa Bebok ◽  
Carmel M. McNicholas-Bevensee ◽  
Lisa Schwiebert ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Surface Expression ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Transmembrane Conductance ◽  
Coated Pits ◽  
Double Mutation ◽  
Carboxyl Terminal ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., Marchase, R. B., and Collawn, J. F. (1999)J. Biol. Chem.274, 3602–3609). Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein half-life. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr1424-X-X-Ile1427where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function.

Chloride channels of intracellular organelles and their potential role in cystic fibrosis.

1992 ◽  
Vol 172 (1) ◽  
pp. 245-266 ◽  
Author(s):  
Q al-Awqati ◽  
J Barasch ◽  
D Landry
Keyword(s):  
Cystic Fibrosis ◽  
Fusion Protein ◽  
Cdna Library ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Kidney Cortex ◽  
Affinity Column ◽  
Channel Activity ◽  
Planar Bilayers ◽  
Intracellular Organelles

Chloride channels were previously purified from bovine kidney cortex membranes using a drug affinity column. Reconstitution of the purified proteins into artificial liposomes and planar bilayers yielded chloride channels. A 64 x 10(3) M(r) protein, p64, identified as a component of this chloride channel, was used to generate antibodies which depleted solubilized kidney membranes of all chloride channel activity. This antibody has now been used to identify a clone, H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B, also depleted solubilized kidney cortex from all chloride channel activity. The predicted amino acid sequence of p64 shows that it contains two and possibly four putative transmembrane domains and potential phosphorylation sites by protein kinases A and C. There was no significant homology to other protein (or DNA) sequences in the data base including other anion channels or the cystic fibrosis transmembrane conductance regulator. The protein is expressed in all cells tested and probably represents the chloride channel of intracellular organelles. Cystic fibrosis (CF) is associated with a defect in a cyclic-AMP-activated chloride channel in secretory epithelia which leads to decreased fluid secretion. In addition, many mucus glycoproteins show decreased sialylation but increased sulfation. We have recently shown that the pH of intracellular organelles is more alkaline in CF cells, an abnormality that is due to defective chloride conductance in the vesicle membranes. We postulate that the defect in the intracellular chloride channel, and hence the alkalization, could explain the glycosylation abnormalities since the pH optimum of Golgi sialyltransferase is acid while that of focusyl- and sulfotransferases is alkaline. Defects in sialyation of glycolipids might also generate receptors for Pseudomonas, which is known to colonize the respiratory tract of CF patients.

scholarly journals Identifying Inhibitors of the Hsp90-Aha1 Protein Complex, a Potential Target to Drug Cystic Fibrosis, by Alpha Technology

2017 ◽  
Vol 22 (7) ◽  
pp. 923-928 ◽  
Author(s):  
Verena Ihrig ◽  
Wolfgang M. J. Obermann
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Chloride Ion ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Transmembrane Conductance ◽  
Culture Cells ◽  
Homogeneous Assay ◽  
Chaperone Complex ◽  
Cystic Fibrosis Transmembrane

Deletion of a single phenylalanine residue at position 508 of the protein CFTR (cystic fibrosis transmembrane conductance regulator), a chloride channel in lung epithelium, is the most common cause for cystic fibrosis. As a consequence, folding of the CFTRΔF508 protein and delivery to the cell surface are compromised, resulting in degradation of the polypeptide. Accordingly, decreased surface presence of CFTRΔF508 causes impaired chloride ion conductivity and is associated with mucus accumulation, a hallmark of cystic fibrosis. Molecular chaperones such as Hsp90 and its co-chaperone partner Aha1 are thought to play a key role in targeting folding-deficient CFTRΔF508 for degradation. Thus, pharmacologic manipulation to inhibit Hsp90-Aha1 chaperone complex formation appears beneficial to inhibit proteolysis of CFTRΔF508 and rescue its residual chloride channel activity. Therefore, we have screened a collection of 14,400 druglike chemical compounds for inhibitors of the Hsp90-Aha1 complex by amplified luminescence proximity homogeneous assay (Alpha). We identified two druglike molecules that showed promising results when we tested their ability to restore chloride channel activity in culture cells expressing the mutant CFTRΔF508 protein. The two molecules were most effective in combination with the corrector VX-809 and may therefore serve as a lead compound that can be further developed into a drug to treat cystic fibrosis patients.

Interaction between 2 extracellular loops influences the activity of the cystic fibrosis transmembrane conductance regulator chloride channel

2014 ◽  
Vol 92 (5) ◽  
pp. 390-396 ◽  
Author(s):  
Steven D. Broadbent ◽  
Wuyang Wang ◽  
Paul Linsdell
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Transmembrane Conductance ◽  
Channel Function ◽  
Extracellular Loops ◽  
Key Sites ◽  
Cystic Fibrosis Transmembrane

Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is thought to be controlled by cytoplasmic factors. However, recent evidence has shown that overall channel activity is also influenced by extracellular anions that interact directly with the extracellular loops (ECLs) of the CFTR protein. Very little is known about the structure of the ECLs or how substances interacting with these ECLs might affect CFTR function. We used patch-clamp recording to investigate the accessibility of cysteine-reactive reagents to cysteines introduced throughout ECL1 and 2 key sites in ECL4. Furthermore, interactions between ECL1 and ECL4 were investigated by the formation of disulfide crosslinks between cysteines introduced into these 2 regions. Crosslinks could be formed between R899C (in ECL4) and a number of sites in ECL1 in a manner that was dependent on channel activity, suggesting that the relative orientation of these 2 loops changes on activation. Formation of these crosslinks inhibited channel function, suggesting that relative movement of these ECLs is important to normal channel function. Implications of these findings for the effects of mutations in the ECLs that are associated with cystic fibrosis and interactions with extracellular substances that influence channel activity are discussed.

scholarly journals Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia

2012 ◽  
Vol 23 (21) ◽  
pp. 4188-4202 ◽  
Author(s):  
Guido Veit ◽  
Florian Bossard ◽  
Julie Goepp ◽  
A. S. Verkman ◽  
Luis J. V. Galietta ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Proinflammatory Cytokine ◽  
Functional Expression ◽  
Innate Immune ◽  
Cytokine Secretion ◽  
Apical Plasma Membrane ◽  
Chronic Obstructive ◽  
Channel Activity ◽  
Bronchial Epithelia

Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of the CF lung disease, yet the existence of and mechanism accounting for the innate immune defect that occurs before infection remain controversial. Inducible expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuated the proinflammatory cytokines interleukin-6 (IL-6), IL-8, and CXCL1/2 in two human respiratory epithelial models under air–liquid but not liquid–liquid interface culture. Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr∆F508/∆F508 immortalized and primary human bronchial epithelia. Similarly, direct but not P2Y receptor–mediated activation of TMEM16A attenuates IL-8 secretion in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport at the apical membrane may contribute to the excessive and persistent lung inflammation in CF and perhaps in other respiratory diseases associated with documented down-regulation of CFTR (e.g., chronic obstructive pulmonary disease). Direct pharmacological activation of TMEM16A offers a potential therapeutic strategy to reduce the inflammation of CF airway epithelia.

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