Antibodies against the cystic fibrosis transmembrane regulator

1992 ◽  
Vol 262 (2) ◽  
pp. C396-C404 ◽  
Author(s):  
C. M. Fuller ◽  
M. B. Howard ◽  
D. M. Bedwell ◽  
R. A. Frizzell ◽  
D. J. Benos
Keyword(s):  
Cystic Fibrosis ◽  
Serum Proteins ◽  
Polyclonal Antibodies ◽  
Chinese Hamster Ovary Cells ◽  
Expression System ◽  
Functional Characterization ◽  
Chinese Hamster ◽  
Cystic Fibrosis Transmembrane Regulator ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

Rabbit polyclonal antibodies have been raised against high-performance liquid chromatography purified synthetic peptides corresponding to two discrete regions of the cystic fibrosis transmembrane regulator (CFTR) protein: the R-domain (residues 785-796) and the extreme COOH-terminus (residues 1467-1480). Antibodies (Ab) to each of these peptides were affinity purified either by passage over a peptide-derivatized agarose matrix (Ab 785) to produce monospecific polyclonal antibodies or by protein A affinity chromatography to purify the immunoglobulin G1 fraction free of other serum proteins (Ab 1467). These antibodies recognize a candidate CFTR protein in the colonic cell line T84, as determined by Western blot analysis and by immunoprecipitation and labeling of the precipitate with [gamma-32P]ATP in the presence of protein kinase A. Both antibodies precipitated CFTR-related polypeptides from four mammalian cell types (HeLa, Bsc-40, HEp-2, and Chinese hamster ovary cells) transfected with the full-length CFTR cDNA clone using a vaccinia T7 protein expression system. Similar results were observed using a yeast CFTR expression system. In each case the Mr values of the bands observed were consistent with that expected for the CFTR protein. These antibodies should be useful probes for the immunocytochemical localization, immunoaffinity purification, and ultimately the functional characterization of the CFTR protein.

scholarly journals Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

2005 ◽  
Vol 16 (5) ◽  
pp. 2154-2167 ◽  
Author(s):  
Silvia M. Kreda ◽  
Marcus Mall ◽  
April Mengos ◽  
Lori Rochelle ◽  
James Yankaskas ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Normal Subjects ◽  
Cell Types ◽  
Cystic Fibrosis Transmembrane Regulator ◽  
Ciliated Cells ◽  
Metabolic Fate ◽  
Specific Expression ◽  
Δf508 Cftr ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

Previous studies in native tissues have produced conflicting data on the localization and metabolic fate of WT and ΔF508 cystic fibrosis transmembrane regulator (CFTR) in the lung. Combining immunocytochemical and biochemical studies utilizing new high-affinity CFTR mAbs with ion transport assays, we examined both 1) the cell type and region specific expression of CFTR in normal airways and 2) the metabolic fate of ΔF508 CFTR and associated ERM proteins in the cystic fibrosis lung. Studies of lungs from a large number of normal subjects revealed that WT CFTR protein localized to the apical membrane of ciliated cells within the superficial epithelium and gland ducts. In contrast, other cell types in the superficial, gland acinar, and alveolar epithelia expressed little WT CFTR protein. No ΔF508 CFTR mature protein or function could be detected in airway specimens freshly excised from a large number of ΔF508 homozygous subjects, despite an intact ERM complex. In sum, our data demonstrate that WT CFTR is predominantly expressed in ciliated cells, and ΔF508 CFTR pathogenesis in native tissues, like heterologous cells, reflects loss of normal protein processing.

scholarly journals Permeability of Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels to Polyatomic Anions

1997 ◽  
Vol 110 (4) ◽  
pp. 355-364 ◽  
Author(s):  
Paul Linsdell ◽  
Joseph A. Tabcharani ◽  
Johanna M. Rommens ◽  
Yue-Xian Hou ◽  
Xiu-Bao Chang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channels ◽  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Cystic Fibrosis Transmembrane

Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (PX/PCl) sequence NO3− > Cl− > HCO3− > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (PX/PCl < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of ∼5.3 Å. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.

PRO–CF–MED, Clinical Proof of concept for a RNA–targeting Oligonucleotide for a Cystic fibrosis–F508del MEDication, Horizon2020

Impact ◽  
2018 ◽  
Vol 2018 (3) ◽  
pp. 52-54
Author(s):  
Nicolas Lamontagne
Keyword(s):  
Cystic Fibrosis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Rna Targeting ◽  
Threatening Condition ◽  
Life Threatening ◽  
Cftr Protein ◽  
Disease Modifying ◽  
Cystic Fibrosis Transmembrane ◽  
Specific Challenge

Cystic fibrosis (CF) is a progressive life–shortening disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene leading to a dysfunctional CFTR protein. The disease affects over 70,000 patients worldwide and while many mutations are known, the F508del mutation affects 90% of all patients. The absence of CFTR in the plasma membrane leads to a dramatic decrease in chloride efflux, resulting in viscous mucus that causes severe symptoms in vital organs like the lungs and intestines. For CF patients that suffer from the life threatening F508del mutation only palliative treatment exist. PRO–CF–MED addresses the specific challenge of this call by introducing the first disease modifying medication for the treatment of the CF patients with F508del mutation. The PRO–CF–MED project has been designed to assess the potential clinical efficacy of QR–010, an innovative disease modifying oligonucleotide–based treatment for F508del patients. Partners within PRO–CF–MED have generated very promising preclinical evidence for QR–010 which allows for further clinical assessment of QR–010 in clinical trials. PRO–CF–MED will enable the fast translation of QR–010 towards clinical practice and market authorisation. PRO–CF–MED has the potential to transform this life–threatening condition into a manageable one.

CPX, a selective A1-adenosine-receptor antagonist, regulates intracellular pH in cystic fibrosis cells

1995 ◽  
Vol 269 (1) ◽  
pp. C226-C233 ◽  
Author(s):  
V. Casavola ◽  
R. J. Turner ◽  
C. Guay-Broder ◽  
K. A. Jacobson ◽  
O. Eidelman ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Receptor Antagonist ◽  
Intracellular Ph ◽  
Adenosine Receptor ◽  
Ph Regulation ◽  
Cystic Fibrosis Transmembrane Regulator ◽  
Wild Type ◽  
A1 Adenosine Receptor ◽  
Adenosine Receptor Antagonist ◽  
Cystic Fibrosis Transmembrane

The selective A1-adenosine-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), has been reported to activate Cl- efflux from cystic fibrosis cells, such as pancreatic CFPAC-1 and lung IB3 cells bearing the cystic fibrosis transmembrane regulator(delta F508) mutation, but has little effect on the same process in cells repaired by transfection with wild-type cystic fibrosis transmembrane regulator (O. Eidelman, C. Guay-Broder, P. J. M. van Galen, K. A. Jacobson, C. Fox, R. J. Turner, Z. I. Cabantchik, and H. B. Pollard. Proc. Natl. Acad. Sci. USA 89: 5562-5566, 1992). We report here that CPX downregulates Na+/H+ exchange activity in CFPAC-1 cells but has a much smaller effect on cells repaired with the wild-type gene. CPX also mildly decreases resting intracellular pH. In CFPAC-1 cells, this downregulation is dependent on the presence of adenosine, since pretreatment of the cells with adenosine deaminase blocks the CPX effect. We also show that, by contrast, CPX action on these cells does not lead to alterations in intracellular free Ca2+ concentration. We conclude that CPX affects pH regulation in CFPAC-1 cells, probably by antagonizing the tonic action of endogenous adenosine.

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