Expression of Cystic Fibrosis Transmembrane Conductance Regulator Alters the Responses to Hypotonic Cell Swelling and ATP of Chinese Hamster Ovary Cells

1998 ◽  
Vol 8 (1-2) ◽  
pp. 61-74 ◽  
Author(s):  
I.E. Thiele ◽  
M.J. Hug ◽  
M. Hübner ◽  
R. Greger
Keyword(s):  
Cystic Fibrosis ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Swelling ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Ovary Cells ◽  
Hypotonic Cell Swelling ◽  
Cystic Fibrosis Transmembrane

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.

scholarly journals Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane

2003 ◽  
Vol 374 (3) ◽  
pp. 793-797 ◽  
Author(s):  
Mohabir RAMJEESINGH ◽  
Jackie F. KIDD ◽  
Ling Jun HUAN ◽  
Yanchun WANG ◽  
Christine E. BEAR
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Biology ◽  
Plasma Membranes ◽  
Chinese Hamster Ovary Cells ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane

CFTR (cystic fibrosis transmembrane conductance regulator) mediates chloride conduction across the apical membrane of epithelia, and mutations in CFTR lead to defective epithelial fluid transport. Recently, there has been considerable interest in determining the quaternary structure of CFTR at the cell surface, as such information is a key to understand the molecular basis for pathogenesis in patients harbouring disease-causing mutations. In our previous work [Ramjeesingh, Li, Kogan, Wang, Huan and Bear (2001) Biochemistry 40, 10700–10706], we showed that monomeric CFTR is the minimal functional form of the protein, yet when expressed in Sf 9 cells using the baculovirus system, it also exists as dimers. The purpose of the present study was to determine if dimeric CFTR exists at the surface of mammalian cells, and particularly in epithelial cells. CFTR solubilized from membranes prepared from Chinese-hamster ovary cells stably expressing CFTR and from T84 epithelial cells migrates as predicted for monomeric, dimeric and larger complexes when subjected to sizing by gel filtration and analysis by non-dissociative electrophoresis. Purification of plasma membranes led to the enrichment of CFTR dimers and this structure exists as the complex glycosylated form of the protein, supporting the concept that dimeric CFTR is physiologically relevant. Consistent with its localization in plasma membranes, dimeric CFTR was labelled by surface biotinylation. Furthermore, dimeric CFTR was captured at the apical surface of intact epithelial cells by application of a membrane-impermeable chemical cross-linker. Therefore it follows from the present study that CFTR dimers exist at the surface of epithelial cells. Further studies are necessary to understand the impact of dimerization on the cell biology of wild-type and mutant CFTR proteins.

scholarly journals Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation

1997 ◽  
Vol 328 (2) ◽  
pp. 353-361 ◽  
Author(s):  
L. Gergely LUKACS ◽  
Gersana SEGAL ◽  
Norbert KARTNER ◽  
Sergio GRINSTEIN ◽  
Fred ZHANG
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Surface ◽  
Protein Phosphorylation ◽  
Chinese Hamster ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Hypertonic Medium ◽  
Cystic Fibrosis Transmembrane

Although the cystic fibrosis transmembrane conductance regulator (CFTR) is primarily implicated in the regulation of plasma-membrane chloride permeability, immunolocalization and functional studies indicate the presence of CFTR in the endosomal compartment. The mechanism of CFTR delivery from the cell surface to endosomes is not understood. To delineate the internalization pathway, both the rate and extent of CFTR accumulation in endosomes were monitored in stably transfected Chinese hamster ovary (CHO) cells. The role of clathrin-dependent endocytosis was assessed in cells exposed to hypertonic medium, potassium depletion or intracellular acid-load. These treatments inhibited clathrin-dependent endocytosis by > 90%, as verified by measurements of 125I-transferrin uptake. Functional association of CFTR with newly formed endosomes was determined by an endosomal pH dissipation protocol [Lukacs, Chang, Kartner, Rotstein, Riordan and Grinstein (1992) J. Biol. Chem. 267, 14568-14572]. As a second approach, endocytosis of CFTR was determined after cell-surface biotinylation with the cleavable sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. Both the biochemical and the functional assays indicated that arresting the formation of clathrin-coated vesicles inhibited the retrieval of the CFTR from the plasma membrane to endosomes. An overall arrest of membrane traffic cannot account for the inhibition of CFTR internalization, since the fluid-phase endocytosis was not effected by the treatments used. Thus the efficient, constitutive internalization of surface CFTR (5% per min) occurs, predominantly by clathrin-dependent endocytosis. Stimulation of protein phosphorylation by cAMP-dependent protein kinase A and by protein kinase C decreased the rate of internalization of cell-surface biotinylated CFTR, and contributed to a substantial diminution of the internal CFTR pool compared with that of unstimulated cells. These results suggest that the rate of CFTR internalization may participate in the determination of the CFTR channel density, and consequently, of the cAMP-stimulated chloride conductance of the plasma membrane.

Malignancy-related characteristics of wild type and drug-resistant chinese hamster ovary cells

Pathology ◽  
1993 ◽  
Vol 25 (3) ◽  
pp. 268-276 ◽  
Author(s):  
Wanda B. Mackinnon ◽  
Marlen Dyne ◽  
Rebecca Hancock ◽  
Carolyn E. Mountford ◽  
Adrienne J. Grant ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Drug Resistant ◽  
Wild Type ◽  
Ovary Cells
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