Phosphorylation-regulated CI− channel in CHO cells stably expressing the cystic fibrosis gene

Tabcharani, Joseph A.; Chang, Xiu-Bao; Riordan, John R.; Hanrahan, John W.

doi:10.1038/352628a0

Cited by 549 publications

(422 citation statements)

References 17 publications

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“…As with PKA, PKC activation may play a role in amplifying chloride secretion during CFTR expression by promoting CFTR accumulation at the cell surface. In this context, it is noteworthy that PKC activation potentiated both the cAMP-mediated CFTR chloride current in inside-out patches on CHO cells and the whole-cell chloride current of pancreatic-duct cells [52,53].…”

Section: Discussionmentioning

confidence: 97%

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

Lukacs

Segal

Kartner

et al. 1997

Biochemical Journal

127

138

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Although the cystic fibrosis transmembrane conductance regulator (CFTR) is primarily implicated in the regulation of plasmamembrane 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 clathrindependent 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 "#&I-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

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Section: Discussionmentioning

confidence: 97%

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

Lukacs

Segal

Kartner

et al. 1997

Biochemical Journal

127

138

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“…Construction, Expression, and Detection of CL3 Mutants-Mutagenesis was performed in the expression vectors pNUT-CFTR and pcDNA3-CFTR as described (Tabcharani et al, 1991;Seibert et al, 1996). The sequence of each polymerase chain reaction fragment was verified after insertion into the vector using the T7 Sequencing Kit (Pharmacia Biotech Inc.).…”

Section: Methodsmentioning

confidence: 99%

“…Patch-clamp Studies of CL3 Mutants-Single CFTR channels were recorded from inside-out patches excised from stably expressing CHO cells, according to Tabcharani et al (1991) and Tabcharani et al (1993). Following patch excision, channels were activated by exposing the cytoplasmic face of the patch to 75 nM catalytic subunit of PKA (Promega) plus 1 mM Na 2 ATP.…”

Section: Methodsmentioning

confidence: 99%

“…Current models predict that CFTR contains 12 hydrophobic transmembrane helices (TMs), which assemble within the lipid bilayer to form the pore of the channel (Riordan et al, 1989). Linked to the TMs and protruding into the cell cytoplasm are three large domains, the two nucleotide binding folds (NBFs) which have Walker A and Walker B motifs (Walker et al, 1982) for interactions with ATP and the highly charged R-domain rich in consensus sequences for phosphorylation by the cAMP-dependent protein kinase (PKA) and protein kinase C. Functional studies showed that these cytoplasmic domains regulate the opening and closing of the channel through ATP binding and ATP hydrolysis as well as through kinase-mediated phosphorylation and phosphatasemediated dephosphorylation (Anderson et al, 1991;Cheng et al, 1991;Tabcharani et al, 1991;Picciotto et al, 1992;Becq et al, 1994;Hwang et al, 1994;Gunderson and Kopito, 1995;Wilkinson et al, 1996).…”

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confidence: 99%

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Cytoplasmic Loop Three of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Regulation of Chloride Channel Activity

Seibert

Linsdell

Loo

et al. 1996

Journal of Biological Chemistry

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104

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To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three pointmutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Chinese hamster ovary cell lines stably expressing wildtype CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein. Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26°C) or incubated in the presence of 10% glycerol. For all mutants, the open probability (P 0 ) of the CFTR channels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the P 0 , whereas the H949Y mutation doubled the P 0 relative to wild-type. The changes in P 0 predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range ؎80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore.

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“…5 Both halves are joined by a specific cytoplasmic regulator (R) domain, whose phosphorylation by protein kinase A activates the outward anionic channel activity. 6,7 To date, Ͼ1860 mutations identified within the human CFTR sequence are listed in the CF mutation database (http://www.genet.sickkids.on.ca/Home.html, last accessed March 8, 2011). These mutations can be divided into six classes, according to the mechanism that disrupts CFTR function 8,9 : absence of the protein at the apical plasma membrane because of i) defective protein synthesis or ii) impaired maturation leading to protein degradation, iii) defective regulation of CFTR channel activity, iv) altered ionic selectivity and conductance, v) lowered CFTR mRNA amount, and vi) decreased protein stability.…”

mentioning

confidence: 99%

Orphan Missense Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator

Fresquet

Clément

Norez

et al. 2011

The Journal of Molecular Diagnostics

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More than 1860 mutations have been found within the human cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence. These mutations can be classified according to their degree of severity in CF disease. Although the most common mutations are well characterized, few data are available for rare mutations. Thus, genetic counseling is particularly difficult when fetuses or patients with CF present these orphan variations. We describe a threestep in vitro assay that can evaluate rare missense CFTR mutation consequences to establish a correlation between genotype and phenotype. By using a green fluorescent protein-tagged CFTR construct, we expressed mutated proteins in COS-7 cells. CFTR trafficking was visualized by confocal microscopy, and the cellular localization of CFTR was determined using intracellular markers. We studied the CFTR maturation process using Western blot analysis and evaluated CFTR channel activity by automated iodide efflux assays. Of six rare mutations that we studied, five have been isolated in our laboratory. The cellular and functional impact that we observed in each case was compared with the clinical data concerning the patients in whom we encountered these mutations. In conclusion, we propose that performing this type of analysis for orphan CFTR missense mutations can improve CF genetic counseling. Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder in the white population, with an incidence of approximately 1 in 3500. It results from mutations in the CF transmembrane conductance regulator (CFTR) gene 1,2 that encodes a chloride channel normally expressed at the apical membrane of epithelial cells. 3,4 CFTR is a member of the ATP-binding cassette transporter family. It contains two repeated units composed of a membrane-spanning domain, which includes six transmembrane helices, and a nucleotide-binding domain, which harbors an ATP-binding and hydrolysis site. 5 Both halves are joined by a specific cytoplasmic regulator (R) domain, whose phosphorylation by protein kinase A activates the outward anionic channel activity. 6,7 To date, Ͼ1860 mutations identified within the human CFTR sequence are listed in the CF mutation database (http://www.genet.sickkids.on.ca/Home.html, last accessed March 8, 2011). These mutations can be divided into six classes, according to the mechanism that disrupts CFTR function 8,9 : absence of the protein at the apical plasma membrane because of i) defective protein synthesis or ii) impaired maturation leading to protein degradation, iii) defective regulation of CFTR channel activity, iv) altered ionic selectivity and conductance, v) lowered CFTR mRNA amount, and vi) decreased protein stability.The most common mutation, F508del, is a class II mutation. It is carried by 90% of patients with CF, on at least one allele, and it leads to a severe CF phenotype. In our laboratory, we use the Elucigene CF30 Kit (Gen-Probe, Inc., San Diego, CA), which has been approved in the French national neonatal CF screening program for rou...

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Phosphorylation-regulated CI− channel in CHO cells stably expressing the cystic fibrosis gene

Cited by 549 publications

References 17 publications

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

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

Cytoplasmic Loop Three of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Regulation of Chloride Channel Activity

Orphan Missense Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator

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