Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells

Schoumacher, Robert; Shoemaker, R. L.; Halm, Dan R.; Tallant, E. Ann; Wallace, Robert W.; Frizzell, Raymond A.

doi:10.1038/330752a0

Cited by 329 publications

(121 citation statements)

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“…In some preparations, outward rectifiers can be activated in cell-attached patches by CAMP and may therefore contribute to apical Cl conductance . This proposed role, and its relevance to CF, is supported by reports that outward rectifiers excised from normal cells can be activated by exposure to A-or C-kinase whereas those from CF cells are not responsive [16,17].…”

Section: The Pathways Responsible For Apical CL Conductancesupporting

confidence: 51%

Low‐conductance chloride channel activated by cAMP in the epithelial cell line T₈₄

et al. 1990

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We have studied the modulation and pharmacological properties of two anion channels in T, ceils by recording single channel and transepithelial currents. One channel had an outwardly rectifying current-voltage Z/V curve, was rarely active in cell-attached patches, and was unaffected by CAMP. The other channel had lower conductance (8.7 pS at 37°C) and a more ohmic Z/V relationship. Exposure to CAMP increased the probability of observing low-conductance channel activity in cell-attached patches > 6-fold. Extracellular DIDS (4,4'-diisothiocyanatostilbene-2,2'disulfonic acid) or IAA-94 (an indanyloxyacetic acid) inhibited the outward rectifier but did not a&t the low-conductance channel or CAMP-stimulated transepithelial current. These results suggest the low-conductance Cl channel may contribute to apical membrane conductance during CAMP-stimulated secretion.

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Section: The Pathways Responsible For Apical CL Conductancesupporting

confidence: 51%

Low‐conductance chloride channel activated by cAMP in the epithelial cell line T₈₄

et al. 1990

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“…Modulation by phosphorylation which is common to many other ion channels, both ligand-and voltage-gated, is rather absolute in the case of CFTR [22] and may be required for propagation of the conformational impact of ligand binding to the pore. Patch-clamp studies with CF epithelial cells in the 1980s before the discovery of CFTR indicated a defective cAMP-stimulated anion current [35,66] and the CFTR sequence later revealed the presence of a large cluster of consensus sites for phosphorylation by cAMPdependent protein kinase A in the R domain [63]. Extensive studies have confirmed that the phosphorylation state of these sites strictly controls the activity of the CFTR chloride channel [12,48].…”

Section: Cftr Control By Phosphorylationmentioning

confidence: 99%

CFTR (ABCC7) is a hydrolyzable-ligand-gated channel

Aleksandrov

Riordan

2006

Pflugers Arch - Eur J Physiol

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As the product of the gene mutated in cystic fibrosis, the most common genetic disease of Caucasians, CFTR is an atypical ABC protein. From an evolutionary perspective, it is apparently a relatively young member of the ABC family, present only in metazoans where it plays a critical role in epithelial salt and fluid homeostasis. Functionally, the membrane translocation process it mediates, the passive bidirectional diffusion of small inorganic anions, is simpler than the vectorial transport of larger more complex substrates ("allocrites") by most ABC transporters. However, the control of the permeation pathway which cannot go unchecked is necessarily more stringent than in the case of the transporters. There is tight regulation by the phosphorylation/dephosphorylation of the unique CFTR R domain superimposed on the basic ABC regulation mode of ATP binding and hydrolysis at the dual nucleotide binding sites. As with other ABCC subfamily members, only the second of these sites is hydrolytic in CFTR. The phosphorylation and ATP binding/hydrolysis events do not strongly influence each other; rather, R domain phosphorylation appears to enable transduction of the nucleotide binding allosteric signal to the responding channel gate. ATP hydrolysis is not required for either the opening or closing gating transitions but efficiently clears the ligand-binding site enabling a new gating cycle to be initiated.

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“…This aspect of epithelial function is essential for rehydration of airway mucus. Abnormalities in the Cl conductance pathway also appear critical to disease processes such as those occurring in cystic fibrosis (11,12).Phenotypic characteristics have been well maintained in SV40-transformed fibroblasts (13-15). In SV40-transformed epithelial cells (16)(17)(18)(19), certain aspects of phenotype are retained better than others (17,18,20,21).…”

mentioning

confidence: 99%

Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.

Gruenert

Basbaum

Welsh

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

211

148

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To facilitate understanding of the mechanisms underlying pulmonary diseases, including lung cancer and cystic fibrosis, we have transformed and characterized cultures of human tracheal epithelial cells. Cells were transfected by calcium phosphate precipitation with a plasmid containing a replication-defective simian virus 40 (SV40) genome. Colonies of cells with enhanced growth potential were isolated and analyzed for transformation-and epithelialspecific characteristics. Precrisis cells were observed to express the SV40 large tumor antigen, produce cytokeratins, have microvilli, and form tight junctions. After crisis, cells continued to express the SV40 large tumor antigen as well as epithelial-specific cytokeratins and to display the apical membrane microvilli. Apical membrane Cl channels were opened in postcrisis cells exposed to 50 ,LM forskolin. These channels showed electrical properties similar to those observed in primary cultures. The postcrisis cells have been in culture for >250 generations and are potentially "immortal." In addition to providing a useful in vitro model for the study of ion transport by human airway epithelial cells, the cells can be used to examine stages of neoplastic progression. (6,7).In contrast to the relatively large number of studies describing cells from other organs (recently reviewed in refs. 1 and 2), only three reports indicate transformation of respiratory tract cells (8-10). Bronchial epithelial cells immortalized with v-Ha-ras display anchorage-independent growth and form tumors in nude mice. However, these cells were poorly differentiated and did not contain tight junctions or keratins characteristic of differentiated epithelial cells. The epithelial origin of the cells was verified by the presence of keratin. A somewhat greater level of differentiation was retained by diethylnitrosamine-transformed fetal tracheal cells, which contained mucin. These cells also expressed carcinoembryonic antigen and wool merokeratin and had enhanced, although limited, growth capacity (9). A third study primarily described methods for transfection and showed expression of the SV40 large tumor antigen (T antigen) three to four subcultures posttransfection (10).A goal of the present study was to develop a transformed human tracheal epithelial (HTE) cell line for detailed studies of the mechanisms regulating Cl ion transport. This aspect of epithelial function is essential for rehydration of airway mucus. Abnormalities in the Cl conductance pathway also appear critical to disease processes such as those occurring in cystic fibrosis (11,12).Phenotypic characteristics have been well maintained in SV40-transformed fibroblasts (13-15). In SV40-transformed epithelial cells (16)(17)(18)(19), certain aspects of phenotype are retained better than others (17,18,20,21). A plasmid containing a SV40 genome defective in the origin of replication (pSVori-) has been shown to enhance the transformation efficiency of human fibroblasts (22). The defective origin of replication precludes background ...

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Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells

Cited by 329 publications

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Low‐conductance chloride channel activated by cAMP in the epithelial cell line T₈₄

Low‐conductance chloride channel activated by cAMP in the epithelial cell line T₈₄

CFTR (ABCC7) is a hydrolyzable-ligand-gated channel

Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.

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Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells

Cited by 329 publications

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Low‐conductance chloride channel activated by cAMP in the epithelial cell line T84

Low‐conductance chloride channel activated by cAMP in the epithelial cell line T84

CFTR (ABCC7) is a hydrolyzable-ligand-gated channel

Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.

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Low‐conductance chloride channel activated by cAMP in the epithelial cell line T₈₄

Low‐conductance chloride channel activated by cAMP in the epithelial cell line T₈₄