Epitope tagging permits cell surface detection of functional CFTR

1995 ◽  
Vol 269 (6) ◽  
pp. C1565-C1576 ◽  
Author(s):  
M. Howard ◽  
M. D. DuVall ◽  
D. C. Devor ◽  
J. Y. Dong ◽  
K. Henze ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Surface Expression ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
External Loop ◽  
Glycosylation Sites ◽  
Apical Membranes ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection for membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. When epitopes were added to the fourth external loop, the N-linked glycosylation sites in that loop were either preserved or they were mutated to produce a deglycosylated CFTR (dgCFTR). Tagged CFTRs were expressed in HeLa cells, and their cAMP-sensitive Cl permeability was assayed using the halide-sensitive fluorophore SPQ. CFTRs containing the M2 epitope showed halide permeability responses to cAMP, whereas cells expressing CFTR with the hemagglutinin (HA) tag showed little or no cAMP response. Xenopus oocytes expressing dgCFTR, with or without the M2 epitope, showed Cl conductance responses that were 20% of the wild-type response, whereas M2-tagged constructs retaining the glycosylation sites responded like wild-type CFTR. External M2-tagged CFTR was detected in the surface membrane of nonpermeabilized cells. The surface expression of the mutant M2-tagged CFTRs correlated with processing of these mutants (Gregory et al. Mol. Cell. Biol. 11:3886-3893, 1991). M2-901/CFTR is a useful reporter for the trafficking of wild-type and mutant CFTRs to the cell surface.

Dynasore inhibits removal of wild-type and ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) from the plasma membrane

2009 ◽  
Vol 421 (3) ◽  
pp. 377-385 ◽  
Author(s):  
Andrew Young ◽  
Martina Gentzsch ◽  
Cynthia Y. Abban ◽  
Ying Jia ◽  
Patricio I. Meneses ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Small Molecule ◽  
Short Exposure ◽  
Growth Media ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Cystic Fibrosis Transmembrane

Dynasore, a small molecule inhibitor of dynamin, was used to probe the role of dynamin in the endocytosis of wild-type and mutant CFTR (cystic fibrosis transmembrane conductance regulator). Internalization of both wild-type and ‘temperature-corrected’ ΔF508 CFTR was markedly inhibited by a short exposure to dynasore, implicating dynamin as a key element in the endocytic internalization of both wild-type and mutant CFTR. The inhibitory effect of dynasore was readily reversible upon washout of dynasore from the growth media. Corr-4 ({2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′]-bithiazolyl-2′-yl}-phenyl-methanonone), a pharmacological corrector of ΔF508 CFTR biosynthesis, caused a marked increase in the cell surface expression of mutant CFTR. Co-incubation of ΔF508 CFTR expressing cells with Corr-4 and dynasore caused a significantly greater level of cell surface CFTR than that observed in the presence of Corr-4 alone. These results argue that inhibiting the endocytic internalization of mutant CFTR provides a novel therapeutic target for augmenting the benefits of small molecule correctors of mutant CFTR biosynthesis.

scholarly journals Calreticulin Negatively Regulates the Cell Surface Expression of Cystic Fibrosis Transmembrane Conductance Regulator

2006 ◽  
Vol 281 (18) ◽  
pp. 12841-12848 ◽  
Author(s):  
Kazutsune Harada ◽  
Tsukasa Okiyoneda ◽  
Yasuaki Hashimoto ◽  
Keiko Ueno ◽  
Kimitoshi Nakamura ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells

2000 ◽  
Vol 279 (2) ◽  
pp. C375-C382 ◽  
Author(s):  
Marybeth Howard ◽  
Xiaosui Jiang ◽  
Donna Beer Stolz ◽  
Warren G. Hill ◽  
Jennifer A. Johnson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Surface Expression ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Madin Darby Canine Kidney ◽  
Cystic Fibrosis Transmembrane ◽  
Camp Stimulation ◽  
Darby Canine Kidney

Channel gating of the cystic fibrosis transmembrane conductance regulator (CFTR) is activated in response to cAMP stimulation. In addition, CFTR activation may also involve rapid insertion of a subapical pool of CFTR into the plasma membrane (PM). However, this issue has been controversial, in part because of the difficulty in distinguishing cell surface vs. intracellular CFTR. Recently, a fully functional, epitope-tagged form of CFTR (M2–901/CFTR) that can be detected immunologically in nonpermeabilized cells was characterized (Howard M, Duvall MD, Devor DC, Dong J-Y, Henze K, and Frizzell RA. Am J Physiol Cell Physiol 269: C1565–C1576, 1995; and Schultz BD, Takahashi A, Liu C, Frizzell RA, and Howard M. Am J Physiol Cell Physiol 273: C2080–C2089, 1997). We have developed replication-defective recombinant adenoviruses that express M2–901/CFTR and used them to probe cell surface CFTR in forskolin (FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells. Virally expressed M2–901/CFTR was functional and was readily detected on the apical surface of FSK-stimulated polarized MDCK cells. Interestingly, at low multiplicity of infection, we observed FSK-stimulated insertion of M2901/CFTR into the apical PM, whereas at higher M2–901/CFTR expression levels, no increase in surface expression was detected using indirect immunofluorescence. Immunoelectron microscopy of unstimulated and FSK-stimulated cells confirmed the M2–901/CFTR redistribution to the PM upon FSK stimulation and demonstrates that the apically inserted M2–901/CFTR originates from a population of subapical vesicles. Our observations may reconcile previous conflicting reports regarding the effect of cAMP stimulation on CFTR trafficking.

scholarly journals Carbon monoxide-releasing molecules inhibit the cystic fibrosis transmembrane conductance regulator Cl− channel

2020 ◽  
Vol 319 (6) ◽  
pp. L997-L1009
Author(s):  
Mayuree Rodrat ◽  
Walailak Jantarajit ◽  
Demi R. S. Ng ◽  
Bartholomew S. J. Harvey ◽  
Jia Liu ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Carbon Monoxide ◽  
Current Flow ◽  
Open Channels ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Flow Through ◽  
Cystic Fibrosis Transmembrane

The gasotransmitter carbon monoxide (CO) regulates fluid and electrolyte movements across epithelial tissues. However, its action on anion channels is incompletely understood. Here, we investigate the direct action of CO on the cystic fibrosis transmembrane conductance regulator (CFTR) by applying CO-releasing molecules (CO-RMs) to the intracellular side of excised inside-out membrane patches from cells heterologously expressing wild-type human CFTR. Addition of increasing concentrations of tricarbonyldichlororuthenium(II) dimer (CORM-2) (1–300 μM) inhibited CFTR channel activity, whereas the control RuCl3 (100 μM) was without effect. CORM-2 predominantly inhibited CFTR by decreasing the frequency of channel openings and, hence, open probability ( Po). But, it also reduced current flow through open channels with very fast kinetics, particularly at elevated concentrations. By contrast, the chemically distinct CO-releasing molecule CORM-3 inhibited CFTR by decreasing Po without altering current flow through open channels. Neither depolarizing the membrane voltage nor raising the ATP concentration on the intracellular side of the membrane affected CFTR inhibition by CORM-2. Interestingly, CFTR inhibition by CORM-2, but not by CFTRinh-172, was prevented by prior enhancement of channel activity by the clinically approved CFTR potentiator ivacaftor. Similarly, when added after CORM-2, ivacaftor completely relieved CFTR inhibition. In conclusion, CORM-2 has complex effects on wild-type human CFTR consistent with allosteric inhibition and open-channel blockade. Inhibition of CFTR by CO-releasing molecules suggests that CO regulates CFTR activity and that the gasotransmitter has tissue-specific effects on epithelial ion transport. The action of ivacaftor on CFTR Cl− channels inhibited by CO potentially expands the drug’s clinical utility.

Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia

2000 ◽  
Vol 279 (5) ◽  
pp. L835-L841 ◽  
Author(s):  
Olafur Baldursson ◽  
Herbert A. Berger ◽  
Michael J. Welsh
Keyword(s):  
Functional Role ◽  
Transmembrane Conductance Regulator ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Dependent Protein ◽  
Rat Thyroid ◽  
Cystic Fibrosis Transmembrane

The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl− current. Mutation of four in vivo phosphorylation sites, Ser660, Ser737, Ser795, and Ser813 (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser660 or Ser813 alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser660 nor Ser813 alone increased current to wild-type levels; both residues were required. Changing Ser737 to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser737 may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.

scholarly journals Influence of Cystic Fibrosis Transmembrane Conductance Regulator on Gene Expression in Response to Pseudomonas aeruginosa Infection of Human Bronchial Epithelial Cells

2005 ◽  
Vol 73 (10) ◽  
pp. 6822-6830 ◽  
Author(s):  
Nina Reiniger ◽  
Jeffrey K. Ichikawa ◽  
Gerald B. Pier
Keyword(s):  
Cystic Fibrosis ◽  
Cell Line ◽  
Cell Lines ◽  
The Other ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Bronchial Epithelial ◽  
Cystic Fibrosis Transmembrane ◽  
In Cells

ABSTRACT Chronic lung infection by Pseudomonas aeruginosa causes significant morbidity in cystic fibrosis patients initiated by the failure of innate immune responses. We used microarray analysis and real-time PCR to detect transcriptional changes associated with cytokine production in isogenic bronchial epithelial cell lines with either wild-type (WT) or mutant cystic fibrosis transmembrane conductance regulator (CFTR) in response to P. aeruginosa infection. The transcription of four NF-κB-regulated cytokine genes was maximal in the presence of WT CFTR: the interleukin-8 (IL-8), IL-6, CXCL1, and intracellular adhesion molecule 1 (ICAM-1) genes. Analysis of protein expression in two cell lines paired for wild-type and mutant CFTR with three P. aeruginosa strains showed IL-6 and IL-8 expressions were consistently enhanced by the presence of WT CFTR in both cell lines with all three strains of P. aeruginosa, although some strains gave small IL-8 increases in cells with mutant CFTR. CXCL1 production showed consistent enhancement in cells with WT CFTR using all three bacterial strains in one cell line, whereas in the other cell line, CXCL1 showed a significant increase in cells with either WT or mutant CFTR. ICAM-1 was unchanged at the protein level in one of the cell lines but did show mild enhancement with WT CFTR in the other cell pair. Inhibitions of NF-κB prior to infection indicated differing degrees of dependence on NF-κB for production of the cytokines, contingent on the cell line. Cytokine effectors of innate immunity to P. aeruginosa were found to be positively influenced by the presence of WT CFTR, indicating a role in resistance to P. aeruginosa infection.

Sign in / Sign up

Export Citation Format

Share Document