Cystic fibrosis transmembrane conductance regulator (CFTR) functionality is dependent on coatomer protein I (COPI)

Ying Yu, Oleksandr Platoshyn, Olga Safrina, Igor Tsigelny, Jason Yuan, Steven H. Keller

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Background Information. Cystic fibrosis results from mutations in the ABC transporter CFTR (cystic fibrosis trans-membrane conductance regulator), which functions as a cAMP-regulated anion channel. The most prevalent mutation in CFTR, the Phe508 deletion, results in the generation of a trafficking and functionally deficient channel. The cellular machineries involved in modulating CFTR trafficking and function have not been fully characterized. In the present study, we identified a role for the COPI (coatomer protein I) cellular trafficking machinery in the development of the CFTR polypeptide into a functional chloride channel. To examine the role of COPI in CFTR biosynthesis, we employed the cell line IdIF, which harbours a temperature-sensitive mutation in ε-COP, a COPI subunit, to inhibit COPI function and then determined whether the CFTR polypeptide produced from the transfected gene developed into a cAMP-regulated chloride channel. Results. When COPI was inactivated in the IdIF cells by an elevated temperature pulse (39°C), the CFTR polypeptide was detected on the cell surface by immunofluorescence microscopy and cell-surface biotinylation. Therefore, CFTR proceeded upstream in the secretory pathway in the absence of COPI function, a result demonstrated previously by others. In contrast, electrophysiological measurements indicated an absence of cAMP-stimulated chloride efflux, suggesting that channel function was impaired. In comparison, expression of CFTR at the same elevated temperature (39°C) in an ε-COP-rescued cell line [IdIF(IdIF)], which has an introduced wild-type ε-COP gene in addition to the mutant ε-COP gene, showed restoration of cAMP-stimulated channel activity, confirming the requirement of COPI for channel function. Conclusions. These results therefore suggest that generation of the folded-functional conformation of CFTR requires COPI.

Original languageEnglish (US)
Pages (from-to)433-444
Number of pages12
JournalBiology of the Cell
Volume99
Issue number8
DOIs
StatePublished - Aug 2007
Externally publishedYes

Fingerprint

Coatomer Protein
Cystic Fibrosis Transmembrane Conductance Regulator
Chloride Channels
Cystic Fibrosis
Peptides
Mutation
Temperature
Genes
Biotinylation
Cell Line
ATP-Binding Cassette Transporters
Secretory Pathway
Protein Subunits
Fluorescence Microscopy
Anions
Chlorides

Keywords

  • Coatomer protein
  • Cystic fibrosis
  • Endoplasmic reticulum
  • IdIF cell
  • Ion-channel trafficking

ASJC Scopus subject areas

  • Cell Biology

Cite this

Cystic fibrosis transmembrane conductance regulator (CFTR) functionality is dependent on coatomer protein I (COPI). / Yu, Ying; Platoshyn, Oleksandr; Safrina, Olga; Tsigelny, Igor; Yuan, Jason; Keller, Steven H.

In: Biology of the Cell, Vol. 99, No. 8, 08.2007, p. 433-444.

Research output: Contribution to journalArticle

Yu, Ying ; Platoshyn, Oleksandr ; Safrina, Olga ; Tsigelny, Igor ; Yuan, Jason ; Keller, Steven H. / Cystic fibrosis transmembrane conductance regulator (CFTR) functionality is dependent on coatomer protein I (COPI). In: Biology of the Cell. 2007 ; Vol. 99, No. 8. pp. 433-444.
@article{0994a044285e446d8994fd79cbd4d7b8,
title = "Cystic fibrosis transmembrane conductance regulator (CFTR) functionality is dependent on coatomer protein I (COPI)",
abstract = "Background Information. Cystic fibrosis results from mutations in the ABC transporter CFTR (cystic fibrosis trans-membrane conductance regulator), which functions as a cAMP-regulated anion channel. The most prevalent mutation in CFTR, the Phe508 deletion, results in the generation of a trafficking and functionally deficient channel. The cellular machineries involved in modulating CFTR trafficking and function have not been fully characterized. In the present study, we identified a role for the COPI (coatomer protein I) cellular trafficking machinery in the development of the CFTR polypeptide into a functional chloride channel. To examine the role of COPI in CFTR biosynthesis, we employed the cell line IdIF, which harbours a temperature-sensitive mutation in ε-COP, a COPI subunit, to inhibit COPI function and then determined whether the CFTR polypeptide produced from the transfected gene developed into a cAMP-regulated chloride channel. Results. When COPI was inactivated in the IdIF cells by an elevated temperature pulse (39°C), the CFTR polypeptide was detected on the cell surface by immunofluorescence microscopy and cell-surface biotinylation. Therefore, CFTR proceeded upstream in the secretory pathway in the absence of COPI function, a result demonstrated previously by others. In contrast, electrophysiological measurements indicated an absence of cAMP-stimulated chloride efflux, suggesting that channel function was impaired. In comparison, expression of CFTR at the same elevated temperature (39°C) in an ε-COP-rescued cell line [IdIF(IdIF)], which has an introduced wild-type ε-COP gene in addition to the mutant ε-COP gene, showed restoration of cAMP-stimulated channel activity, confirming the requirement of COPI for channel function. Conclusions. These results therefore suggest that generation of the folded-functional conformation of CFTR requires COPI.",
keywords = "Coatomer protein, Cystic fibrosis, Endoplasmic reticulum, IdIF cell, Ion-channel trafficking",
author = "Ying Yu and Oleksandr Platoshyn and Olga Safrina and Igor Tsigelny and Jason Yuan and Keller, {Steven H.}",
year = "2007",
month = "8",
doi = "10.1042/BC20060114",
language = "English (US)",
volume = "99",
pages = "433--444",
journal = "Biology of the Cell",
issn = "0248-4900",
publisher = "Portland Press Ltd.",
number = "8",

}

TY - JOUR

T1 - Cystic fibrosis transmembrane conductance regulator (CFTR) functionality is dependent on coatomer protein I (COPI)

AU - Yu, Ying

AU - Platoshyn, Oleksandr

AU - Safrina, Olga

AU - Tsigelny, Igor

AU - Yuan, Jason

AU - Keller, Steven H.

PY - 2007/8

Y1 - 2007/8

N2 - Background Information. Cystic fibrosis results from mutations in the ABC transporter CFTR (cystic fibrosis trans-membrane conductance regulator), which functions as a cAMP-regulated anion channel. The most prevalent mutation in CFTR, the Phe508 deletion, results in the generation of a trafficking and functionally deficient channel. The cellular machineries involved in modulating CFTR trafficking and function have not been fully characterized. In the present study, we identified a role for the COPI (coatomer protein I) cellular trafficking machinery in the development of the CFTR polypeptide into a functional chloride channel. To examine the role of COPI in CFTR biosynthesis, we employed the cell line IdIF, which harbours a temperature-sensitive mutation in ε-COP, a COPI subunit, to inhibit COPI function and then determined whether the CFTR polypeptide produced from the transfected gene developed into a cAMP-regulated chloride channel. Results. When COPI was inactivated in the IdIF cells by an elevated temperature pulse (39°C), the CFTR polypeptide was detected on the cell surface by immunofluorescence microscopy and cell-surface biotinylation. Therefore, CFTR proceeded upstream in the secretory pathway in the absence of COPI function, a result demonstrated previously by others. In contrast, electrophysiological measurements indicated an absence of cAMP-stimulated chloride efflux, suggesting that channel function was impaired. In comparison, expression of CFTR at the same elevated temperature (39°C) in an ε-COP-rescued cell line [IdIF(IdIF)], which has an introduced wild-type ε-COP gene in addition to the mutant ε-COP gene, showed restoration of cAMP-stimulated channel activity, confirming the requirement of COPI for channel function. Conclusions. These results therefore suggest that generation of the folded-functional conformation of CFTR requires COPI.

AB - Background Information. Cystic fibrosis results from mutations in the ABC transporter CFTR (cystic fibrosis trans-membrane conductance regulator), which functions as a cAMP-regulated anion channel. The most prevalent mutation in CFTR, the Phe508 deletion, results in the generation of a trafficking and functionally deficient channel. The cellular machineries involved in modulating CFTR trafficking and function have not been fully characterized. In the present study, we identified a role for the COPI (coatomer protein I) cellular trafficking machinery in the development of the CFTR polypeptide into a functional chloride channel. To examine the role of COPI in CFTR biosynthesis, we employed the cell line IdIF, which harbours a temperature-sensitive mutation in ε-COP, a COPI subunit, to inhibit COPI function and then determined whether the CFTR polypeptide produced from the transfected gene developed into a cAMP-regulated chloride channel. Results. When COPI was inactivated in the IdIF cells by an elevated temperature pulse (39°C), the CFTR polypeptide was detected on the cell surface by immunofluorescence microscopy and cell-surface biotinylation. Therefore, CFTR proceeded upstream in the secretory pathway in the absence of COPI function, a result demonstrated previously by others. In contrast, electrophysiological measurements indicated an absence of cAMP-stimulated chloride efflux, suggesting that channel function was impaired. In comparison, expression of CFTR at the same elevated temperature (39°C) in an ε-COP-rescued cell line [IdIF(IdIF)], which has an introduced wild-type ε-COP gene in addition to the mutant ε-COP gene, showed restoration of cAMP-stimulated channel activity, confirming the requirement of COPI for channel function. Conclusions. These results therefore suggest that generation of the folded-functional conformation of CFTR requires COPI.

KW - Coatomer protein

KW - Cystic fibrosis

KW - Endoplasmic reticulum

KW - IdIF cell

KW - Ion-channel trafficking

UR - http://www.scopus.com/inward/record.url?scp=34547970891&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34547970891&partnerID=8YFLogxK

U2 - 10.1042/BC20060114

DO - 10.1042/BC20060114

M3 - Article

VL - 99

SP - 433

EP - 444

JO - Biology of the Cell

JF - Biology of the Cell

SN - 0248-4900

IS - 8

ER -