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 language | English (US) |
|---|---|
| Pages (from-to) | 433-444 |
| Number of pages | 12 |
| Journal | Biology of the Cell |
| Volume | 99 |
| Issue number | 8 |
| DOIs | |
| State | Published - Aug 2007 |
| Externally published | Yes |
Fingerprint
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 journal › Article
}
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 -