Low temperature and chemical rescue affect molecular proximity of ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC)

Yawar J. Qadri, Estelle Cormet-Boyaka, Arun K. Rooj, William Lee, Vladimir Parpura, Cathy M. Fuller, Bakhrom K. Berdiev

Research output: Contribution to journalArticle

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Abstract

An imbalance of chloride and sodium ion transport in several epithelia is a feature of cystic fibrosis (CF), an inherited disease that is a consequence of mutations in the cftr gene. The cftr gene codes for a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Some mutations in this gene cause the balance between Cl- secretion and Na + absorption to be disturbed in the airways; Cl-secretion is impaired, whereas Na+absorption is elevated. Enhanced Na + absorption through the epithelial sodium channel (ENaC) is attributed to the failure of mutated CFTR to restrict ENaC-mediated Na + transport. The mechanism of this regulation is controversial. Recently, we have found evidence for a close association of wild type (WT) CFTR and WT ENaC, further underscoring the role of ENaC along with CFTR in the pathophysiology of CF airway disease. In this study, we have examined the association of ENaC subunits with mutated ΔF508-CFTR, the most common mutation in CF. Deletion of phenylalanine at position 508 (ΔF508) prevents proper processing and targeting of CFTR to the plasma membrane. When ΔF508-CFTR and ENaC subunits were co-expressed in HEK293T cells, we found that individual ENaC subunits could be co-immunoprecipitated with ΔF508-CFTR, much like WT CFTR. However, when we evaluated the ΔF508-CFTR and ENaC association using fluorescence resonance energy transfer (FRET), FRET efficiencies were not significantly different from negative controls, suggesting that ΔF508-CFTR and ENaC are not in close proximity to each other under basal conditions. However, with partial correction of ΔF508-CFTR misprocessing by low temperature and chemical rescue, leading to surface expression as assessed by total internal reflection fluorescence (TIRF) microscopy, we observed a positive FRET signal. Our findings suggest that the ΔF508 mutation alters the close association of CFTR and ENaC.

Original languageEnglish
Pages (from-to)16781-16790
Number of pages10
JournalJournal of Biological Chemistry
Volume287
Issue number20
DOIs
Publication statusPublished - May 11 2012
Externally publishedYes

Fingerprint

Epithelial Sodium Channels
Cystic Fibrosis Transmembrane Conductance Regulator
Temperature
Fluorescence Resonance Energy Transfer
Cystic Fibrosis
Mutation
Genes
Fluorescence microscopy
Ion Transport
Cell membranes
Phenylalanine
Fluorescence Microscopy
Sodium Chloride

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Low temperature and chemical rescue affect molecular proximity of ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC). / Qadri, Yawar J.; Cormet-Boyaka, Estelle; Rooj, Arun K.; Lee, William; Parpura, Vladimir; Fuller, Cathy M.; Berdiev, Bakhrom K.

In: Journal of Biological Chemistry, Vol. 287, No. 20, 11.05.2012, p. 16781-16790.

Research output: Contribution to journalArticle

Qadri, Yawar J. ; Cormet-Boyaka, Estelle ; Rooj, Arun K. ; Lee, William ; Parpura, Vladimir ; Fuller, Cathy M. ; Berdiev, Bakhrom K. / Low temperature and chemical rescue affect molecular proximity of ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC). In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 20. pp. 16781-16790.
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abstract = "An imbalance of chloride and sodium ion transport in several epithelia is a feature of cystic fibrosis (CF), an inherited disease that is a consequence of mutations in the cftr gene. The cftr gene codes for a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Some mutations in this gene cause the balance between Cl- secretion and Na + absorption to be disturbed in the airways; Cl-secretion is impaired, whereas Na+absorption is elevated. Enhanced Na + absorption through the epithelial sodium channel (ENaC) is attributed to the failure of mutated CFTR to restrict ENaC-mediated Na + transport. The mechanism of this regulation is controversial. Recently, we have found evidence for a close association of wild type (WT) CFTR and WT ENaC, further underscoring the role of ENaC along with CFTR in the pathophysiology of CF airway disease. In this study, we have examined the association of ENaC subunits with mutated ΔF508-CFTR, the most common mutation in CF. Deletion of phenylalanine at position 508 (ΔF508) prevents proper processing and targeting of CFTR to the plasma membrane. When ΔF508-CFTR and ENaC subunits were co-expressed in HEK293T cells, we found that individual ENaC subunits could be co-immunoprecipitated with ΔF508-CFTR, much like WT CFTR. However, when we evaluated the ΔF508-CFTR and ENaC association using fluorescence resonance energy transfer (FRET), FRET efficiencies were not significantly different from negative controls, suggesting that ΔF508-CFTR and ENaC are not in close proximity to each other under basal conditions. However, with partial correction of ΔF508-CFTR misprocessing by low temperature and chemical rescue, leading to surface expression as assessed by total internal reflection fluorescence (TIRF) microscopy, we observed a positive FRET signal. Our findings suggest that the ΔF508 mutation alters the close association of CFTR and ENaC.",
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AU - Qadri, Yawar J.

AU - Cormet-Boyaka, Estelle

AU - Rooj, Arun K.

AU - Lee, William

AU - Parpura, Vladimir

AU - Fuller, Cathy M.

AU - Berdiev, Bakhrom K.

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