Low temperature and chemical rescue affect molecular proximity of DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC).

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 ?F508-CFTR, the most common mutation in CF. Deletion of phenylalanine at position 508 (?F508) prevents proper processing and targeting of CFTR to the plasma membrane. When ?F508-CFTR and ENaC subunits were co-expressed in HEK293T cells, we found that individual ENaC subunits could be co-immunoprecipitated with ?F508-CFTR, much like WT CFTR. However, when we evaluated the ?F508-CFTR and ENaC association using fluorescence resonance energy transfer (FRET), FRET efficiencies were not significantly different from negative controls, suggesting that ?F508-CFTR and ENaC are not in close proximity to each other under basal conditions. However, with partial correction of ?F508-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 ?F508 mutation alters the close association of CFTR and ENaC.

SUBMITTER: Qadri YJ 

PROVIDER: S-EPMC3351335 | biostudies-other | 2012 May

REPOSITORIES: biostudies-other