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Probing of the CFTR and an epithelial Na channel association by Fluorescence Resonance Energy Transfer imaging and biochemical approaches
Author(s) -
Berdiev Bakhrom K.,
CormetBoyaka Estelle,
Meltzer Robert H.,
Anderson Sue J.,
Fuller Cathy M.,
Benos Dale J.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a797-b
Subject(s) - epithelial sodium channel , förster resonance energy transfer , cystic fibrosis transmembrane conductance regulator , chemistry , cystic fibrosis , hek 293 cells , secretion , transfection , microbiology and biotechnology , medicine , biology , fluorescence , biochemistry , gene , physics , organic chemistry , quantum mechanics , sodium
Cystic Fibrosis (CF) is a genetic disease that results from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The mutations cause inadequate functioning of CFTR, which in turn leads to severe disruption in transport function of several epithelia. Decreased Cl secretion and increased Na absorption are the main contributing factors in the pathogenesis of CF lung disease and gradual lung failure is the most major life limiting factor in patients with CF. Decreased Cl secretion is a consequence of defective CFTR, while increased Na absorption results from the failure of CFTR to restrict Na absorption through an Epithelial Na + Channel (ENaC). The mechanism by which CFTR regulates ENaC is still obscure. We have used Fluorescence Resonance Energy Transfer (FRET) imaging and biochemical approaches to study the molecular events underlying reciprocal CFTR and ENaC coupling. HEK cells co‐transfected with vectors encoding CFTR N terminally tagged with CFP and αβγ‐ENaC (α C‐terminally tagged with YFP) exhibited higher FRET than the negative control (CFP and YFP co‐transfected cells), while CFP‐CFTR co‐expressed with αENaC YFP (without β and γ ENaC) does not exhibit strong FRET. When wild type CFTR and human ENaC subunits were over‐expressed in HEK293T cells, we found that β ENaC could be co‐immunoprecipitated with CFTR. Our results favor the possibility that CFTR might influence ENaC activity by means of a direct intermolecular interaction. Supported by NIH 2RO1‐DK37206‐15, NIH P50 DK53090‐05. Drs. Berdiev and Meltzer are the recipients of the UAB Cell Adhesion and Matrix Research FRET Microscopy Workshop Scholarship