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Visualizing the CFTR and ENaC association in living cells
Author(s) -
Berdiev Bakhrom K,
CormetBoyaka Estelle,
OosterveldHut Ria,
Tousson Albert,
Kovacs Gergely Gy,
Qadri Yawar,
Fuller Cathy,
Lukacs Gergely,
Benos Dale J
Publication year - 2007
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.21.5.a547-b
Subject(s) - förster resonance energy transfer , immunoprecipitation , chemistry , epithelial sodium channel , hek 293 cells , biophysics , fluorescence , biochemistry , biology , gene , sodium , physics , organic chemistry , quantum mechanics
The molecular events underlying CFTR‐ENaC coupling, two major transport proteins implicated in pathophysiology of Cystic Fibrosis, is obscure. In an attempt to examine the intermolecular interaction of these proteins we used fluorescence resonance energy transfer (FRET) microscopy. FRET is distance‐dependent imaging approach to measure protein‐protein interactions. To measure FRET we used acceptor bleaching, and monitored the donor's fluorescence lifetime by frequency domain fluorescence lifetime imaging microscopy (FLIM). These FRET measurements were complemented by coimmunoprecipitation experiments. Apparent FRET efficiencies ( E ) averaged ~8% but values as high as ~17% were observed, significant when compared to the negative control and comparable to E reported in other FRET studies. No appreciable FRET signal was observed when ECFP‐ClC1 was co‐transfected with αβ γ‐ENaC (<1%). When CFTR and ENaC subunits were over‐expressed in HEK293T cells, we found that β‐ENaC could be co‐immunoprecipitated with CFTR. Under the same condition, we were unable to co‐immunoprecipitate β‐ENaC with ECFP‐ClC1. Our results place CFTR and ENaC in close proximity to each other, suggestive of direct interaction between these two proteins. Supported by NIH 2RO1‐DK37206‐15, NIH P50 DK53090‐05.