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Mapping Critical Residues in the Epithelial Sodium Channel (LB292)
Author(s) -
Hayford Corey,
StewartEbert Lauren,
Booth Rachell
Publication year - 2014
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.28.1_supplement.lb292
Subject(s) - epithelial sodium channel , mutant , yeast , microbiology and biotechnology , western blot , sodium , chemistry , biology , biochemistry , gene , organic chemistry
The epithelial sodium channel (ENaC) is responsible for sodium re‐absorption in the kidneys, lungs, and other epithelial tissues, which contributes to overall fluid homeostasis. While the intracellular termini of each of the three ENaC subunits (α, β, and γ) are necessary for insertion and retrieval of functional ENaCs, the role of the extracellular loop region is rudimentary. Expression of functional ENaC in yeast cells induces a salt sensitive phenotype, which was used to screen mutant ENaCs for function. In an effort to identify critical residues in the extra‐cellular region, error‐prone polymerase chain reaction (EP‐PCR) was used to generate random mutations within this region. Twelve mutant αENaCs were expressed in yeast and screened in a yeast pronging assay for function. Three αENaC mutants (CH3, CH5, and CH7) demonstrated no salt sensitivity (i.e. loss of functional ENaC). Expression of ENaC mutants in yeast lacking salt sensitivity was verified by western blot and mutant ENaC DNA was sequenced. Further characterization of these three mutant ENaCs is currently underway. Grant Funding Source : Supported by NIH R15 GM86798 (R. Booth & W. David), Welch Foundation Departmental Grant AI‐0045, NSF CHE‐1156579, and Research Corporation CC6531 (R. Booth)