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The development of a quick screen in yeast for functional epithelial sodium channels (ENaC)
Author(s) -
Ybanez Raquel V,
Boswell Mikki G,
Booth Rachell E
Publication year - 2009
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.23.1_supplement.698.6
Subject(s) - epithelial sodium channel , yeast , mutant , saccharomyces cerevisiae , acid sensing ion channel , biology , microbiology and biotechnology , sodium , chemistry , biochemistry , ion channel , gene , receptor , organic chemistry
The epithelial sodium channel (ENaC) is essential for sodium re‐absorption in the distal nephron of the kidney, thus, plays a critical role in fluid maintenance. In an effort to identify critical regions within the channel subunits, we have developed a quick screen for functional ENaC in yeast. In this system, ENaC genes were co‐transformed into Saccharomyces cerevisiae strain S1InsE4A on individual yeast expression vectors with different selectable markers . Western blot analysis was used to confirm ENaC production, immunocytochemistry was used to demonstrate membrane localization of subunits, and survival pronging dilutions were used to assay salt sensitivity. Five different αENaC mutants with various levels of expected function were then screened for salt sensitivity. Our results demonstrated various levels of salt sensitivity indicating that when expressed in yeast, survival pronging can be used to detect variations in ENaC mutant function. Additionally, we found that αENaC with a C‐terminal tag is not functional (i.e. no salt sensitivity) in yeast; however, function seems unaffected by an tag at the N‐terminal. This yeast system will enable quick screening of additional mutations in ENaC subunits that may affect function. Funding has been provided by Research Corporation, the Welch Foundation, and Texas State University‐San Marcos.