The Development of a Quick Screen in Yeast for Functional Epithelial Sodium Channels
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Epithelial sodium channels (ENaC) are voltage independent Na+ channels found in the lining of the lungs, salivary glands, and kidneys. Situated within the apical membrane of epithelial cells, ENaC works in conjunction with a Na+/K+ ATPase to transport Na+ through the cell. In water permeable cells the movement of Na+ triggers the movement of water through the cell as well. This function is especially critical in the distal portion of the kidney tubule where Na+ and water must be reabsorbed in order to maintain appropriate blood pressure levels (1). Anomalous ENaC activity can lead to disorders such as Liddle’s syndrome, which is due to overactive ENaC and is characterized by severe hypertension, or pseudohypoaldosteronism type I, which is caused by decreased ENaC activity and results in hypotension (2).
Understanding the structure of ENaC as it relates to function is essential to understanding ENaC’s physiological role. We report development of an expression system in yeast that allows us to quickly screen the level of activity for various ENaC mutants. The screen takes advantage of ENaC’s ability to transport Na+ into the cell and cause growth inhibition (3). Yeast cells expressing wild type ENaC display a salt sensitivity phenotype when grown on high salt media, but cells expressing an ENaC mutant that is no longer functional do not. These results can easily be visually compared through the use of a survival pronging dilution assay. This will allow for future identification of loss of function mutants, which can then be further examined to determine exactly how and why they are critical for function.
Additionally, viability for utilizing the assay to screen strains from the Saccaromyces Genome Deletion Project was also examined. We report the identification of four genes required for proper ENaC function in yeast: SUR4, ERV14, EMP24, and ERV25.
CitationYbanez, R. V. (2009). The development of a quick screen in yeast for functional epithelial sodium channels (Unpublished thesis). Texas State University-San Marcos, San Marcos, Texas.
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