Identification of Residues Critical to Epithelial Sodium Channel Assembly
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The Epithelial Sodium Channel (ENaC) is a major determinant in fine-tuning the volume and pressure of blood within the cardiovascular system. Understanding the regulation, structural assembly, and arrangement of ENaC is needed. This study describes a method for identifying residues that participate in structural stability interactions between and within subunits as well as those critical to function. A novel yeast screen was used to describe salt-sensitive phenotypes observed in S. cerevisiae that express ENaC and show growth inhibition. Error prone polymerase chain reaction (EP-PCR) was employed to promote random mutagenesis in the extracellular loop of alpha-ENaC. The levels of growth inhibition were monitored in the yeast strain S1InsE4A and subsequently characterized. The location of the point mutations and the corresponding amino acid transitions cause varying degrees of function. This study successfully illustrates a method for inducing mutations in areas of interest in the gene and screening the resulting mutants. We also identify several mutations of the extracellular domain of alpha ENaC which are critical to function.