Elucidation of Critical Residues Within the Extracellular Loop of the Epithelial Sodium Channel
Ramsdell, Kimberly J.
The epithelial sodium channel (ENaC) is a membrane protein complex located in the distal tubule of the nephron in the kidney which is responsible for the reabsorption of the final 3-5% of sodium from the urine back into the blood. ENaC is a constitutively open ion channel located in the apical membrane of epithelial cells which works in conjunction with sodium/potassium pumps and aquaporins to regulate ion concentration and fluid balance within the blood. Genetic disorders that cause mutations in ENaC have been correlated to imbalances in blood pressure making it greatly important to study the structure of this protein as it relates to function. This investigation aimed to elucidate critical residues within the ENaC extracellular loop that are critical for optimal functionality. To identify critical residues, previous work was conducted to induce random mutations through the use of error prone PCR. Mutant genes were transformed into yeast cells and preliminary functional screens were conducted. Analysis of previous yeast screening was conducted and mutants of interest were selected for further study. For this investigation, the mutant genes of interest were transformed into Saccharomyces cerevisiae yeast strain S1INsE4A and subjected to a novel yeast screen to observe changes in protein function based on growth inhibition. Mutant gene products exhibiting changes in functionality when compared to wild type ENaC were then sequenced and characterized based on number and location of point mutations and changes in amino acid sequence. Mutants containing single amino acid changes leading to an increase in ENaC function in yeast screenings were cloned into a mammalian vector. Chinese hamster ovary cells were transfected with mutant vectors and subjected to electrophysiological studies to see if increased ENaC functionality was also exhibited in this model system. The correlations of functional change in ENaC in both model systems support that the position at which the amino acid changes occurred were critical to proper ENaC function.
Epithelial sodium channel, Trans-membrane protein, ENaC, Alpha ENaC
Ramsdell, K. J. (2016). <i>Elucidation of critical residues within the extracellular loop of the epithelial sodium channel</i> (Unpublished thesis). Texas State University, San Marcos, Texas.