The role of the Epithelial Sodium Channel N-termini in regulation of activity
Berman, Jonathan Max
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The absorption of sodium through the Epithelial Sodium Channel in the nephron impacts whole body water and sodium balance. The regulation of this absorption is important and incompletely understood. This dissertation addresses this basic question through two avenues: an investigation of a poorly described alternatively spiced isoform of the ENaC alpha subunit with an extended N-terminus, and an investigation of the mechanisms which underlie proteolytic activation of ENaC. The first approach reveals that the isoform of alpha with a long N-terminus is highly active despite decreased membrane density, decreased response to proteolysis, and similar single channel conductance to the short isoform. The investigation of the mechanism of proteolytic activation demonstrates that the N termini of the alpha and gamma subunits are retained post-proteolysis and that they retain an important functional role in the regulation of activity. Together these data indicate that internalization of the alpha and gamma N termini is a rate limiting determinant of ENaC activity that may underlie a great deal of variation in sodium absorption at the population and individual level. This is a novel observation with explanatory power for a number of previous observations, including the difficulty of detecting the N terminal fragment at the membrane, multiple ubiquitination sites and an internalization motif in the N terminus, and a lower portion of electrically active channels would be expect based on protein levels.