The epithelial sodium channel (ENaC) is the limiting entry point for Na+ reabsorption in the distal kidney nephron and is regulated by numerous hormones, including the mineralocorticoid hormone aldosterone. Previously we identified ankyrin G (AnkG), a cytoskeletal protein involved in vesicular transport, as a novel aldosterone-induced protein that can alter Na+ transport in mouse cortical collecting duct cells. AnkG is highly expressed in the kidney, particularly in the distal nephron. Increasing AnkG expression increases ENaC activity while depleting AnkG reduces ENaC-mediated Na+ transport. The underlying mechanism presiding over this effect; however, was unknown. Here we report that AnkG expression directly regulates Na+ transport by altering ENaC activity in the apical membrane. These changes are due to a change in ENaC directly rather than through alterations to the Na+ driving force created by Na+K+ATPase. Using a constitutively open mutant of ENaC and surface biotinylation, we demonstrate that the augmentation of Na+ transport is caused predominantly by increasing the number of ENaCs at the surface rather than alterations to open probability. To determine the mechanism of AnkG action on ENaC surface number, changes in rates of internalization, recycling, and membrane delivery were investigated. AnkG did not alter ENaC delivery to the membrane from biosynthetic pathways or removal by endocytosis; however, AnkG did alter ENaC insertion from constitutive recycling pathways. We also investigated the potential role of a putative AnkG binding domain in the C-terminus of âENaC, and whether single-site mutations of a charged residue and two regulatory phosphorylation sites could disrupt AnkG augmentation of ENaC current. We did not find any significant evidence that this region is essential for AnkG-ENaC interaction. These findings provide a mechanism to account for the role of AnkG in the regulation of Na + transport in the distal kidney nephron.
|Advisor:||Kleyman, Thomas R.|
|Commitee:||Butterworth, Michael B., DeFranco, Donald B., Devor, Daniel C., Kwiatkowski, Adam V.|
|School:||University of Pittsburgh|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 79/01(E), Dissertation Abstracts International|
|Subjects:||Biology, Cellular biology, Physiology|
|Keywords:||Anlyun G, ENaC, Epithelial traffiking, Kidney|
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