The level of expression of epithelial sodium channels (ENaC) is the main determinant of the net sodium reabsorption by the distal nephron. The first hypothesis to be evaluated is that expression of ENaC at the cell surface is actively regulated by clathrin-mediated endocytosis which, under basal conditions, maintains a low number of channels at the apical membrane. Hormones and environmental factors can increase the expression of channels by modifying the rate of their internalization. This is achieved by modifications of amino acids located in the vicinity of the endocytic signals that change the interaction between the channel proteins and the endocytic machinery. To evaluate the role of endocytosis on ENaC expression we will transfect MDCK cells with wild-type or mutant channels lacking the endocytic signals. The level of expression of channels and the rate of endocytosis will be assessed by functional assays such as short- circuit current, and biochemical ones such as biotinylation of cell surface channels. The effects on the rate of endocytosis of channels induced by aldosterone, insulin, ADH, and luminal sodium concentration will be specifically examined. The second hypothesis to be examined is that the rate limiting step in the delivery of newly synthesized channels is the assembly of subunits. To evaluate this process we will determine the rate of delivery of newly synthesized channels to the plasma membrane; what subunit combinations result in delivery of functional channels to the plasma membrane; the half-life of the subunits in cells expressing varied subunit combinations, and the domains that participate in subunit recognition and assembly. The experimental approaches to investigate subunit interactions will consist on co-immunoprecipitation experiments, sedimentation in sucrose gradients and the two-hybrid system in yeast. These studies will provide insight into mechanisms important to the regulation of ENaC expression and sodium reabsorption by the cortical collecting tubule, and the pathogenesis of salt-sensitive hypertension.

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Yale University
New Haven
United States
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