Regulated Na+ reabsorption at the distal nephron, in part, controls blood pressure in humans and other terrestrial vertebrates. Activity of the aldosterone-sensitive epithelial Na+ channel (ENaC) is limiting for Na+ transport here. Dysfunction and inappropriate regulation of ENaC result in blood pressure disorders and improper salt handling by the kidney, colon and lungs. This proposal is a competitive renewal of an R01 investigating aldosterone regulation of ENaC that has had much success during its initial funding period. Results from this initial funding period, as well as, newer preliminary results strongly suggest that phospholipid kinases, including phosphatidylinositide 3-kinase (PI3-K), and their product phosphatidylinositides are critical determinants of ENaC activity. Two physiologically important hormones that increase ENaC activity, aldosterone and insulin, increase P3-K activity and synthesis of its product phosphatidylinositol, phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), in distal nephron epithelia. The cellular mechanism underpinning this increase remains unknown. The extent to which PI3-K is the primary arbiter of aldosterone and insulin actions on ENaC, in addition, remains unexplored. Moreover, possible mechanisms and physiological consequences of PI(3,4,5)P3 modulation of ENaC are not fully appreciated. Putative molecular determinants within ENaC responsive to PI(3,4,5)P3 signaling, similarly, remain to be identified. The general hypothesis that PI3-K and its phosphatidylinositide products are central to regulation of ENaC unites the experiments and ideas of this proposal. This line of inquiry is a logical extension of my earlier studies, and I expect testing it to result in novel and significant findings. Here, I address three specific aims: 1) Delineate and assign physiological significance to PI3-K and PI(3,4,5)P3 signaling pathways targeting ENaC;2) Determine the cellular mechanism of PI(3,4,5)P3 regulation of ENaC;and 3) Establish the molecular determinants within ENaC of PI(3,4,5)P3 regulation. I test my hypotheses with a comprehensive experimental design structured with complementary experiments to provide an integrative understanding of ENaC regulation by PI3-K from the molecule to the whole tissue. Proper Na+ reabsorption at the distal renal nephron in humans and other terrestrial vertebrates is central to regulation of systemic Na+ balance and thus, blood pressure. Activity of the aldosterone-sensitive epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption here. The current proposal continues our investigation of the cellular and molecular mechanisms controlling ENaC activity in response to aldosterone.