Hypertension affects millions of people in the United States and worldwide. Improved understanding of the molecular and cellular origins of hypertension will improve the efficacy of treatment and diagnosis. The Epithelial Na+ Channel, ENaC, is the final arbiter of Na+ excretion in the kidneys. As such, discretionary control of ENaC fine-tunes renal excretion. Appropriate renal excretion is a key factor in the normal regulation of arterial blood pressure. Consequently, dysfunction of ENaC and its upstream modulators cause dysregulation of blood pressure due to abnormal excretion. Casein kinase 2 (CK2) is known to phosphorylate ENaC. The physiological importance of this, though, is obscure. Our preliminary results demonstrate that phosphorylation by CK2 is necessary for normal ENaC activity and renal Na+ excretion. The CK2 phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. This site in ENaC shares similarity to CK2 phosphorylation sites in the unrelated NaV and KCNQ channels, which also lie within “anchor” motifs. Phosphorylation of NaV and KCNQ channels by CK2 acts as a molecular “switch” favoring the binding of ankyrin-3 (Ank-3). The binding of Ank-3 facilitates the proper membrane localization of these channels increasing their activity. The targeted deletion of Ank-3 in principal cells (PC) significantly decreased ENaC activity in our PC-Ank-3 KO mouse. In consideration of our strong preliminary results and the possible convergent evolution shaping regulation of ENaC, NaV and KCNQ by CK2, we propose testing the premise that phosphorylation of ENaC by CK2 within “anchor” motifs is necessary and sufficient for Ank-3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion. We will test these ideas using a multidisciplinary approach that includes novel thinking and tools, including PC-specific CK2 and Ank-3 KO mice, and a high degree of rigor in conjunction with a research design that is broad in scope asking questions about molecular and cellular mechanisms as well as whole animal physiological consequences. The following specific aims will be used to test our ideas: 1) To determine the cellular and molecular mechanisms of CK2 regulation of ENaC activity; 2) To quantify the physiological function of CK2 regulation of ENaC; and 3) To determine if CK2 regulation of ENaC is conserved in humans. If CK2 regulation of ENaC is to be of clinic and physiological importance such regulation must be conserved across phyla particularly in humans. Our pioneering efforts to quantify ENaC activity in tubules from healthy donor human kidneys allows us to test our ideas in the most relevant setting possible: the human principal cell within the native collecting duct. After accomplishing these aims, we will know if, how and when CK2 phosphorylation of ENaC functions as a “switch” to favor Ank-3 binding to increase channel activity to include having a d...