This project's long-term goal is a molecular understanding of the renal sites and mechanisms that regulate body K homeostasis. Recent work has identified a role for the "With No Lysine" kinases (WNK4, WNK1, and KD-WNK1) in regulating the surface expression of the K channel, ROMK. We will test the hypothesis that WNK expression and location within segments and cells is regulated such that multiple WNK isoforms function together to coordinate renal K secretion. Changes in WNK isoform abundance due to altered K and Na diet will be evaluated by immunoblot and RT-PCR. Immunolocalizations, including electron microscopy, will be used to assess each isoform's location. Segmental changes due to diet will also be assessed by immunolocalizations and by RT-PCR of isolated segments. Co-expression of WNK isoforms and ROMK in Xenopus oocytes and cultured epithelial cells will be used to assess the effect of WNK isoforms on ROMK surface expression and trafficking pathways. Studies will examine the functional mechanisms whereby WNK4 and WNK1 regulate ROMK. These studies will also test the hypothesis that KD-WNK1 may function to block the ability of WNK1 to inhibit ROMK and thereby promote K secretion by the kidney. By evaluating the possible effects of WNK isoforms on ROMK endocytosis and exocytosis, these studies will provide critical tests needed to provide a mechanistic foundation for the role of WNK isoforms in K homeostasis. To determine the role of different domains of WNK1 we will evaluate selective mutations and deletions to determine which elements are critical to the ability of WNK1 to inhibit ROMK. We will also exploit yeast twohybrid methods to map the specific sites in ROMK and WNK isoforms that mediate their interactions. In summary our aims are: 1) Does variation in K and Na diet cause changes in WNK isoform protein and mRNA expression and localization in different segments consistent with a role for them in regulating renal K secretion? 2) By what mechanism(s) do WNK isoforms affect ROMK surface expression in oocytes and cultured epithelial cells? 3) What is the molecular basis for WNK-ROMK interactions? Together these studies of the WNK kinases are designed to unravel critical details of how renal K transport is coordinated in health and disease. [unreadable] [unreadable] [unreadable]