H+-ATPase And AQP2: Regulation Of Targeting And Recycling In IMCD Cells

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DESCRIPTION (provided by applicant): IMCD cells in vivo and in culture secrete H+, mediated by an H+-ATPase, and absorb H2O mediated by aquaporin-2 (AQP2). In cultured rat IMCD cells and in intact IMCD segments, transport is regulated by SNARE mediated exocytic insertion and endocytic retrieval of vesicles carrying either the H+-ATPase or AQP2. Although exocytosis of H+-ATPase and AQP2 are independently regulated, they utilize a similar SNARE system for membrane targeting-fusion. In this proposal we will characterize the targeting process for these proteins and identify the disparate effects of SNARE regulators on H+-ATPase versus AQP2 membrane cycling. We will test the hypothesis that the B1 subunit of the plasma membrane H+-ATPase contains molecular information for targeting of the assembled H+-ATPase to the apical membrane. B1 is the isoform of the B subunit that is present in the H+-ATPase of cells specialized for proton secretion. The other isoform, B2, is present in the vesicular membrane H+-ATPase of all cells. We predict that the targeting information for the plasma membrane H+-ATPase is encoded in either the N or the C-terminal amino acids, the two regions of ATP6V1B1 that are most dissimilar from B2. Our studies will also determine the role of Munc 18-2 and snapin as SNARE regulators for H+- ATPase and AQP2. Munc 18-2, through PKC signaling, controls the apical targeting and insertion of H+-ATPase in IMCD cells by regulating its interaction with syntaxin. We hypothesize that Munc 18-2 controls plasma membrane AQP2 very differently by regulating both AQP2 exocytosis and endocytosis. We propose the following cycle: Munc prevents formation of a fusion complex by association with vesicular AQP2-VAMP. Upon PKA phosphorylation of AQP2, Munc dissociates from AQP2 and VAMP and vesicle fusion occurs delivering AQP2, VAMP and Munc to the plasma membrane. Upon dephosphorylation of plasma membrane AQP2, Munc reassociates with AQP2, concentrates AQP2 and VAMP in clathrin-coated pits where this complex of Munc-AQP2-VAMP is endocytosed into the cell interior. Lastly, the role of snapin, a recently identified SNAP and adenylate cyclase binding protein, will be evaluated. We propose that snapin regulates exocytosis of both H+-ATPase and AQP2 in IMCD cells. Upon snapin binding to SNAP-23, an increased number of SNAP-syntaxin complexes are formed which are the t-SNARE receptor for the vesicular VAMP, delivering H+-ATPase to the apical membrane. Snapin may also regulate AQP2 exocytosis by amplifying the vasopressin second message (cAMP) since it has been shown that upon PKA phosphorylation, snapin binds and activates adenylate cyclase. We believe these studies will provide novel paradigms for the regulation of these apical membrane transporters and provide newer modalities for the treatment of fluid and acid-base disorders. PUBLIC HEALTH RELEVANCE These studies will provide important new information concerning the mechanisms by which the kidney regulates water and acid-base balance and how these mechanisms are disrupted by genetic and acquired disease states. An enhanced understanding of these abnormalities could lead to the development of improved therapeutic interventions.