Function of SNAP-23 in H+-ATPase Trafficking in the IMCD

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DESCRIPTION (provided by applicant): The inner medullary collecting duct is a major location of non-volatile acid/base. The IMCD is also responsible for aquaporin- 2 (AQP-2) mediated water reabsorption from the urine. Both the proton pump (H+-ATPase) and AQP-2 are apically targeted in the IMCD cells. In this model of exocytosis, the specific association of a protein(s) on the surface of an H+-ATPase or AQP2 containing vesicle termed v-SNARE (VAMP), and its cognate partner, a membrane protein, termed t-SNARE (Syntaxin). These proteins interact to form a 7S complex, and then, with the interaction of the cytosolic proteins, NSF and SNAP form a 20S fusion complex. Both processes are rate regulated by the balance of exocytic insertion and endocytic retrieval of either the proton pump or the AQP-2 channel. The long term objectives are to elucidate the mechanisms which confer specificity to protein trafficking - which proteins are responsible for the differential regulation of AQP-2 versus H+-ATPase. Further, the role of the ubiquitous protein SNAP-23 has not been elucidated. Utilizing IMCD cell lines in culture, the role of SNAP-23 will be investigated. GFP attached SNAP-23 will be overexpressed in an IMCD cell line. Their response to different pH challenges, compared to wild-type IMCD cells, will be characterized by quantitative mRNA analysis, western blot analysis, immunocytochemical techniques and confocal microscopy. Further, Syntaxin IA and SNAP-23 trafficking and binding characteristics will be examined.