| Research Abstract |
Overall, this research has proceeded very well and we believe most of our intended objects have been attained. 1) Clarification of regulation mechanisms of membrane transport proteins : We found that phosphorylation of the C-terminal of AQP2 is a determinant of its intracellular trafficking, and serine 256 is the site for phosphorylation. We observed that the AQP2 C-terminal binds to PDZ domains, and several PDZ proteins are present in the collecting duct cells. We are now identifying which one actually binds and how it alters AQP2's functions. We also isolated several cDNA clones that bind to CLC-3 and-5 by using 2-hybrid system. Their physiological functions are now under extensive examinations. 2) Transport protein diseases and their cellular pathophysiology : Gene targeting of CLC-K1 causing its knockout showed nephrogenic diabetes insipidus, confirming CLC-K1 as a chloride channel in thin ascending limb of Henle. We also analyzed gene mutations in human hereditary diseases and examined their in vitro functional expression for the cases of CLC-5 and Dent disease, AQP2 and NDI, and EnaC and Liddle syndrome. The results identified the presence of many novel regulation mechanisms of membrane proteins. 3) Kidney nephron specific expression : ClC-K1, ClC-K2, and AQP2 show kidney and nephronspecific expression. Understanding of these expressional regulation is critical for future nephron specific therapeutic manipulations. We made transgenic mice harboring promoters of ClC-K1, ClC-K2, and AQP2, and succeeded to show Henle loop-specific expression by ClC-K2. We are preparing to prepare whole nephron segments-specific promoters.
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