Molecular Cell Biological Studies of Kidney Membrane Transporter Diseases.

总的来说，这项研究进展得很好，我们相信我们的大部分预期目标已经实现。1)阐明膜转运蛋白的调控机制：我们发现AQP 2的C端磷酸化是其胞内运输的决定因素，丝氨酸256是磷酸化位点。我们观察到，AQP 2的C-末端结合PDZ结构域，和几个PDZ蛋白存在于集合管细胞。我们现在正在确定哪一个实际上结合，以及它如何改变AQP 2的功能。我们还利用双杂交系统分离了几个与CLC-3和CLC-5结合的cDNA克隆。他们的生理功能正在接受广泛的检查。2)转运蛋白疾病及其细胞病理生理学：CLC-K1的基因靶向导致其敲除，显示了肾源性尿崩症，证实了CLC-K1是Henle细升支中的氯离子通道。我们还分析了人类遗传性疾病中的基因突变，并检测了CLC-5和Dent病、AQP 2和NDI以及EnaC和Liddle综合征病例的体外功能表达。结果表明，存在许多新的膜蛋白的调节机制。3)肾脏肾单位特异性表达：ClC-K1、ClC-K2和AQP 2显示肾脏和肾单位特异性表达。了解这些表达调控是未来肾单位特异性治疗操作的关键。我们制作了携带ClC-K1、ClC-K2和AQP 2启动子的转基因小鼠，并成功地显示出ClC-K2的Henle环特异性表达。我们正在准备制备全肾单位片段特异性启动子。

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.