Molecular Cell Biological Studies of Kidney Membrane Transporter Diseases.

总体而言，这项研究进展顺利，我们相信大部分预期目标都已经达到。1)阐明膜转运蛋白的调控机制：我们发现AQP2 C端的磷酸化是其细胞内转运的决定因素，而丝氨酸256是其磷酸化的位点。我们观察到AQP2的C-末端与PDZ结构域结合，在集合管细胞中存在几种PDZ蛋白。我们现在正在确定哪一个实际上是结合的，以及它是如何改变AQP2的S功能的。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。他们的生理功能现在正在接受广泛的检查。2)转运蛋白疾病及其细胞病理生理：基因打靶导致ClC-K1基因敲除后发现肾源性尿崩症，证实ClC-K1是Henle细长升支的氯离子通道。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、AQP2和NDI以及ENAC和利德尔综合征患者的体外功能表达。这些结果证实了许多新的膜蛋白调控机制的存在。3)肾单位特异性表达：CLC-K1、ClC-K2、AQP2呈肾脏和肾脏特异性表达。了解这些表达调控对于未来的肾单位特异性治疗操作是至关重要的。我们制备了含有ClC-K1、ClC-K2和AQP2启动子的转基因小鼠，并成功地通过ClC-K2表达了Henle环特异性表达。我们正准备准备完整的肾单位节段特异性启动子。

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.