Development of membrane permeation inhibitors which are specific for kidney collecting duct, based on protein structure-function analysis.

本研究旨在利用酵母表达系统阐明肾脏集合管水通道AQP 2的结构与功能关系。新开发的系统使用缺乏内源蛋白酶的酵母菌株BJ 3505。将酵母细胞在30 ℃下培养，用蔗糖密度梯度法获得胞内囊泡。Western blot分析证实了该组分中AQP 2蛋白的选择性表达。用停流法测定了这些囊泡的渗透水渗透性（Pf）。AQP 2的表达使Pf增加了22倍，建立了适合于分析AQP 2结构-功能关系的系统。结果表明，当在环C和环E之间引入突变时，Pf严重受损。囊泡中AQP 2蛋白的量在突变体之间没有差异。因此，我们可以确定结构域，这是至关重要的水通道AQP 2。我们目前正在缩小这一区域1 r分离关键残基，这一信息将直接适用于膜转运抑制剂的开发。

In this research, intended to clarify structure-function relationship of kidney collecting duct water channel, AQP2, by using yeast expression system. The newly developved system used a yeast strain BJ3505 which lacks endogenous protease. Yeast cells were incubated at 30 degree, a-id intracellular vesicle fration was obtained by sucrose density gradient. Western blot analysis confirmed the selective expression of AQP2 proteins in this fraction. Osmotic water permeability (Pf) of these vesicles was determined by stoped flow method. Expression of AQP2 induced 22-fold increase of Pf, demonstrating the establishment of the suitable system for analysis of structure-function relationship of AQP2.Using this system, we measured Pf of mutant AQP2s ; mutations were introduced along the aminoacid sequence. The results showed that when mutations were introduced between Loop C and Loop E, Pf was severely impaired. The amount AQP2 protein in the vesicles were not different among the mutants. Thus, we could determine the domain which is critically important for aquaous route of AQP2. We are currently narrowing this region 1r isolate critical residues, this information would be directly applicable in the development of inhibitors of membrane transport.