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

基于蛋白质结构功能分析，开发肾集合管特异性膜渗透抑制剂。

• 批准号: 08557066
• 负责人: UCHIDA Shinichi
• 金额: $ 7.81万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557066/
• 关键词: Aquaporin; Water transport; Yeast; Kidney; Collecting duct; Membrane transport; Protein Structure; AQP2; バゾプレシン; 利尿剤; 尿濃縮機構; 集合尿細管

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.

本研究拟利用酵母表达系统阐明肾集水管水通道AQP2的结构-功能关系。新开发的系统使用缺乏内源性蛋白酶的酵母菌BJ3505。酵母细胞在30度下孵育，通过蔗糖密度梯度获得a-id胞内囊泡。Western blot分析证实了AQP2蛋白在该部位的选择性表达。用止流法测定了这些囊泡的渗透透水性（Pf）。表达AQP2可诱导Pf增加22倍，表明建立了适合AQP2结构-功能关系分析的体系。利用该系统，我们测量了AQP2s突变体的Pf；突变沿着氨基酸序列引入。结果表明，当在环C和环E之间引入突变时，Pf严重受损。AQP2蛋白在囊泡中的含量在突变体中没有差异。因此，我们可以确定对AQP2水生路径至关重要的区域。我们目前正在缩小这一区域以分离出关键残基，这一信息将直接适用于膜运输抑制剂的开发。

项目成果

Fushimi,K.,et al.: "Isolation of a gene encoding nodulin-like intrinsic protein of escherichia coli." Biochemi.Mol.Biol.Int.41. 995-1003 (1997)

Fushimi,K.,et al.：“编码大肠杆菌结节蛋白样内在蛋白的基因的分离。”

Uchida,S.,et al.: "Regulation of aquaporin-2gene transcription by gate-3." Biochemi.Biophys.Res.Commun. 232. 65-68 (1997)

Uchida,S.,et al.：“gate-3 对水通道蛋白 2 基因转录的调节”。

Fushimi, K., et al.: "lsolation of a gene encoding nodulin-like intrinsic protein of escherichia coli." Biochemi Mol.Biol.Int.41. 995-1003 (1997)

Fushimi, K. 等人：“编码大肠杆菌结节蛋白样内在蛋白的基因的分离。”

Fushimi,K.: "Membrane Proteins : Structure,Function and Expression control" Karger,Basel,Switze-land, 413 (1997)

Fushimi，K.：“膜蛋白：结构、功能和表达控制”Karger，巴塞尔，瑞士，413（1997）

Ishibashi,K.,et al.: "Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water,glycerol and urea." J.Biol.Chem.272. 20782-20786 (1997)

Ishibashi,K.等人：“在可渗透水、甘油和尿素的睾丸中大量表达的新水通道的克隆和功能表达。”