Structural analysis and functional expression of vasopressin water channel

应用分子生物学方法研究水通道蛋白-2加压素水通道的分子结构。水通道蛋白-2的部分潜在关键结构被其他MIP家族通道的相应部分取代，包括E.Coli的甘油促进剂GlpF和晶状体的非特异性通道MIP。在非洲爪哇卵母细胞和哺乳动物上皮细胞中表达嵌合体，并分析了AQP2部分结构与功能的关系。结果表明，AQP2的水孔是由六个排列整齐的α螺旋结构跨膜段组装而成。AQP2羧基末端的第三个和第四个亲水环分别位于胞外和内质侧，形成了一个只通过水分子的收缩，从而解释了选择性水的通透性。此外，功能表达研究表明，AQP2羧基末端的内质结构域的结构对AQP2的质膜表达至关重要。特别是，研究表明，羧基末端在加压素调节的AQP2转位中起着至关重要的作用，而AQP2是集合管细胞中加压素抗利尿的关键特征。AQP2羧基末端的丝氨酸256被蛋白激酶A磷酸化，而丝氨酸256的突变降低了加压素调节的AQP2的胞吐作用。因此，证明了AQP2的血管加压调节转运需要丝氨酸256的磷酸化。这一发现很有趣，因为囊泡运输系统不仅受囊泡相关蛋白的调节，而且还通过对囊泡的主要运输物质AQP2的修饰来调节。

Methods of molecular biotechnology has been applied to study molecular structure of aquaporin-2 vasopressin water channel. Parts of potentially critical structure of aquaporin-2 were replaced with corresponding parts of other MIP family channels, including GlpF,a glycerol facilitator of E.Coli, and MIP,a non-specific channel of lens. Chimeras were expressed in Xenopus oocytes and mammalian epithelial cells, and structure-function relations of parts of AQP2 were analyzed. It was shown that the aqueous pore of AQP2 was assembled with aligned six transmembrane segments in alpha-helical structure. A constriction that passes only water molecules, thus accounting for selective water permeability, was created with the third and fourth hydrophilic loops in ectoplasmic and endoplasmic side respectively.In addition, functional expression studies have elucidated that the structure of the endoplasmic domain of AQP2 carboxyl terminus is critically important for plasma membrane expression of AQP2. Especially, it was shown that the carboxyl terminus plays crucial roles in vasopressin-regulated translocation of AQP2, a key feature of vasopressin antidiuresis in the collecting duct cells as revealed in previous studies. The serine 256 in AQP2 carboxyl terminus is phosphorylated by protein kinase A and a mutation in the serine 256 diminished vasopressin-regulated exocytosis of AQP2. Thus, it was proved that phosphorylation of the serine 256 is required for vasopression-regulated trafficking of AQP2. This finding is interesting in that vesicle trafficking system is not only regulated by vesicle-associated proteins but also regulated through modifications on AQP2, a major cargo of vesicles.