Structural analysis and functional expression of vasopressin water channel

应用分子生物技术的方法研究了水通道蛋白-2抗利尿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所取代，包括大肠杆菌的甘油促进剂GlpF和透镜体的非特异性通道MIP。嵌合体在爪蟾卵母细胞和哺乳动物上皮细胞中表达，并分析部分AQP2的结构与功能关系。结果表明，AQP2的水孔由排列整齐的6个跨膜片段组成，呈α -螺旋结构。仅通过水分子的收缩，从而说明选择性水渗透性，分别由外质侧和内质侧的第三和第四亲水环产生。此外，功能表达研究表明，AQP2羧基端内质区结构对AQP2的质膜表达至关重要。特别是羧基末端在抗利尿激素调控的AQP2易位中起着至关重要的作用，这是既往研究揭示的抗利尿激素在集管细胞中的关键特征。AQP2羧基末端的丝氨酸256被蛋白激酶A磷酸化，丝氨酸256的突变减少了抗利尿激素调节的AQP2胞吐。因此，证明了丝氨酸256的磷酸化是血管抑制调节AQP2运输所必需的。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，还通过对AQP2的修饰进行调节，AQP2是囊泡的主要货物。

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.