Liu Kun, Nagase Hiroaki, Huang Chunyi George, Calamita Giuseppe, Agre Peter
Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
Biol Cell. 2006 Mar;98(3):153-61. doi: 10.1042/BC20050026.
Aquaporins (AQPs) are a family of channels permeable to water and some small solutes. In mammals, 13 members (AQP0-AQP12) have been found. AQP8 is widely distributed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to water, urea and ammonium, but no direct characterization had yet been reported.
We expressed recombinant rAQP8, hAQP8 and mAQP8 (rat, human and mouse AQP8 respectively) in yeast, purified the proteins to homogeneity and reconstituted them into proteoliposomes. Although showing high sequence similarity, AQP8 proteins from the three species had to be purified with different detergents prior to reconstitution. In stopped-flow studies, all three AQP8 proteoliposomes showed water permeability, which was inhibited by mercuric chloride and rescued by 2-mercaptoethanol. rAQP8 and hAQP8 proteoliposomes did not transport glycerol or urea but were permeable to formamide, which was also inhibited by mercuric chloride. In the oocyte transport assay, hAQP8-injected oocytes showed significantly higher [14C]methylammonium uptake than water-injected oocytes.
In the present study, we successfully purified rAQP8, hAQP8 and mAQP8 proteins and characterized their biochemical and biophysical properties. All three AQP8 proteins transport water. rAQP8 and hAQP8 are not permeable to urea or glycerol. Moreover, hAQP8 is permeable to ammonium analogues (formamide and methylammonium). Our results suggest that AQP8 may transport ammonium in vivo and physiologically contribute to the acid-base equilibrium.
水通道蛋白（AQPs）是一类对水和一些小分子溶质具有通透性的通道家族。在哺乳动物中，已发现13个成员（AQP0 - AQP12）。AQP8广泛分布于许多组织和器官中。先前在蛙卵母细胞中的研究表明，AQP8对水、尿素和铵具有通透性，但尚未有直接的特性描述报道。
我们在酵母中表达了重组的rAQP8、hAQP8和mAQP8（分别为大鼠、人类和小鼠的AQP8），将蛋白质纯化至同质，并将它们重构成蛋白脂质体。尽管三种物种的AQP8蛋白具有高度的序列相似性，但在重构之前必须用不同的去污剂对其进行纯化。在停流研究中，所有三种AQP8蛋白脂质体均表现出水通透性，这被氯化汞抑制并被2 - 巯基乙醇挽救。rAQP8和hAQP8蛋白脂质体不转运甘油或尿素，但对甲酰胺具有通透性，这也被氯化汞抑制。在卵母细胞转运试验中，注射hAQP8的卵母细胞比注射水的卵母细胞显示出显著更高的[14C]甲基铵摄取。
在本研究中，我们成功纯化了rAQP8、hAQP8和mAQP8蛋白，并对其生化和生物物理特性进行了表征。所有三种AQP8蛋白都能转运水。rAQP8和hAQP8对尿素或甘油不通透。此外，hAQP8对铵类似物（甲酰胺和甲基铵）具有通透性。我们的结果表明，AQP8可能在体内转运铵，并在生理上有助于酸碱平衡。
