Regulation, Structure and Function of Aquaporin-6

描述（由申请人提供）：水和酸碱代谢的调节是哺乳动物最重要的稳态功能之一。最近，我们从大鼠肾脏中鉴定并鉴定了一种新的水通道蛋白家族同源物水通道蛋白6（AQP 6）。AQP 6与水通道蛋白家族的其他成员有很大的不同。AQP 6蛋白仅存在于肾集合管泌酸闰细胞的胞内囊泡膜上，与H+-ATP酶共定位。AQP 6作为pH门控阴离子通道而不是水通道发挥作用。利用AQP 6基因敲除小鼠研究其酸碱调节、尿浓缩和稀释能力，并探讨AQP 6介导的离子转运的意义。AQP 6的结构-功能关系的解决，以阐明AQP 6的门控和离子渗透的独特机制。表达并纯化AQP 6蛋白用于结构研究。这些转基因动物生理学，分子生物学，电生理学和生物物理学的多种方法可能有助于在分子水平上定义AQP 6的生物学和生理学相关性，希望为水和酸碱平衡问题提供新的见解。 艾姆岛AQP 6缺失小鼠的表型分析。基因靶向方法已被应用于创建缺乏AQP 6的小鼠。将在代谢笼中在不同条件下进行肾清除率研究，包括水负荷或剥夺以及慢性酸中毒或酸中毒。将进行离体灌注肾小管研究，以检查AQP 6在水、质子和碳酸氢盐转运中的作用。 Aim II. AQP 6的结构与功能关系 将引入定点突变以确定AQP 6离子渗透和门控的关键残基。突变体的功能将通过非洲爪蟾卵母细胞的渗透水渗透性和离子电导率来评估。将进行膜片钳分析以表征AQP 6的单通道特性，并检查一氧化氮对哺乳动物细胞中AQP 6的影响。异源表达系统将用于结构研究。

DESCRIPTION (provided by applicant): Regulation of water and acid-base metabolism is one of the most important homeostatic functions in mammals. We have recently identified and characterized a new homolog of aquaporin family, aquaporin-6 (AQP6) from rat kidney. AQP6 is quite different from the other members of the aquaporin family. AQP6 protein resides exclusively in the membranes of intracellular vesicles of acid secreting intercalated cells in renal collecting duct where it colocalizes with H+-ATPase. AQP6 functions as a pH-gated anion channel rather than a water channel. AQP6-null mice are used in studies of acid-base regulation, urinary concentrating and diluting capacity, and to investigate the significance of AQP6-mediated ion transport. The structure-function relationships of AQP6 are addressed to elucidate the unique mechanisms of gating and ion permeation by AQP6. AQP6 proteins are expressed and purified for structural studies. These multiple approaches in transgenic animal physiology, molecular biology, electrophysiology, and biophysics may facilitate definition of the biological and physiological relevance of AQP6 at a molecular level in hopes of providing new insight into problems in water and acid-base homeostasis. Aim I. Phenotypic analysis of AQP6-null mice. A gene targeting approach has been applied to create a mouse lacking AQP6. Renal clearance studies will be performed in metabolic cages under different conditions including water loading or deprivation and chronic acidosis or alkalosis. Isolated perfused renal tubule studies will be performed to examine the role of AQP6 in water, proton, and bicarbonate transport. Aim II. Structure-function relationship of AQP6. Site-directed mutations will be introduced to identify the key residues for ion permeation and gating of AQP6. Functions of mutants will be assessed by osmotic water permeability and ion conductance in Xenopus laevis oocytes. Patch-clamp analysis will be performed to characterize single-channel properties of AQP6 and to examine the effects of nitric oxide on AQP6 in mammalian cells. Heterologous expression system will be used for structural studies.