Regulation, Structure and Function of Aquaporin-6

描述(申请人提供)：水和酸碱代谢的调节是哺乳动物最重要的体内平衡功能之一。我们最近从大鼠肾脏中鉴定了一个水通道蛋白家族的新同源物，水通道蛋白-6(AQP6)。AQP6与水通道蛋白家族的其他成员有很大的不同。AQP6蛋白与H+-ATPase共定位，仅存在于肾集合管内分泌酸性的细胞内囊泡膜上。AQP6是一种pH门控的阴离子通道，而不是水通道。AQP6基因缺失的小鼠被用于研究酸碱调节、尿液浓缩和稀释能力，并探讨AQP6介导的离子转运的意义。对AQP6的结构与功能的关系进行了研究，以阐明AQP6的门控和离子渗透的独特机制。表达和纯化AQP6蛋白用于结构研究。这些在转基因动物生理学、分子生物学、电生理学和生物物理学方面的多种方法可能有助于在分子水平上定义AQP6的生物学和生理学相关性，希望为水和酸碱平衡的问题提供新的见解。 目的I.AQP6基因缺失小鼠的表型分析。一种基因打靶的方法已经被应用于创造一种缺乏AQP6的小鼠。肾脏清除研究将在代谢笼中进行，在不同的条件下进行，包括水负荷或缺水以及慢性酸中毒或碱中毒。将进行分离的灌流肾小管研究，以检查AQP6在水、质子和碳酸氢盐运输中的作用。 目的II.AQP6的结构与功能关系。将引入定点突变来确定AQP6离子渗透和门控的关键残基。突变体的功能将通过非洲爪哇卵母细胞的渗透水透性和离子电导来评估。膜片钳分析将用于表征AQP6的单通道特性，并检测一氧化氮对哺乳动物细胞中AQP6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.