Regulation, Structure and Function of Aquaporin-6

Aquaporin-6 的调控、结构和功能

• 批准号: 7459890
• 负责人: Peter C Agre
• 金额: $ 29.72万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-15 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7459890
• 关键词: ATP phosphohydrolase; Acidosis; Acids; Address; Alkalosis; Anions; Bicarbonates; Biological; Biophysics; Cell membrane; Cells; Chronic; Computers; Condition; Duct (organ) structure; Electron Microscopy; Electrophysiology (science); Family; Functional disorder; Gene Targeting; Green Fluorescent Proteins; Homeostasis; Homologous Gene; Human Identifications; In Situ Hybridization; Insecta; Intercalated Cell; Intracellular Membranes; Ion Transport; Ions; Kidney; Knockout Mice; Mammalian Cell; Mammals; Mediating; Membrane; Metabolic; Metabolism; Modeling; Molecular; Molecular Biology; Mus; Mutation; Nitric Oxide; Oocytes; Permeability; Physiological; Physiology; Property; Proteins; Proton-Translocating ATPases; Protons; Rattus; Regulation; Renal Tissue; Renal clearance function; Renal tubule structure; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Sequence Analysis; Site; Structural Models; Structural Protein; Structure; Structure-Activity Relationship; System; Tissues; Transgenic Animals; Vesicle; Water; Wild Type Mouse; Xenopus laevis; Yeasts; aquaporin-6; base; deprivation; insight; member; mutant; patch clamp; programs; proteoliposomes; reconstitution; urinary; water channel

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.

描述（由申请人提供）：水和酸碱代谢的调节是哺乳动物中最重要的稳态功能之一。我们最近确定并描述了Rat Neidney的Aquaporin家族Aquaporin-6（AQP6）的新同源物。 AQP6与Aquaporin家族的其他成员完全不同。 AQP6蛋白仅驻留在肾脏收集导管中酸分泌的互化细胞的细胞内囊泡的膜中，并与H+-ATPase共定位。 AQP6充当pH门控阴离子通道，而不是水通道。 AQP6-NULL小鼠用于酸碱调节，尿液浓缩和稀释能力的研究，并研究AQP6介导的离子转运的显着性。 AQP6的结构功能关系是为了阐明AQP6的门控和离子渗透的独特机制。 AQP6蛋白被表达并纯化用于结构研究。这些在转基因动物生理，分子生物学，电生理学和生物物理学中的多种方法可能有助于在分子水平上定义AQP6的生物学和生理相关性，以期为水和酸碱稳态提供新的见解。 AIM I. AQP6无效小鼠的表型分析。已经应用了一种基因靶向方法来创建缺乏AQP6的小鼠。肾清除研究将在不同条件下的代谢笼中进行，包括水负荷或剥夺以及慢性酸中毒或碱性病。将进行分离的灌注肾小管研究，以检查AQP6在水，质子和碳酸氢盐转运中的作用。 目标II。 AQP6的结构功能关系。 将引入定点突变，以识别AQP6离子渗透和门控的关键残基。突变体的功能将通过渗透性水的渗透性和离子电导率评估。将进行贴片钳分析以表征AQP6的单通道特性，并检查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。

项目成果

Aquaporin 6 binds calmodulin in a calcium-dependent manner.

• DOI: 10.1016/j.bbrc.2009.03.128
• 发表时间: 2009-05-22
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Rabaud, Nicole E.;Song, Linhua;Wang, Yiding;Agre, Peter;Yasui, Masato;Carbrey, Jennifer M.
• 通讯作者: Carbrey, Jennifer M.