Regulation, Structure and Function of Aquaporin-6

描述（由申请人提供）：调节水和酸碱代谢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发现并鉴定了水通道蛋白家族的一个新的同源物——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蛋白仅存在于肾集管分泌酸插层细胞的胞内囊泡膜中，与H+- atp酶共定位。AQP6作为ph门控阴离子通道而不是水通道。我们利用aqp6缺失小鼠进行酸碱调节、尿浓缩和稀释能力的研究，并探讨aqp6介导的离子转运的意义。研究了AQP6的结构-功能关系，阐明了AQP6独特的门控和离子渗透机制。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途径，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，希望为水和酸碱稳态问题提供新的认识。

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.