Regulation, Structure and Function of Aquaporin-6

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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.