Aquarporin Water Channel Proteins in Eye

描述：(申请人提供)：许多生理和 病理生理过程包括水在细胞内的运输。 膜。水转运蛋白的分子特性随着 膜水通道水通道蛋白家族的发现。十个中的四个 已知的哺乳动物水通道蛋白在眼前节表达。这 应用解决人类调节功能的分子机制 AQPO、AQP1、AQP3、AQP5及其功能障碍的临床研究 眼科疾病。 目的I.正常人眼内水通道蛋白的分析。新的试剂将是 制备包括编码野生型、定点突变体和 表位标记的人AQPO、AQP1、AQP3和AQP5。针对N-的抗体- 和人类蛋白的C末端将在兔子和 亲和力-纯化。人类水通道蛋白的生物物理功能将是 在非洲爪哇卵母细胞中的表达及其基线和后分析 激活。人水通道蛋白将在酵母中表达并纯化用于 重组成蛋白质脂质体用于渗透研究，进入平面 用于电生理特性分析的双层膜，并进入膜 用于结构研究的晶体。这些水通道蛋白的分布将是 在正常的人眼中定义的。 目的II.水通道蛋白在眼科临床疾病中的分析。这个 人类水通道蛋白的分布将被定义在患者的组织中 白内障或干燥综合征。AQPO和AQP5可能通过的基本机制 将寻求造成这些紊乱的因素，包括水中的缺陷和 溶质渗透、膜迁移、亚基齐聚和 内化，生理缺陷也将在啮齿动物模型中进行评估 AQPO降解和AQP5缺乏。

DESCRIPTION: (provided by applicant): Numerous physiological and pathophysiological processes involve the transport of water across cell membranes. The molecular identity of water transporters became known with discovery of the aquaporin family of membrane water channels. Four of the ten known mammalian aquaporins are expressed in anterior segment of eye. This application addresses the molecular mechanisms regulating function of human AQPO, AQP1, AQP3, and AQP5 and dysfunction of these proteins in clinical disorders of eye. Aim I. Analysis of aquaporin proteins in normal human eye. New reagents will be prepared including plasmids encoding wild-type, site-directed mutant, and epitope-tagged human AQPO, AQP1, AQP3, and AQP5. Antibodies specific for the N- and C-termini of the human proteins will be raised in rabbits and affinity-purified. The biophysical functions of human aquaporins will be expressed in Xenopus laevis oocytes and analyzed at baseline and after activation. Human aquaporins will be expressed in yeast and purified for reconstitution into proteoliposomes for permeation studies, into planar bilayers for analysis of electrophysiological properties, and into membrane crystals for structural studies. The distribution of these aquaporins will be defined in normal human eye. Aim II. Analysis of aquaporin proteins in clinical disorders of eye. The distribution of human aquaporins will be defined in tissues from patients with cataract or Sjogren's syndrome. Basic mechanisms by which AQPO and AQP5 may contribute to these disorders will be sought including defects in water and solute permeation, membrane trafficking, subunit oligomerization, and internalization, Physiological deficits will also be evaluated in rodent models of AQPO degradation and AQP5 deficiency.