Structure-Function Studies of Aquaporin 0 in Lens Development and Physiology

项目摘要 白内障，即眼睛透镜的混浊，是全世界失明的主要原因。 水通道蛋白0（AQP 0）是透镜中最丰富的膜蛋白，其功能是调节晶状体的生长。 水通道和作为粘附蛋白。AQP 0的缺陷可导致白内障， 并且对透镜的形成具有不利影响。尽管它在透镜中起着关键作用 在生理学上，AQP 0的功能尚未完全了解。我们提出的研究 旨在促进我们对AQP 0水渗透率（Pf）的理解， 其控制是维持透镜透明度所必需的，由Ca 2+和质子调节， 其浓度取决于透镜内的AQP 0位置。拟议 研究还试图鉴定对Pf调节至关重要的氨基酸残基， 对于AQP 0的粘附功能，通过蛋白-蛋白和/或蛋白-膜 相互作用，并确定AQP 0的遗传修饰对透镜的影响 生理和发育。为了实现这些目标，我们将采用一种紧密耦合的多- 学科方法，在水通道蛋白研究领域内是独一无二的， 技术范围从原子/分子到细胞和有机体 水平具体而言，在目标1中，我们建议使用体外非洲爪蟾卵母细胞渗透性 对一组哺乳动物和鱼类AQP 0突变体进行测量，以评估 特定残基对AQP 0 Pf的贡献及其调节，沿着粘附 对来自含有野生型和突变型Aqp 0的斑马鱼的透镜纤维细胞的测定。这些 目标1中的实验方法将通过计算机模拟多μs分子 动力学模拟，通过与实验Pf测量比较验证， 阐明了Ca 2+通过钙调蛋白结合，pH， 包括各种策略性选择的突变对Pf的影响， 哺乳动物和鱼类的水通道蛋白。目标1中提出的计算机模拟还将 解决蛋白质-蛋白质和蛋白质-膜相互作用在蛋白质中的相对作用 AQP 0的粘附功能。AIM 2将使用转基因斑马鱼来确定 水通道蛋白0对透镜的结构和功能的作用及其在 vivo.我们的工作将提高对透镜生理学和分子生物学的理解。 水通道门控机制及其受Ca 2+和pH的调节，并揭示 可以为治疗策略的未来发展提供信息的基本原则 用于延迟或消除白内障形成。

Project Summary Cataract, the opacification of the eye lens, is the leading cause of blindness worldwide. Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, functions as a water channel and as an adhesive protein. Defects in AQP0 can produce cataract, as well as have adverse effects on lens development. Despite its critical role in lens physiology, the functions of AQP0 are not fully understood. Our proposed research seeks to advance our understanding of how AQP0 water permeability (Pf), the exquisite control of which is required to maintain lens clarity, is regulated by Ca2+ and protons, whose concentrations depend on the AQP0 location within the lens. The proposed studies also seek to identify the amino acid residues that are crucial for Pf regulation and for the adhesive function of AQP0, through protein-protein and/or protein-membrane interactions, and to determine the effects of genetic modifications of AQP0 on lens physiology and development. To these ends, we will employ a tightly coupled, multi- disciplinary approach, unique within the field of aquaporin research, which employs techniques ranging in scale from the atomic/molecular to the cellular and organismal level. Specifically, in Aim 1 we propose to use in vitro Xenopus oocyte permeability measurements on a panel of mammalian and fish AQP0 mutants to assess the contribution of particular residues to AQP0 Pf and its regulation, along with adhesion assays on lens fiber cells from zebrafish containing wild-type and mutant Aqp0s. These experimental approaches will be complemented in Aim 1 with in silico multi-µs molecular dynamics simulations, validated by comparison with experimental Pf measurements, to elucidate mechanistic aspects of the influence of Ca2+ via calmodulin binding, pH, including the effects of a variety of strategically chosen mutations on the Pf of mammalian and fish AQP0s. The computer simulations proposed in Aim 1 will also address the relative role of protein-protein and protein-membrane interactions in the adhesive function of AQP0. Aim 2 will use genetically modified zebrafish to determine how AQP0 contributes to the structure and function of the lens and its development in vivo. Our work will improve understanding of lens physiology and the molecular mechanisms of water channel gating and its regulation by Ca2+ and pH, and uncover fundamental principles that could inform the future development of therapeutic strategies for delaying or eliminating cataract formation.