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

项目摘要 白内障，即眼睛晶状体混浊，是世界范围内导致失明的主要原因。 水通道蛋白0(AQP0)是晶状体中含量最丰富的膜蛋白，其功能是 水通道和作为一种黏附蛋白。AQP0的缺陷会导致白内障，因为 也会对晶状体发育产生不利影响。尽管它在镜头中扮演着关键的角色 生理学方面，AQP0的功能还不完全清楚。我们建议的研究 力求增进我们对AQP0如何透水性(PF)、细腻 它的控制是维持晶状体清晰度所必需的，由钙离子和质子调节， 其浓度取决于AQP0在晶状体中的位置。建议数 研究还试图确定对PF调节和功能至关重要的氨基酸残基。 对于AQP0的黏附功能，通过蛋白质-蛋白质和/或蛋白质-膜 相互作用，并确定AQP0基因修饰对晶状体的影响 生理学和发育。为此，我们将采用紧密结合的、多方面的 在水通道蛋白研究领域独一无二的学科方法，它使用 从原子/分子到细胞和有机体的各种技术 水平。具体地说，在目标1中，我们建议使用非洲爪哇卵母细胞的体外渗透性 对一组哺乳动物和鱼类AQP0突变体的测量以评估 特定残基对AQP0-PF的贡献及其调节，以及黏附 含有野生型和突变型Aqpos的斑马鱼晶状体纤维细胞的检测。这些 实验方法将与目标1中的多微米S分子相辅相成 动力学模拟，通过与实验PF测量进行比较验证，以 阐明钙通过钙调素结合、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AQP0。在目标1中提出的计算机模拟也将 阐述蛋白质-蛋白质相互作用和蛋白质-膜相互作用在 AQP0的粘接功能。Aim 2将使用转基因斑马鱼来确定 AQP0如何对晶状体的结构和功能及其发展做出贡献 活着。我们的工作将提高对晶状体生理学和分子生物学的理解 水通道门控机制及其受钙和pH的调节 可为治疗策略的未来发展提供信息的基本原则 用于延缓或消除白内障的形成。

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.