Structure and Function of Lens Channels

镜头通道的结构和功能

• 批准号: 7418287
• 负责人: GUIDO A ZAMPIGHI
• 金额: $ 29.4万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-08-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7418287
• 关键词: AQP1 gene; Ablation; Active Biological Transport; Anterior; Antibiotics; Biochemical; Blood Vessels; Blood capillaries; Brain; Caliber; Cardiovascular system; Cations; Cell membrane; Cells; Chemicals; Cholesterol; Complex; Connexin 43; Connexins; Crystallins; Cytoplasm; Diffusion; Elements; Environment; Epithelium; Equilibrium; Exhibits; Extracellular Space; Eye; Family; Fiber; Freeze Fracturing; Gap Junctions; Genetic; Glucose; Homeostasis; Immune; Intercellular Fluid; Investigation; Ions; Knockout Mice; Label; Lateral; Life; Light; Liver; MIP gene; Mediating; Membrane Lipids; Membrane Microdomains; Membrane Potentials; Metabolic; Metabolism; Methods; Modeling; Molecular; Morphology; Movement; Mus; Na(+)-K(+)-Exchanging ATPase; Neuraxis; Nutrient; Organ; Organelles; Osmotic Pressure; Pathway interactions; Phorbol Esters; Phosphorylation; Protein Isoforms; Protein Kinase C; Proteins; Range; Rattus; Refractive Indices; Regulation; Research; Resolution; Rest; Retina; Retrieval; Side; Signal Transduction; Structure; Surface; System; Testing; Thick; Tissues; Vascular blood supply; Vesicle; Waste Products; Water; Xenopus laevis; Xenopus oocyte; Yeasts; base; capillary; caveolin 1; density; extracellular; fascinate; injured; lens; lens transparency; light scattering; novel; protein kinase C gamma; protein structure; radius bone structure; research study; size; small molecule; success; trafficking; water channel

DESCRIPTION (provided by applicant): One of the most fascinating and clinically important questions in Eye Research is how the lens can maintain transparency and homeostasis in the absence of blood supply and with a limited metabolic capacity. Current models propose that the lens establishes a primitive "circulatory system" that move ions, water and small molecules throughout the lens. Critical to understanding the mechanisms responsible for this system is the identification and characterization of the channels and transporters comprising the pathways followed by the small molecules, ions and water through the lens. This proposal is directed at unveiling the structure and mechanisms of regulation of the extensive cell-to-cell pathway that communicates directly the cytoplasm of fibers in the lens surface and interior. Such an extensive pathway carries the implicit danger that damage of a single fiber injures the entire lens. To avoid this danger, the pathway is tightly regulated at the cellular and molecular levels. Because of the unique morphology and limited metabolism of the fibers, it is unlikely that regulation involves the opening/closing of cell-to-cell channels or the insertion/retrieval of hemi-channels into the plasma membrane. Consequently, we will test an alternative regulatory mechanism involving protein kinase C phosphorylation, disassembly of channels into hemi-channels and re-distribution into lipid rafts. The principal advantages of this mechanism are the clustering of proteins in the cytoplasmic side of the plasma membrane, amplification of the signaling cascade as well as the fact that lipid rafts formation is dynamic and reversible. Specifically, we propose to identify and quantify connexin43 (Cx43), connexin46 (Cx46), connexin50 (Cx50), caveolin-1 and 2 and the protein kinase C gamma isoform in gap junctions, lipid rafts and the plasma membrane. We will use lenses of rats where phosphorylation has been stimulated with phorbol esters and mice where the PKC has been genetically ablated. We will combine biochemical and freeze-fracture-immuno-labeling (FRIL), a method that identifies proteins in their native environment, with high spatial resolution (approximately 3 nm) and in a quantitative manner. We also propose to study the distribution and interaction of connexin and aquaporin channels in the native fiber environment and in the Xenopus oocyte expression system.

描述（由申请人提供）：眼科研究中最引人入胜且临床上最重要的问题之一是晶状体如何在缺乏血液供应且代谢能力有限的情况下保持透明度和稳态。目前的模型表明，晶状体建立了一个原始的“循环系统”，可以在整个晶状体中移动离子、水和小分子。了解该系统机制的关键是识别和表征通道和转运蛋白，这些通道和转运蛋白包括小分子、离子和水通过晶状体所遵循的路径。该提案旨在揭示直接沟通晶状体表面和内部纤维细胞质的广泛细胞间通路的结构和调节机制。如此广泛的路径存在潜在的危险，即单根光纤的损坏会损害整个晶状体。为了避免这种危险，该途径在细胞和分子水平上受到严格调控。由于纤维独特的形态和有限的代谢，调节不太可能涉及细胞间通道的打开/关闭或半通道插入/收回质膜。因此，我们将测试另一种调节机制，涉及蛋白激酶 C 磷酸化、通道分解为半通道以及重新分布为脂筏。该机制的主要优点是蛋白质在质膜的细胞质侧聚集、信号级联放大以及脂筏形成是动态且可逆的。具体来说，我们建议鉴定和定量缝隙连接、脂筏和质膜中的 connexin43 (Cx43)、connexin46 (Cx46)、connexin50 (Cx50)、caveolin-1 和 2 以及蛋白激酶 C γ 同工型。我们将使用佛波酯刺激磷酸化的大鼠晶状体和 PKC 已被基因消除的小鼠晶状体。我们将结合生物化学和冷冻断裂免疫标记 (FRIL)，这是一种以高空间分辨率（约 3 nm）定量方式识别天然环境中的蛋白质的方法。我们还建议研究连接蛋白和水通道蛋白通道在天然纤维环境和非洲爪蟾卵母细胞表达系统中的分布和相互作用。