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）：眼睛研究中最迷人和临床上最重要的问题之一是，在没有血液供应和代谢能力有限的情况下，镜头如何保持透明度和稳态。当前的模型表明，镜头建立了一个原始的“循环系统”，该镜头在整个镜头中移动离子，水和小分子。了解该系统负责的机制至关重要的是识别和表征包括途径的通道和转运蛋白，然后通过镜头分子，离子和水。该提案针对广泛的细胞到细胞途径的调节的结构和机理，该途径直接传达了镜头表面和内部纤维的细胞质。如此广泛的途径带来了隐含的危险，即单个纤维的损害会伤害整个镜头。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节。由于纤维的独特形态和有限的新陈代谢，调节不太可能涉及开放/关闭细胞间通道或将半通道插入/检索到质膜中。因此，我们将测试一种涉及蛋白激酶C磷酸化的替代调节机制，将通道拆卸为半通道，然后将其重新分布到脂质筏中。该机制的主要优点是蛋白质在质膜的细胞质侧的聚类，信号级联的扩增以及脂质筏的形成是动态且可逆的事实。具体而言，我们建议识别和量化connexin43（CX43），Connexin46（CX46），Connexin50（CX50），Caveolin-1和2以及差距连接，脂质柱和血浆膜中的蛋白激酶C伽马型。我们将使用大鼠的透镜，其中磷酸化已被植物酯和小鼠刺激，而PKC已在遗传上消融。我们将结合生化和冷冻裂缝 - 免疫标记（FRIL），这种方法可以鉴定其天然环境中的蛋白质，具有高空间分辨率（约3 nm），并且以定量方式。我们还建议研究在天然纤维环境和爪蟾卵母细胞表达系统中连接蛋白和水通道通道的分布和相互作用。