MECHANISM OF CALCIUM SPIKING IN SIGNAL TRANSDUCTION

信号转导中的钙尖峰机制

• 批准号: 3384995
• 负责人: LUBERT STRYER
• 金额: $ 17.56万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3384995
• 关键词: biological signal transduction; calcium channel; calcium flux; calmodulin; cyclic AMP; enzyme substrate; fluorescence microscopy; fluorescent dye /probe; immunoelectron microscopy; inositol phosphates; laboratory rat; liver cells; membrane reconstitution /synthesis; memory; neoplastic cell culture for noncancer research; neural information processing; neurotransmitters; phosphatidylinositols; pinocytosis; protein tyrosine kinase

Many cells exhibit calcium spikes (periodic transient increases in cytosolic CA2+) when stimulated by a neurotransmitter, hormone, or growth factor. The overall goal of this research is to elucidate the molecular mechanism of calcium spiking and delineate its role in signal transduction. The rat basophilic leukemic cell (RBL cell) will be studied as a model secretory cell, the rat hepatocyte as an integrator of diverse stimuli, and the PC12 cell as a model neuron. Calcium spiking will be monitored by photon-counting fluorescence microscopy of single cells containing an indicator such as fluo-3. Biochemical, fluorescence, and ultrastructural studies of intact cells, permeabilized cells, purified proteins, and reconstituted membrane assemblies will be carried out to answer: (1) How are calcium spikes generated? We will test a molecular model for calcium spiking that is based on four elements: cooperativity and positive feedback between inositol 1,4,5- trisphosphate (IP3) and cytosolic Ca2+, delayed deactivation by mitochondrial uptake of Ca2+, and reactivation by refilling of the endoplasmic reticulum Ca2+ store. Specific inhibitors will pinpoint the contributions of particular processes to spike generation. (2) How is calcium spiking modulated? The effects of calcium influx into the cell, intracellular pH, the level of phosphoinositides, and phosphorylation state of components of the spike generator will be determined. (3) How is spiking altered by the interplay of the phosphoinositide cascade with other signal transduction pathways? The modulatory actions of the cyclic AmP cascade, growth factors, voltage-sensitive calcium channels, and lithium ion will be investigated. (4) How do calcium spikes trigger effector events such as exocytosis and memory? The distribution of F- actin and myosin in stimulated RBL cells will be determined by fluorescence and immunoelectron microscopy to determine whether individual spikes induce discrete cytoskeletal rearrangements and to relate them to granule release. The Ca2+/calmodulin-dependent protein kinase of PC12 cells will be studied as a model memory protein. We will measure how calcium spikes switch this protein between different functional states as expressed by their degree of phosphorylation- autophosphorylation activity, and kinase activity for exogenous substrates such as tyrosine hydroxylase. A deeper understanding of calcium spiking is likely to reveal how digital logic is used within cells to process information and achieve timing control (as in circadian rhythms). Some neuropsychiatric disorders may arise from kinetic mismatches of components of the spike generator. Information about spiking should lead to a better understanding of the therapeutic action of lithium ion in manic-depressive disorders.

许多细胞表现出钙尖峰(周期性的一过性增加 细胞内钙离子)，当受到神经递质、激素或 生长因子。这项研究的总体目标是阐明 钙离子尖峰的分子机制及其在信号转导中的作用 转导。大鼠嗜碱性白血病细胞(RBL细胞) 作为模型分泌细胞的大鼠肝细胞作为整合子被研究 以PC12细胞为模型神经元。钙 尖峰将通过光子计数荧光显微镜进行监测 包含诸如FLOO-3的指示剂的单个细胞。生化的， 渗透的完整细胞的荧光和超微结构研究 细胞、纯化蛋白和重组膜组件将被 答案是：(1)钙离子尖峰是如何产生的？我们会 测试钙离子尖峰的分子模型，该模型基于四个 元素：肌醇1，4，5-之间的协同作用和正反馈 三磷酸(IP3)和胞内钙离子，延迟失活 线粒体对Ca~(2+)的摄取和通过再灌流而重新激活 内质网钙离子储存库。特定的抑制剂将精确地定位 特定过程对尖峰产生的贡献。(2)情况如何？ 钙离子尖峰调节？钙离子流入细胞的影响， 细胞内pH、肌醇磷脂水平和磷酸化 将确定尖峰发生器组件的状态。(3)如何 磷脂酰肌醇与磷脂酰肌醇的相互作用是否改变了尖峰 其他信号转导途径？的调节作用 循环放大级联，生长因子，电压敏感钙通道， 和锂离子将被研究。(4)钙离子峰值是如何触发的 效应器事件，如胞吐和记忆？F-的分布 受刺激的RBL细胞中的肌动蛋白和肌球蛋白将通过 荧光和免疫电子显微镜来确定 单个棘波诱导离散的细胞骨架重排并 将它们与颗粒剂释放联系起来。钙/钙调蛋白依赖蛋白 将PC12细胞的激酶作为模型记忆蛋白进行研究。我们会 测量钙离子是如何在不同的 通过它们的磷酸化程度表达的功能状态- 外源蛋白的自磷酸化活性和蛋白激酶活性 底物，如酪氨酸羟基酶。更深入地了解 钙离子峰值很可能揭示数字逻辑是如何在 细胞处理信息并实现定时控制(如在昼夜节律中 节奏)。一些神经精神障碍可能是由运动性 尖峰发生器的组件不匹配。有关以下内容的信息 尖峰应有助于更好地理解治疗作用 锂离子在躁郁症中的作用。