CALCIUM DYNAMICS AND INTRACELLULAR SIGNALING

钙动力学和细胞内信号传导

• 批准号: 2609667
• 负责人: DAVID D FRIEL
• 金额: $ 10.68万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2609667
• 关键词: Rana; caffeine; calcium flux; calmodulin dependent protein kinase; cell membrane; enzyme activity; intracellular; intracellular transport; ion transport; membrane transport proteins; neurons; neuropharmacology; ryanodine; sympathetic nervous system

Ca2+ is a critical link between many physiological stimuli and their intracellular effectors. In neurons, stimulus-evoked changes in the cytosolic free Ca2+ concentration ([Ca2+]i) influence enzyme activity, membrane excitability, exocytosis, and gene expression, and cytotoxic [Ca2+]i levels contribute to cell death in neurologic disease. Functional and molecular studies have described Ca2+ channels, pumps and exchangers that regulate [Ca2+]i, and enzymes whose activities depend on [Ca2+]i. However, little is known about how Ca2+ transporters define the time course of [Ca2+]i during stimulation, or the way time-varying [Ca2+] signals influence enzyme activity. The proposed research will examine how various Ca2+ transport systems shape the time course of [Ca2+]i during stimulation, and how a Ca2+-sensitive enzyme is influenced by [Ca2+]i dynamics. Bullfrog sympathetic neurons (BFSN's) will provide the model cell, Ca2+/calmodulin-dependent protein kinase II (CaMKII) will serve as the model enzyme, and Ca2+ indicator dyes, electrophysiological techniques, and protein kinase activity assays will be employed to examine the following topics: * Plasma membrane Ca2+ transport during stimulation. During membrane depolarization, [Ca2+]i rises under the influence of voltage-dependent Ca2+ entry, but the time-course of [Ca2+]i reflects other Ca2+ transporters as well. The net Ca2+ flux across the plasma membrane will be separated into contributions from Ca2+ entry through channels and Ca2+ extrusion via pumps and exchangers. Transport rates and their dependence on Ca2+ concentration, membrane potential and time will be examined to establish macroscopic transport rate laws. * Contributions from intracellular pools. I have proposed that a caffeine- and ryanodine-sensitive store (CSS) modulates [Ca2+]i responses to depolarization in BFSN's. The net Ca2+ flux between the CSS and cytosol will be separated into contributions from Ca2+ uptake and release, including Ca2+-induced Ca2+ release. Measurements will also be made of changes in the free Ca2+ level within the CSS during stimulation and the underlying Ca2+ fluxes. Along with information about plasma membrane transport. these data will elucidate how the time course of [Ca2+] during stimulation is defined, and its sensitivity to transport regulation. * Temporal control of CaMKII by [Ca2+]i. CaMKII is a ubiquitous [Ca2+]i- sensitive enzyme, and depolarization changes its activity by elevating (Ca2+]i. The kinetics of enzyme activation and deactivation will be examined in cell populations following imposed changes in [Ca2+]i. Predicted effects on CaMKII activity of temporal patterns of stimulation and Ca2+ transport modulation will also be tested.

Ca2+ 是许多生理刺激及其作用之间的关键联系 细胞内效应器。在神经元中，刺激引起的变化 胞质游离 Ca2+ 浓度 ([Ca2+]i) 影响酶活性， 膜兴奋性、胞吐作用、基因表达和细胞毒性 [Ca2+]i 水平导致神经系统疾病中的细胞死亡。功能性 分子研究描述了 Ca2+ 通道、泵和交换器 调节 [Ca2+]i 的酶，以及其活性依赖于 [Ca2+]i 的酶。 然而，人们对 Ca2+ 转运蛋白如何定义时间知之甚少 刺激期间 [Ca2+]i 的过程，或随时间变化的 [Ca2+] 方式 信号影响酶活性。拟议的研究将探讨如何 各种 Ca2+ 转运系统塑造 [Ca2+]i 的时间过程 刺激，以及 Ca2+ 敏感酶如何受到 [Ca2+]i 的影响 动力学。牛蛙交感神经元（BFSN）将提供模型 细胞中，Ca2+/钙调蛋白依赖性蛋白激酶 II (CaMKII) 将充当 模型酶和 Ca2+ 指示剂染料，电生理学 技术和蛋白激酶活性测定将用于检查 以下主题： * 刺激期间质膜 Ca2+ 转运。膜期间 去极化，[Ca2+]i 在电压依赖性影响下上升 Ca2+ 进入，但 [Ca2+]i 的时间进程反映了其他 Ca2+ 运输商也是如此。穿过质膜的净 Ca2+ 通量将为 分为通过通道进入 Ca2+ 和 Ca2+ 的贡献 通过泵和交换器挤出。运输费率及其依赖性 将检查 Ca2+ 浓度、膜电位和时间 建立宏观运输费率规律。 *来自细胞内库的贡献。我建议咖啡因- 兰尼碱敏感库 (CSS) 调节 [Ca2+]i 响应 BFSN 中的去极化。 CSS 和细胞质之间的净 Ca2+ 通量 将分为 Ca2+ 吸收和释放的贡献， 包括 Ca2+ 诱导的 Ca2+ 释放。还将进行测量 刺激期间 CSS 内游离 Ca2+ 水平的变化 潜在的 Ca2+ 通量。以及有关质膜的信息 运输。这些数据将阐明 [Ca2+] 的时间进程如何 刺激被定义，及其对运输调节的敏感性。 * [Ca2+]i 对 CaMKII 的时间控制。 CaMKII 是一种普遍存在的 [Ca2+]i- 敏感酶，去极化通过提高其活性来改变 (Ca2+]i。酶激活和失活的动力学为 在 [Ca2+]i 发生变化后对细胞群进行检查。 刺激时间模式对 CaMKII 活性的预测影响 Ca2+运输调节也将被测试。