CALCIUM DYNAMICS AND INTRACELLULAR SIGNALING

钙动力学和细胞内信号传导

基本信息

批准号：
2839371
负责人：
DAVID D FRIEL
金额：
$ 11.1万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-12-01 至 2001-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2839371
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

项目摘要

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。