INTRACELLULAR CONTROL OF NEURONAL CALCIUM CURRENTS

神经元钙电流的细胞内控制

基本信息

批准号：
2393103
负责人：
WILLIAM L. BYERLEY
金额：
$ 9.39万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-04-01 至 2002-03-31
项目状态：
已结题

项目摘要

The long-term objective of this project is to understand the mechanisms by which neurons control the voltage-activated Ca2+ currents in their surface membranes. These Ca2+ currents are directly responsible for the intracellular Ca2+ signals that trigger neurotransmitter release, modulate membrane excitability and synaptic efficacy, and control neurite growth. The project is focused on two phenomena that strongly change the function of Ca2+ channels, the "block" of Ca2+ channels by intracellular Ca2+ and the "rundown" of Ca2+ channels when exposed to artificial internal environments. The goal is to determine the molecular mechanisms underlying these phenomena, testing the hypothesis that common mechanisms underlie both. During the first period of this project it was found that submicromolar concentrations of Ca2+ rapidly blocked Ca2+ channels, indicating few molecular steps between Ca2+ binding and channel block. Both block by intracellular Ca2+ and rundown were found to involve the local cytoskeleton and not to depend on dephosphorylation, as has been commonly assumed. The proposal for the next period of this project is to further characterize the interaction between cytoskeleton and Ca2+ channels and to more reliably quantify the relation between intracellular Ca2+ and channel function. The role of the cytoskeleton in Controlling Ca2+ channels under more physiological conditions (without rundown) will be studied, and cytoskeletal proteins involved in Ca2+ channel control will be identified. Photolyzable Ca2+ chelators and ratiometric Ca2+ monitoring will be used to complete the quantitative study of the relationship between intracellular Ca2+ and channel function. Patch-clamp physiological techniques will be applied to acutely isolated neurons from the snail Lymnaea. Rundown will primarily be studied in inside-out patches, so that non-permeant drugs, enzymes and other proteins can be applied with certainty. Studies of Ca2+ channel block by intracellular Ca2+ will be done in two ways: direct perfusion of inside- out patches with solutions of well-buffered Ca2+ concentrations, or flash photolysis of Ca2+ chelators (DM-nitrophen, nitrophenyl-EGTA, diazo-4) in whole cells, while monitoring intracellular Ca2+ with fluorescent probes (fura-2, furaptra). This project will provide direct measurements of two of the most important components of neuronal Ca2+ regulation, the cell's ability to handle Ca2+ released inside the cell and the effect of intracellular Ca2+ on membrane Ca2+ influx. These components of Ca2+ regulation are central to understanding the Ca2+-induced cell death common to human pathologies as diverse as cerebral stroke and Alzheimer's disease.

该项目的长期目标是了解哪些神经元控制着表面的电压激活的Ca2+电流膜。这些CA2+电流直接负责触发神经递质释放的细胞内Ca2+信号调节膜兴奋性和突触功效，并控制神经突的生长。该项目的重点是强烈改变功能的两个现象 Ca2+通道，细胞内Ca2+的Ca2+通道的“块”和 Ca2+通道的“破产”暴露于人造内部环境。目标是确定基础的分子机制这些现象，检验了以下假设：公共机制是基础的两个都。在这个项目的第一阶段，发现 Ca2+迅速阻断Ca2+通道的亚摩尔摩尔浓度，表明Ca2+结合和通道块之间的分子步数很少。两者都发现细胞内Ca2+和缩小均涉及局部细胞骨架，不依赖于去磷酸化通常假定。该项目下一个时期的建议是进一步表征细胞骨架和Ca2+之间的相互作用通道和更可靠地量化细胞内的关系 CA2+和通道功能。细胞骨架在控制中的作用在更多的生理条件下（无需摘要）的CA2+通道将研究，参与Ca2+通道控制的细胞骨架蛋白将被确定。可光溶剂CA2+螯合剂和比率计量法Ca2+ 监视将用于完成对细胞内Ca2+与通道功能之间的关系。贴片夹生理技术将应用于急性分离蜗牛淋巴结的神经元。摘要将主要研究内而外的斑块，因此非份额药物，酶和其他蛋白质可以肯定地应用。研究CA2+通道块细胞内Ca2+将通过两种方式进行：直接灌注带有缓冲良好的Ca2+浓度的解决方案或闪光灯 Ca2+螯合剂的光解（DM-硝基苯基，硝基苯基-EGTA，Dizo-4）在全细胞，同时用荧光探针监测细胞内Ca2+ （Fura-2，furaptra）。该项目将提供两个直接测量在神经元Ca2+调节的最重要组成部分中，该细胞的成分能够处理细胞内释放的Ca2+的能力以及细胞内Ca2+在膜Ca2+涌入上。这些CA2+的成分调节对于理解Ca2+诱导的细胞死亡常见至关重要像脑卒中和阿尔茨海默氏症一样多样化的人类病理疾病。