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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.

本项目的长期目标是通过以下方式了解这些机制：哪些神经元控制着它们表面的电压激活的钙电流膜。这些Ca 2+电流直接负责细胞内Ca 2+信号触发神经递质释放，调节膜兴奋性和突触功效，并控制神经突生长。该项目的重点是两个现象，强烈改变功能钙通道的“阻断”，细胞内钙离子对钙通道的“阻断”，当暴露于人工内毒素时，环境.我们的目标是确定潜在的分子机制这些现象，测试的假设，共同机制的基础，两者在该项目的第一阶段，发现，亚微摩尔浓度的Ca 2+迅速阻断Ca 2+通道，表明在Ca 2+结合和通道阻断之间几乎没有分子步骤。细胞内Ca 2+的阻断和细胞内Ca 2+的流失都涉及到细胞内Ca 2+的释放。局部细胞骨架，而不是依赖于去磷酸化，因为已经通常假设。该项目下一阶段的建议是，进一步表征细胞骨架与Ca 2+之间的相互作用更可靠地量化细胞内 Ca 2+和通道功能。细胞骨架在控制细胞凋亡中的作用在更多生理条件下（没有下降）的Ca 2+通道将研究，和细胞骨架蛋白参与钙离子通道的控制将被识别。光解Ca 2+螯合剂和比率Ca 2 + 监测将用于完成定量研究的细胞内Ca 2+与通道功能的关系。膜片钳生理技术将应用于急性孤立的蜗牛的神经元。Rundown将主要在由内而外的补丁，使非渗透性药物，酶和其他蛋白质可以肯定地应用。钙通道阻滞剂的研究细胞内Ca 2+将以两种方式进行：直接灌注内- 用缓冲良好的Ca 2+浓度的溶液冲洗贴片，或快速冲洗钙离子螯合剂（DM-nitrophen，nitrophenyl-EGTA，diazo-4）在水中的光解全细胞，同时用荧光探针监测细胞内Ca 2 + （fura-2，furaptra）。该项目将提供直接测量两个神经元钙调节的最重要的组成部分，细胞的处理细胞内释放的Ca 2+的能力以及细胞内Ca ~（2+）对膜Ca ~（2+）内流的影响。Ca 2+的这些成分调控是理解钙诱导细胞死亡的核心，人类的各种疾病，如脑中风和老年痴呆症，疾病