INTRACELLULAR CONTROL OF NEURONAL CALCIUM CURRENTS

神经元钙电流的细胞内控制

• 批准号: 2266964
• 负责人: WILLIAM L. BYERLEY
• 金额: $ 11.77万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2266964
• 关键词: Gastropoda; actins; barium; calcium channel; calcium channel blockers; calcium flux; calcium indicator; chelating agents; cytoskeleton; endopeptidases; enzyme activity; flash photolysis; fluorine; intracellular; magnesium; neurons; neuropharmacology; osmotic pressure; phospholipids; potassium channel; protease inhibitor; sodium channel; tubulin; voltage /patch clamp; voltage gated channel

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通过以下方式了解其机制： 哪些神经元控制其表面的电压激活 Ca2+ 电流 膜。这些 Ca2+ 电流直接负责 细胞内 Ca2+ 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。 该项目重点关注两种强烈改变功能的现象 Ca2+ 通道的“阻断”是细胞内 Ca2+ 和 当暴露于人工体内时 Ca2+ 通道的“耗尽” 环境。目标是确定潜在的分子机制 这些现象，检验了共同机制背后的假设 两个都。在该项目的第一阶段，我们发现 亚微摩尔浓度的 Ca2+ 迅速阻断 Ca2+ 通道， 表明 Ca2+ 结合和通道阻断之间的分子步骤很少。 发现细胞内 Ca2+ 阻断和耗尽都涉及 局部细胞骨架，而不依赖于去磷酸化，正如以前所报道的那样 通常假设。该项目下一阶段的建议是 进一步表征细胞骨架和 Ca2+ 之间的相互作用 通道并更可靠地量化细胞内之间的关系 Ca2+ 与通道功能。细胞骨架在控制中的作用 Ca2+ 通道在更多的生理条件下（没有流失）会 待研究，细胞骨架蛋白参与 Ca2+ 通道控制 将被识别。可光解 Ca2+ 螯合剂和比率 Ca2+ 监测将用于完成定量研究 细胞内Ca2+与通道功能的关系。 膜片钳生理学技术将应用于急性分离 来自 Lymnaea 蜗牛的神经元。 Rundown 将主要研究于 由内而外的贴片，使非渗透性药物、酶和其他蛋白质 可以肯定地应用。 Ca2+通道阻断的研究 细胞内 Ca2+ 将通过两种方式进行：直接灌注内部 - 使用缓冲良好的 Ca2+ 浓度的溶液或闪存进行贴片 Ca2+ 螯合剂（DM-硝基苯、硝基苯基-EGTA、diazo-4）的光解作用 全细胞，同时用荧光探针监测细胞内 Ca2+ （fura-2，furaptra）。该项目将提供两个的直接测量 神经元 Ca2+ 调节最重要的组成部分，细胞的 处理细胞内释放的 Ca2+ 的能力以及 细胞内Ca2+在膜上Ca2+流入。 Ca2+的这些成分 调节对于理解 Ca2+ 诱导的细胞死亡常见现象至关重要 脑中风和阿尔茨海默病等多种人类疾病 疾病。