INTRACELLULAR CONTROL OF NEURONAL CALCIUM CURRENTS

该项目的长期目标是通过以下方式了解这些机制 哪些神经元控制其表面的电压激活的钙电流 膜。这些钙电流直接导致了 细胞内钙离子信号触发神经递质释放，调节 膜兴奋性和突触效能，并控制轴突生长。 该项目的重点是两个强烈改变功能的现象 在钙离子通道中，细胞内钙离子和钙离子通道的“阻断” 人工内固定时钙离子通道的“衰败” 环境。其目标是确定潜在的分子机制。 这些现象，检验了共同机制所依据的假设 两者都有。在这个项目的第一阶段，人们发现 亚微摩尔浓度的钙离子迅速阻断钙离子通道， 表明钙离子结合和通道阻断之间的分子步骤很少。 被细胞内钙离子阻断和衰弱都被发现涉及到 局部细胞骨架，而不是像以前那样依赖去磷酸化 通常认为是。该项目下一阶段的建议是 进一步表征细胞骨架与钙离子的相互作用 并更可靠地量化细胞内 钙离子和通道功能。细胞骨架在调控中的作用 在更多生理条件下(没有衰竭)的钙离子通道将 被研究，细胞骨架蛋白参与钙通道调控 将会被确认。光解钙离子螯合剂与比例钙离子 将利用监测来完成对 细胞内钙离子与通道功能的关系 膜片钳生理学技术将应用于急性隔离 蜗牛淋巴线虫的神经元。概要将主要研究在 由内而外的贴片，使非本意的药物、酶和其他蛋白质 可以肯定地应用。钙离子通道阻滞剂的研究 细胞内钙离子将通过两种方式实现：直接灌流细胞内- 用缓冲良好的钙离子浓度或闪光溶液贴出贴片 Ca~(2+)螯合剂(DM-硝基苯酚、硝基苯基-EGTA、重氮-4)的光解 整个细胞，同时用荧光探针监测细胞内钙离子 (Fura-2，Furaptra)。该项目将提供对两个 在神经元钙调节的最重要成分中，细胞的 处理细胞内释放的钙离子的能力和 细胞内钙离子对膜上钙离子内流的影响。这些钙离子的成分 调控是理解常见的钙离子诱导的细胞死亡的核心 人类的各种病理疾病，如中风和阿尔茨海默氏症 疾病。

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.