INTRACELLULAR CONTROL OF NEURONAL CALCIUM CURRENT

神经元钙电流的细胞内控制

• 批准号: 3415019
• 负责人: WILLIAM L. BYERLEY
• 金额: $ 6.87万
• 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 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3415019
• 关键词: Cephalopoda; Gastropoda; axon; calcium channel; cell membrane; hormone regulation /control mechanism; intracellular transport; membrane permeability; neurons; neurotransmitter biosynthesis; neurotransmitter metabolism; phosphorylation; potassium channel; synapses; tissue /cell culture; voltage /patch clamp

The long-term goal of this project is to understand the mechanisms by which neurons control the Ca currents in their surface membranes. The Ca currents are directly responsible for the intracellular Ca signals that trigger neurotransmitter release, modulate membrane excitability and control neurite growth. This project uses molluscan neurons, which provide excellent models for studying transmitter release (e.g. squid giant synapse) and the cellular basis of behavior (e.g. Aplysia learning). The large molluscan neurons allow the application of a broad range of biophysical techniques; accurate measurement of membrane currents is possible either when intracellular environment is controlled (internal perfusion and patch clamp techniques) or minimally disturbed (two-electrode voltage clamp). All studies proposed for this period use isolated neurons from the snail Lymnaea stagnalis. The intensity and time course of the intracellular Ca2+ signal depends on two properties of the Ca channels, their activity and their distribution. The first part of this project focuses on intracellular control of the activity of Ca channels. Molluscan Ca currents are blocked by intracellular Ca2+ (Ca-dependent inactivation) and are very liable when exposed to an artificial intracellular solution (washout). The hypothesis that block by intracellular Ca2+ and washout of Ca currents is mediated by dephosphorylation of the channel will be tested. The inactivation of Ca current in patches will be studied to determine if Ca channels have to be clustered to exhibit Ca-dependent inactivation. Photorelease of Ca2+ will be used to measure both the concentration dependence and time course of block of Ca current by intracellular Ca2+. The second part of the project focuses on the distribution of Ca channels in the neuronal membrane and the relation of Ca-activated channels to the Ca channels. The microscopic distribution of Ca channels will be determined by simultaneous measurements of patch capacitance and patch Ca current. Fura-2 imaging will be used to measure spatial gradients of Ca2+ during internal perfusion of neurons and the apparent redistribution of Ca channels that occurs in cultured cells. The relative location of Ca- activated K channels will be studied, and the role of Ca-activated divalent permeable channel in controlling intracellular Ca2+ will be clarified.

该项目的长期目标是了解 神经元控制其表面膜中的CA电流。 CA 电流直接负责细胞内CA信号 触发神经递质释放，调节膜兴奋性和 控制神经突的生长。 该项目使用软体动物神经元，该神经元提供 研究发射器释放的出色模型（例如鱿鱼巨人 突触）和行为的细胞基础（例如垂直学习）。 这 大型软体动物神经元允许应用广泛的范围 生物物理技术；膜电流的准确测量是 可能会在控制细胞内环境时（内部 灌注和斑块夹技术）或最小的干扰（两电极 电压夹）。 在此期间提出的所有研究都使用孤立的神经元 来自蜗牛lymnaea stagnalis。 细胞内Ca2+信号的强度和时间过程取决于 CA通道的两个特性，其活动和分布。 该项目的第一部分重点是对 CA通道的活动。 软体动物Ca电流被阻止 细胞内Ca2+（CA依赖性灭活），当 暴露于人工细胞内溶液（洗涤）。 假设 通过细胞内Ca2+的阻滞和Ca电流的冲洗是由 将测试通道的去磷酸化。 CA的失活 将研究贴片中的电流，以确定CA通道是否必须为 聚集以表现出CA依赖性失活。 Ca2+的光弹性将 用于测量集中依赖性和时间过程 细胞内Ca2+的CA电流块。 该项目的第二部分重点是CA渠道的分布 在神经元膜上以及CA激活通道与 CA通道。 CA通道的显微镜分布将是 通过同时测量贴片电容和斑块Ca确定 当前的。 Fura-2成像将用于测量Ca2+的空间梯度 在神经元的内部灌注和CA的明显重新分布期间 在培养细胞中发生的通道。 CA-的相对位置 将研究激活的k通道，并将CA激活的二价作用 控制细胞内Ca2+的渗透通道将被阐明。