INTRACELLULAR CONTROL OF NEURONAL CALCIUM CURRENT

这个项目的长期目标是了解 神经元控制其表面膜上的钙电流。案例 电流直接负责细胞内的钙信号， 触发神经递质释放，调节膜兴奋性和 控制轴突生长。这个项目使用了软体动物的神经元，它为 研究递质释放的优秀模型(例如鱿鱼巨型 突触)和行为的细胞基础(例如海兔的学习)。这个 大型软体动物神经元允许广泛的应用 生物物理技术；膜电流的准确测量 当细胞内环境受到控制时(内部 灌流和膜片钳技术)或最小干扰(双电极 电压钳位)。所有为这一时期提出的研究都使用分离的神经元。 来自蜗牛Lymneea stagnalis。 细胞内钙信号的强度和时间进程取决于 钙通道的两个特性，它们的活性和分布。 这个项目的第一部分集中在细胞内控制 钙通道的活动。软体动物钙电流被以下物质阻断 细胞内钙离子(钙依赖失活)，并在以下情况下非常敏感 暴露在人工细胞内溶液中(冲刷)。假说 细胞内钙离子的阻断和钙电流的洗脱是由 将测试该通道的去磷酸化。钙离子的失活 将研究斑块中的电流，以确定钙通道是否必须 聚集在一起，表现出钙依赖的失活。钙离子的光释放会 可用于测量浓度依赖关系和时间进程 细胞内钙离子阻断钙电流。 项目的第二部分重点是钙离子通道的分布 在神经细胞膜上的分布及钙激活通道与 CA频道。钙通道的微观分布将是 通过同时测量贴片电容和贴片面积来确定 电流。Fura-2成像将用于测量钙离子的空间梯度 在神经元内灌流和钙的表观再分布过程中 出现在培养细胞中的通道。钙离子的相对位置 将研究激活的钾通道，以及钙激活的二价钾通道的作用 控制细胞内钙离子的通透性通道将被阐明。

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.