INTRACELLULAR CONTROL OF NEURONAL CALCIUM CURRENT

本项目的长期目标是了解 神经元控制其表面膜中的Ca电流。 的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， 控制神经突的生长 这个项目使用软体动物神经元， 研究发射物释放的极好模型（例如， 突触）和行为的细胞基础（例如，学习障碍）。 的 大的软体动物神经元允许应用广泛的 生物物理技术;膜电流的准确测量是 当细胞内环境受到控制（内部 灌注和膜片钳技术）或最小干扰（双电极 电压箝位）。 在此期间提出的所有研究都使用了孤立的神经元 来自于一种叫Stagnalis的蜗牛 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，它们的活性和分布。 本项目的第一部分重点是细胞内控制的 Ca通道的活性。 软体动物钙电流被阻断 细胞内Ca2+（钙依赖性失活），当 暴露于人工细胞内溶液（洗脱）。 的假设 通过细胞内Ca 2+的阻断和Ca电流的洗脱是由 将测试通道的去磷酸化。 Ca的失活 将研究贴片中的电流，以确定钙通道是否必须 聚集以显示Ca依赖性失活。 Ca2+的光释放将 用于测量浓度依赖性和时间过程， 通过细胞内Ca 2+阻断Ca电流。 该项目的第二部分重点关注Ca通道的分布 以及钙激活通道与神经细胞膜钙通道的关系 钙通道。 Ca通道的微观分布将是 通过同时测量贴片电容和贴片Ca 电流 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钙的明显重新分布过程中， 在培养的细胞中发生的通道。 的相对位置- 激活的K通道将被研究，钙激活的二价 细胞内Ca~（2+）调控的渗透性通道。

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.