CA CHANNEL INACTICATION AND MOLECULAR CONTROL OF TRANSMITTER RELEASE

CA 通道失活和递质释放的分子控制

• 批准号: 5214777
• 负责人: RICHARD W TSIEN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5214777
• 关键词: Xenopus oocyte; calcium; calcium channel; calcium channel blockers; clone cells; electrophysiology; intermolecular interaction; membrane potentials; molecular cloning; neural plasticity; neural transmission; protein structure function; radionuclides; synapses; synaptosomes; tissue /cell culture; voltage /patch clamp; voltage gated channel

The overall objective of this project is to understand the molecular interactions that appear to exist between specific types of Ca2+ channels and components of the protein complex supporting transmitter release. The general background is growing knowledge about the diversity of voltage- gated Ca2+ channels, including a novel channel labelled Q-type, and the structural determinants of key processes such as Ca2+ channel inactivation, the shutting off of the channel during a maintained depolarization. In the proposed experiments, we will focus on interactions between synataxin, a key component of the fusion machinery, and N- and Q-type Ca2+ channels which act in concert at CA3-CA1 hippocampal synapses to trigger synaptic transmission. The immediate impetus is our recent observation that coexpression of syntaxin 1A with N-type channels in Xenopus oocytes promotes inactivation of the channels by causing a about 20 m V hyperpolarizing shift in the voltage-dependence of their availability. Like N-type channels, Q-type channels were also affected by syntaxin, whereas inactivation of l-type channels was unchanged. Previous biochemical studies had already demonstrated t hat syntaxin is capable of binding to N-type Ca2+ channels, but this is the first evidence for any effect on the channels' functional properties and any interaction with Q- type channels. Thus, our data raise the possibility that syntaxin may modulate Ca2+ entry via key presynaptic Ca2+ channels, over and above its putative role as a docking site for synaptic vesicles. During the grant support period, we intend to (1) analyze syntaxin action according to the modulated receptor hypothesis and determine which types of Ca2+ channels can be influenced by syntaxin, (2) delineate regions of Ca2+ channels that allow the interaction, with specific attention to those domains that are dominant in governing the speed or extent of inactivation, (3) characterize the molecular determinants that allow syntaxin to influence Ca2+ channels, (4) find out whether syntaxin's actions on Ca2+ channels in oocytes can be prevented by the botulinum toxin BoTx/C1, which selectively cleaves syntaxin, or by other protein components (e.g. cysteine string proteins, synaptotagmin, VAMP, n-secl, alpha-SNAP, NSF etc. (5) determine the importance of syntaxin -Ca2+ channel signalling in nerve terminals by testing effects of BoTx/C1 on Ca2+ influx on synaptosomes. It is interesting to ask whether syntaxin effects on the gating of N- and Q- type channels might represent a novel signaling pathway from exocytotic machinery to Ca2+ channels that would allow Ca2+ entry at sites where vesicles have been readied for exocytosis. This would be an efficient system for controlling transmitter release while minimizing possible Ca2+ overload and associated neurotoxicity.

这个项目的总体目标是理解分子 似乎存在于特定类型的钙通道之间的相互作用 以及支持递质释放的蛋白质复合体的成分。这个 大背景是关于电压多样性的不断增长的知识- 门控钙通道，包括一种标记为Q-型的新通道，以及 关键过程的结构性决定因素，如钙离子通道失活， 在维持的去极化过程中关闭通道。在 计划中的实验，我们将重点研究Synataxin，一种 融合机制的关键部件，以及N型和Q型钙离子通道 它们协同作用于CA3-CA1海马区突触以触发突触 变速箱。直接的推动力是我们最近观察到的 突触素1A与N型通道在非洲爪哇卵母细胞中的共表达 通过引起约20 mV的电压来促进通道的失活 它们的可用性的电压依赖关系中的超极化变化。 与N型通道一样，Q型通道也受Synaxin的影响， 而L类通道失活无明显变化。上一首 生物化学研究已经证明，合成素能够 结合到N型钙通道，但这是第一个证据 对通道的功能特性以及与Q-的任何相互作用的影响 键入Channels。因此，我们的数据增加了Synaxin可能 通过关键的突触前钙通道调节钙离子内流 可能的作用是作为突触小泡的对接地点。 在资助支持期间，我们打算(1)分析语法蛋白的作用 根据调制受体假说确定哪些类型的 钙通道可受Synaxin的影响，(2)勾画钙离子区域 允许互动的渠道，并特别关注那些 在控制失活速度或程度方面占主导地位的域， (3)描述使Synaxin能够 影响Ca~(2+)通道，(4)了解Synaxin是否对Ca~(2+)起作用 卵母细胞中的通道可以被肉毒毒素BoTx/C1阻止，BoTx/C1是一种 选择性地切割合成素，或通过其他蛋白质成分(如半胱氨酸)切割 字符串蛋白、突触素、VAMP、n-secl、α-SNAP、NSF等(5) 确定合成素-钙通道信号在神经中的重要性 通过检测BoTx/C1对突触体上钙离子内流的影响来检测终末。它 有趣的是，句法蛋白是否影响N-和Q-的门控？ 类型通道可能代表胞吐的一条新的信号通路 允许钙离子进入下列部位的钙离子通道的机械 囊泡已经为胞吐作用做好了准备。这将是一种高效的 控制发射机释放同时将可能的钙离子降至最低的系统 超负荷和相关的神经毒性。