CA SENSING FOR EXOCYTOSIS

胞吐作用的 CA 传感

• 批准号: 6612611
• 负责人: Kevin D Gillis
• 金额: $ 18.13万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6612611
• 关键词: animal tissue; calcium flux; cell line; divalent cations; exocytosis; gene mutation; nerve /myelin protein

DESCRIPTION: (Applicant's Abstract) The secretion of neurotransmitter and hormones from neurons and neuroendocrine cells is a highly regulated process. It is now widely accepted that a rise in intracellular [Ca2+] rapidly triggers secretion from excitable cells. However, it has recently become clear that Ca2+ also slowly modulates ("primes") release, in part through activation of protein kinase C (PKC), which, in turn, accelerates the rate that secretory vesicles become ready to be released. Therefore it is likely that there are multiple fast (triggering) and slow (modulating) Ca2+ sensors for exocytosis. A long-range goal of the investigator is to understand how Ca2+ triggers exocytosis from excitable cells and how exocytosis is regulated by Ca2+ and other second messengers. The goal of this project is to characterize fast and slow Ca2+ sensing for exocytosis in individual cells using optical and electrophysiological techniques which allow both fine control of [Ca2+] and high-time-resolution measurements of exocytosis. The 3 aims are: Aim I. To determine how the protein SNAP-25 is involved in Ca2+ priming and triggering steps. The effect of mutations of SNAP-25 on exocytosis will be measured to test the hypothesis that the C-terminus of the protein participates in both Ca2+-priming and triggering steps. Aim II. To determine how fast Ca2+ can prime exocytosis. Experiments will elevate [Ca2+]i in 2 steps to sequentially prime and trigger secretion to test the hypothesis that Ca2+ priming occurs in less than 1 second. Aim III. To quantify the ionic selectivity of the Ca2+ trigger for exocytosis. Multivalent cations such as Sr2+, Ba2+, and Pb2+ can act as "Ca2+ surrogates" in triggering exocytosis and other Ca2+-activated cellular responses. The ability of Ca2+ surrogates to rapidly trigger exocytosis will be measured to provide clues about the approximate size, flexibility and accessibility of the Ca2+-binding cavity of the triggering Ca2+ sensor. Achieving these aims will provide new insights into the mechanisms whereby secretion is regulated. Such basic knowledge is essential to understand complex processes such as short-term memory formation in the brain, the modulation of insulin secretion by glucagon in the endocrine pancreas, and the neurotoxicity of Pb2+ in the central nervous system.

描述：（申请人摘要） 神经元和神经内分泌神经递质和激素的分泌 细胞是一个高度调控的过程。现在人们普遍认为， 细胞内[Ca2+]迅速触发可兴奋细胞的分泌。然而， 最近发现 Ca2+ 也会缓慢调节（“启动”） 释放，部分是通过激活蛋白激酶 C (PKC)，反过来， 加速分泌囊泡准备释放的速度。 因此很可能存在多个快（触发）和慢 （调节）用于胞吐作用的 Ca2+ 传感器。研究者的长期目标 是了解 Ca2+ 如何触发可兴奋细胞的胞吐作用以及如何 胞吐作用受 Ca2+ 和其他第二信使的调节。此举的目标 该项目的目的是表征胞吐作用的快速和慢速 Ca2+ 传感 使用光学和电生理技术的单个细胞可以 [Ca2+] 的精细控制和高时间分辨率测量 胞吐作用。这 3 个目标是： 目标 I. 确定蛋白质 SNAP-25 如何参与 Ca2+ 启动和 触发步骤。 SNAP-25 突变对胞吐作用的影响将是 测量以检验蛋白质 C 末端参与的假设 在 Ca2+ 启动和触发步骤中。 目标二。确定 Ca2+ 启动胞吐作用的速度。实验将 分 2 步升高 [Ca2+]i，依次启动并触发分泌以进行测试 Ca2+ 启动发生在不到 1 秒的假设。 目标三。量化 Ca2+ 触发胞吐作用的离子选择性。 Sr2+、Ba2+ 和 Pb2+ 等多价阳离子可以充当“Ca2+ 替代物” 触发胞吐作用和其他 Ca2+ 激活的细胞反应。这 Ca2+替代物快速触发胞吐作用的能力将被测量 提供有关大致大小、灵活性和可访问性的线索 触发 Ca2+ 传感器的 Ca2+ 结合腔。 实现这些目标将为了解机制提供新的见解 分泌受到调节。这些基础知识对于理解复杂的问题至关重要 大脑中短期记忆的形成、调节等过程 内分泌胰腺中胰高血糖素分泌胰岛素以及神经毒性 中枢神经系统中的 Pb2+。