CA SENSING FOR EXOCYTOSIS

描述：(申请人摘要) 神经元和神经内分泌的神经递质和激素分泌 细胞是一个高度受调控的过程。现在人们普遍接受的是， 细胞内的[Ca~(2+)]迅速触发可兴奋细胞的分泌。然而， 最近发现，钙离子也能缓慢地调节(“质数”)。 释放，部分通过激活蛋白激酶C(PKC)，进而， 加快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)胞吐作用的钙离子传感器。调查员的远大目标 是为了了解钙离子如何触发可兴奋细胞的胞吐以及如何 胞吐作用受钙离子和其他第二信使的调节。这样做的目的是 项目是表征快速和缓慢的钙离子对胞吐作用的感知 使用光学和电生理技术的单个细胞 [Ca~(2+)]的精细控制和高时间分辨率的测量 胞吐。这三个目标是： 目的I.确定SNAP-25蛋白如何参与钙启动和 触发步骤。SNAP-25突变对胞吐作用的影响将是 测量以检验蛋白质的C-末端参与的假设 在钙离子启动和触发步骤中。 目的II.确定钙离子启动胞吐的速度。实验将会 分两步升高[Ca~(2+)]i，按顺序启动并触发分泌进行测试 认为钙离子启动发生在不到1秒内的假设。 目的III.量化钙离子触发胞吐作用的离子选择性。 Sr2+、Ba2+、Pb2+等多价阳离子可作为“钙离子替代物” 在触发胞吐和其他钙离子激活的细胞反应中。这个 钙离子替代物快速触发胞吐的能力将被测量为 的大致大小、灵活性和可访问性提供线索 触发钙离子传感器的钙离子结合腔。 实现这些目标将为以下机制提供新的见解 分泌是受调节的。这些基本知识对于理解复杂的事物是必不可少的 大脑中的短期记忆形成等过程，调制 胰腺内分泌高血糖素的胰岛素分泌及其神经毒性 中枢神经系统中的Pb2+。

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.