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 细胞是一个高度调节的过程。现在人们普遍认为， 胞内[Ca 2 +]迅速触发可兴奋细胞的分泌。然而，在这方面， 最近已经清楚的是，Ca 2+也缓慢地调节（“引发”） 释放，部分通过激活蛋白激酶C（PKC），反过来， 加速了分泌囊泡准备释放的速度。 因此，很可能存在多个快速（触发）和慢速 用于胞吐作用的（调节）Ca 2+传感器。研究者的长期目标 是了解Ca 2+如何触发可兴奋细胞的胞吐作用， 胞吐作用受Ca 2+和其他第二信使调节。这个目标 该项目是表征细胞胞吐的快速和慢速Ca 2+传感 使用光学和电生理学技术， [Ca 2 +]的精细控制和高时间分辨率测量 胞吐作用这三个目标是： 艾姆岛为了确定蛋白SNAP-25如何参与Ca 2+引发和 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Ca 2+引发和触发步骤中。 Aim II.以确定Ca 2+引发胞吐作用的速度。实验将 分2步升高[Ca 2 +]i，依次启动和触发分泌，以检测 假设Ca 2+引发发生在不到1秒内。 Aim III.定量胞吐作用的Ca 2+触发剂的离子选择性。 多价阳离子如Sr 2+、Ba 2+和Pb 2+可充当“Ca 2+替代物”。 在触发胞吐和其他钙激活的细胞反应。的 将测量Ca 2+替代物快速触发胞吐作用的能力， 提供了有关的大致大小，灵活性和可访问性的线索， 触发Ca 2+传感器的Ca 2+结合腔。 实现这些目标将使人们对各种机制有新的认识， 分泌受到调节。这些基本知识对于理解复杂的 大脑中的短期记忆形成， 内分泌胰腺中胰高血糖素的胰岛素分泌，以及神经毒性 中枢神经系统中的Pb 2+。

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.