DYNAMICS OF CALMODULIN IN NEURONS

神经元中钙调蛋白的动力学

• 批准号: 6637343
• 负责人: Sally Ann Kim
• 金额: $ 0.43万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6637343
• 关键词: PC12 cells; calcium flux; calcium indicator; calcium ion; calmodulin; confocal scanning microscopy; diffusion; enzyme mechanism; fluorescence spectrometry; immunocytochemistry; intermolecular interaction; neurons; protein binding; protein localization; radioimmunoassay; time resolved data

One of the fundamental questions of Cat+-mediated signal transduction is how activation of multiple enzymes by changes in a diffusible second messenger, calcium, leads to temporally and spatially restricted effects. The objective of this research is to examine further the spatial and temporal dynamics of the Cat+-binding protein calmodulin by measuring its diffusion utilizing a combination of biochemical and imaging approaches. Preliminary experiments and previous work (Persechini & Cronk, 1999; Luby-Phelps et al., 1995; Tansey et al., 1994) suggest that calmodulin is limiting in cells. If found true for neurons, this discovery imparts important constraints on how neuronal calmodulin-dependent enzymes are activated and poses profound implications on the role of calmodulin in acting as the detection system for Ca2+ transients to regulate cellular processes: The specific aims of this research project are: 1) to determine the amount of diffusible and bound calmodulin in PC12 cells at differing Ca2+ concentrations using a biochemical approach; 2) to characterize the compartmentalization and localization of calmodulin in PC12 cells and neurons using immunocytochemistry and confocal microscopy; and 3) to determine the diffusion of calmodulin in PC12 cells and neurons using both multiphoton fluorescence photobleaching recovery (MPFPR) and fluorescence correlation spectroscopy (MPFCS). Through the quantitative analysis of diffusion and spatial localization of calmodulin at different levels of Cat+, a more complete understanding between Ca2+ signaling and calmodulin availability in neurons will be elucidated. These studies will provide essential information for understanding how neurons process information through the Ca2+/calmodulin-dependent signaling pathway.

Cat+介导的信号转导的基本问题之一是如何通过可扩散的第二信使钙的变化激活多个酶，从而导致时间和空间上的限制效应。这项研究的目的是通过结合生化和成像方法测量其扩散，进一步检测Cat+结合蛋白钙调蛋白的空间和时间动力学。初步实验和以前的工作(Persechini&Cronk，1999；Luby-Pps等人，1995；Tansey等人，1994)表明，钙调蛋白在细胞中是有限的。如果对神经元成立，这一发现为神经元钙调蛋白依赖酶的激活提供了重要的限制，并对钙调蛋白在作为钙瞬变检测系统调节细胞过程中的作用产生了深远的影响：本研究项目的具体目标是：1)使用生化方法确定不同钙浓度下PC12细胞中可扩散和结合的钙调蛋白的数量；2)使用免疫细胞化学和共聚焦显微镜表征钙调蛋白在PC12细胞和神经元中的区隔和定位；以及3)使用多光子荧光光漂白恢复(MPFPR)和荧光相关光谱(MPFCS)来确定钙调蛋白在PC12细胞和神经元中的扩散。通过对钙调素在不同Cat+水平上的扩散和空间定位的定量分析，将更全面地了解钙信号与神经元中钙调素的可用性之间的关系。这些研究将为理解神经元如何通过钙/钙调蛋白依赖的信号通路处理信息提供必要的信息。