DYNAMICS OF CALMODULIN IN NEURONS

神经元中钙调蛋白的动力学

• 批准号: 6298675
• 负责人: Sally Ann Kim
• 金额: $ 2.21万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6298675
• 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+介导的信号转导的一个基本问题是，多种酶如何通过可扩散的第二信使钙的变化而激活，从而导致时间和空间上的限制效应。本研究的目的是通过结合生化和成像方法测量Cat+结合蛋白钙调素的扩散，进一步研究其时空动态。初步实验和前期工作（被害希尼和克朗克，1999；卢比-菲尔普斯等，1995；Tansey等，1994）表明，钙调素在细胞中是有限的。如果这一发现对神经元是正确的，这一发现对神经元钙调素依赖酶是如何被激活的重要限制，并对钙调素在作为Ca2+瞬态检测系统调节细胞过程中的作用产生深远的影响：本研究项目的具体目的是：1)使用生化方法确定不同Ca2+浓度下PC12细胞中扩散和结合钙调素的数量；2)利用免疫细胞化学和共聚焦显微镜研究钙调素在PC12细胞和神经元中的区隔化和定位；3)利用多光子荧光光漂白恢复（MPFPR）和荧光相关光谱（MPFCS）测定钙调素在PC12细胞和神经元中的扩散。通过定量分析钙调素在不同Cat+水平下的扩散和空间定位，将更全面地了解Ca2+信号传导与神经元钙调素可用性之间的关系。这些研究将为理解神经元如何通过Ca2+/钙调素依赖的信号通路处理信息提供必要的信息。