Changes/SNARE Proteins/Neural Transmission/FRET

变化/SNARE 蛋白质/神经传递/FRET

• 批准号: 6876665
• 负责人: ROBERT STEPHEN ZUCKER
• 金额: $ 17.58万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6876665
• 关键词: binding proteins; cell membrane; conformation; endocytosis; fluorescence resonance energy transfer; green fluorescent proteins; hippocampus; laboratory rat; method development; neural transmission; neurons; protein binding; protein protein interaction; protein structure; tissue /cell culture; transfection; voltage /patch clamp

DESCRIPTION (provided by applicant): The specific goals of this project are: 1) Development of methods and procedures for detecting conformational changes in SNARE proteins during and after secretion. This is a developmental/exploratory proposal for a project whose chief goal is the development of fluorescence resonance transfer (FRET) as a dynamic measure of changes in the structure and binding of SNARE proteins during and following the release of transmitter in neurons. Cultured hippocampal cells will be transfected with green fluorescent protein (GFP)-Iabeled presynaptic SNARE proteins, and procedures will be developed and perfected to detect changes in the spatial relationships and interactions between these proteins during and following secretion that will be triggered by depolarization. 2) Application of FRET to detecting SNARE complex assembly and disassembly. The N-termini of SNAP-25 and VAMP become closely associated during SNARE complex formation, and dissociate when SNARE complexes are disassembled. These changes will be monitored by FRET following secretion of the readily releasable pool of vesicles, when new vesicles dock and SNARE complexes assemble, and when SNARE complexes of previously exocytosed vesicles are disassembled prior to endocytosis. 3) Application of FRET to detection SNARE complex reorientation following vesicle fusion. The C-termini of VAMP and syntaxin come together on the external surface of the plasma membrane on vesicle fusion. We will attempt to detect this reorientation during secretion, and measure its lifetime before vesicle recovery by endocytosis. These are novel applications of the FRET technique to studies of synaptic transmission, and require solution of numerous technical obstacles. Success will open wide possibilities for the study of dynamic changes in protein structure during a variety of cell activities, and pave the way for the study of protein structural alterations in diseased tissue.

描述（由申请人提供）： 本项目的具体目标是：1）发展检测SNARE蛋白在分泌过程中和分泌后构象变化的方法和程序。这是一个项目的发展/探索性建议，其主要目标是开发荧光共振转移（FRET）作为神经元中释放递质期间和之后SNARE蛋白质结构和结合变化的动态测量。将用绿色荧光蛋白（GFP）标记的突触前SNARE蛋白转染培养的海马细胞，并且将开发和完善程序以检测在由去极化触发的分泌期间和之后这些蛋白之间的空间关系和相互作用的变化。 2)FRET在检测SNARE复杂组装和拆卸中的应用SNAP-25和VAMP的N-末端在SNARE复合物形成过程中紧密结合，并在SNARE复合物分解时解离。这些变化将通过FRET监测后分泌的囊泡容易释放的池，当新的囊泡码头和陷阱复合物组装，当陷阱复合物的先前exocytosed囊泡被拆卸之前的内吞作用。 3)应用FRET检测囊泡融合后SNARE复合物的重定向。VAMP和突触融合蛋白的C-末端在囊泡融合时在质膜的外表面上聚集在一起。我们将尝试检测分泌过程中的这种重新定位，并测量囊泡通过内吞作用恢复之前的寿命。 这些是FRET技术在突触传递研究中的新应用，需要解决许多技术障碍。成功将为研究蛋白质结构在各种细胞活动中的动态变化开辟广阔的可能性，并为研究病变组织中的蛋白质结构改变铺平道路。