Neurochemistry and Genetics of Drosophila CaMKII

果蝇 CaMKII 的神经化学和遗传学

基本信息

批准号：
8460044
负责人：
Leslie C Griffith
金额：
$ 31.64万
依托单位：
BRANDEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-05-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8460044
关键词：
5&apos Splice Site Address Adult Affect Affinity Alternative Splicing Animals Area Axon Behavior Behavioral Binding Biochemical Biochemical Pathway Biochemical Reaction Biological Assay C-terminal Calcium Cells Complementary DNA Complex Dendrites Drosophila genus Drug Design Drug Targeting Ethers Gene Chips Gene Expression Genes Genetic Goals Homeostasis In Vitro Individual Ion Channel Ions Kinetics Knock-in Mouse Knock-out Location Mass Spectrum Analysis Measures Membrane Membrane Potentials Messenger RNA Modification Molecular Molecular Conformation Motor Motor output Multiprotein Complexes Mutate Mutation Nervous system structure Neurons Output Phosphorylation Phosphotransferases Potassium Channel Process Protein Binding Proteins RNA Editing RNA Interference Regulation Role Scaffolding Protein Sequence Analysis Signal Transduction Signaling Protein Site Solutions Structure Subcellular structure Synapses Synaptic plasticity System Testing Therapeutic Intervention Transgenes Transgenic Organisms Voltage-Gated Potassium Channel Work calmodulin-dependent protein kinase II deep sequencing density fly homologous recombination in vivo information processing insight mutant neurochemistry novel postsynaptic prevent public health relevance research study response scaffold

项目摘要

DESCRIPTION (provided by applicant): The computational power of the nervous system relies on the ability of individual cells to integrate multiple inputs and generate an output targeted to the appropriate partner(s). These functions would be impossible without the asymmetric localization of proteins within the cell. For example, many of the proteins that are in the axon are specialized for functions like transport and electrical conduction. In contrast, many of the proteins in the dendrites are specialized for receiving information from other cells. Restriction of localization is important for limiting the activity of a particular protein to a specific cellular compartment, but it can also be an important regulator of biochemical reactions. Many proteins behave differently when localized to a multiprotein complex due to the ability of direct binding partners to alter their conformation. This function of localization has important implications for how neurons process information that is relevant to many therapeutic interventions since it means that the behavior or activity of a protein cannot always be predicted from in vitro studies. This proposal has the goal of understanding how CaMKII and two proteins that directly bind to it, dCASK, a scaffold protein, and Eag, a potassium channel, are regulated by localization and cellular context. To achieve an understanding of how cellular context can influence the activity of these proteins we propose two sets of experiments. In Aim #1 we will use a transfected cell system to understand how dCASK and CaMKII interact to change the location, activity and potential substrates of CaMKII in response to calcium influx. We will do a parallel set of experiments in dCASK mutant transgenic flies that express a YFP tagged dCASK transgene that restores normal behavior. We will determine where in the cell dCASK has to reside and what proteins it has to complex with to carry out its behavioral function. Aim #2 addresses how Eag, a voltage-gated potassium channel, is localized and regulated by other proteins that bind to it. We have discovered a novel role for RNA editing in localization that has implications for many other genes that undergo this type of modification. We will also investigate the function of an alternative splice product of the eag gene that does not conduct ions, but instead functions as a scaffold for CaMKII and other signaling proteins. We address these issues by using homologous recombination to create mutations in the eag gene that selectively alter each of these processes and assess the impact on neuronal gene expression, excitability and behavior.

描述（由申请人提供）：神经系统的计算能力依赖于单个细胞整合多个输入并生成针对适当伙伴的输出的能力。如果细胞内蛋白质不对称定位，这些功能就不可能实现。例如，轴突中的许多蛋白质专门用于运输和导电等功能。相比之下，树突中的许多蛋白质专门用于接收来自其他细胞的信息。定位限制对于将特定蛋白质的活性限制在特定的细胞区室中非常重要，但它也可以是生化反应的重要调节剂。由于直接结合配偶体能够改变其构象，许多蛋白质在定位于多蛋白复合物时表现不同。这种定位功能对于神经元如何处理与许多治疗干预相关的信息具有重要意义，因为这意味着蛋白质的行为或活性不能总是从体外研究中预测。该提案的目标是了解 CaMKII 和直接与其结合的两种蛋白质（支架蛋白 dCASK 和钾通道 Eag）如何受到定位和细胞环境的调节。为了了解细胞环境如何影响这些蛋白质的活性，我们提出了两组实验。在目标 #1 中，我们将使用转染的细胞系统来了解 dCASK 和 CaMKII 如何相互作用，从而改变 CaMKII 的位置、活性和潜在底物以响应钙流入。我们将在 dCASK 突变转基因果蝇中进行一组平行实验，这些果蝇表达 YFP 标记的 dCASK 转基因，可恢复正常行为。我们将确定 dCASK 必须驻留在细胞中的何处以及它必须与哪些蛋白质复合才能执行其行为功能。目标#2 解决了 Eag（一种电压门控钾通道）如何被与其结合的其他蛋白质定位和调节。我们发现了 RNA 编辑在定位中的新作用，这对许多其他经历此类修饰的基因具有影响。我们还将研究 eag 基因的替代剪接产物的功能，该剪接产物不传导离子，而是充当 CaMKII 和其他信号蛋白的支架。我们通过使用同源重组在 eag 基因中产生突变来解决这些问题，这些突变选择性地改变每个过程并评估对神经元基因表达、兴奋性和行为的影响。