Structural and Catalytic Functions of CaMKII in Neurons

神经元中 CaMKII 的结构和催化功能

• 批准号: 7212598
• 负责人: K. Ulrich Bayer
• 金额: $ 33.4万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-30 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7212598
• 关键词: Behavior; Binding; Biological Assay; Biological Models; Brain; Calcium/calmodulin-dependent protein kinase; Calmodulin; Cell Death; Cell model; Cessation of life; Chromosome Pairing; Count; Cultured Cells; DLG4 gene; Data; Development; Event; Future; Glucose; Glutamate Receptor; Glutamates; Goals; Hippocampus (Brain); Holoenzymes; Impairment; In Vitro; Injury; Ischemia; Learning; Mediating; Mediator of activation protein; Memory; Modeling; Mutation; N-Methyl-D-Aspartate Receptors; Neuroglia; Neurons; Numbers; Outcome; Oxygen; Personal Satisfaction; Phosphotransferases; Point Mutation; Population; Predisposition; Property; Protein Isoforms; Protein Kinase M; Protein Overexpression; Proteins; RNA Interference; Rattus; Regulation; Research Personnel; Role; Sequence Homology; Signal Transduction; Site; Staging; Structural Protein; Structure; Synapses; Synaptic plasticity; Testing; Thinking; age related; calmodulin-dependent protein kinase II; deprivation; excitotoxicity; inhibitor/antagonist; mutant; neuron loss; neuronal excitability; neurotoxic; novel therapeutics; postsynaptic; presynaptic density protein 95; prevent; programs; reconstitution; tool

DESCRIPTION (provided by applicant): The Ca2????dependent protein kinase II (CaMKII) is required for forms of learning and memory. In brain, CaMKII is expressed at levels reminiscent of structural proteins (1-2% of total protein), and its 12meric holoenzyme structure allows multiple simultaneous protein interactions. However, structural roles of CaMKII or functions of its interactions with other protein are understood poorly at best. The major excitatory neuro- transmitter in mammalian brain, glutamate, induces two types of CaMKII translocation in hippocampal neurons: To postsynaptic sites upon brief stimulation (0.5-2 min) and to extra-synaptic clusters after extended exposure (3-5 min). Both types of translocation require the multivalent CaMKII holoenzyme structure (unpublished observations). Brief glutamate treatment can induce forms of synaptic plasticity thought to underlie learning and memory, while extended exposure leads to excitotoxicity, a cellular model for ischemic cell death. The goal of this proposal is to test our hypotheses that synaptic translocation requires binding to the NMDA-type glutamate receptor subunit NR2B and is involved in synaptic plasticity, while formation of extrasynaptic clusters is mediated by CaMKII self-association and is involved in ischemic cell death. Elucidating the mechanisms and functions of CaMKII targeting will aid our understanding of synaptic principles underlying higher brain functions and behavior, and provide new therapeutic avenues for ischemic damage. We will accomplish our goal in the following aims (using rat hippocampal and cortical cultures as main model systems for studies in neurons): (1) Determine protein interactions required for subcellular CaMKII localization in neurons. We will identify CaMKII mutations and inhibitors that prevent NR2B-binding or self-association in vitro and determine their effect on GFP-CaMKII localization in neurons. (2) Determine neuroprotective versus neurotoxic effects of CaMKII activity, synaptic targeting, and extrasynaptic clustering. We will make use of an inhibitor of CaMKII activity and clustering, knockdown of expression by RNAi, and overexpression of CaMKII wildtype and mutants with specific impairments in activity, regulation and targeting. (3) Determine functions of CaMKII in regulation of synapse number and strength. We will make use of the same tools as in (2) and determine effects on synapse number and strength in established assays.

描述（由申请人提供）：Ca 2？依赖性蛋白激酶II（CaMKII）是学习和记忆形式所必需的。在大脑中，CaMKII以结构蛋白的水平表达（占总蛋白的1-2%），其12聚体全酶结构允许多种蛋白质同时相互作用。然而，CaMKII的结构作用或其与其他蛋白质相互作用的功能知之甚少。哺乳动物脑中的主要兴奋性神经递质谷氨酸盐在海马神经元中诱导两种类型的CaMK II易位：在短暂刺激（0.5-2分钟）后易位至突触后位点，以及在延长暴露（3-5分钟）后易位至突触外簇。两种类型的易位都需要多价CaMKII全酶结构（未发表的观察结果）。短暂的谷氨酸治疗可以诱导突触可塑性的形式，被认为是学习和记忆的基础，而长期暴露会导致兴奋性毒性，这是缺血性细胞死亡的细胞模型。这个建议的目的是测试我们的假设，突触易位需要结合到NMDA型谷氨酸受体亚基NR 2B，并参与突触可塑性，而突触外簇的形成是由CaMKII自协会介导的，并参与缺血性细胞死亡。阐明CaMKII靶向的机制和功能将有助于我们理解更高脑功能和行为背后的突触原理，并为缺血性损伤提供新的治疗途径。我们将在以下目标中完成我们的目标（使用大鼠海马和皮层培养物作为神经元研究的主要模型系统）：（1）确定神经元中亚细胞CaMK II定位所需的蛋白质相互作用。我们将确定CaMKII突变和抑制剂，防止NR 2B结合或自缔合在体外，并确定其对GFP-CaMKII定位在神经元的影响。(2)确定神经保护与神经毒性作用的CaMKII活性，突触靶向，和突触外群集。我们将利用CaMKII活性和聚集的抑制剂，通过RNAi敲低表达，以及过表达CaMKII野生型和在活性、调节和靶向方面具有特异性损伤的突变体。(3)确定CaMKII在调节突触数量和强度中的功能。我们将使用与（2）中相同的工具，并在已建立的测定中确定对突触数量和强度的影响。