Structural and Catalytic Functions of CaMKII in Neurons

钙/钙调蛋白依赖的蛋白激酶II(CaMKII)是学习和记忆所必需的。在……里面 在大脑中，CaMKII的表达水平与结构蛋白(占总蛋白的1-2%)及其12meric类似 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 或者，它与其他蛋白质相互作用的功能充其量也是知之甚少。主要的兴奋性神经- 哺乳动物大脑中的递质谷氨酸诱导海马区两种类型的CaMKII易位 神经元：短暂刺激(0.5-2分钟)后到达突触后部位，之后到达突触外簇 延长暴露时间(3-5分钟)。这两种易位都需要多价CaMKII全酶 结构(未发表的观察结果)。短暂的谷氨酸处理可诱导多种形式的突触可塑性 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，这是一种细胞模型 用于缺血性细胞死亡。这项提议的目标是测试我们的假设，即突触易位 需要与NMDA型谷氨酸受体亚单位NR2B结合，并参与突触可塑性， 而突触外簇的形成是由CaMKII自关联介导的，并参与了脑缺血 细胞死亡。阐明CaMKII靶向的机制和功能将有助于我们理解 大脑高级功能和行为的突触原理，并提供了新的治疗途径 缺血性损伤。我们将在以下方面实现我们的目标(使用大鼠海马区和大脑皮层 培养作为神经元研究的主要模型系统)：(1)确定所需的蛋白质相互作用 CaMKII在神经元中的亚细胞定位。我们将确定CaMKII突变和抑制物，以防止 NR2B在体外结合或自结合，并确定它们对GFP-CaMKII在神经元中定位的影响。 (2)确定CaMKII活性、突触靶向和 突触外聚集。我们将利用一种抑制CaMKII活性和聚集的抑制剂，敲除 RNAi表达及CaMKII野生型和突变体的过表达 活动、监管和目标确定。 (3)确定CaMKII在突触数目和强度调节中的作用。我们将利用 与(2)中相同的工具，并在已建立的分析中确定对突触数量和强度的影响。

The Ca2+/calmodulin-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 Both types of translocation require the multivalent CaMKII holoenzyme structure (unpublished observations).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 overexpressionof 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.