Mechanisms of CaMKII Binding to GluN2B and its Role in Synaptic Plasticity and Me

CaMKII 与 GluN2B 结合的机制及其在突触可塑性和 Me 中的作用

• 批准号: 8648004
• 负责人: Kelsey Marie Barcomb
• 金额: $ 2.82万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8648004
• 关键词: Address; Affect; Affinity; Behavioral; Binding; Calmodulin; Cell Death; Cell physiology; Cells; Co-Immunoprecipitations; Complex; Data; Disease; Dissociation; Dose; Drug Addiction; Epilepsy; Glutamate Receptor; Hippocampus (Brain); Impairment; In Vitro; Lead; Learning; Long-Term Potentiation; Maintenance; Measurement; Measures; Mediating; Mediation; Mediator of activation protein; Memory; Molecular; Mus; N-Methylaspartate; Neurons; Nucleotides; Peptides; Pharmacology; Phase; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Proteins; Resolution; Role; Signal Transduction; Site; Slice; Specificity; Staurosporine; Stimulus; Stroke; Synapses; Synaptic plasticity; System; Testing; Therapeutic; Training; Wild Type Mouse; addiction; calmodulin-dependent protein kinase II; clinically relevant; density; genetic manipulation; in vitro Assay; information gathering; inhibitor/antagonist; interest; long term memory; memory acquisition; memory retention; morris water maze; mouse model; nervous system disorder; prevent; protein function; public health relevance; response

DESCRIPTION (provided by applicant): Long-term potentiation (LTP) is a cellular process important in the mediation of synaptic strength, which is thought to underlie learning and memory. Ca2+/Calmodulin-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor subunit 2B (GluN2B) are two important proteins in LTP. The interaction between the proteins is additionally required for normal induction of LTP. The functions of these proteins have been associated with a number of neurological disorders such as epilepsy, stroke, and addiction. Of particular interest is the role f CaMKII in the maintenance phase of LTP. While this protein has a well- defined role in LTP induction, its function in maintenance is unclear. Many studies have demonstrated that enzymatic activity of CaMKII is not required; however, it has recently been suggested that a structural interaction between CaMKII and the NMDAR-complex may have an important role. It is further speculated that CaMKII-GluN2B binding specifically mediates this effect due to a few key aspects of that interaction. Namely, translocation of CaMKII to GluN2B is induced by LTP stimuli, the interaction maintains CaMKII in an active state after stimuli have subsided, and binding also persists post-stimuli. Therefore, GluN2B maintains CaMKII activity and localization after LTP induction, providing a theoretical explanation for the structural role of CaMKII in LTP maintenance. The current study will further investigate the role of CaMKII-GluN2B binding in LTP maintenance in three aims, specifically hypothesizing that the interaction is required for maintenance as well as the behavioral correlate of that process, i.e. memory storage. Aim 1: How does enzymatic activity of CaMKII effect GluN2B binding? Hypothesis: Enzymatic activity of CaMKII is not required for its interaction with GluN2B. Aim will be addressed using two ATP competitive inhibitors of CaMKII, H7 and staurosporine (ST), using the following approaches: (i) measurement of CaMKII-GluN2B binding in an in vitro assay using purified proteins, and (ii) stimulus-induced translocation of CaMKII to GluN2B in heterologous cellular expression systems and in primary neuron cultures. Aim 2: Is LTP maintenance mediated by the persistent association of CaMKII-GluN2B? Hypothesis: Sustained binding of CaMKII to GluN2B after LTP stimuli is required for LTP maintenance. Aim will be addressed by comparing wild type (WT) and CaMKII-GluN2B binding incompetent knockin mice (KI) with respect to (i) reduction in LTP maintenance measured under the treatment with the CaMKII inhibitor tatCN21 and (ii) reduction in CaMKII-NMDAR complexes under tatCN21 treatment. Aim 3: Are CaMKII-NMDAR complexes required for memory consolidation? Hypothesis: Memory storage is dependent on the persistent association of CaMKII-NMDAR-complexes. Aim will be addressed by training WT mice on the Morris Water Maze (MWM) and then testing their memory retention after administration of tatCN21, expecting that the inhibitor will reverse memory consolidation.

描述（由申请人提供）： 长期增强（LTP）是一个在突触强度介导的细胞过程中，被认为是学习和记忆的基础。 Ca2+/钙调蛋白依赖性蛋白激酶II（CAMKII）和NMDA型谷氨酸受体亚基2B（GLUN2B）是LTP中的两个重要蛋白质。蛋白质之间的相互作用是正常诱导LTP所需的。这些蛋白质的功能与多种神经系统疾病有关，例如癫痫，中风和成瘾。特别令人感兴趣的是f camkii在LTP的维护阶段中的作用。尽管该蛋白质在LTP诱导中具有明确的作用，但其维护功能尚不清楚。许多研究表明，不需要CAMKII的酶活性。但是，最近有人提出，CaMKII和NMDAR复合物之间的结构相互作用可能具有重要作用。进一步推测，由于该相互作用的一些关键方面，CAMKII-GLUN2B结合特异性介导了这种效果。即，LTP刺激诱导了CAMKII向Glun2b的易位，这种相互作用在刺激后保持CAMKII处于活跃状态，并且结合在刺激后也持续存在。因此，Glun2b在LTP诱导后保持CAMKII活性和定位，为CAMKII在LTP维持中的结构作用提供了理论上的解释。当前的研究将进一步研究CAMKII-GLUN2B结合在LTP维持中的三个目标中的作用，特别是假设这种相互作用是维护所必需的，以及该过程的行为相关性，即存储器存储。 AIM 1：CAMKII的酶活性如何效应Glun2b结合？假设：CAMKII的酶活性与Glun2b的相互作用不需要。使用以下方法，将使用CAMKII，H7和Staurosporine（ST）的两个ATP竞争抑制剂来解决目标：（ i）使用纯化的蛋白质的体外测定中CaMKII-GLUN2B结合的测量，以及（II）刺激诱导的camkkii to glun2b in neur interology intressods andologolodolodolodology od opricanos in opricanologolosology andologolodology andologolodology andologolodology andologology。 AIM 2：LTP维护是否是由CAMKII-GLUN2B持续关联介导的？假设：LTP维护需要LTP刺激后CAMKII与Glun2b的持续结合。与（i）在Camkii-Nmdar Complexts降低CAMKII-NMDAR Complects在TATCN2治疗的情况下，在CAMKII抑制剂TATCN21和（II）降低CAMKII抑制剂Tatcn21和（II）下，将野生型（WT）和CAMKII-GLUN2B结合无能力的敲门蛋白小鼠（KI）进行比较，将解决目标。 AIM 3：记忆合并需要CAMKII-NMDAR复合体吗？假设：存储器存储取决于CAMKII-NMDAR复合物的持续关联。 AIM将通过在Morris Water Maze（MWM）上训练WT小鼠，然后在给药TatCN21后测试其记忆力，并期望抑制剂会逆转内存巩固。