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）： 长时程增强（LTP）是一种在突触强度调节中起重要作用的细胞过程，被认为是学习和记忆的基础。Ca 2 +/钙调素依赖性蛋白激酶II（CaMKII）和NMDA型谷氨酸受体亚基2B（GluN 2B）是LTP中的两个重要蛋白。蛋白质之间的相互作用对于LTP的正常诱导是额外需要的。这些蛋白质的功能与许多神经系统疾病如癫痫、中风和成瘾有关。特别令人感兴趣的是钙调素激酶II在LTP维持阶段的作用。虽然这种蛋白质在LTP诱导中具有明确的作用，但其在维持中的功能尚不清楚。许多研究表明，CaMKII的酶活性是不需要的，然而，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CaMKII-GluN 2B结合特异性介导这种作用，这是由于该相互作用的几个关键方面。也就是说，易位的CaMKII的GluN 2B诱导的LTP刺激，相互作用保持CaMKII在一个活跃的状态后，刺激已经消退，和绑定也持续刺激后。因此，GluN 2B在LTP诱导后维持CaMKII的活性和定位，为CaMKII在LTP维持中的结构作用提供了理论解释。本研究将进一步研究CaMKII-GluN 2B结合在三个目标中的LTP维持中的作用，特别是假设这种相互作用是维持所需的，以及该过程的行为相关性，即记忆储存。目的1：CaMK II的酶活性如何影响GluN 2B结合？假设：CaMK II的酶活性不是其与GluN 2B相互作用所必需的。目的将使用CaMKII的两种ATP竞争性抑制剂H7和星形孢菌素（ST），使用以下方法来解决：（i）使用纯化的蛋白质在体外测定中测量CaMKII-GluN 2B结合，和（ii）在异源细胞表达系统和原代神经元培养物中刺激诱导的CaMKII向GluN 2B的易位。目的2：LTP的维持是否由CaMKII-GluN 2B的持续结合介导？假设：在LTP刺激后，CaMK II与GluN 2B的持续结合是LTP维持所必需的。目的将通过比较野生型（WT）和CaMKII-GluN 2B结合缺陷型敲入小鼠（KI）在以下方面来解决：（i）在用CaMKII抑制剂tatCN 21处理下测量的LTP维持的减少和（ii）在tatCN 21处理下CaMKII-NMDAR复合物的减少。目的3：记忆巩固需要CaMKII-NMDAR复合物吗？假设：记忆存储依赖于CaMKII-NMDAR复合物的持续关联。通过在Morris水迷宫（MWM）上训练WT小鼠，然后在施用tatCN 21后测试它们的记忆保持，期望抑制剂将逆转记忆巩固来解决目的。