The NMDA Receptor Interaction with CaMKII

NMDA 受体与 CaMKII 的相互作用

• 批准号: 6895243
• 负责人: JOHANNES W HELL
• 金额: $ 28.03万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-20 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6895243
• 关键词: NMDA receptors; binding sites; calcium flux; calmodulin dependent protein kinase; dendrites; fluorescence microscopy; glutamate receptor; hippocampus; laboratory mouse; laboratory rat; learning; long term potentiation; memory; neurons; phosphorylation; polymerase chain reaction; protein binding; protein protein interaction; receptor binding; synapses; tissue /cell culture; voltage /patch clamp

DESCRIPTION (provided by applicant): Synapses are central to neuronal signaling and key targets for drug treatments of neurological disorders. Ca2+ influx through NMDA receptors and subsequent activation of CaMKII are critical events in the induction of LTP and in learning and memory. The NMDA receptor interacts with CaMKII in a complex and highly regulated manner. This interaction places CaMKII at a strategically ideal location, where it is most effectively activated by Ca2+ influx through the NMDA receptor and where it is close to its substrates at the postsynaptic site (e.g., GluRl in LTP). Biochemical details and the functional relevance of this interaction, which is currently unproven, will be determined. Preliminary results will be scrutinized that indicate that the CaMKII, calmodulin, and a-actinin binding sites on the NR1 subunit of the NMDA receptor overlap and that Ca2+/calmodulin promotes CaMKII binding to NR1 by displacing a-actinin. Whether binding of CaMKII to the NR1 and perhaps NR2B subunit per se changes NMDA receptor activity as observed earlier for Ca2+/calmodulin binding to NR1 will be investigated with different electrophysiological methods including recording from excised inside-out patches perfused with purified CaMKII, calmodulin and a-actinin in various combinations and whole cell patch clamping using HEK293 cells transfected with NR1, NR2A, NR2B and CaMKII in different combinations. Whether CaMKII binding to the NMDA receptor is critical for recruiting CaMKII to the postsynaptic site will be tested in primary hippocampal cultures by imaging of GFP-tagged CaMKII in the absence and presence of peptides (membrane-permeable or injected) that inhibit NR1 or NR2B binding of CaMKII. The importance of the NMDA receptor - CaMKII interaction for the induction of LTP will be evaluated by intracellular recording from hippocampal slices with and without the binding-inhibiting peptides. Phosphorylation of GluR1 by CaMKII likely contributes to LTP and will be quantified in slices with biochemical methods. Overstimulation of glutamatergic synapses has been implicated in neuropathologies due to stroke, status epilepticus, and brain trauma. NMDA receptor-mediated Ca2+ influx and CaMKII activation are critical for neuronal damage caused by ischaemia. The postsynaptic anchoring of CaMKII by the NMDA receptor constitutes, therefore, a potentially very important target for drugs that can specifically disrupt this interaction and thereby alleviate neuropathologies due to overactivation of glutamate receptors.

描述（由申请人提供）： 突触是神经元信号传导和神经系统疾病药物治疗的关键靶标。 Ca2+通过NMDA受体的涌入以及随后的CAMKII激活是LTP以及学习和记忆中的关键事件。 NMDA受体以复杂且高度调节的方式与CAMKII相互作用。这种相互作用使CAMKII处于战略性理想的位置，在该位置，通过NMDA受体CA2+涌入最有效地激活了CAMKII，并且它接近其后突触部位的底物（例如，LTP中的Glurl）。将确定这种相互作用的生化细节和功能相关性，该相互作用将被确定。初步结果将被审查，以表明NMDA受体重叠的NR1亚基上的CAMKII，钙调蛋白和a-肌动蛋白结合位点，并且Ca2+/钙调蛋白通过替代A-肌酸会促进CAMKII与NR1促进CAMKII结合。是否将Ca2+/钙调蛋白与NR1结合的早期观察到的CaMKII与NR1和NR2B亚基的结合是否会改变NMDA受体的活性，并使用不同的电生理方法进行研究NR1，NR2A，NR2B和CAMKII不同组合。 CAMKII是否与NMDA受体结合对于将CAMKII募集到突触后部位至关重要，将在原发性海马培养物中测试GFP标记的CAMKII在不存在和存在肽（膜 - 可渗透或注射）的情况下，抑制了NR1或NR1或NR2BINR2BI的CAMKKII。 NMDA受体-CAMKII相互作用对LTP的诱导的重要性将通过有或没有具有结合抑制肽的海马切片的细胞内记录来评估。 CAMKII对GLUR1的磷酸化可能有助于LTP，并将在具有生化方法的切片中进行量化。谷氨酸能突触的过度刺激已与中风，癫痫持续性和脑外伤引起的神经病理有关。 NMDA受体介导的Ca2+流入和CAMKII激活对于缺血引起的神经元损伤至关重要。因此，NMDA受体对CAMKII的突触后锚定构成了可能非常重要的药物靶标的药物，可以特异性破坏这种相互作用，从而减轻由于谷氨酸受体的过度激活而导致的神经病理学。