The NMDA Receptor Interaction with CaMKII

描述(由申请人提供)： 突触是神经元信号传递的中心，也是神经疾病药物治疗的关键靶点。钙离子通过NMDA受体内流和随后激活CaMKII是LTP诱导和学习记忆过程中的关键事件。NMDA受体与CaMKII以一种复杂和高度调控的方式相互作用。这种相互作用将CaMKII置于一个具有战略意义的理想位置，在那里，通过NMDA受体的钙离子内流最有效地激活CaMKII，并且它靠近突触后部位的底物(例如，LTP中的GluRl)。生化细节和这种相互作用的功能相关性将被确定，这一点目前尚未得到证实。将仔细研究的初步结果表明，NMDA受体NR1亚单位上的CaMKII、钙调素和α-肌动蛋白结合部位重叠，钙/钙调蛋白通过取代α-肌动蛋白促进CaMKII与NR1的结合。CaMKII与NR1或NR2B亚基本身的结合是否会改变NMDA受体的活性，就像前面观察到的钙/钙调蛋白与NR1结合一样，将用不同的电生理学方法来研究，包括从内向外记录不同组合中纯化的CaMKII、钙调素和α-肌动蛋白，以及使用以不同组合转染NR1、NR2A、NR2B和CaMKII的HEK293细胞进行全细胞膜片钳。CaMKII与NMDA受体的结合是否是将CaMKII重新招募到突触后位置的关键，将在原代海马培养中通过在没有或存在抑制CaMKII与NR1或NR2B结合的多肽(膜通透性或注射)的情况下进行GFP标记的CaMKII成像来测试。NMDA受体-CaMKII相互作用对LTP诱导的重要性将通过细胞内记录结合抑制肽和不结合抑制肽来评估。CaMKII对GluR1的磷酸化可能有助于LTP，并将在切片中用生化方法进行定量。谷氨酸能突触的过度刺激与中风、癫痫持续状态和脑创伤的神经病理有关。NMDA受体介导的钙内流和CaMKII激活在脑缺血所致的神经元损伤中起关键作用。因此，NMDA受体对CaMKII的突触后锚定构成了一个潜在的非常重要的药物靶点，可以特异性地破坏这种相互作用，从而减轻由于谷氨酸受体过度激活而引起的神经病理。

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.