The NMDA Receptor Interaction with CaMKII

描述（由申请人提供）： 突触是神经元信号传导的中心，也是神经系统疾病药物治疗的关键靶点。通过NMDA受体的Ca 2+内流和随后的CaMKII激活是LTP诱导和学习记忆中的关键事件。NMDA受体与CaMKII以复杂和高度调节的方式相互作用。这种相互作用将CaMKII置于战略上理想的位置，在那里它被通过NMDA受体的Ca 2+内流最有效地激活，并且在那里它在突触后位点靠近其底物（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，这是目前未经证实的。初步结果将被仔细检查，表明NMDA受体NR 1亚基上的CaMKII、钙调蛋白和α-辅肌动蛋白结合位点重叠，并且Ca 2 +/钙调蛋白通过置换α-辅肌动蛋白促进CaMKII与NR 1结合。CaMK II与NR 1和可能的NR 2B亚基本身的结合是否改变了NMDA受体活性，如先前观察到的Ca 2 +/钙调蛋白与NR 1的结合，将用不同的电生理学方法研究，包括从用纯化的CaMK II、钙调蛋白和α-辅肌动蛋白以各种组合灌注的切除的由内而外的膜片记录，以及使用用NR 1、NR 2A、NR 2B和CaMKII的不同组合。将在原代海马培养物中通过在不存在和存在抑制CaMKII的NR 1或NR 2B结合的肽（膜可渗透的或注射的）的情况下对GFP标记的CaMKII进行成像来测试CaMKII与NMDA受体的结合是否对于将CaMKII募集到突触后位点至关重要。NMDA受体- CaMKII相互作用对于诱导LTP的重要性将通过来自具有和不具有结合抑制肽的海马切片的细胞内记录来评估。CaMK II对GluR 1的磷酸化可能有助于LTP，并将用生化方法在切片中定量。神经元能突触的过度刺激与中风、癫痫持续状态和脑外伤引起的神经病理学有关。NMDA受体介导的Ca 2+内流和CaMKII激活是缺血引起的神经元损伤的关键。因此，NMDA受体对CaMKII的突触后锚定构成了药物的一个潜在的非常重要的靶点，该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活化而引起的神经病理学。

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.