Calcium/Calmodulin-dependent Protein Kinase â…¡(Camkâ…¡)Mediated Serine Phosphorylation Of Glur6and Its Regulation To Receptor Function Of Glur6

Background Although studies have shown that GluR6serine phosphorylation plays a important role in brain ischemia/reperfusion (I/R) mediated neuronal injury, little is known about the precise regulating mechanisms of serine phosphorylation of GluR6. At present, relationship between Ca2+/calmodulin-dependent protein kinase II (CaMKII) and specific regulating mechanism of GluR6serine phosphorylation induced by brain ischemia/reperfusion is also still unknown.Objectives This study has three main purposes,1) To investigate CaMKII whether can facilitate GluR6serine phosphorylation through CaMKIIGluR6PSD-95signaling module assembly, and further activate downstream JNK signaling pathway and lead to cell injury and death in cerebral I/R injury.2) To probe effects of CaMKII antisense oligodeoxynucleotides (ODNs) on the assembly of CaMKⅡ·GluR6·PSD-95signaling module, the GluR6serine phosphorylation and the activation of c-Jun N-terminal kinase (JNK), and further to reveal the neuroprotective mechanisms of CaMKII antisense ODNs against neuronal cell death induced by cerebral I/R.3) To identify the possible sites of GluR6serine phosphorylation mediated by CaMKⅡ.Methods:Transient cerebral ischemia (15min) and reperfusion rats animal model was established by four-vessel occlusion method. MK-801, Nifedipine and CaMKⅡ antisense ODNs used as tools medicine. GluR6mutants were constructed by Gene site-directed mutagenesis kit (Stratagene, USA). GluR6mutants and CaMKⅡ plasmid were cotransfected to HEK293cells by the method of liposome-mediated. The analyses of immunoprecipitation and immunoblotting were used to detect the situation of CaMKⅡ·GluR6·PSD-95signaling module assembly, GluR6serine phosphorylation and JNK3activation in rat hippocampus following transient cerebral I/R. The immunochemistry and histochemistry were used to detect the situation of cell injury and death after cerebral ischemia-reperfusion.Results:1) The CaMKⅡ·GluR6·PSD-95signaling module assembly and GluR6serine phosphorylation can be induced by transient cerebral I/R, and change trends of the two have obvious consistency.2) MK801, Nifedipine and KN-93can significantly suppress the assembly of CaMKⅡ·GluR6·PSD-95signaling module, the GluR6serine phosphorylation and the activation of JNK3induced by cerebral I/R.3) CaMKII antisense ODNs have important neuroprotective effects on neuronal cell injury and death in hippocampal CA1subfield of rats induced by cerebral ischemia/reperfusion.4) CaMKII antisense ODNs can knockdown the expression of CaMKII at gene levels; and further to suppress the increased of CaMKⅡ·GluR6·PSD-95signaling module assembly, GluR6serine phosphorylation, as well as the activation of JNK signaling pathway induced by cerebral I/R.5) constructed nine serine mutants of GluR6in intracellular side (serine mutanted to alanine).6) Serine phosphorylation sites of GluR6mediated by CaMKII most likely are the599and868sites of serine residue of GluR6.Conclusions:1) NMDA receptors and L-VGCCs depended-CaMKII could functionally modulate GluR6serine phosphorylation through the assembly of CaMKⅡ·GluR6·PSD-95signaling module after ischemic brain injury.2) CaMKII antisense ODNs can exert important neuroprotection against neuronal cell death induced by cerebral ischemia-reperfusion; Its possible molecular mechanism is CaMKII antisense ODNs can inhibit the expression of CaMKII, and further suppress CaMKⅡ·GluR6·PSD-95signal module assembly, GluR6serine phosphorylation as well as the activation of JNK signaling pathway induced by cerebral I/R.3) The possible sites of serine phosphorylation of GluR6mediated by CaMKII were identified preliminarily. This study also can provide new, potential and better treatment strategies for clinical cerebrovascular disease.