Dynorphin Modulation of N-Methyl-D-Aspartate (NMDA) Receptor Function

描述（由申请人提供）：每年有成千上万的人因脊髓损伤而丧生。如果设计出阻断幸存组织的创伤后降解的治疗方法，这些患者经历的运动缺陷和疼痛的很大一部分可以预防。脊髓的创伤引发了一系列生化事件，加剧了损伤。延长的损伤导致运动功能的额外下降和感觉障碍，如慢性疼痛。作为这些过程的一部分，阿片肽强啡肽浓度增加，并在创伤后时期在脊髓中释放。强啡肽先前被证明增强N-甲基-D-天冬氨酸（NMDA）受体功能，从而在脊髓中产生神经元损伤。因此，强啡肽可能是损伤后退行性事件的重要参与者。本研究将检验强啡肽与不具有蛋白质外显子5编码区（NRla）的NR 1剪接变体结合以暴露先前无活性或低活性NMDA受体的假设。将被检验的第二个假设是强啡肽诱导NMDA受体的内部储存迁移到质膜。为了验证这些假设，提出了三个目标。在第一个目的中，Hek-293细胞将用32对可能的NR 1剪接变体和NR 2亚基瞬时转染。将通过使用高通量96孔测定法测量乳酸脱氢酶释放到培养基中来评价各种亚基组合对强啡肽和NMDA诱导的兴奋性毒性的敏感性。在第二个目标中，将使用全细胞膜片钳和细胞表面标记技术检查对强啡肽最敏感的NR 1和NR 2亚基的组合（从目标1确定），以确定强啡肽如何影响受体的功能。在最终的目标中，将使用免疫共沉淀、2D电泳、免疫组织化学和RT-PCR来确定脊髓中表达的NR 1剪接变体和NR 2亚基对。这些实验将提供脊髓中强啡肽增强的NMDA受体的亚基身份和强啡肽如何增强NMDA受体功能的机制。希望这些信息将提供一个有价值的药理学靶点，中断强啡肽参与脊髓创伤后神经退行性变。

DESCRIPTION (provided by applicant): Thousands of lives are devastated each year by spinal cord injury. A significant portion of the motor deficits and pain experienced by these patients could be prevented if therapies were devised to block posttrauma degradation of the surviving tissue. Trauma to the spinal cord initiates a cascade of biochemical events that exacerbate the injury. The extended damage leads to an additional decline in motor function and to sensory disturbances such as chronic pain. As part of these processes the opioid peptide dynorphin increases in concentration and is released in the spinal cord during the post-trauma period. Dynorphin was previously demonstrated to enhance N-methyl-D-aspartate (NMDA) receptor function to produce neuronal damage in the spinal cord. Thus, dynorphin is likely to be a significant participant in the post-insult degenerative events. This study will test the hypothesis that dynorphin binds to NR1 splice variants that do not possess the exon-5 coded region of the protein (NRla) to expose previously inactive or low activity NMDA receptors. A second hypothesis that will be tested is that dynorphin induces migration of internal stores of NMDA receptors to the plasma membrane. To test these hypotheses three aims are proposed. In the first aim Hek-293 cells will be transiently transfected with the 32 possible pairs of NR1 splice variants and NR2 subunits. The sensitivity of the various subunit combinations to dynorphin and NMDA induced excitotoxicity will be evaluated by measuring the release of lactate dehydrogenase into the culture media using a high throughput 96 well assay. In the second aim the combinations of NR1 and NR2 subunits that are most sensitive to dynorphin (determined from aim 1) will be examined using whole cell patch clamp and cell surface labeling techniques to determine how dynorphin influences the receptor's function. In the final aim the pairs of NR1 splice variants and NR2 subunits expressed in the spinal cord will be determined using co-immunoprecipitation, 2D-electrophoresis, immunohistochemistry and RT-PCR. These experiments will provide the subunit identity of the dynorphin enhanced NMDA receptors in the spinal cord and the mechanism for how dynorphin enhances NMDA receptor function. It is hoped that this information will provide a valuable pharmacological target for interrupting dynorphin's participation in post spinal trauma neurodegeneration.