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

强啡肽调节 N-甲基-D-天冬氨酸 (NMDA) 受体功能

• 批准号: 6964766
• 负责人: ROBERT M CAUDLE
• 金额: $ 31.5万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6964766
• 关键词: NMDA receptors; cell membrane; dynorphins; gel electrophoresis; high throughput technology; immunocytochemistry; immunoprecipitation; laboratory rat; lactate dehydrogenases; polymerase chain reaction; protein structure function; receptor expression; spinal cord; tissue /cell culture; transfection; voltage /patch clamp

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.

描述（由申请人提供）：每年有数千人因脊髓损伤而丧生。如果设计出治疗方法来阻止幸存组织的创伤后退化，那么这些患者所经历的大部分运动缺陷和疼痛是可以预防的。脊髓损伤会引发一系列生化事件，从而加剧损伤。长期损害会导致运动功能进一步下降和慢性疼痛等感觉障碍。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。强啡肽先前被证明可以增强 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，强啡肽可能是侮辱后退化事件的重要参与者。这项研究将测试强啡肽与不具有蛋白质外显子 5 编码区 (NRla) 的 NR1 剪接变体结合的假设，以暴露先前不活跃或低活性的 NMDA 受体。将要测试的第二个假设是强啡肽诱导 NMDA 受体内部储存迁移到质膜。为了检验这些假设，提出了三个目标。第一个目标是用 32 对可能的 NR1 剪接变体和 NR2 亚基瞬时转染 Hek-293 细胞。各种亚基组合对强啡肽和 NMDA 诱导的兴奋性毒性的敏感性将通过使用高通量 96 孔测定法测量乳酸脱氢酶释放到培养基中来评估。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 NR1 和 NR2 亚基组合（根据目标 1 确定），以确定强啡肽如何影响受体的功能。在最终目标中，将使用免疫共沉淀、2D 电泳、免疫组织化学和 RT-PCR 确定脊髓中表达的 NR1 剪接变体对和 NR2 亚基对。这些实验将提供脊髓中强啡肽增强 NMDA 受体的亚基身份以及强啡肽如何增强 NMDA 受体功能的机制。希望这一信息将为中断强啡肽参与脊柱创伤后神经变性提供有价值的药理学靶点。