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）：每年脊髓损伤每年都会遭受数千人的生活。如果设计疗法可以阻止幸存组织的创伤后降解，则可以预防这些患者经历的运动缺陷和疼痛的很大一部分。脊髓的创伤引发了一系列生化事件，加剧了伤害。扩展的损害导致运动功能的额外下降和感觉干扰，例如慢性疼痛。作为这些过程的一部分，阿片类肽驱肽的浓度增加，并在创伤后时期在脊髓中释放。先前证明dynorphin可以增强N-甲基-D-天冬氨酸（NMDA）受体功能，以在脊髓中产生神经元损伤。因此，Dynorphin可能是冥想后退化事件的重要参与者。这项研究将检验以下假设：dynorphin与不具有蛋白质（NRLA）外显子5编码区的NR1剪接变体结合以暴露以前无活性或低活性NMDA受体。将要检验的第二个假设是，测能引起NMDA受体内部存储到质膜的迁移。为了检验这些假设，提出了三个目标。在第一个目标中，HEK-293细胞将用32对NR1剪接变体和NR2亚基瞬时转染。通过使用高通量96井测定法测量乳酸脱氢酶在培养基中释放到培养基中，可以评估各种亚基组合和NMDA诱导的兴奋性毒素的敏感性。在第二个目标中，将使用全细胞贴片夹和细胞表面标记技术来检查最敏感的NR1和NR2亚基的组合（根据AIM 1确定），以确定Dynorphin如何影响受体的功能。在最终目的中，将使用共免疫沉淀，2D-电泳，免疫组织化学和RT-PCR确定脊髓中表达的NR1剪接变体和NR2亚基。这些实验将提供脊髓中增强的NMDA受体的亚基身份，以及Dynorphin如何增强NMDA受体功能的机制。希望这些信息将为中断Dynorphin参与脊柱创伤后神经变性的参与提供宝贵的药理目标。