Mechanism and physiology of NMDA receptor desensitization

项目摘要 NMDA受体功能减退与神经精神疾病有关，如精神分裂症， 在美国有超过220万患者患有这种致残性精神疾病。NMDA受体的一种形式 功能减退是异常门控，即受体将谷氨酸结合转化为开放的过程。 相关的离子通道。调节功能减退的一种途径是NMDA受体脱敏， 门控配置，其中受体是配体结合的，但离子通道是不导电的。但其 生理和机械基础的定义很差，部分原因是缺乏研究它的工具。 我将利用新的工具-单位点突变，选择性地改变特定的功能， NMDA受体脱敏最近在Wollerland实验室发现-以及尖端 技术，以解决机制和生理学的NMDA受体脱敏。 而非NMDA受体的脱敏几乎完全依赖于重排， 在配体结合域（LBD）二聚体界面中，这种机制在NMDA受体中不太重要 所有的结构域，尤其是跨膜结构域（TMD），都与NMDA受体有关 脱敏。在目的1中，我将测试一般假设，即NMDA受体的机制， 失敏作用与非NMDA受体的失敏作用根本不同，强烈依赖于 离子通道的构象。为了检验这个假设，我将利用新发现的单站点 突变，膜片钳电生理学以及调节亚基组成的技术， NMDA受体和光活化非天然氨基酸。这些实验将有助于确定 控制NMDA受体脱敏的机制，有助于开发新的治疗方法 它可以通过特异性靶向脱敏选择性调节NMDA受体活性。 在目标2中，我将检验NMDA受体脱敏导致兴奋性信号减少的假设 在高活动时的突触。事实上，我的初步数据表明，快速应用谷氨酸引起 在这些突变体中，电流衰减程度更高，表明NMDA脱敏的潜在作用 机能减退为了更严格地验证这一假设，我将表达突变的NMDA受体， 改变器官型海马切片培养物的脱敏特性，以解决它们如何改变 突触动力学，包括Ca 2+内流和突触可塑性。这些实验将解决NMDA 受体脱敏有助于突触生理学。 本提案中的实验所获得的信息将提供对机制的深入了解 NMDA受体脱敏及其在突触动力学中的作用。我的实验将有助于 开发通过特异性地调节NMDA受体活性的新疗法 靶向脱敏

PROJECT SUMMARY NMDA receptor hypofunction has been implicated in neuropsychiatric disorders such as schizophrenia, a disabling mental disorder that affects over 2.2 million patients in the US. One modality for NMDA receptor hypofunction is aberrant gating, the process where the receptor converts glutamate binding into opening of the associated ion channel. One pathway to regulate hypofunction is NMDA receptor desensitization, a gating configuration where the receptor is ligand-bound, but the ion channel is non-conducting. However, its physiological and mechanistic basis is poorly defined in part because of the lack of tools to study it. In this proposal I will take advantage of new tools – single site mutations that selectively alter specific features of NMDA receptor desensitization recently discovered in the Wollmuth lab – as well as cutting edge technologies to address the mechanism and physiology of NMDA receptor desensitization. Whereas desensitization in non-NMDA receptors depends almost exclusively on the rearrangement of the ligand-binding domain (LBD) dimer interface, such a mechanism is less significant in NMDA receptors and all domains, most notably the transmembrane domain (TMD), have been implicated in NMDA receptor desensitization. In Aim 1, I will test the general hypothesis that the mechanism of NMDA receptor desensitization is fundamentally different from that of non-NMDA receptors, depending strongly on the conformation of the ion channel. To test this hypothesis, I will take advantage of newly identified single-site mutations, patch clamp electrophysiology as well as techniques to regulate the subunit composition of NMDA receptors and light-activated unnatural amino acids. These experiments will help define the structural mechanisms that govern NMDA receptor desensitization, aiding in the development of novel therapeutics that can selectively modulate NMDA receptor activity by specifically targeting desensitization. In Aim 2, I will test the hypothesis that NMDA receptor desensitization leads to decreased excitatory signaling at synapses during high activity. Indeed, my preliminary data show that fast applications of glutamate caused a higher degree of current decay in these mutants, suggesting a potential role of desensitization in NMDA hypofunction. To test this hypothesis more rigorously, I will express mutant NMDA receptors that have altered desensitization properties in organotypic hippocampal slice cultures to address how they change synaptic dynamics including Ca2+ influx and synaptic plasticity. These experiments will address how NMDA receptor desensitization contributes to synaptic physiology. The information gained by the experiments in this proposal will provide insight into the mechanism of NMDA receptor desensitization and its role in synaptic dynamics. My experiments will aid in the development of novel therapeutics that selectively modulates NMDA receptor activity by specifically targeting desensitization.