Mechanism and physiology of NMDA receptor desensitization

项目总结 NMDA受体功能低下与神经精神障碍有关，如精神分裂症， 致残精神障碍在美国影响着超过220万患者。NMDA受体的一种模式 功能减退是异常门控，是受体将谷氨酸结合转化为开放的过程。 关联的离子通道。调节功能减退的一个途径是NMDA受体脱敏，一种 门控结构，其中受体是配体结合的，但离子通道不导电。然而，它的 生理和机制基础的定义不明确，部分原因是缺乏研究它的工具。在这 提案I将利用新工具--选择性地改变特定特征的单部位突变 最近在Wollmuth实验室发现的NMDA受体脱敏-以及尖端 研究NMDA受体脱敏的机制和生理学的技术。 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受体中意义不大 所有的结构域，尤其是跨膜结构域(TMD)都与NMDA受体有关 脱敏。在目标1中，我将检验一般假设，即NMDA受体的机制 脱敏与非NMDA受体有根本的不同，强烈依赖于 离子通道的构象。为了验证这一假设，我将利用新确定的单站点 突变、膜片钳电生理学以及调节细胞亚基组成的技术 NMDA受体和光激活的非天然氨基酸。这些实验将有助于定义结构 控制NMDA受体脱敏的机制，有助于开发新的治疗药物 它可以通过特异性靶向脱敏来选择性地调节NMDA受体的活性。 在目标2中，我将验证NMDA受体脱敏导致兴奋性信号减少的假设 在高活动期的突触。事实上，我的初步数据显示，快速应用谷氨酸会导致 这些突变体中的电流衰减程度更高，这表明NMDA中可能存在脱敏作用 功能减退。为了更严格地检验这一假设，我将表达突变的NMDA受体 在器官型海马片培养中改变脱敏特性以解决它们如何变化的问题 突触动力学包括钙离子内流和突触可塑性。这些实验将解决NMDA如何 受体脱敏与突触生理学有关。 这项建议中的实验所获得的信息将为深入了解这一机制提供帮助。 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 通过特异性地选择性调节NMDA受体活性的新疗法的开发 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.