Structure and Function of NMDA Receptors

描述(申请人提供)：N-甲基D-天冬氨酸受体，是谷氨酸受体的一个亚型，在哺乳动物中枢神经系统中介导兴奋性信号传递。它们的主要功能是将化学信号转化为电信号，即谷氨酸与受体中的胞外区域结合，触发受体中阳离子可渗透跨膜通道的形成。鉴于这些受体在调节许多生理过程中的重要性以及在疾病状态下调节其功能的必要性，主要问题是激动剂如何激活蛋白质以及如何调节这一机制。为了解决这个问题，我们建议在激动剂和调节剂存在的情况下，结合光谱和生化方法来确定激动剂结合域和N末端调节剂结合域的完整构象。我们的假说是基于NMDA受体和密切相关的AMPA受体的分离成分的结构和电生理测量，认为受体的活性受受体中处于闭合裂隙构象的激动剂结合域和N-端域的部分控制。我们建议通过单分子和系综FRET研究来验证这一假设，以探索构象变化，然后将其与单通道和全细胞电流记录确定的功能结果相关联。此外，我们假设激动剂甘氨酸和谷氨酸之间的负协同作用是由受体二聚体结构中二聚体内部和之间的界面接触所介导的。为了验证这一假说，我们将研究稳定和不稳定二聚体界面以及激动剂结合结构域中的二聚体间界面对激动剂解离速率所衡量的功能的影响。基于功能的研究将通过使用LRET测量亚基上的距离变化来进一步从结构上得到证实。这些功能和结构的研究将为激动剂和调节剂介导NMDA受体功能的机制提供全面的理解。 与公共健康相关：NMDA受体是谷氨酸受体的一个亚型，在人类中枢神经系统中介导兴奋性反应，并在控制认知和运动反应方面发挥重要作用。在这里，我们建议确定激活和调节NMDA受体功能的机制和结构变化。这些研究将在分子水平上提供关于NMDA受体功能如何改变的见解，从而帮助治疗与其相关的临床状况。。

DESCRIPTION (provided by applicant): N-methyl D-aspartate receptors, are a subtype of glutamate receptors that mediate excitatory signal transmission in the mammalian central nervous system. Their primary function involves converting the chemical signal into an electrical signal, i.e. glutamate binding to an extracellular domain in the receptor triggers the formation of cation permeable transmembrane channels in the receptor. Given the importance of these receptors in mediating a number of physiological processes and the need to modulate their function in disease states, the primary questions are how does the agonist activate the protein and how can this mechanism be modulated. To address this question, we propose to use a combination of spectroscopic and biochemical methods to identify the complete conformational landscape of the agonist binding domain and the N-terminal modulator binding domain, in the presence of agonists and modulators that induce a large spectrum of activity. Our hypothesis based on the structures of the isolated components and the electrophysiological measurements of the NMDA receptor and the closely related AMPA receptors is that the activity of the receptor is controlled by the fraction of the agonist binding domain and the N-terminal domain that are in a closed cleft conformation in the receptor. We propose to test this hypothesis by using single molecule and ensemble FRET investigations to probe the conformational changes, which will then be correlated to functional consequences as determined by single channel and whole cell current recordings. Additionally, we hypothesize that the negative cooperativity between the agonist glycine and glutamate is mediated by the interface contacts within and across the dimers, in the dimer of dimer structure of the receptor. To test this hypothesis we will investigate the effect of stabilizing and destabilizing the dimer interface as well as the inter dimer interface in the agonist binding domain on the function as measured by the rates of agonist dissociation. The function based studies will be further corroborated structurally by measuring distance changes across the subunit using LRET. These functional and structural investigations will provide a comprehensive understanding of the mechanism by which agonists and modulators mediate NMDA receptor function. PUBLIC HEALTH RELEVANCE: NMDA receptors are a subtype of the glutamate receptor that mediate excitatory responses in the human central nervous system, and play an important role in controlling the cognitive and motor responses. Here we propose to determine the mechanistic and structural changes that underlie the activation and modulation of the function of the NMDA receptors. These investigations will provide insight at a molecular level as to how NMDA receptor function can be altered and hence aid in the treatment of the clinical conditions associated with it. .