Structure and Function of NMDA Receptors

描述（由申请人提供）：N-甲基D-天冬氨酸受体是谷氨酸受体的一种亚型，在哺乳动物中枢神经系统中介导兴奋性信号传递。它们的主要功能涉及将化学信号转化为电信号，即谷氨酸与受体中的细胞外结构域结合触发受体中阳离子可渗透跨膜通道的形成。鉴于这些受体在介导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需要，主要问题是激动剂如何激活蛋白质以及如何调节这种机制。为了解决这个问题，我们建议使用光谱和生物化学方法的组合，以确定激动剂结合结构域和N-末端调节剂结合结构域的完整构象景观，在激动剂和调节剂的存在下，诱导大范围的活动。我们的假设的基础上分离的组件和NMDA受体和密切相关的AMPA受体的电生理测量的结构是，受体的活性控制的激动剂结合结构域和N-末端结构域的分数是在一个封闭的裂缝构象的受体。我们建议测试这一假设，通过使用单分子和合奏FRET调查，以探测构象变化，这将是相关的功能后果，确定由单通道和全细胞电流记录。此外，我们假设，在受体的二聚体结构的二聚体中，激动剂甘氨酸和谷氨酸之间的负协同性是由二聚体内和跨二聚体的界面接触介导的。为了检验这一假设，我们将研究稳定和不稳定的二聚体界面以及激动剂结合结构域中的二聚体间界面对通过激动剂解离速率测量的功能的影响。基于功能的研究将通过使用LRET测量亚基之间的距离变化在结构上得到进一步证实。这些功能和结构的调查将提供一个全面的了解的机制，激动剂和调节剂介导的NMDA受体功能。 公共卫生相关性：NMDA受体是谷氨酸受体的一种亚型，在人类中枢神经系统中介导兴奋性反应，并且在控制认知和运动反应中起重要作用。在这里，我们建议确定的机制和结构的变化，背后的激活和调节的功能的NMDA受体。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，从而有助于治疗与之相关的临床疾病。

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. .