Structure and function of hetero-multimeric ligand-gated ion channels

项目总结 这个项目的目标是深入了解多个异构体离子通道的机理， N-甲基-D-天冬氨酸受体(NMDAR)，受不同化合物、剪接变异体和亚型调节。 这些受体属于离子型谷氨酸受体(IGluRs)家族，该受体介导大多数 兴奋性突触传递和在基本大脑功能、发育和神经学中发挥重要作用 疾病，如癫痫、中风、抑郁症和精神分裂症，以及帕金森和阿尔茨海默氏症 疾病。NMDAR是由GluN1和GluN2和/或 GluN3亚基。GluN1和GluN3亚基结合包括甘氨酸和D-丝氨酸在内的共同激动剂，而 GluN2亚基与神经递质谷氨酸结合。每个亚基蛋白质由一个氨基末端组成。 结构域(ATD)、配体结合结构域(LBD)、跨膜结构域(TMD)和羧基末端结构域 (CTD)。GluN1-GluN2NMDAR在与甘氨酸和 谷氨酸，而GluN1-GluN3 NMDAR仅由甘氨酸激活。我们最近解决了第一个结构 对完整的异四聚体大鼠GluN1-GluN2B NMDARs，最初通过X射线结晶学，后来通过冷冻- 电子显微镜。尽管最近取得了进展，但关于 竞争性拮抗剂的激活、脱敏、抑制和功能多样性的机制 通过在GluN1和不同亚基组合(GluN1-GluN2A-D和GluN3A-B)中的选择性剪接。这个 GluN3 NMDAR的结构仅限于GluN3A LBD的结构，因此限制了我们对 这个未被研究的亚型是如何形成离子通道和中介功能的。因此，拟议项目的目标是 是研究不同物种的复合结合、蛋白质构象和亚基排列的模式 NMDAR的功能状态和亚型。为了实现这些目标，我们将进行以下研究：目标1 确定激动剂和拮抗剂的激活、脱敏和抑制机制；目标2 定义剪接体中多胺改变pH敏感性和失活速度的潜在机制- 目标3首次揭示了GluN1-GluN3 NMDAR的结构。 这三个目标将通过获得GluN1-GluN2B NMDAR的结构信息在 激动剂和拮抗剂的存在，带有和不带有精胺的剪接变异体，以及GluN1-GluN3A NMDAR由X射线结晶学和低温电子显微镜相结合。基于结构的功能假说将 通过涉及电生理学的实验进行测试。成功完成拟议的研究将提供 深入了解NMDAR亚型和剪接变体的复杂功能，由 复合结合之后是多个亚基和结构域的重排。这些发现将支持 治疗破坏性神经疾病和疾病的治疗策略的发展 如上所述。

PROJECT SUMMARY The goal of this project is to gain an in-depth mechanistic understanding about multi-heteromeric ion channels, N-methyl-D-aspartate receptors (NMDARs), regulated by different compounds, splice variants, and subtypes. These receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of excitatory synaptic transmission and play significant roles in basic brain functions, development, and neurological disorders such as seizures, strokes, depression, and schizophrenia, as well as Parkinson’s and Alzheimer’s diseases. NMDARs are hetero-multimeric ligand-gated ion channels composed of GluN1 and GluN2 and/or GluN3 subunits. The GluN1 and GluN3 subunits bind co-agonists including glycine and D-serine, whereas the GluN2 subunits bind the neurotransmitter, glutamate. Each subunit protein is composed of an amino terminal domain (ATD), a ligand-binding domain (LBD), a transmembrane domain (TMD), and a carboxyl terminal domain (CTD). The GluN1-GluN2 NMDARs open their transmembrane ion channels upon binding of glycine and glutamate, whereas the GluN1-GluN3 NMDARs activate by glycine alone. We recently solved the first structure of the intact hetero-tetrameric rat GluN1-GluN2B NMDARs, initially by x-ray crystallography and later by cryo- electron microscopy. Despite recent advances, there are many fundamental questions remaining regarding the mechanism of activation, desensitization, inhibition by competitive antagonists, and functional diversity elicited by alternative splicing in GluN1 and different subunit combinations (GluN1-GluN2A-D and GluN3A-B). The structure of the GluN3 NMDARs is limited to that of the GluN3A LBD, thus, restricting our understanding about how this under-studied subtype form ion channels and mediate functions. Thus, the goal of the proposed project is to investigate patterns of compound bindings, protein conformations and subunit arrangements in various functional states and subtypes of NMDARs. To achieve these goals, we will conduct research aimed at: Aim 1 determining the mechanisms of activation, desensitization, and inhibition by agonists and antagonists; Aim 2 defining the underlying mechanism for altered pH-sensitivity and deactivation speeds by polyamines in a splice- variant-specific manner; and Aim 3 revealing the architecture of the GluN1-GluN3 NMDAR for the first time. These three aims will be achieved by obtaining the structural information of the GluN1-GluN2B NMDAR in the presence of agonists and antagonists, splice variants with and without spermine, and the GluN1-GluN3A NMDAR by a combination of x-ray crystallography and cryo-EM. The structure-based functional hypotheses will be tested by experiments involving electrophysiology. Successful completion of the proposed studies will provide in-depth information into the sophisticated function of NMDAR subtypes and splice variants mediated by compound bindings followed by rearrangement of multiple subunits and domains. These findings will support the development of therapeutic strategies used to treat the devastating neurological disorders and diseases mentioned above.