NSF/MCB-BSF: Collaborative Research: Towards development of the structural determinants of the Glutamate receptor gating regulation by auxiliary membrane anchored proteins

高级认知功能，包括定量推理、学习和记忆，起源于人类大脑，是通过突触连接的复杂神经元网络之间交流的结果。这种突触连接的关键分子元件是嗜离子性谷氨酸受体（iGluR），它是突触后神经元膜上的一种蛋白质，负责传导电信号，传递突触前神经元的信息。这一神经传递过程是由多种与谷氨酸受体结合的辅助蛋白调节的。该项目将阐明不同的辅助蛋白如何与谷氨酸受体相互作用，以及彼此之间如何启动和调节神经元之间的通信。这项研究增强了我们对脑功能分子基础的认识。该研究将涉及本科生和研究生，为大脑研究的前沿提供教育环境和培训机会。此外，还计划开展包括当地高中和大学在内的外展活动，以促进STEM领域的多元化参与。人们越来越认识到，大多数蛋白质在细胞中动态形成和重组的复合物中起作用。这对于信号受体蛋白尤其重要，比如大脑中的离子受体，它们通过突触后膜传导电流，促进神经元之间的快速通信。AMPA受体（AMPAR）是iGluRs的一种亚型，在哺乳动物大脑中介导最快的兴奋性神经传递。相应地，AMPAR运输、定位和功能的调节是决定突触强度和可塑性的关键机制，而突触强度和可塑性是学习和记忆等高认知脑功能的基础。最近发现突触ampar与许多辅助蛋白作为复合物起作用。本项目旨在表征AMPAR与cyso结AMPAR调节蛋白（ckamp）的分子相互作用及其与跨膜AMPAR调节蛋白（TARPs）的相互作用。大量的AMPAR和CKAMP突变体将用于鉴定参与受体调节的相互作用域和特定残基。包括对接和分子动力学模拟在内的计算结构建模将用于指导进一步确定蛋白质-蛋白质界面，这些界面将通过电生理实验进行测试，并生成和分析候选结构，以便通过冷冻电镜成功确定结构。这种合作的努力，结合了互补的方法和持续的信息和试剂交换，可能会提供必要的协同作用，以达到对AMPAR调控的新水平。除了提供对AMPAR门控和调控的基本见解外，本研究的结果还将为研究AMPAR介导的突触生理学过程提供路线图。该奖项由分子和细胞生物科学部、综合有机系统部和生命规则风险基金共同资助。这个美国/以色列合作项目由美国国家科学基金会和以色列两国科学基金会支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

High cognitive functions, including quantitative reasoning, learning and memory, originate in the human brain as a result of communication between complex networks of neurons connected through synapses. The key molecular element of this synaptic connection is the ionotropic glutamate receptor (iGluR), a protein in the postsynaptic neuronal membrane that conducts electrical signals to deliver messages from the presynaptic neuron. This process of neurotransmission is modulated by a variety of auxiliary proteins that bind to glutamate receptors. This project will illuminate how different auxiliary proteins interact with the glutamate receptor and with each other to initiate and modulate communication between neurons. This study enhances our knowledge of the molecular underpinnings of brain function. The research will involve undergraduate and graduate students, providing an educational environment and training opportunities at the forefront of brain research. Outreach activities involving local high schools and colleges are planned as well to promote diverse participation in STEM fields.It is increasingly recognized that the majority of proteins operate in cells within dynamically formed and re-formed complexes. This is especially important for signaling receptor proteins, such as ionotropic receptors in the brain that conduct electrical current through postsynaptic membranes to facilitate fast communication between neurons. The AMPA receptors (AMPAR) - a subtype of iGluRs - mediate the fastest excitatory neurotransmission in the mammalian brain. Correspondingly, regulation of AMPAR trafficking, localization and function is the key mechanism determining synaptic strength and plasticity that underlies high cognitive brain functions, such as learning and memory. It has been discovered recently that synaptic AMPARs function as complexes with a multitude of auxiliary proteins. This project aims to characterize molecular interactions of AMPARs with the cys-knot AMPAR modulating proteins (CKAMPs) and their interplay with the transmembrane AMPAR regulatory proteins (TARPs). A large collection of AMPAR and CKAMP mutants will be employed to identify interaction domains and specific residues involved in receptor regulation. Computational structural modeling including docking and molecular dynamics simulations will be employed to guide further determination of the protein-protein interfaces, which will be tested by electrophysiology experiments, and to generate and analyze candidate constructs for successful structure determination by cryo-EM. This collaborative effort, combining complementary approaches and continuous exchange of information and reagents, may provide the synergy needed to reach a new level of understanding of AMPAR regulation. In addition to providing fundamental insights into AMPAR gating and regulation, the results of this study will also serve as a roadmap for investigating AMPAR-mediated processes in synaptic physiology.This award was co-funded by the Division of Molecular and Cellular Biosciences, the Division of Integrative Organismal Systems, and the Rules of Life Venture Fund.This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.