Understanding higher-order glycine receptor assemblies in neurotransmission

概况.细胞间的电信号传输是人类几乎所有生理过程的基础， 从心跳到学习和记忆神经递质受体以微米大小的簇状组织 细胞间的机制，即突触，在我们的神经系统，使信号传输。 几十年的深入研究已经确定了大多数个体受体的工作机制。然而，在这方面， 由于技术上的困难，受体簇仍然是个谜--无论是结构还是功能， 意义是已知的。在这个提案中，我的实验室将使用最先进的方法和重组系统， 系统地描述了突触甘氨酸受体的结构，这是最后一种主要的神经递质 在我们的研究之前，这些受体的结构仍然难以捉摸。我们还将开发新技术， 甘氨酸受体和其他蛋白质在2D环境中的高阶组装的定量表征 脂质膜。以下是拟议工作的简要说明。 甘氨酸受体及其与桥蛋白骨架的聚集。甘氨酸受体（GlyR）属于半胱氨酸环 五聚体配体门控离子通道家族。成体组织中的GlyR是由以下组成的异聚体受体： α和β亚基，它们通过特异性的 β亚基和桥蛋白之间的相互作用。GlyR信号传导障碍是罕见的 与慢性神经性疼痛和自闭症谱系障碍有关的先天性疾病。的 异聚GlyR的结构以及它们如何与桥蛋白形成簇是非常有限的-即使是α：β亚基 化学计量已经争论了几十年。我们发现了一种意想不到的亚基组成， GlyR，解释了异聚GlyR的独特功能，并清除了长期的混乱。我们将使用这个 建立了一个平台来表征所有主要类型的GlyR以及它们如何与桥蛋白相互作用。 开发表征脂质膜中蛋白质簇的新技术。我的实验室将开发一种小说 相关增强全内反射荧光显微镜（TIRF）和电生理重建 系统表征离子通道/受体簇。该系统具有高信噪比和单通道的特点， 分子灵敏度，并允许进行复杂的电生理学实验 与此同时，成像。我们将学习集群是如何形成的，它们是如何被调节的，以及集群是否 产生功能效应并调节生理活动。在这里获得的知识将指导 使用cryo-EM单颗粒和/或 层析成像方法这些新方法将允许空间组织的定量表征， 集群的功能意义，以及组装和功能的机制。我的实验室 将应用这些新的方法来研究GlyR-桥卟啉簇，作为课堂上的第一个例子，并打开新的 研究方向在分析其他受体集群发现无处不在的生物学信号传输。

Overview. Electrical signal transmission between cells underlies almost all physiological processes in human, from heartbeat, to learning and memory. Neurotransmitter receptors are organized in clusters at micron-sized inter-cellular machineries, namely synapses, throughout our nervous system to enable signal transmission. Decades of intense research have characterized the working mechanism of most individual receptors. However, due to technical difficulties, the clusters of receptors remain enigmatic – neither the structure, nor the functional significance is known. In this proposal, my lab will use state-of-the-art methods and a reconstitution system to systematically characterize the architecture of synaptic glycine receptors, which is the last major neurotransmitter receptors whose architecture remained elusive before our work. We will also develop novel technologies for quantitative characterization of higher-order assemblies of glycine receptors, and other proteins in the 2D setting of lipid membranes. Following is a brief description of the proposed work. Glycine receptors and its clustering with gephyrin scaffold. Glycine receptor (GlyR) belongs to the Cys-loop family of pentameric ligand-gated ion channels. GlyRs in adult tissue are heteromeric receptors composed of both the α and β subunits, which form clusters with scaffold protein gephyrin at synapses through a specific interaction between the β subunit and gephyrin. Disfunction of GlyR signaling is the major cause of the rare congenital disease hyperekplexia and related to chronical neurological pain and autism spectrum disorders. The architecture of heteromeric GlyRs and how they form clusters with gephyrin is very limited – even the α:β subunit stoichiometry has been under debate for decades. We have discovered an unexpected subunit composition of GlyR, explaining the unique function of heteromeric GlyRs and clearing long-lasting confusion. We will use this established platform to characterize all major types of GlyRs and how they interact with gephyrin. Develop novel technologies for characterizing protein clusters in lipid membrane. My lab will develop a novel correlated raised-total internal refection fluorescence microscopy (TIRF) and electrophysiology reconstitution system to characterize ion channel/receptor clusters. This system has the high signal/noise ratio and single- molecule sensitivity as traditional TIRF, and allow complex electrophysiological experiments to be performed simultaneously with imaging. We will learn how clusters form, how they are regulated, and whether clustering gives rise to functional effects and regulate physiological activities. The knowledge gained here will guide the reconstitution of functional clusters for structural characterization using cryo-EM single particle and/or tomography methods. These new methods will allow quantitative characterization of the spatial organization and functional significance of clustering, as well as the mechanisms underlying assembly and functionality. My lab will apply these novel methods to investigate GlyR-gephyrin clusters as the first example in class, and open new research directions in analyzing other receptor clusters found ubiquitously in biology for signal transmission.