Single Channel Properties and Structure of Glutamate Receptors

谷氨酸受体离子通道(IGluRs)参与从快速突触到脑功能的各种活动 传递到依赖活动的变化，这些变化是某些形式的学习和记忆的基础。这些 受体还与多种兴奋性毒性病理和神经退行性疾病有关。这个 IGluRs的AMPA亚型(AMPAR)引起兴奋性突触后电流的快速成分 (EPSCs)几乎所有的大脑突触都被检查过，但很少有对这些特性的直接测量 单个通道分子。此外，没有关于决定因素的直接结构数据 影响突触受体定位的蛋白质-蛋白质相互作用。 我们提出了一系列生物物理研究，将通过以下方式表征AMPAR的么正特性 通过自然通道和重组通道分析单通道电流。重组人的研究进展 通道将与GluR2的配体结合域的结晶学研究相结合 亚基(GluR2-S1S2)。其他的结晶学研究将进一步定义分子决定因素 Ampar与重要的运输、支架和细胞骨架蛋白的相互作用。该提案有四个方面 主要目标。 1)在目标1中，我们将测定来自小脑的单通道斑块中天然AMPAR的动力学 原位神经元。这些结果将提供对评估受体特性对 突触传递。 2.)目标2将使用单通道记录来阐明潜在的分子机制 减少AMPAR稳态脱敏的突变的影响并决定在多大程度上 突变也会改变激活门控。 3.)在目标3中，将使用单通道记录和x射线结晶学来了解详细程度 结合裂隙内的相互作用影响结合裂隙闭合的稳定性，进而影响亲和力和亲和力 受体激动剂的疗效。 4.)突触上AMPAR的数量和位置是决定突触获得性和保真度的关键因素 大脑中的信息传递。目标4将使用x射线结晶学来确定结构基础 调节受体运输的重要蛋白质-蛋白质相互作用。

Glutamate-receptor ion channels (iGluRs) participate in brain functions that range from fast synaptic transmission to activity-dependent changes that underlie certain forms of learning and memory. These receptors are also implicated in a variety of excitotoxic pathologies and neurodegenerative diseases. The AMPA subtype of iGluRs (AMPARs) gives rise to the fast component of excitatory postsynaptic currents (EPSCs) at virtually all brain synapses examined, but there are few direct measurements of the properties of individual channel molecules. In addition, there are no direct structural data on the determinants of protein-protein interactions that influence localization of the receptors at synapses. We propose a series of biophysical studies that will characterize the unitary properties of AMPARs by analyzing single-channel currents through native and recombinant channels. The studies of recombinant channels will be combined with crystallographic investigations of the ligand-binding domain of the GluR2 subunit (GluR2-S1S2). Other crystallographic studies will further define the molecular determinants of AMPAR interactions with important trafficking, scaffolding, and cytoskeletal proteins. The proposal has four main goals. 1.) In Aim 1 we will determine the kinetics of native AMPARs in one channel patches from cerebellar neurons in situ. The results will provide information crucial to evaluating the impact of receptor properties on synaptic transmission. 2.) Aim 2 will employ single-channel recording to elucidate the molecular mechanisms underlying the effect of mutations that reduce steady-state desensitization of AMPARs and determine to what extent the mutations also alter activation gating. 3.) In Aim 3, single-channel recording and x-ray crystallography will be used to understand how detailed interactions within the binding cleft influence the stability of binding cleft closure and in turn the affinity and efficacy of receptor agonists. 4.) The number and location of AMPARs at synapses are key determinants of the gain and fidelity of information transfer in the brain. Aim 4 will use x-ray crystallography to determine the structural basis of important protein-protein interactions that regulate receptor trafficking.