Single Channel Properties and Structure of Glutamate Receptors

描述（由申请人提供）：谷氨酸受体离子通道（iGluRs）参与从快速突触传递到活动依赖性变化的大脑功能，这些变化是某些学习和记忆形式的基础。这些受体也与多种兴奋性毒性病理和神经退行性疾病有关。iGluRs的AMPA亚型（AMPARs）在几乎所有被检测的脑突触中产生兴奋性突触后电流（EPSCs）的快速成分，但很少有对单个通道分子特性的直接测量。此外，对于影响突触受体定位的蛋白质-蛋白质相互作用的决定因素，没有直接的结构数据。我们提出了一系列生物物理研究，通过分析原生和重组通道的单通道电流来表征AMPARs的单一性。重组通道的研究将结合GluR2亚基（GluR2- s1s2）配体结合域的晶体学研究。其他晶体学研究将进一步确定AMPAR与重要的运输、支架和细胞骨架蛋白相互作用的分子决定因素。该提案有四个主要目标。1)。在Aim 1中，我们将原位确定来自小脑神经元的一个通道斑块中天然ampar的动力学。该结果将为评估受体特性对突触传递的影响提供至关重要的信息。2)。目的2将采用单通道记录来阐明突变降低ampar稳态脱敏效应的分子机制，并确定突变在多大程度上也改变了激活门控。3)。在Aim 3中，将使用单通道记录和x射线晶体学来了解结合间隙内的详细相互作用如何影响结合间隙闭合的稳定性，进而影响受体激动剂的亲和力和有效性。4)。突触中ampar的数量和位置是大脑中信息传递的增益和保真度的关键决定因素。目的4将使用x射线晶体学来确定调节受体运输的重要蛋白质-蛋白质相互作用的结构基础。

DESCRIPTION (provided by applicant): 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.