Single Channel Properties and Structure of Glutamate Receptors

谷氨酸受体离子通道（iGluRs）参与大脑功能，从快速突触， 传递到活动依赖的变化，这些变化是某些形式的学习和记忆的基础。这些 受体还涉及多种兴奋性毒性病理和神经变性疾病。的 AMPA亚型的iGluRs（AMPAR）产生兴奋性突触后电流的快成分 （EPSC）在几乎所有的大脑突触检查，但很少有直接测量的性质 单个的通道分子。此外，没有关于以下因素决定因素的直接结构性数据 影响突触处受体定位的蛋白质-蛋白质相互作用。 我们提出了一系列的生物物理研究，将表征AMPAR的单一属性， 分析通过天然和重组通道的单通道电流。重组的研究 通道将与GluR 2配体结合域的晶体学研究相结合， 亚基（GluR 2-S1 S2）。其他的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的相互作用。该提案有四个 主要目标。 1.）的人。在目的1中，我们将确定来自小脑的单通道斑中的天然AMPAR的动力学。 原位神经元。这些结果将为评估受体特性对细胞增殖的影响提供重要信息。 突触传递 2.）的情况。目的2将采用单通道记录来阐明 减少AMPAR稳态脱敏的突变的影响，并确定在何种程度上， 突变也改变激活门控。 3.）第三章在目标3中，单通道记录和X射线晶体学将用于了解 结合缝内的相互作用影响结合缝闭合的稳定性，进而影响亲和力和 受体激动剂的功效。 4.）突触处AMPAR的数量和位置是神经元的增益和保真度的关键决定因素。 大脑中的信息传递。AIM 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。

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.