Control of AMPA receptor function by phosphorylation

描述（由申请人提供）：ampa型谷氨酸受体是配体门控的阳离子通道，介导大脑中的快速兴奋性神经传递，因此在认知、运动、学习和记忆等脑功能的各个方面都有重要作用。突触后AMPA受体的功能和数量受到动态调节，从而控制突触连接的强度，这种可塑性是学习和记忆细胞模型的关键特征。触发突触可塑性的信号导致蛋白激酶磷酸化AMPA受体，这种磷酸化控制AMPA受体的运输和功能。蛋白激酶C （PKC）或Ca2+/钙调蛋白依赖激酶II （CamKII）磷酸化位于GluR1亚基上的细胞内丝氨酸残基（Ser831）可增强AMPA受体功能，从而在长时程增强（LTP）表达过程中增加突触强度，LTP是突触可塑性的一种模型。虽然之前的研究发现CamKII磷酸化GluR1增强了单通道电导，但这种独特的离子通道调节形式没有概念或结构机制。本实验的目的是了解GluR1 Ser831磷酸化如何在功能、结构和概念上增强AMPA受体的功能。我们将重点关注GluR1中的Ser831，因为其独特的增强机制（增加单一电导），并将扩大研究范围，首次评估三个附近的磷酸化位点（Ser818, Thr840, Ser845）如何在功能上与phospho-Ser831相互作用。此外，我们将测试phospho-Ser831的作用是否反映了phospho-Ser831与受体细胞内部分之间的蛋白内相互作用，或phospho-Ser831与GluR1结合伙伴之间的蛋白间相互作用。这些研究的完成将对突触可塑性的一个未被研究的特征——磷酸化介导的突触后AMPA通道功能的变化——提供全面的功能和结构理解。提出的实验解决了三个问题：1。磷酸化调节AMPA受体功能的机制是什么？将记录单通道电流，以确定Ser831磷酸化如何控制GluR1功能。我们还将评估Ser831与附近磷酸化位点的相互作用，并在神经元中验证我们的结论。2. 磷酸化丝氨酸调控AMPA受体功能的结构基础是什么？我们将确定细胞内GluR1残基作为磷酸丝氨酸831氢键伙伴。我们还将寻找依赖于Ser831磷酸化的涉及GluR1的蛋白间相互作用。3. 独立亚基门控模型能否描述AMPA受体磷酸化调控？我们将分析具有一个活性GluR1通道（加上stargazin）的贴片对快速应用最大有效浓度谷氨酸的响应。这些数据将用于开发一种新的亚基门控模型，该模型可以通过磷酸化Ser831来解释GluR1通道功能的增强。

DESCRIPTION (provided by applicant): The AMPA-type glutamate receptors are ligand-gated cation channels that mediate fast excitatory neurotransmission in the brain, and thus are critically involved in all aspects of brain function including cognition, movement, learning, and memory. The function and number of postsynaptic AMPA receptors are dynamically regulated to control the strength of synaptic connections, and this plasticity is a key feature of cellular models of learning and memory. Signals that trigger synaptic plasticity lead to phosphorylation of AMPA receptors by protein kinases, and this phosphorylation controls AMPA receptor trafficking and function. Phosphorylation by protein kinase C (PKC) or Ca2+/calmodulin dependent kinase II (CamKII) of an intracellular serine residue (Ser831) located on the GluR1 subunit enhances AMPA receptor function to increase synaptic strength during expression of long-term potentiation (LTP), one model of synaptic plasticity. Although previous studies observed that CamKII phosphorylation of GluR1 enhances the single channel conductance, no conceptual or structural mechanism has been described for this unique form of ion channel regulation. The goal of the experiments proposed here is to understand functionally, structurally, and conceptually how phosphorylation of GluR1 Ser831 potentiates AMPA receptor function. We will focus on Ser831 in GluR1 because of the unique mechanism of potentiation (increased unitary conductance), and will expand the study to evaluate for the first time how three nearby phosphorylation sites (Ser818, Thr840, Ser845) might functionally interact with phospho-Ser831. Furthermore, we will test whether the effects of phospho-Ser831 reflect either intra-protein interactions between the phospho-Ser831 and intracellular portions of the receptor, or inter-protein interactions between phospho-Ser831 and GluR1 binding partners. Completion of these studies will provide a comprehensive functional and structural understanding of an under-studied feature of synaptic plasticity-phosphorylation mediated changes in postsynaptic AMPA channel function. The proposed experiments address three questions: 1. What is the mechanism by which phosphorylation regulates AMPA receptor function? Single channel currents will be recorded to determine how phosphorylation of Ser831 controls GluR1 function. We will also evaluate the interactions of Ser831 with nearby phosphorylation sites, and validate our conclusions in neurons. 2. What is the structural basis for phospho-serine regulation of AMPA receptor function? We will identify intracellular GluR1 residues as phospho-Ser831 hydrogen bonding partners. We will additionally search for inter-protein interactions involving GluR1 that depend on the phosphorylation of Ser831. 3. Can models of independent subunit gating describe AMPA receptor regulation by phosphorylation? We will analyze the response of patches with one active GluR1 channel (plus stargazin) to the rapid application of a maximally effective concentration of glutamate. These data will be used to develop a novel model of subunit gating that can account for the potentiation of GluR1 channel function by phosphorylation of Ser831.