Control of AMPA receptor function by phosphorylation

描述(申请人提供)：AMPA型谷氨酸受体是配体门控的阳离子通道，介导大脑中快速的兴奋性神经传递，因此关键涉及大脑功能的各个方面，包括认知、运动、学习和记忆。突触后AMPA受体的功能和数量是动态调节的，以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通过蛋白激酶导致AMPA受体的磷酸化，这种磷酸化控制AMPA受体的运输和功能。在突触可塑性的一种模型--长时程增强(LTP)的表达过程中，蛋白激酶C(PKC)或钙/钙调蛋白依赖的蛋白激酶II(CaMKII)对位于GluR1亚基上的丝氨酸残基(Ser831)的磷酸化增强了AMPA受体的功能，从而增强了突触的强度。虽然以前的研究观察到GluR1的CaMKII磷酸化增强了单通道电导，但对于这种独特的离子通道调节形式，概念上或结构上的机制还没有被描述。这些实验的目的是从功能、结构和概念上了解GluR1Ser831的磷酸化如何增强AMPA受体的功能。我们将重点关注GluR1中的Ser831，因为它具有独特的增强机制(单位电导增加)，并将扩大研究范围，首次评估附近的三个磷酸化位点(Ser818、Thr840、Ser845)如何与磷酸化Ser831功能相互作用。此外，我们将测试磷酸化Ser831的作用是否反映了磷酸化Ser831和受体的细胞内部分之间的蛋白质内相互作用，或者磷酸Ser831和GluR1结合伙伴之间的蛋白质间相互作用。这些研究的完成将提供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通道功能变化的全面的功能和结构的理解。拟议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？单通道电流将被记录，以确定Ser831的磷酸化如何控制GluR1的功能。我们还将评估Ser831与附近的磷酸化位点的相互作用，并在神经元中验证我们的结论。2.磷酸化丝氨酸调节AMPA受体功能的结构基础是什么？我们将确定细胞内的GluR1残基是磷酸化的Ser831氢键伙伴。此外，我们还将寻找与GluR1有关的蛋白质间相互作用，这依赖于Ser831的磷酸化。3.独立亚单位门控模型能否描述AMPA受体通过磷酸化的调节？我们将分析具有一个活跃的GluR1通道(加上Stargazin)的贴片对快速应用最有效浓度的谷氨酸的反应。这些数据将被用来开发一个新的亚基门控模型，该模型可以解释Ser831的磷酸化对GluR1通道功能的增强。与公共健康相关：AMPA受体调节中枢神经系统神经元之间的通讯，因此在几乎所有大脑功能中都发挥着重要作用。AMPA受体由四个不同的亚基组成(GluR1-4)。其中，GluR1亚单位在活性依赖的突触可塑性中发挥着独特的作用。GluR1的C末端被多种激酶磷酸化，这种磷酸化可以影响膜的转运和AMPA受体的功能。在这个提案中，我们研究了GluR1-Ser831的CaMKII磷酸化。CaMKII的激活被证明是某些形式的突触可塑性的关键步骤，可能是通过GluR1亚单位的磷酸化来实现的。GluR1-Ser831的磷酸化以一种未知的机制增加单通道电导。这项建议描述了三个系列的实验，这些实验将评估GluR1-Ser831的磷酸化对受体功能的影响的潜在功能和结构机制。了解AMPA受体的功能是如何由细胞内信号通路塑造的，这是理解突触可塑性机制的重要一步，突触可塑性可能是学习和记忆等高阶功能的基础。

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. PUBLIC HEALTH RELEVANCE: AMPA receptors mediate communication between neurons in the central nervous system, and thus play an important role in virtually all brain functions. The AMPA receptors are comprised of four different subunits (GluR1-4). Among these, the GluR1 subunit has been shown to play a unique role in activity-dependent synaptic plasticity. GluR1 is subject to C-terminal phosphorlyation by a variety of kinases, and this phosphorylation can influence trafficking to the membrane and AMPA receptor function. In this proposal we examine CamKII phosphorylation of GluR1-Ser831. CamKII activation has been shown to be a critical step in some forms of synaptic plasticity, presumably through phosphorylation of the GluR1 subunit. Phosphorylation of GluR1-Ser831 increases single channel conductance by an unknown mechanism. This proposal describes three series of experiments that will evaluate the underlying functional and structural mechanisms of the effects on receptor function following phosphorylation at GluR1-Ser831. Understanding how AMPA receptor function is sculpted by intracellular signaling pathways is an important step towards understanding the mechanisms of synaptic plasticity, which likely underlie higher order functions such as learning and memory.