Control of AMPA receptor function by phosphorylation

通过磷酸化控制 AMPA 受体功能

• 批准号: 8213435
• 负责人: Stephen F Traynelis
• 金额: $ 33.23万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8213435
• 关键词: AMPA Receptors; Accounting; Address; Agonist; Binding; Biochemical; Brain; C-terminal; Calmodulin; Cations; Cell model; Chemosensitization; Cognition; Communication; Coupling; Data; Elements; Event; Excitatory Synapse; Glutamate Receptor; Glutamates; Goals; Hippocampus (Brain); Hydrogen Bonding; Individual; Infection; Ion Channel; Lead; Learning; Ligands; Long-Term Potentiation; Mediating; Membrane; Memory; Modeling; Movement; Mutagenesis; Neuraxis; Neurons; Peptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Probability; Property; Protein Kinase; Protein Kinase C; Proteins; Recombinants; Regulation; Role; Scanning; Series; Serine; Signal Pathway; Signal Transduction; Structural Models; Synapses; Synaptic plasticity; System; Testing; Time; Work; base; functional mimics; ionic bond; neurotransmission; novel; polypeptide; postsynaptic; public health relevance; receptor; receptor function; research study; response; stargazin; trafficking

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.

描述（由申请人提供）：AMPA 型谷氨酸受体是配体门控阳离子通道，介导大脑中的快速兴奋性神经传递，因此关键参与大脑功能的各个方面，包括认知、运动、学习和记忆。突触后 AMPA 受体的功能和数量受到动态调节，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+/钙调蛋白依赖性激酶 II (CamKII) 对 GluR1 亚基上的细胞内丝氨酸残基 (Ser831) 进行磷酸化可增强 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，突触可塑性的一种模型。尽管之前的研究观察到 CamKII 的 GluR1 磷酸化增强了单通道电导，但尚未描述这种独特形式的离子通道调节的概念或结构机制。这里提出的实验的目的是从功能、结构和概念上理解 GluR1 Ser831 磷酸化如何增强 AMPA 受体功能。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）如何与磷酸化 Ser831 进行功能性相互作用。此外，我们将测试磷酸化 Ser831 的作用是否反映磷酸化 Ser831 和受体细胞内部分之间的蛋白质内相互作用，或磷酸化 Ser831 和 GluR1 结合伴侣之间的蛋白质间相互作用。这些研究的完成将为突触可塑性磷酸化介导的突触后 AMPA 通道功能变化的一个尚未研究的特征提供全面的功能和结构理解。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？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. AMPA受体功能的磷酸丝氨酸调节的结构基础是什么？我们将鉴定细胞内 GluR1 残基作为磷酸-Ser831 氢键伙伴。我们还将另外寻找涉及依赖于 Ser831 磷酸化的 GluR1 的蛋白质间相互作用。 3. 独立亚基门控模型能否描述磷酸化对AMPA受体的调节？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的谷氨酸的反应。这些数据将用于开发一种新的亚基门控模型，该模型可以解释 Ser831 磷酸化对 GluR1 通道功能的增强作用。公共健康相关性：AMPA 受体介导中枢神经系统神经元之间的通信，因此在几乎所有大脑功能中发挥着重要作用。 AMPA 受体由四个不同的亚基 (GluR1-4) 组成。其中，GluR1 亚基已被证明在活动依赖性突触可塑性中发挥独特的作用。 GluR1 会受到多种激酶的 C 末端磷酸化影响，这种磷酸化会影响向膜的运输和 AMPA 受体功能。在本提案中，我们检查了 GluR1-Ser831 的 CamKII 磷酸化。 CamKII 激活已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 亚基的磷酸化实现的。 GluR1-Ser831 的磷酸化通过未知机制增加单通道电导。该提案描述了三个系列的实验，这些实验将评估 GluR1-Ser831 磷酸化后对受体功能影响的潜在功能和结构机制。了解 AMPA 受体功能是如何通过细胞内信号通路塑造的，是了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级功能的基础。