Phospho-regulation as a driver of inhibitory synapse function

磷酸调节作为抑制性突触功能的驱动因素

• 批准号: 10371876
• 负责人: Alicia Mae Purkey
• 金额: $ 1.75万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371876
• 关键词: Biochemical; Biological Assay; Biotin; Biotinylation; Brain; Brain Diseases; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Computer software; Coupled; Cyclic AMP-Dependent Protein Kinases; Data; Dependence; Electrophysiology (science); Event; Excitatory Synapse; Foundations; GABA Receptor; Genome engineering; Glutamates; Hippocampus (Brain); Image; Inhibitory Synapse; Knowledge; Label; Laboratories; Ligase; Link; Literature; Mass Spectrum Analysis; Methodology; Methods; Modification; Molecular; Neurons; Outcome; Output; Phosphorylation; Phosphorylation Site; Phosphotransferases; Positioning Attribute; Protein Kinase; Proteins; Proteome; Regulation; Role; Shapes; Signal Transduction; Synapses; Synaptic Transmission; Translations; Work; base; behavior influence; calmodulin-dependent protein kinase II; casein kinase I; experience; experimental study; gamma-Aminobutyric Acid; genetic approach; in vivo; information processing; neural network; neuronal cell body; novel; optogenetics; phosphoproteomics; protein function; synaptic function; tool; transmission process

Phosphorylation has long been studied as a way to shape protein function after translation. In the brain, phosphorylation has emerged as one of the most efficient ways to transduce activity-dependent information to altered synaptic function. Extensive literature supports phosphorylation as an essential component of signal transduction at glutamatergic excitatory postsynapses within the CNS. Emerging evidence, however, demonstrates that GABAergic inhibitory postsynapses also undergo activity dependent plasticity. Based on limited candidate studies, a handful of GABAergic proteins have recently been shown to be phosphorylated at inhibitory synapses. While this supports phosphorylation as a likely mechanism to modulate inhibitory synapses, an in-depth analysis of how phosphorylation is coupled to inhibitory synapses function has not been attempted. Moreover, it is still unclear as to which kinases are present and therefore act at inhibitory synapses. Thus, the inhibitory synaptic phospho-proteome is currently a “black box”, which is a significant barrier to understanding how activity modifies inhibition important for shaping experience-dependent brain function. Here, I propose to use a cutting edge in vivo chemico-genetic approach to identify phosphorylated proteins at inhibitory synapses in combination with CRISPR-depletion, optogenetics, electrophysiology and imaging to understand how kinases orchestrate the complex yet fundamental workings of the inhibitory synapse.

长期以来，磷酸化一直被研究为翻译后塑造蛋白质功能的一种方式。在大脑中，磷酸化已经成为将依赖活动的信息转化为改变的突触功能的最有效方式之一。大量文献支持磷酸化是中枢神经系统谷氨酸能兴奋性突触信号转导的重要组成部分。然而，新的证据表明，GABA能抑制性突触后也经历了活动依赖的可塑性。基于有限的候选研究，最近发现少数GABA能蛋白在抑制性突触处被磷酸化。虽然这支持磷酸化作为调节抑制性突触的可能机制，但还没有人试图深入分析磷酸化是如何耦合到抑制性突触功能的。此外，目前还不清楚哪些激酶存在并因此作用于抑制性突触。因此，抑制性突触磷酸蛋白质组目前是一个“黑匣子”，这是理解活动如何修改抑制对塑造经验依赖型大脑功能至关重要的一个重要障碍。在这里，我建议使用一种先进的在体化学遗传学方法来鉴定抑制性突触上的磷酸化蛋白，并结合CRISPR耗竭、光遗传学、电生理学和成像来了解激酶是如何协调抑制性突触复杂而基础的工作的。