GABARAP在突触传递过程中调控GABAA受体转运和功能研究

该数据集2019YFA0508402-011内容为GABARAP介导的GABAA受体转运的结构基础及其在GABA能突触传递中的功能研究，GABARAP最早被鉴定为与GABAA受体γ2亚基相互作用的蛋白，是参与GABAA受体细胞膜定位过程的重要蛋白之一。过往的研究发现，GABARAP可以提高GABAA受体在COS-7细胞膜和海马神经元细胞膜上的分布水平，但其具体作用途径仍然不清晰。在此项研究中，我们通过体外生化实验发现γ2亚基的TM3-TM4胞内段一段18个氨基酸的短肽（γ2-GIM）能直接结合GABARAP，并利用多种生化手段进行了定量的相互作用分析发现γ2-GIM结合GABARAP和GABARAPL1的特异性，解析了GABARAPL1/γ2-GIM复合物的三维结构，确定了复合物组装的分子机制。我们通过电生理手段发现GABARAP显著提高了GABAA介导的氯离子电流。这种作用可以被膜蛋白内吞抑制剂进一步增强；但当加入细胞外泌抑制剂后GABAA介导的氯离子电流则明显降低。最后，我们利用课题组此前开发的Ankyrin来源的干扰性多肽工具，内源性的破坏小鼠脑内GABAA受体和GABARAP的相互结合后，发现运动皮层神经元的微小抑制性突触后电流的发放（mIPSCs）显著降低。这些结果表明GABARAP的作用途径为促进受体的转运过程，揭示了GABARAP调节GABAA受体在细胞膜上动态分布水平的分子机理，为相关的人类神经系统疾病的治疗和高效特异性药物的研发提供了充实的结构信息和分子基础。本数据集共包含八个文件夹和一个PDF文件，共6.31MBThis dataset, with the grant number 2019YFA0508402-011, documents a study on the structural basis of GABARAP-mediated GABAA receptor trafficking and its functions in GABAergic synaptic transmission. GABARAP was first identified as a protein that interacts with the γ2 subunit of GABAA receptors, and it is one of the key proteins involved in the plasma membrane localization of GABAA receptors. Previous studies have revealed that GABARAP enhances the surface expression of GABAA receptors on COS-7 cell membranes and hippocampal neuron membranes, yet the exact underlying mechanism remains unclear. In this study, we conducted in vitro biochemical experiments and found that an 18-amino-acid peptide (γ2-GIM) within the intracellular TM3-TM4 loop of the γ2 subunit directly binds to GABARAP. Through quantitative interaction assays using multiple biochemical approaches, we confirmed the binding specificity of γ2-GIM for GABARAP and GABARAPL1. We also resolved the three-dimensional structure of the GABARAPL1/γ2-GIM complex, elucidating the molecular mechanism underlying complex assembly. Using electrophysiological assays, we observed that GABARAP significantly potentiates GABAA-mediated chloride currents. This potentiation can be further enhanced by inhibitors of membrane protein endocytosis; however, treatment with inhibitors of cellular exocytosis markedly reduces GABAA-mediated chloride currents. Finally, using an Ankyrin-derived interfering peptide tool previously developed by our team, we endogenously disrupted the interaction between GABAA receptors and GABARAP in mouse brains, and found that the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in motor cortical neurons was significantly reduced. Collectively, these results demonstrate that GABARAP functions to promote receptor trafficking, uncovering the molecular mechanism by which GABARAP regulates the dynamic surface expression of GABAA receptors. This work provides solid structural and molecular foundations for the treatment of related human neurological disorders and the development of highly specific therapeutic agents. This dataset contains eight folders and one PDF file, with a total size of 6.31 MB.

搜集汇总

背景与挑战

背景概述

该数据集聚焦于GABARAP在突触传递中调控GABAA受体转运和功能的研究，通过结构解析和功能实验揭示了其分子机制。研究发现GABARAP能增强受体介导的电流，并促进受体转运，为神经系统疾病治疗和药物研发提供了结构基础。