Molecular mechanisms of GLURl trafficking and insertion

描述(由申请人提供)： 这一研究提案的主要焦点是阐明分子 在两种发育过程中参与AMPA受体运输的机制 和活动依赖范式。这项研究将进一步加深对 突触前信号如何导致急性以及脑内长期变化 突触功效。这项建议的优点是利用了广泛的 解决所提出的重要问题的技术。使用以下组合 电生理、生化、分子和显微镜方法， GluR1AMPA受体亚单位与细胞内突触的相互作用 蛋白质可能是突触功能中的关键角色。一个主要的 新皮质培养系统将被用作功能性神经元的模型。 网络。野生型或突变型荧光标记AMPA的过表达 Sindbis病毒的受体相互作用蛋白SAP97/hDLG和蛋白4.1 表达系统将使这些蛋白质的研究成为可能 对突触传递的影响。慢性(&gt；24 HR)过表达的影响 将通过测量AMPA介导的mEPSCs来评估SAP97和Protein 4.1的表达。 此外，GluR1的表面表达也将通过生化方法进行测量 在这些蛋白质过度表达后。最后，使用一种新的分析方法 正常或过度表达培养神经元的长时程增强 SAP97和蛋白质4.1的突变形式将被用来理解这一作用 这些蛋白质的突触可塑性。

DESCRIPTION (provided by applicant): The main focus of this research proposal is to elucidate the molecular machinery involved in trafficking of AMPA receptors during both developmental and activity dependent paradigms. This study will further the understanding of how pre synaptic signals can lead to acute as well was as long-term changes in synaptic efficacy. This proposal has the advantage of utilizing a wide range of techniques to address the important questions posed. Using a combination of electrophysiological, biochemical, molecular, and microscopy methods, interactions of the GluR1 AMPA receptor subunit with intracellular synaptic proteins can be implicated as key players in synaptic function. A primary neocortical culture system will be used as a model for a functional neuronal network. Overexpression of the wildtype or mutant fluorescent-tagged AMPA receptor interacting proteins SAP97/hDLG and Protein 4.1 by the Sindbis viral expression system will enable the study of these proteins with regard to their effect on synaptic transmission. The effect of chronic (>24 HR) overexpression of SAP97 and Protein 4.1 will be assessed by measuring AMPA mediated mEPSCs. Also, surface expression of GluR1 will also be measure by biochemical methods after overexpression of these proteins. Finally, using a novel assay for long-term potentiation in cultured neurons overexpressing either normal or mutant forms of SAP97 and Protein 4.1 will be employed to understand the role of these proteins in synaptic plasticity.