Molecular mechanisms of GLURl trafficking and insertion

描述（由申请人提供）： 这项研究计划的主要重点是阐明 在发育过程中参与AMPA受体运输的机制 和依赖于活动的范例。这项研究将进一步了解 突触前信号如何导致急性以及长期的变化， 突触效能该提案的优点是利用了广泛的 技术来解决提出的重要问题。结合使用 电生理学、生物化学、分子和显微镜方法， GluR 1 AMPA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参与者。主 新皮层培养系统将被用作功能性神经元的模型 网络野生型或突变体荧光标记AMPA的过表达 Sindbis病毒的受体相互作用蛋白SAP 97/hDLG和蛋白4.1 表达系统将使这些蛋白质的研究，就其 对突触传递的影响慢性（>24 HR）过表达的影响 将通过测量AMPA介导的mEPSC来评估SAP 97和蛋白4.1的表达。 此外，GluR 1的表面表达也将通过生物化学方法测量 这些蛋白质的过度表达。最后，使用一种新的测定方法， 在培养的神经元中，长时程增强过度表达正常或 SAP 97和蛋白质4.1的突变形式将被用来理解 这些蛋白质在突触可塑性中的作用

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.