EXPRESSION AND REGULATION OF KAINATE RECEPTORS

谷氨酸是哺乳动物中枢神经系统中的主要兴奋性神经递质， 门三种主要类型的离子型受体，命名为NMDA，AMPA和 红藻氨酸盐，基于它们的药理学和分子特性， 受体亚单位该提案将集中在红藻氨酸受体的 到目前为止，已经克隆了这五个亚基：GluR 5，GluR 6，GluR 7，KA 1和 KA2尽管这些亚基在整个大脑中表达，但快速- 脱敏红藻氨酸受体仅在背根中发现 神经节（Huettner，1990）。在这项研究中，我们将研究亚基 天然红藻氨酸受体的组成，生物物理特性如何 可以通过不同的亚基形式进行调节， 磷酸化在调节通道特性中的作用。红藻氨酸受体 被认为在介导快速突触传递中起着关键作用 并可能在发育过程中调节Ca 2+内流。因此，更好的 了解它们的结构、功能和调节将提供 深入了解它们在突触可塑性和细胞死亡中的作用。 红藻氨酸受体亚单位RNA和蛋白的发育表达 将在小脑颗粒细胞中进行研究，以确定哪些亚型 存在于这种单一的神经元类型中。膜片钳实验将 重组确定这些通道的功能特性 在HEK 293细胞中表达。在这个项目中，我们将主要使用克隆 红藻氨酸受体cDNA稳定整合到基因组中或瞬时整合到基因组中 转染HEK 293细胞。稳定的表达系统提供了 小脑颗粒细胞的额外和必要的信息，因为 可以在没有 其他谷氨酸受体基因产物。影响的生化研究 磷酸化对红藻氨酸受体表达的影响将在 稳定的细胞系，以及小脑颗粒细胞，以确定 功能差异。稳定细胞和小脑的优势 颗粒细胞的一个重要特征是， 获得的生化分析的水平的调节， 表面表达和磷酸化。两人合作， 这些方法可以提供对分子机制的有力见解， 这是中枢神经系统中红藻氨酸受体功能的基础。

Glutamate, the major excitatory neurotransmitter in the mammalian CNS, gates three major types of ionotropic receptors, named NMDA, AMPA and kainate, based on their pharmacology and molecular properties of the receptor subunits. This proposal will focus on the kainate receptor of which five subunits have been cloned so far: GluR5, GluR6, GluR7, KA1 and KA2. Although these subunits are expressed throughout the brain, fast- desensitizing kainate receptors have only been found in the dorsal root ganglion (Huettner, 1990). In this study, we will investigate the subunit composition of native kainate receptors, how the biophysical properties can be regulated by different subunit forms, and the role of phosphorylation in the modulation of channel properties. Kainate receptors are believed to play a key role in mediating fast synaptic transmission and may regulate Ca2+ entry during development. Therefore, a better understanding of their structure, function and modulation will provide insight into their role in synaptic plasticity and cell death. The developmental expression of kainate receptor subunit RNA and protein will be studied in cerebellar granule cells to identify which isoforms are present in this single neuronal type. Patchclamp experiments will determine the functional properties of these channels recombinantly expressed in HEK293 cells. For this project, we will primarily use cloned kainate receptor cDNAs stably integrated into the genome or transiently transfected into HEK293 cells. The stable expression system provides additional and necessary information to cerebellar granule cells because a single population of identical channels can be studied in the absence of other glutamate-receptor gene products. Biochemical studies on the effect of phosphorylation on kainate receptor expression will be performed on the stable cell lines, as well as cerebellar granule cells to identify functional differences. The advantage of stable cells and cerebellar granule cells is that large quantities of kainate receptors can be obtained for biochemical analysis of the regulation of the level of surface expression and phosphorylation. Working together, the two approaches can provide powerful insights into the molecular mechanisms that underlie kainate receptor function in the CNS.