CHARACTERIZATION OF RECOMBINANT GLUTAMATE RECEPTORS

本文提出了一种实验方法， 重组谷氨酸受体 α-氨基-3-羟基-5-甲基异恶唑-4-丙酸酯（AMPA）和红藻氨酸盐 亚型 天然和重组受体的功能特性 将使用电生理学方法比较在非洲爪蟾卵母细胞中表达的 方法. 脑内AMPA或红藻氨酸受体亚单位的水平 衍生的RNA将通过聚合酶链反应（PCR）定量， 将在功能特性和可能的 亚基结构 这些相关性将通过测量 从相同的细胞表达的重组受体的功能特性 在大脑mRNA制备物中发现的这些亚基的量。 针对肽的亚基特异性抗体的开发 发现在克隆的氨基酸序列也提出了。 有用 抗体将有助于直接探测天然的亚基结构， 蛋白质印迹分析和高分辨率 免疫定位 鉴定和表征激动剂， 单个亚单位的亚结构内的拮抗剂结合位点， 将引入对亚单位结构的有目的的改变 使用重组DNA技术。 这些变化对 激动剂和拮抗剂的效力将用两个 爪蟾卵母细胞的电极电压钳。 使用快速的动力学研究 膜片钳成纤维细胞瞬时应用技术 表达受体亚基的基因将被用来分析 配体结合和解结合的机制。 这些研究旨在 增加我们对神经元AMPA和红藻氨酸受体的了解， 从而导致开发作用于 这些受体 AMPA和红藻氨酸受体系统与 在许多病理过程中， 脑缺血、帕金森氏症、癫痫和精神病。

Experiments are proposed to study the structure and function of recombinant glutamate receptors of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate subtypes. The functional properties of native and recombinant receptors expressed in Xenopus oocytes will be compared using electrophysiological methods. The levels of AMPA or kainate receptor subunits in brain derived RNAs will be quantitated by polymerase chain reaction (PCR) and correlations will be made between functional properties and possible subunit structure. These correlations will be tested by measuring the functional properties of recombinant receptors expressed from identical amounts of the those subunits found in brain mRNA preparations. Development of subunit-specific antibodies directed against peptides found in the cloned amino acid sequences is also proposed. Useful antibodies will help directly probe the subunit structure of native receptors by Western blot analysis and high resolution immunolocalization. To identify and characterize the agonist and antagonist binding sites within the substructures of individual subunits, purposeful alterations in structure of the subunits will be introduced using recombinant DNA technology. The effects of these alterations on the potencies of agonists and antagonists will be measured with two electrode voltage clamp of Xenopus oocytes. Kinetic studies using rapid application techniques in patch clamped fibroblasts transiently expressing receptor subunits will be used to dissect the molecular mechanism of ligand binding and unbinding. These studies are designed to increase our understanding of neuronal AMPA and kainate receptors and lead toward the development of useful therapeutic compounds that act on these, receptors. The AMPA and kainate receptor systems are implicated in a number of pathological processes including neuronal damage resulting from cerebral ischemia, Parkinson's disease, epilepsy and psychosis.