RECOMBINANT GLUTAMATE RECEPTORS

建议通过实验来研究其结构和功能 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 在非洲爪蟾卵母细胞中表达的情况将使用电生理学进行比较 方法。 大脑中 AMPA 或红藻氨酸受体亚基的水平 衍生的 RNA 将通过聚合酶链式反应 (PCR) 进行定量 将在功能属性和可能的属性之间建立相关性 亚基结构。 这些相关性将通过测量 相同表达的重组受体的功能特性 大脑 mRNA 制剂中发现的这些亚基的数量。 开发针对肽的亚基特异性抗体 还提出了在克隆的氨基酸序列中发现的。 有用的 抗体将有助于直接探测天然的亚基结构 通过蛋白质印迹分析和高分辨率检测受体 免疫定位。 识别和表征激动剂 各个亚基的亚结构内的拮抗剂结合位点， 将有目的地改变子单元的结构 使用重组DNA技术。 这些改变的影响 激动剂和拮抗剂的效力将用两种方法测量 非洲爪蟾卵母细胞的电极电压钳。 使用快速动力学研究 膜片钳成纤维细胞瞬时应用技术 表达受体亚基将用于剖析分子 配体结合和解除结合的机制。 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 导致开发有用的治疗化合物，作用于 这些，受体。 AMPA 和红藻氨酸受体系统参与其中 在许多病理过程中，包括导致的神经元损伤 脑缺血、帕金森病、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.