CHARACTERIZATION OF RECOMBINANT GLUTAMATE RECEPTORS

重组谷氨酸受体的表征

• 批准号: 2268921
• 负责人: TODD A VERDOORN
• 金额: $ 18.89万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2268921
• 关键词: Xenopus oocyte; antibody formation; cerebellum; cerebral cortex; chimeric proteins; excitatory aminoacid; glutamate receptor; glutamates; hippocampus; kainate; laboratory rabbit; laboratory rat; messenger RNA; molecular cloning; molecular site; peptides; polymerase chain reaction; prosencephalon; protein sequence; protein structure; protein structure function; receptor binding; receptor expression; recombinant DNA; recombinant proteins; site directed mutagenesis; spinal cord; voltage /patch clamp; western blottings

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.

提出了实验研究的结构和功能