CHEMICAL SYNAPSES, BIOPHYSICAL STUDIES

化学突触、生物物理学研究

• 批准号: 3394577
• 负责人: Henry A. Lester
• 金额: $ 15.07万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-12-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3394577
• 关键词: acetylcholine; biophysics; electrophysiology; fresh water environment; gamma aminobutyrate; genetic library; membrane permeability; molecular cloning; neural conduction; saltwater environment; stoichiometry; synapses

This project concerns the function of neurotransmitter receptors that directly gate ion channels. Nucleic acid techniques will be used in conjunction with electrophysiological measurements (voltage clamp, patch clamp) to test hypotheses about the role of specific polypeptide domains and of individual amino acids in agonist binding and channel gating. The molecular biological manipulations will employ cDNA clones for the receptors; site-specific mutations of these clones will be constructed. Available enzymatic methods will be used to transcribe the normal and mutated clones. The mRNA will be introduced by microinjection or by membrane fusion into Xenopus oocytes or mammalian cells in culture. The target cells will express the receptors in the cell membrane; the functional ion channels will be studied electrophysiologically. The work will include both the nicotinic acetylcholine receptor of Torpedo electric organ (clones now available and under study) and the receptor from mouse muscle (clones expected soon). Analogous studies will be pursued on the GABA receptor-channel complex of rat and chick brain. Work will continue on assaying fractionated mRNA preparations that express GABA channels and on screening cDNA clones constructed from the active fractions. When sequenced clones are available, voltage-clamp and patch-clamp analysis of specifically constructed mutant channels will begin.

该项目涉及神经递质受体的功能， 直接控制离子通道。 核酸技术将用于 结合电生理测量（电压钳、贴片） 钳）来测试有关特定多肽结构域作用的假设 以及激动剂结合和通道门控中的单个氨基酸。 的 分子生物学操作将采用cDNA克隆， 受体;将构建这些克隆的位点特异性突变。 可用的酶促方法将用于转录正常和 变异的克隆人 mRNA将通过显微注射或通过 在培养物中将膜融合到非洲爪蟾卵母细胞或哺乳动物细胞中。 的 靶细胞将在细胞膜中表达受体; 功能性离子通道将被电生理学研究。 工作 将包括烟碱乙酰胆碱受体和电鳐电 小鼠器官（克隆现已可用并正在研究中）和受体 肌肉（克隆预计很快）。 类似的研究将在GABA受体通道复合物上进行， 老鼠和小鸡的大脑 将继续进行分析分级mRNA的工作 表达GABA通道的制剂和筛选cDNA克隆 由活性组分组成。 当测序克隆被 可用电压钳和膜片钳分析具体 构建的突变通道将开始。