CHEMICAL MODIFICATIONS & ACETYLCHOLINE RECEPTOR FUNCTION

化学修饰

• 批准号: 3405785
• 负责人: MARK G MCNAMEE
• 金额: $ 11.52万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3405785
• 关键词: acetylcholine; chemical binding; chromatography; complementary DNA; egg /ovum; electrophoresis; fresh water environment; ligands; membrane permeability; messenger RNA; molecular biology; molecular cloning; nucleic acid sequence; radioassay; saltwater environment

The long-term goal is to characterize the molecular basis of nicotinic acetylcholine receptor (AChR) function. The approach to be used in the proposed research involves the use of chemical modifications and site-directed mutagenesis to probe the role of free sulhydryl groups in the activation and inactivation phases of AChR-mediated ion channel function. The proposed experiments provide a unique opportunity for this laboratory to exploit the new molecular biology techniques for modifying protein structure while continuing to extend our ongoing chemical modification studies of receptor function. Specific goals of the proposed research include: 1). To identify the subunit(s) and peptide fragment(s) in purified ACHR from electric organ of the electric ray Torpedo californica that contain the sulfhydryl groups that are specifically labeled by hydrophobic maleimides, such as N-phenylmaleimide. 2). To alter the structure of the AChR by converting the DNA coding sequence for one or more of the sulfhydryl group(s) identified in (1) to a sequence for a different, non-reative amino acid (such as serine) using site-directed mutagenesis. The altered AChR will be expressed in Xenopus oocytes by injecting the oocytes with altered mRNA obtained using the SP6 system to produce the mRNA from AChR cDNA. 3). To characterize the effects of AChR sequence alterations on AChR function by measuring both the ligand binding and ion permeability control properties of the nicotinic AChR in the Xenopus oocytes. Voltage clamp techniques will be used to monitor the channel activity of both normal and altered AChR in the oocytes.

长期目标是表征烟碱的分子基础 乙酰胆碱受体（ACHR）功能。 在 拟议的研究涉及化学修饰和 定点诱变，以探测游离磺胺基团在 ACHR介导的离子通道功能的激活和灭活阶段。 拟议的实验为这个实验室提供了独特的机会 利用用于修饰蛋白质的新分子生物学技术 在继续扩展我们正在进行的化学修饰的同时结构 受体功能的研究。 拟议研究的具体目标包括： 1）。识别纯化的ACHR中的亚基和肽片段 来自包含电动射线鱼雷的电动器官 由疏水特异性标记的硫基团 马来酰亚胺，例如N-苯基胺。 2）。通过转换DNA编码来改变ACHR的结构 在（1）中鉴定到A的一个或多个硫酸组的序列 使用不同的非晶氨基酸（例如丝氨酸）的序列 定向诱变。 改变的ACHR将在Xenopus中表达 卵母细胞通过使用SP6获得的卵母细胞注射改变的mRNA 从ACHR cDNA产生mRNA的系统。 3）。表征ACHR序列改变对ACHR的影响 通过测量配体结合和离子通透性控制来功能 异爪蟾卵母细胞中烟酸ACHR的性质。 电压夹 技术将用于监视正常和 卵母细胞中的ACHR改变。