ALPHA1 GLYCINE RECEPTOR STRUCTURE AND FUNCTION

ALPHA1 甘氨酸受体结构和功能

• 批准号: 2022941
• 负责人: MICHAEL CASCIO
• 金额: $ 9.4万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022941
• 关键词: Baculoviridae; Sf9 cell line; chimeric proteins; circular dichroism; crosslink; cysteine; gel filtration chromatography; glycine receptors; glycosylation; human genetic material tag; liposomes; phosphorylation; protein reconstitution; protein structure function; recombinant proteins; site directed mutagenesis; ultrafiltration

DESCRIPTION: (adapted from Applicant's Abstract) The glycine receptor (GlyR), a glycine-gated Cl- channel, is the major inhibitory neurotransmitter channel of the central nervous system. The overall goal of this project is to determine the secondary structure, topology, and functional role of domains and specific residues of the homomeric recombinant human alpha1 GlyR overexpressed in a baculovirus expression system. These investigations of GlyR structure may provide structure- function information at a molecular level on neurological disorders such as myoclonus, spasticity, familial startle disease or other diseases in which deficiencies of GlyR have been implicated. Additionally, this receptor is a member of the ligand-gated superfamily, which include the homologous nicotinic acetylcholine receptor, the 5-HT3 serotonin receptor, and the GABA receptor, all of which act in rapid mediation of signal transduction at the synapse. These investigations may provide insight into the general conserved mechanisms used in channel design. More specifically, the aims include utilizing the previously developed expression system (Cascio et al., 1993) to produce sufficient quantities of recombinant protein and the amino-terminal domain (NGly) for reconstitution, gross characterization, and subsequent analyses. Crosslinking studies and ultrafiltration studies coupled with gel filtration will be conducted in order to determine the aggregation state of purified recombinantly produced alpha1 GlyR and NGly proteins. The effects of phosphorylation and glycosylation on activity in reconstituted liposomes and single channel measurements in black lipid membranes will be determined. Circular dichroism (CD) spectroscopic studies of the amino-terminal (NGly) domain or on the membrane-bound domain remaining after proteolysis will provide information not only on the folding of this domain, but, by comparison with studies of reconstituted GlyR, will allow assignation of the secondary structure of the remaining portion of the molecule and provide the first quantitation of GlyR secondary structure and a template for subsequent modeling. In addition, the sensitivity of CD to small changes in secondary structure will allow determinations of the effects of ligand binding on protein secondary structure. Site-directed mutagenesis coupled with covalent modification will be used to probe channel topology. In addition, the construction of chimeric channel proteins will allow structure- function maps to be constructed for this ion channel.

描述：(改编自申请者摘要)甘氨酸受体 甘氨酸门控的氯离子通道(GlyR)是主要的抑制物 中枢神经系统的神经递质通道。整体而言 本项目的目标是确定二级结构、拓扑、 同聚体的结构域和特定残基的功能作用 重组人α1-GlyR在杆状病毒中的高效表达 系统。这些对GlyR结构的研究可能提供结构- 在分子水平上关于神经系统疾病的功能信息 如肌阵挛、痉挛、家族性惊厥病或其他疾病 与GlyR的哪些缺陷有关。此外，这一点 受体是配体门控超家族的一员，包括 同源烟碱型乙酰胆碱受体，5-HT3 5-羟色胺 受体和GABA受体，所有这些都在快速调节 突触的信号转导。这些调查可能会提供 洞察渠道设计中使用的一般保守机制。 更具体地说，目标包括利用以前开发的 表达系统(Cascio等人，1993)以产生足够数量的 重组蛋白和氨基末端结构域(NGly) 重建、总体特征和随后的分析。 凝胶联用的交联研究和超滤研究 将进行过滤，以确定聚合状态 纯化的重组表达的α1-GlyR和NGly蛋白。这个 磷酸化和糖基化对肌动蛋白活性的影响 重组脂质体及其在黑色脂类中的单通道测量 膜将被确定。圆二色(CD)光谱 氨基末端(NGly)结构域或膜结合区的研究 蛋白质降解后残留的结构域将不仅提供关于 这一领域的折叠，但与 重组的GlyR，将允许分配二级结构 分子的剩余部分并提供第一个 GlyR二级结构的定量和后续的模板 模特儿。此外，Cd对微小变化的敏感性 二级结构将允许确定配体的影响 结合在蛋白质二级结构上。定点突变 结合共价修饰将被用来探测通道 拓扑学。此外，嵌合通道蛋白的构建 将允许为该离子构建结构-功能图 频道。