ALPHA1 GLYCINE RECEPTOR STRUCTURE AND FUNCTION

说明：（改编自申请人的摘要）甘氨酸受体 甘氨酸门控氯离子通道（GlyR）是主要的抑制性通道， 中枢神经系统的神经递质通道。 整体 这个项目的目标是确定二级结构，拓扑结构， 和功能作用的结构域和特定残基的同源 在杆状病毒表达中过表达的重组人α 1 GlyR 系统 GlyR结构的这些研究可以提供结构- 在分子水平上对神经系统疾病的功能信息， 如肌阵挛、痉挛、家族性惊吓疾病或其他疾病， 这与GlyR的缺陷有关。 而且这个 受体是配体门控超家族的成员，其包括 同源烟碱乙酰胆碱受体，5-羟色胺3 受体和GABA受体，所有这些都在快速介导 突触的信号传导 这些调查可以提供 深入了解通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，1993年），生产出足够数量的 重组蛋白的氨基末端结构域（NGly） 重构、大体表征和后续分析。 交联研究和超滤研究结合凝胶 将进行过滤以确定聚集状态 纯化的重组产生的α 1 GlyR和NGly蛋白。 的 磷酸化和糖基化对 重构脂质体和黑色脂质中的单通道测量 膜将被确定。 圆二色性（CD）光谱 氨基末端（NGly）结构域或膜结合蛋白的研究 蛋白水解后剩余的结构域不仅提供了 这个领域的折叠，但是，通过与研究相比， 重组GlyR，将允许二级结构的分配 分子的剩余部分，并提供第一个 GlyR二级结构的定量和随后的模板 建模 此外，CD对微小变化的敏感性 二级结构将允许确定配体的作用 结合蛋白质二级结构。 定点诱变 结合共价修饰将用于探测通道 topology. 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

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.