Structural Studies of the Glycine Receptor

描述(由申请人提供)： 甘氨酸受体(GIyR)是烟碱受体超家族的一员，包括同源的烟碱型乙酰胆碱受体、5-HT3-5-羟色胺受体和GABAA受体，所有这些受体都在突触中起快速的信号转导作用。该受体是一种甘氨酸门控阴离子通道，是脊髓和下脑的主要抑制性神经递质通道。这项CEBRA建议的总体目标是在高分辨率下确定人类α1 GlyR结构。这些对GlyR结构的研究旨在提供分子水平的结构-功能信息。由于这个受体家族是麻醉药、酒精、吸入性溶剂和其他麻醉剂的靶标，这些结构的确定将影响我们对离子通道抑制、脱敏和激活的基本机制的理解。CEBRA的供资机制特别重要，因为在没有可衍射微晶体的情况下，通常无法获得资金。然而，像本提案中所描述的那些致力于高风险的努力需要成功地高分辨率确定膜蛋白结构的支持。拟议的研究利用了先前开发的表达系统(Cascio等人，1993)。高聚体GlyR的成功过表达和重组(Cascio等人，2001年)为进行GlyR的结构研究提供了一个独特的机会。初步研究发现，GlyR的构象发生了依赖胆固醇的变化。此外，我们的蛋白质分解和质谱学研究(Leite等人，2000)和重组GlyR的光谱研究(Cascio等人，2001)已经确定尼古丁受体的四跨膜螺旋模型可能是不合适的。我们建议利用膜蛋白结晶学的最新进展，利用脂类立方中间相，与单抗共结晶和/或结晶学研究受体配体结合域的可溶形式。考虑到用结晶学方法确定膜蛋白的高分辨率结构的困难，我们还建议通过确定实验限制条件来进一步完善受体的拓扑结构。这些距离限制将使用EDTA-士的宁试剂或结合化学修饰研究和质谱学的定点半胱氨酸突变来确定。总体而言，这些研究旨在深入了解尼古丁通道的一般保守结构。

DESCRIPTION (provided by applicant): The glycine receptor (GIyR), is a member of the nicotinicoid receptor superfamily, which include the homologous nicotinic acetylcholine receptor, the 5-HT3 serotonin receptor, and the GABAA receptor, all of which act in rapid mediation of signal transduction at the synapse. This receptor is a glycine-gated anionic channel, and is the major inhibitory neurotransmitter channel in spinal cord and lower brain. The overall goal of this CEBRA proposal is the determination of human alpha1 GlyR structure at high resolution. These investigations of GlyR structure aim to provide structure-function information at a molecular level. Since this receptor family is targeted by anesthetics, alcohols, inhaled solvents and other narcotics, these structural determinations will impact on our understanding of the basic mechanisms underlying ion channel inhibition, desensitization, and activation. The CEBRA mechanism of funding is especially relevant since in the absence of diffractable microcrystals, funding is typically unavailable. Yet, dedicated higher-risk efforts such as those described in this proposal require support for successful high-resolution determination of membrane protein structure. The proposed studies exploit the previously developed expression system (Cascio et al., 1993). Successful overexpression and reconstitution of homomeric GlyR (Cascio et al., 2001) presents a unique opportunity to undertake structural studies of the GlyR. Preliminary studies noted a cholesterol-dependent conformational change in GlyR. Additionally, our coupled proteolysis and mass spectrometry studies (Leite et al., 2000) and spectroscopic studies of reconstituted GlyR (Cascio et al., 2001) have determined that the four-transmembrane helix model for the nicotinicoid receptors may be inappropriate. We propose to use recent advances in membrane protein crystallography exploiting lipidic cubic mesophases, co-crystallizations with monoclonal antibodies and/or crystallographic studies of a soluble form of the ligand-binding domain of the receptor. Given the difficulties in determining high-resolution structures of membrane proteins by crystallographic methods, we also alternatively propose to further refine receptor topology via determination of experimental constraints. These distance constraints will be determined using an EDTA-strychnine reagent or site-directed Cys mutagenesis coupled with chemical modification studies and mass spectrometry. Overall these investigations aim to provide insight into the general conserved structure of nicotinicoid channels.