Structural Studies of the Glycine Receptor

描述（由申请人提供）： 甘氨酸受体（GlyR）是烟碱受体超家族的成员，其包括同源烟碱乙酰胆碱受体、5-HT 3 5-羟色胺受体和GABAA受体，所有这些都在突触处快速介导信号转导中起作用。该受体是甘氨酸门控的阴离子通道，并且是脊髓和下脑中的主要抑制性神经递质通道。该CEBRA提案的总体目标是以高分辨率测定人α 1 GlyR结构。这些研究的GlyR结构的目的是提供在分子水平上的结构-功能信息。由于该受体家族被麻醉剂、醇类、吸入溶剂和其他麻醉剂靶向，这些结构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的供资机制尤其重要，因为在没有可衍射微晶的情况下，通常无法获得资金。然而，专门的高风险的努力，如本提案中描述的那些需要支持成功的高分辨率膜蛋白结构的测定。所提出的研究利用了先前开发的表达系统（Cascio等人，1993年）。同源GlyR的成功过表达和重建（Cascio等人，2001）提供了一个独特的机会，进行结构研究的GlyR。初步研究指出，胆固醇依赖性构象变化的GlyR。此外，我们的偶联蛋白水解和质谱研究（Leite等人，2000）和重构GlyR的光谱研究（Cascio等人，2001）已经确定烟碱受体的四跨膜螺旋模型可能是不合适的。我们建议使用膜蛋白晶体学的最新进展，利用cardidic立方中间相，与单克隆抗体和/或晶体学研究的可溶性形式的受体的配体结合结构域的共结晶。由于晶体学方法难以确定膜蛋白的高分辨率结构，我们还建议通过确定实验约束进一步完善受体拓扑结构。这些距离限制将使用EDTA-士的宁试剂或定点Cys诱变结合化学修饰研究和质谱法来确定。总的来说，这些调查的目的是提供深入了解烟碱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.