Determination of Glycine Receptor Structure Using FT-ESR

描述（由申请人提供）：广泛的长期目标：发起跨学科的研究奋进，致力于测量甘氨酸受体（GlyR）的门控机制，以及结构与功能的相关性。具体目标：在这项研究中，我们将确定：1）距离，侧链动力学和浸入深度的限制，以位点特异性地映射GlyR的静息状态。(2)距离，侧链动力学，和浸入深度的限制，以位点特异性地映射GlyR的脱敏状态。(3)距离约束以位点特异性地映射GlyR的开放状态。ESR数据将提供分子运动的位点特异性细节，以确定在GlyR中产生离子通量的结构域运动的身份、方向和幅度。我们也将能够确定如何在胞外结构域的配体结合导致跨膜结构域的通道开放的细节。方法：将产生在选定位点具有半胱氨酸的功能性GlyR突变体并进行自旋标记。电子自旋共振将提供intersubunit和intrasubunit接近，和当地的结构constants在每个变构状态的GlyR。这些结构限制，加上分子建模将使我们能够开发一个详细的分子运动的图片，打开和脱敏的GlyR。健康相关性：确定GlyR的门控机制将有助于了解许多神经疾病的分子基础，如痉挛的发病机制，与肌萎缩性侧索硬化症和帕金森综合征相关的运动控制丧失，遗传性先天性肌阵挛和家族性惊吓病（hyperekplexia）。它还将增强我们对与GlyR相互作用的许多药物的作用机制的理解，例如麻醉剂，酒精和用于治疗各种神经系统疾病的各种麻醉剂和治疗剂。

DESCRIPTION (provided by applicant): Broad Long-Term Objectives: Initiate an interdisciplinary reseach endeavor, dedicated to the measurement of the gating mechanism of the Glycine Receptor (GlyR), and the correlation of structure with function. Specific Aims: In this research we will determine: 1) distances, side-chain dynamics, and depths of immersion constraints to site-specifically map the resting state of the GlyR. (2) distances, side-chain dynamics, and depths of immersion constraints to site-specifically map a desensitized state of the GlyR. (3) distance constraints to site-specifically map an open state of the GlyR . The ESR data will provide site-specific details of molecular movements to determine the identities, directions, and amplitudes of domain movements that generate ion-flux in the GlyR. We will also be able to determine the details of how ligand binding in the extracellular domain leads to channel opening in the transmembrane domain. Method: Functional GlyR mutants with cysteines at selected sites will be generated and spin-labeled. Electron spin resonance will provide intersubunit and intrasubunit proximities, and local structural constaints in each allosteric state of the GlyR. These structural constraints, coupled with molecular modeling will allow us to develop a detailed picture of molecular movements that open and desensitize the GlyR. Health Relatedness: Determination of the gating mechanism of the GlyR will lead to an understanding of the molecular basis for many nuerological diseases, such as pathogenesis of spasticity, loss of motor control associated with amyotrophic lateral sclerosis and Parkinsonism, inherited congenital myoclonus, and familial startle disease (hyperekplexia). It will also enhance our understanding of mechanisms of action of many drugs that interact with the GlyR, such as anesthetics, alcohols, and various narcotics and therapeutic agents used for the treatment of a wide range of neurologic disorders.