Determination of Glycine Receptor Structure Using FT-ESR

描述(由申请人提供)：广泛的长期目标：发起跨学科的研究努力，致力于测量甘氨酸受体(Glyine Receptor，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，以定位特定的GlyR的静止状态。(2)距离、侧链动力学和浸入深度的限制，以定位地映射GlyR的脱敏状态。(3)位置特定的GlyR开放状态的距离限制。ESR数据将提供特定位置的分子运动细节，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。我们还将能够确定细胞外域的配体结合如何导致跨膜域的通道开放的细节。方法：在选定的位点产生半胱氨酸的功能性GlyR突变体，并进行自旋标记。电子自旋共振将在GlyR的每个变构状态中提供亚基间和亚基内的接近，以及局部结构约束。这些结构限制，再加上分子建模，将使我们能够开发出打开和减敏GlyR的分子运动的详细图像。与健康相关：确定GlyR的门控机制将有助于了解许多神经系统疾病的分子基础，如痉挛的发病机制，与肌萎缩侧索硬化症和帕金森病相关的运动控制丧失，遗传性肌阵挛，以及家族性惊厥病(多动症)。它还将加深我们对许多与GlyR相互作用的药物的作用机制的了解，如麻醉剂、酒精以及用于治疗广泛神经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.