Probing the structure and function of the intracellular domain of Cys-loop recept

描述(申请人提供)：属于Cys-loop基因超家族的受体，也称为五聚体配体门控离子通道(PLGIC)，包括乙酰胆碱、5-羟色胺(5-HT3)、GABAA、GABAR和甘氨酸受体。PLGIC在神经系统中介导快速突触传递。这些受体是目前临床使用的药物的靶点，包括抗抑郁药、抗癫痫药、止吐药、抗精神病药物、麻醉药、肌肉松弛药、痉挛药、镇静剂和治疗药物滥用的药物。一些家族成员也在不可兴奋的细胞中被发现，在那里他们代表着治疗各种炎症性疾病的新的治疗靶点，如动脉粥样硬化、阿尔茨海默氏症、糖尿病、炎症性肠病和脓毒症。后生动物中所有的Cys-loop受体家族成员都包含三个结构域：胞外区、跨膜区和胞内区(ICD)。对胞外和跨膜结构域进行了详细的功能研究，确定了它们的三维结构。最近在细菌中发现了Cys-loop受体同源物，这将结构知识推向了原子分辨。然而，原核生物成员缺乏胞内结构域。有趣的是，动物的Cys-loop受体的胞内结构域 在长度和氨基酸组成方面，王国是最多样化的结构域。因此，细胞内结构域是开发亚型选择性药物的一个有吸引力的靶点，与目前的药物相比，副作用更少，这些药物都针对高度保守的细胞外或跨膜结构域。这个项目将专注于探索迄今为止被忽视的胞内结构域的结构和功能。我们将使用我们通过将来自选择的阴离子和阳离子pLGIC的ICD插入到来自紫球藻(GLIC)的原核pLGIC中而产生的功能嵌合体，GLIC是一种研究得很好的同源物，由ECD和TMD组成，缺乏ICD，已被建立为研究pLGIC的工具。在目标1中，我们将使用电压钳、膜片钳和双层记录来详细表征特定的阴离子和阳离子pLGIC对ICD的功能贡献。在目标2中，我们将研究pLGIC ICD中存在的分子决定因素，这些分子决定因素介导了与胆碱酯酶抑制剂(RIC-3)抗性伴侣蛋白的相互作用。在目标3中，我们将研究和比较野生型和嵌合型受体的相互作用，并利用FT-IR和CD光谱以及SDSL-EPR的副残基分析来探索嵌合体中ICD的整体三维结构。我们的研究将具有重要意义，因为它将完成对Cys-loop受体所有三个结构域的结构-功能-相互作用的理解。它还将评估细胞内结构域作为新的药物靶点，然后可用于设计不基于传统激动剂/拮抗剂方法的创新药物。

DESCRIPTION (provided by applicant): Receptors belonging to the Cys-loop gene superfamily, also called pentameric ligand-gated ion channels (pLGIC), include acetylcholine, serotonin (5-HT3), GABAA, GABAr and glycine receptors. pLGIC mediate fast synaptic transmission in the nervous system. These receptors are targeted by current clinically used drugs that include antidepressants, antiepileptics, antiemetics, antipsychotics, anesthetics, muscle relaxants, spasmolytics, tranquillizers, and drugs for the treatment of substance abuse. Some family members are also found in non-excitable cells where they represent novel therapeutic targets to treat inflammatory diseases as diverse as atherosclerosis, Alzheimer's, diabetes, inflammatory bowel disease, and sepsis. All Cys-loop receptor family members in metazoans contain three domains: an extracellular domain, a transmembrane domain, and an intracellular domain (ICD). The extracellular and transmembrane domains have been functionally studied in great detail and their three-dimensional structures have been determined. The recent identification of Cys-loop receptor homologues in bacteria has propelled the structural knowledge into atomic resolution. However, the prokaryotic members lack the intracellular domain. Interestingly, the intracellular domain of Cys-loop receptors from the animal kingdom is the most diverse domain with respect to both length and amino-acid composition. The intracellular domain therefore represents an attractive target for developing subtype-selective drugs with the promise of fewer side effects than current drugs, which all target the highly- conserved extracellular or transmembrane domains. This project will focus on probing the structure and function of the so-far neglected intracellular domain. We will use functional chimeras that we have generated by inserting the ICD from select anionic and cationic pLGIC into the prokaryotic pLGIC from Gloeobacter violaceus (GLIC), a well-studied homologue that consists of ECD and TMD and lacks an ICD, and that has been established as a tool to study pLGIC. In Aim 1 we will characterize in detail the functional contributions of ICDs from specific anionic and cationic pLGIC using voltage-clamp, patch-clamp and bilayer recordings. In Aim 2 we will investigate the molecular determinants present in pLGIC ICDs that mediate interaction with the chaperone protein resistance to inhibitors of choline esterase (RIC-3). In Aim 3 we will study and compare the interactomes of wild-type and chimeric receptors and probe the overall three-dimensional structure of the ICD in the chimeras with FT-IR and CD spectroscopy, and the by-residue analysis with SDSL-EPR. Our study will be of significance because it will complete the understanding of the structure-function-interplay of all three domains of Cys-loop receptors. It will also evaluate the intracellular domain as a new drug target that can then be utilized to design innovative drugs that are not based on the conventional agonist / antagonist approach.