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

Cys环受体胞内结构域的结构和功能探讨

• 批准号: 9240679
• 负责人: Michaela Jansen
• 金额: $ 32.9万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9240679
• 关键词: Acetylcholine; Adverse effects; Affect; Agonist; Alzheimer' s Disease; Amino Acid Sequence; Amino Acids; Anesthetics; Animals; Antidepressive Agents; Antiemetics; Antiepileptic Agents; Antipsychotic Agents; Anxiety; Atherosclerosis; Attention deficit hyperactivity disorder; Bacteria; Binding; Biochemical; Biological Assay; Butyric Acids; Cations; Cell membrane; Cell physiology; Cells; Cellular biology; Charge; Chimera organism; Choline; Clinical; Development; Diabetes Mellitus; Disease; Drug usage; Electrodes; Elements; Engineering; Epilepsy; Extracellular Domain; Extracellular Structure; Family; Family member; Future; Gated Ion Channel; Genes; Glycine Receptors; Goals; Health; Homologous Gene; Homologous Protein; Human; Inflammatory; Inflammatory Bowel Diseases; Ion Channel; Ion Channel Gating; Kinetics; Knowledge; Length; Ligands; Link; Lipid Bilayers; Mammalian Cell; Manuscripts; Mediating; Mental Depression; Methods; Molecular; Molecular Chaperones; Muscle relaxants; Mutation; Myasthenia Gravis; N-terminal; Nervous system structure; Nicotine Dependence; Nicotinic Receptors; Parkinson Disease; Peptides; Pharmaceutical Preparations; Phosphorylation; Play; Positioning Attribute; Preparation; Property; Proteins; Publishing; Receptor Cell; Receptor Gene; Reporting; Research; Resistance; Resolution; Roentgen Rays; Role; Schizophrenia; Sepsis; Serotonin; Site; Spasmolytics; Spectrum Analysis; Spin Labels; Structure; Subcellular structure; Synaptic Transmission; Testing; Tranquilizing Agents; Transmembrane Domain; Vertebral column; Xenopus oocyte; alpha helix; design; esterase; experimental study; gamma-Aminobutyric Acid; improved; inhibitor/antagonist; innovation; member; neglect; new therapeutic target; novel; patch clamp; public health relevance; receptor; receptor function; reconstitution; serotonin receptor; small molecule; substance abuse treatment; therapeutic target; three dimensional structure; tool; trafficking; voltage clamp

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.

描述（申请人提供）：属于Cys环基因超家族的受体，也称为五聚体配体门控离子通道（pLGIC），包括乙酰胆碱、血清素（5-HT3）、GABAA、GABAr和甘氨酸受体。 pLGIC 介导神经系统中的快速突触传递。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、麻醉药、肌肉松弛药、解痉药、镇静药和治疗药物滥用的药物。一些家族成员也存在于非兴奋性细胞中，它们代表了治疗动脉粥样硬化、阿尔茨海默病、糖尿病、炎症性肠病和败血症等多种炎症性疾病的新治疗靶点。后生动物中的所有 Cys 环受体家族成员均包含三个结构域：细胞外结构域、跨膜结构域和细胞内结构域 (ICD)。细胞外和跨膜结构域的功能已得到详细研究，并且已确定了它们的三维结构。最近对细菌中半胱环受体同源物的鉴定将结构知识推向了原子分辨率。然而，原核成员缺乏细胞内结构域。有趣的是，来自动物的 Cys 环受体的细胞内结构域 就长度和氨基酸组成而言，Kingdom 是最多样化的结构域。因此，细胞内结构域代表了开发亚型选择性药物的一个有吸引力的靶标，其副作用比目前的药物更少，目前的药物都针对高度保守的细胞外或跨膜结构域。该项目将重点探讨迄今为止被忽视的细胞内结构域的结构和功能。我们将使用通过将来自选定的阴离子和阳离子 pLGIC 的 ICD 插入来自紫地杆菌 (GLIC) 的原核 pLGIC 中而生成的功能嵌合体，GLIC 是一种经过充分研究的同源物，由 ECD 和 TMD 组成，缺乏 ICD，并且已被确立为研究 pLGIC 的工具。在目标 1 中，我们将使用电压钳、膜片钳和双层记录详细描述来自特定阴离子和阳离子 pLGIC 的 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与伴侣蛋白对胆碱酯酶抑制剂 (RIC-3) 的抗性的相互作用。在目标 3 中，我们将研究和比较野生型和嵌合受体的相互作用，并利用 FT-IR 和 CD 光谱探测嵌合体中 ICD 的整体三维结构，并利用 SDSL-EPR 进行残基分析。我们的研究具有重要意义，因为它将完成对半胱环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。