Probing the structure and function of the intracellular domain of cys-loop receptors

探讨cys环受体胞内结构域的结构和功能

• 批准号: 10363881
• 负责人: Michaela Jansen
• 金额: $ 38.25万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-15 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10363881
• 关键词: Address; Alzheimer' s Disease; Amino Acids; Anesthetics; Anti-Anxiety Agents; Antiemetics; Antiepileptic Agents; Anxiety; Anxiety Disorders; Architecture; Atherosclerosis; Attention; Binding; Biochemical; Biological Assay; Cations; Cause of Death; Cell Line; Cell membrane; Characteristics; Chimera organism; Choline; Cholinergic Receptors; Clinical; Coupled; Cryoelectron Microscopy; Dementia; Diabetes Mellitus; Disease; Drug Design; Drug Targeting; Electrophysiology (science); Environment; Epilepsy; Eukaryota; Extracellular Domain; Family; Family member; Foundations; Funding; Future; Glycine; Heart Diseases; Homo; Human; Immobilization; Inflammatory; Inflammatory Bowel Diseases; Investigation; Ion Channel; Ion Channel Gating; Knowledge; Length; Ligands; Link; Mediating; Membrane Transport Proteins; Mental Depression; Methods; Modification; Molecular; Molecular Chaperones; Molecular Conformation; Muscle relaxants; Myasthenia Gravis; Nerve Degeneration; Neurologic; Neurotransmitter Receptor; Nicotine Dependence; Pain; Parkinson Disease; Peptides; Pharmaceutical Preparations; Pharmacology; Plant Resins; Proteins; Publishing; Resistance; Role; Schizophrenia; Sepsis; Serotonin; Shapes; Sodium Chloride; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Surface; Tertiary Protein Structure; Testing; Transmembrane Domain; Western Blotting; base; brain tissue; burden of illness; clinical effect; conformational conversion; design; disability; esterase; experimental study; gamma-Aminobutyric Acid; inhibitor; innovation; insight; maltose-binding protein; nervous system disorder; neuropsychiatric disorder; neuropsychiatry; novel; overexpression; prevent; protein protein interaction; receptor; smoking cessation; targeted treatment; therapeutic development; therapy design

Project Summary Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotes, include the receptors for acetylcholine, serotonin, GABA and glycine. They are involved in numerous neuropsychiatric, neurologic, and inflammatory diseases. All Cys-loop receptor family members in metazoans contain three structural domains: an extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular domain (ICD). The ECD and TMD are architecturally conserved between receptor families. The ICD is poorly conserved in both length and amino-acid composition and, for many subunits, contains large regions of predicted structural disorder. Due to the challenge of working with the ICDs of pentameric receptors, they have been essentially overlooked in terms of rigorous mechanistic characterization and direct exploration as pharmacological targets. The ICD is involved in finetuning plasma membrane expression levels, targeting, and function, in part mediated by protein-protein interactions (PPI) for example with chaperones. We envision that mechanistic knowledge of chaperone-mediated modulation will uncover new PPI drug targets. Through our published studies of the ICD we defined a linker that allowed for structure determination of the first in class structures of homo- and heteropentameric GABAA receptors. We also established that the ICD alone assembles into pentamers, establishing a novel role for the ICD in oligomeric assembly. Here, we propose a multi-layered approach leveraging both soluble ICD chimeras and full-length receptors together with results obtained during the previous funding period in careful consideration of recently-published pLGIC structures. We discovered that the resistance to inhibitors of choline esterase (Ric-3) chaperone binds to a 24-amino acid L1-MX segment of 5-HT3A subunits. With the new proposed specific aims (SA) we will: (SA1) determine the role of the L1-MX segment in RIC-3 modulation of cationic pLGIC assembly, (SA2) identify novel protein-protein interactions mediated via cationic ICDs, and (SA3) characterize the mechanism by which opening of cation-conducting pLGICs involves translocation of the L1-MX segment through the MA-helix framed portals. In each aim, we will use biochemical and electrophysiological methods, coupled with overexpressed and purified proteins or proteins in their cellular environment. We anticipate that our studies will provide the basis for a novel class of targets for therapeutic development, PPI modulators for pentameric channel intracellular domains.

项目摘要 五聚体配体门控离子通道（pLGIC），在真核生物中也称为Cys环受体，包括 乙酰胆碱、血清素、GABA和甘氨酸的受体。他们参与了许多神经精神疾病， 神经系统和炎症性疾病。后生动物中的所有Cys环受体家族成员都含有三个 结构域：胞外结构域（ECD）、跨膜结构域（TMD）和胞内结构域 （ICD）。ECD和TMD在受体家族之间是结构保守的。ICD保存不佳 在长度和氨基酸组成方面，对于许多亚基，包含预测结构的大区域， disorder.由于与五聚体受体的ICD一起工作的挑战，它们基本上是 在严格的机制表征和作为药理学靶点的直接探索方面被忽视。 ICD参与微调质膜表达水平、靶向和功能，部分介导 通过蛋白质-蛋白质相互作用（PPI）例如与分子伴侣。我们设想， 分子伴侣介导的调节将揭示新的PPI药物靶点。通过我们发表的ICD研究， 我们定义了一个连接器，它允许确定homo-和 异五聚体GABAA受体。我们还确定了ICD单独组装成五聚体， 建立了ICD在寡聚体组装中的新作用。在这里，我们提出了一个多层次的方法， 利用可溶性ICD嵌合体和全长受体以及在研究期间获得的结果， 在仔细考虑最近公布的pLGIC结构的情况下，我们发现 对胆碱酯酶（Ric-3）抑制剂的抗性伴侣与5-HT 3A的24个氨基酸的L1-MX片段结合 亚单位。根据新提出的具体目标（SA），我们将：（SA 1）确定L1-MX部分在以下方面的作用： RIC-3对阳离子pLGIC组装的调节，（SA 2）鉴定了通过以下途径介导的新型蛋白质-蛋白质相互作用： 阳离子ICD和（SA 3）表征了阳离子传导pLGIC的开放机制， L1-MX片段通过MA-螺旋框架门户的易位。在每个目标中，我们将使用生物化学 和电生理学方法，结合过表达和纯化的蛋白质或其细胞中的蛋白质， 环境我们预计，我们的研究将为一类新的治疗靶点提供基础。 开发，五聚体通道细胞内结构域的PPI调节剂。