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: 10540405
• 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/10540405
• 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; Brain; 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; Neurodegenerative Disorders; Neurologic; Neurotransmitter Receptor; Nicotine Dependence; Pain; Parkinson Disease; Peptides; Pharmaceutical Preparations; 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; Tissues; Transmembrane Domain; Western Blotting; alpha helix; burden of illness; clinical effect; conformational conversion; design; disability; esterase; experimental study; extracellular; gamma-Aminobutyric Acid; inhibitor; innovation; insight; maltose-binding protein; nervous system disorder; neuropsychiatric disorder; novel; overexpression; pharmacologic; prevent; protein protein interaction; protein purification; 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 五聚体门控离子通道（PLGIC），也称为真核生物中的Cys-loop受体，包括 乙酰胆碱，5-羟色胺，GABA和甘氨酸的受体。他们参与了许多神经精神病学， 神经系统和炎症性疾病。后生动物中的所有Cys-loop受体家庭成员都包含三个 结构域：细胞外域（ECD），跨膜结构域（TMD）和细胞内结构域 （ICD）。 ECD和TMD在受体家族之间是建筑保守的。 ICD保守不佳 在长度和氨基酸组成中，对于许多亚基，都包含大量的预测结构区域 紊乱。由于挑战与五聚体受体的ICD，它们本质上是 在严格的机械表征和直接探索作为药理目标方面被忽视。 ICD参与鉴定质膜表达水平，靶向和功能，部分介导 通过蛋白质 - 蛋白质相互作用（PPI），例如与伴侣。我们设想了对 伴侣介导的调节将发现新的PPI药物靶标。通过我们对ICD的发表研究 我们定义了一个链接器，该链接允许结构确定同型和同类的类结构中的第一个结构 异源性GABAA受体。我们还确定，仅ICD将ICD组装成五个人， 建立ICD在低聚组装中的新作用。在这里，我们提出了一种多层方法 利用可溶性ICD嵌合体和全长受体以及在该期间获得的结果 以前的资金期是仔细考虑最近出版的PLGIC结构。我们发现 抗胆碱酯酶抑制剂（RIC-3）伴侣的抗性与5-HT3A的24-氨基酸L1-MX段结合 亚基。使用新提出的特定目标（SA），我们将：（SA1）确定L1-MX段在 RIC-3调节阳离子PLGIC组装，（SA2）鉴定通过通过 阳离子ICD和（SA3）表征了阳离子传导PLGIC涉及的机制 L1-MX段通过MA-HELIX框架门户转运。在每个目标中，我们都将使用生化 和电生理方法，并在其细胞中与过表达和纯化的蛋白质或蛋白质结合 环境。我们预计我们的研究将为治疗的新型目标提供基础 开发，PPI调节剂的细胞内域。