Structural Basis of Nociceptor Channel TRPM3 gating and pharmacology

伤害感受器通道 TRPM3 门控和药理学的结构基础

• 批准号: 10735377
• 负责人: Juan Du
• 金额: $ 77.08万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735377
• 关键词: 3-Dimensional; Action Potentials; Afferent Neurons; Affinity; Affinity Chromatography; Analgesics; Antibodies; Architecture; Binding; Binding Proteins; Binding Sites; Biochemical; Brain; CRISPR/Cas technology; Calcium; Canis familiaris; Cations; Cell membrane; Chemicals; Complex; Cryoelectron Microscopy; Data; Detection; Detergents; Development; Disease; Dose; Electrophysiology (science); Ensure; Environment; Epilepsy; Epitopes; Fire - disasters; Foundations; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; Glucose; Human; Inflammatory; Injury; Insulin; Intellectual functioning disability; Ion Channel; Ions; Knock-in; Knowledge; Length; Letters; Ligand Binding; Ligands; Lipids; Mediating; Medical; Membrane Proteins; Miconazole; Mission; Molecular; Molecular Mechanisms of Action; Molecular Structure; Monoclonal Antibodies; Mus; Negative Staining; Neurons; Nociceptors; Outcome; Pain; Pathologic; Pathway interactions; Peripheral; Permeability; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Physiological; Play; Primidone; Property; Protein Biochemistry; Proteins; Protocols documentation; Public Health; Recombinant Proteins; Recombinants; Research; Research Project Grants; Resolution; Role; Sensory; Signal Pathway; Solid; Source; Stimulus; Structure; Structure of beta Cell of islet; System; TRP channel; TRPV1 gene; Temperature; Therapeutic; Thermal Hyperalgesias; Tissues; Transgenic Mice; United States National Institutes of Health; Work; chemical association; chronic pain management; comorbidity; desensitization; epileptic encephalopathies; experimental study; inhibitor; nanodisk; neurosteroids; novel; novel therapeutics; pain model; pain perception; pain sensation; particle; patch clamp; pharmacologic; pregnenolone sulfate; pressure; protein complex; protein purification; protein structure; receptor; response; sensor; small molecule; somatosensory; stoichiometry; success

PROJECT SUMMARY The detection and rapid avoidance of harmful external stimuli via pain perception is crucial for survival. The sensors of pain pathways are nociceptors, specialized peripheral sensory neurons. Nociceptors detect external extreme stimuli such as temperature, pressure and chemicals associated with injury via sensory ion channels expressed on the plasma membrane. In response to such stimuli, they fire action potentials and convey the information to the brain, thus alerting us to potential injury and allowing us to take proper actions. Transient receptor potential melastatin 3 (TRPM3) is one of three molecular pain sensors that, together with TRPV1 and TRPA1, detect noxious heat. TRPM3 is a calcium-permeable nonselective cation channel belonging to the melastatin subfamily of the TRP superfamily. It is expressed in a large subset of somatosensory neurons and is activated by heat and by a variety of chemical ligands, including the endogenous neurosteroid pregnenolone sulfate (PS). Both heat and PS-induced activation of TRPM3 evoke pain. TRPM3-deficient mice show a deficit in the development of inflammatory thermal hyperalgesia. Inhibition of TRPM3 by low doses of primidone in PS- and heat-induced pain models showed analgesic effects in mice. The activity of TRPM3 in somatosensory neurons is also negatively modulated by Gβγ protein which is released upon activation of several G-protein- coupled receptors, thereby reducing TRPM3-mediated pain. Because pain is a major unresolved medical problem and because pharmacotherapies targeting GPCR have many and severe unwanted effects, pharmacological targeting of the downstream TRPM3 may have great potential to influence several signaling pathways relevant in pain sensation and in the development of new and alternative analgesic drugs. TRPM3 is also highly expressed in pancreatic beta cells and has application in glucose-induced insulin release. Moreover, TRPM3 is implicated in developmental and epileptic encephalopathies which are a heterogeneous group of disorders characterized by epilepsy with comorbid intellectual disability. At present, the physiological and pathological properties of TRPM3 are still poorly understood. The focus of this proposal is to determine the first TRPM3 structures in different functional states at high-resolution, and to understand the molecular architecture, gating mechanism, and pharmacology of TRPM3 by combining single-particle cryo electron microscopy (cryo-EM) and patch-clamp electrophysiology. We will also initiate the purification of TRPM3 from native tissues using monoclonal antibodies that recognize three-dimensional epitopes of TRPM3 as well as knocked-in affinity tag to TRPM3 gene in mice as there is growing evidence that proteins purified from native sources may display different assembly, complex composition, and functional properties relative to proteins from recombinant systems. The strong preliminary data we provide will ensure the success of this proposal, and the outcome of this proposal will pave a solid foundation for the development of novel analgesics.

项目摘要 通过疼痛感知检测和快速避免有害的外部刺激对于存活至关重要。 疼痛途径的伤害感受器是伤害感受器，专门的外围感觉神经元。伤害感受器检测到外部 极端刺激，例如温度，压力和与损伤通过感觉离子通道相关的化学物质 在质膜上表达。为了应对这种刺激，它们发动了动作潜力并传达 向大脑提供信息，从而提醒我们潜在的伤害并允许我们采取适当的行动。瞬态 受体电位曲霉素3（TRPM3）是三个分子疼痛传感器之一，与TRPV1和TRPV1一起 trpa1，检测有毒的热量。 TRPM3是属于钙的非选择性阳离子通道 TRP超家族的Melastatin亚家族。它在大量的体感神经元中表达， 被热量和多种化学配体激活 硫酸盐（PS）。热和PS诱导的TRPM3激活引起了疼痛。 TRPM3缺陷小鼠显示防御 通过低剂量的初次酮抑制TRPM3 PS和热诱导的疼痛模型在小鼠中显示出镇痛作用。 TRPM3在体感中的活性 神经元也受到Gβγ蛋白的负面调节，该蛋白在激活几种G蛋白时释放 耦合受体，从而减少TRPM3介导的疼痛。因为疼痛是一种未解决的主要医学 问题，并且因为针对GPCR的药物治疗具有许多严重的不良影响，所以 下游TRPM3的药理靶向可能具有影响多种信号的巨大潜力 与疼痛感觉以及新的和替代性镇痛药的发展有关的途径。 trpm3是 也在胰腺β细胞中高表达，并在葡萄糖诱导的胰岛素释放中应用。 此外，TRPM3在发育和癫痫性脑病中实施，这是一种异质 以合并症智力障碍为特征的一组疾病。目前，生理 TRPM3的病理特性仍然鲜为人知。该提议的重点是确定 高分辨率的不同功能状态中的第一个TRPM3结构，并了解分子 通过组合单粒子冷冻电子设备，TRPM3的结构，门控机制和药物 显微镜（冷冻EM）和斑板钳电生理学。我们还将从 使用单克隆抗体识别TRPM3的三维表位的天然组织 在小鼠中敲入与TRPM3基因的亲和力标签，因为越来越多的证据表明蛋白质是从天然纯化的 来源可能显示不同的组件，复杂组成和功能性能相对于蛋白质 来自重组系统。我们提供的强大初步数据将确保该提案的成功， 这项提案的结果将为发展新型镇痛药的发展奠定坚实的基础。