Structural Basis of Nociceptor Channel TRPM3 gating and pharmacology

项目总结 通过疼痛感知检测和快速避免有害的外部刺激是生存的关键。这个 痛觉通路的传感器是伤害性感受器，即专门的外周感觉神经元。伤害性感受器检测到外部 通过感觉离子通道与损伤相关的极端刺激，如温度、压力和化学物质 在质膜上表达。作为对这种刺激的反应，它们激发动作电位并传递 信息传递给大脑，从而提醒我们潜在的伤害，并允许我们采取适当的行动。瞬变 受体潜能Melastatin 3(TRPM3)是三种分子痛觉传感器之一，与TRPV1和TRPV1一起， TRPA1，探测到有害热量。TRPM3是一种钙离子通透性非选择性阳离子通道，属于 Melastatin是色氨酸超家族中的一个亚家族。它在躯体感觉神经元的大亚群中表达， 被热和各种化学配体激活，包括内源性神经类固醇孕烯醇酮 硫酸盐(PS)。热和PS诱导的TRPM3的激活都会引起疼痛。TRPM3缺陷小鼠表现出缺陷 在炎症性热痛觉过敏的发展过程中。小剂量扑米酮对细胞内TRPM3的抑制作用 PS和热诱导的小鼠疼痛模型均表现出镇痛作用。TRPM3在躯体感觉中的活性 神经元也受到G-βγ蛋白的负调节，该蛋白在激活几种G-蛋白后释放- 偶联受体，从而减少TRPM3介导的疼痛。因为疼痛是一种尚未解决的重大医学问题 问题和因为以gpcr为靶点的药物疗法有许多严重的不良影响， 下游TRPM3的药理靶向可能有很大的潜力影响几个信号转导 与痛觉相关的通路以及新的和替代止痛药的开发。TRPM3是 在胰岛β细胞中也高表达，并在葡萄糖诱导的胰岛素释放中有应用。 此外，TRPM3与发育和癫痫脑病有关，这是一种异质性的疾病。 以癫痫为特征并伴有智力残疾的一组障碍。目前，生理学的 目前对TRPM3的病理性质仍知之甚少。这项提案的重点是确定 首先在高分辨率下研究TRPM3在不同功能状态下的结构，并了解其分子 结合单粒子低温电子的TRPM3的结构、门控机制和药理学 显微镜(冷冻-EM)和膜片钳电生理学。我们还将启动从 使用识别TRPM3三维表位的单抗以及 小鼠TRPM3基因的敲入亲和标记，因为越来越多的证据表明，从天然的 来源可能表现出与蛋白质不同的组装、复杂的组成和功能特性 来自重组系统。我们提供的强有力的初步数据将确保这项提议的成功， 这一建议的结果将为新型止痛药的开发奠定坚实的基础。

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.