Structural Basis of Nociceptor Channel TRPM3 gating and pharmacology

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生存至关重要。的 疼痛通路的传感器是伤害感受器，即专门的外周感觉神经元。伤害感受器检测外部 极端刺激，如温度、压力和化学物质，通过感觉离子通道与损伤相关 在质膜上表达。在对这些刺激的反应中，它们激发动作电位，并传递信号。 它可以将信息传递给大脑，从而提醒我们潜在的伤害，并允许我们采取适当的行动。瞬态 受体电位melastatin 3（TRPM 3）是三种分子疼痛感受器之一，与TRPV 1和 TRPA 1，探测有害热。TRPM 3是一种钙渗透性非选择性阳离子通道，属于 TRP超家族的melastatin亚家族。它在一大部分躯体感觉神经元中表达， 通过加热和多种化学配体激活，包括内源性神经类固醇双烯醇酮 硫酸盐（PS）。热和PS诱导的TRPM 3激活都引起疼痛。TRPM 3缺陷小鼠表现出缺陷 在炎症性热痛觉过敏的发展中起着重要作用。低剂量扑米酮对TRPM 3的抑制作用 PS和热诱导的疼痛模型在小鼠中显示出镇痛作用。TRPM 3在躯体感觉神经元中的活性 神经元也受到Gβγ蛋白的负调节，Gβγ蛋白在几种G蛋白激活后释放， 偶联受体，从而减轻TRPM 3介导的疼痛。因为疼痛是一个主要的未解决的医疗问题 问题，并且因为靶向GPCR的药物疗法具有许多和严重的不希望的作用， 下游TRPM 3的药理学靶向可能具有影响几种信号传导的巨大潜力 与疼痛感觉相关的通路以及新的和替代镇痛药物的开发。TRPM 3是 也在胰腺β细胞中高度表达，并在葡萄糖诱导的胰岛素释放中具有应用。 此外，TRPM 3与发育性和癫痫性脑病有关，这是一种异质性的神经系统疾病。 一组以癫痫伴智力残疾为特征的疾病。目前，生理 TRPM 3的病理学特性仍然知之甚少。本提案的重点是确定 第一个TRPM 3结构在不同的功能状态在高分辨率，并了解分子 TRPM 3的结构、门控机制和药理学，通过结合单粒子冷冻电子 显微镜（cryo-EM）和膜片钳电生理学。我们还将启动TRPM 3的纯化， 使用识别TRPM 3的三维表位的单克隆抗体以及 敲入小鼠TRPM 3基因的亲和标签，因为越来越多的证据表明，从天然来源纯化的蛋白质 相对于蛋白质，来源可以显示不同的组装、复杂的组成和功能特性 重组系统。我们提供的强有力的初步数据将确保这一提议的成功， 该研究成果将为新型镇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.