HCN ion channels and pain

疼痛是生存的关键，正如少数没有痛感的人的身体损伤和短暂的生命所表明的那样。然而，疼痛也有不利的一面，因为慢性疼痛会严重限制患者的行动能力和生活质量。疼痛可分为三种模式：急性疼痛，由初始损伤引起；炎症性疼痛，组织损伤释放炎症介质，增强痛感；神经病理性疼痛，由直接神经损伤引起。急性疼痛对于保护我们免受伤害是必不可少的。炎症性疼痛在保护受损区域免受进一步伤害方面是有益的，但当慢性疼痛时，它可能会导致生活质量的大幅下降。神经性疼痛没有明显的保护功能，对那些遭受这种疼痛的人来说是一种严重的障碍。这也是最鲜为人知的疼痛模式，也是最难治疗的。申请实验室最近的工作发现，表达在伤害性(疼痛敏感)神经元表面膜上的单个离子通道HCN_2控制着它们对炎症介质反应的动作电位的放电速率。在伤害性感受器的子集中选择性地基因缺失HCN2可以消除炎症性疼痛中的热敏感性，也可以消除神经病理性疼痛。这项工作首次表明，两种形式的疼痛都是由一个单一的分子实体构成的，此外，它还通过开发选择性的HCN受体阻滞剂，为疼痛的新治疗开辟了可能性。这是一个特别受欢迎的发展，因为目前所有常用的止痛药都有主要的副作用。本申请旨在通过扩大我们对HCN参与疼痛的科学理解来建立在这一进展的基础上。我们已经确定了一组特定的伤害性感受器，其中HCN2的表达对疼痛至关重要。我们用来实现基因缺失的方法告诉我们，这些神经元必须表达电压依赖的钠通道NaV1.8。这些神经元的其他电生理学和组织学特性是什么使它们对疼痛至关重要？我们将对这些神经元进行基因标记，并将它们分离出来进行进一步研究。在接下来的两条调查路线中，我们将调查哪些炎性介质对调节hcn2至关重要。我们之前的工作已经确定前列腺素E2就是这样一种介质，但我们有强烈的迹象表明，其他介质也很重要，特别是对神经病理性疼痛。我们将在分离的神经元中测试候选介体，并阐明它们的细胞内信号通路。然后，我们将在活体实验中测试这些介体或它们的信号通路的抑制剂。这项工作可能会为通过攻击hcn2的调制而不是直接阻断通道来控制疼痛打开治疗的可能性。第四个领域将研究hcn2在神经元中的表达及其在神经元膜上的表达如何通过生长因子家族的炎症介质(如NGF和GDNF)以更长期的方式进行调节。有证据表明，这些因子是在神经损伤后释放的，它们可能在神经病理性疼痛的长期维持中起重要作用。上述工作将为我们理解HCN在疼痛中的作用奠定坚实的基础。HCN离子通道家族的另外两个成员，HCN3和HCN4，仍有待研究(我们小组之前的工作已经排除了HCN1是疼痛的主要贡献者)。在项目的最后阶段，我们将构建这两个离子通道基因缺失的动物，并将检查缺失对疼痛的影响。这项工作将以直截了当的方式告诉我们，这两个渠道中的任何一个在炎症性疼痛或神经病理性疼痛中都发挥着重要作用。

Pain is critical for survival, as shown by the bodily damage and short lives of the rare humans who lack a sensation of pain. Pain also has a downside, however, because chronic pain causes severe limitations in mobility and quality of life of those who suffer from it. Three modes of pain can be distinguished: acute pain, caused by an initial injury; inflammatory pain, in which tissue damage releases inflammatory mediators which enhance the sensation of pain; and neuropathic pain, caused by direct nerve damage. Acute pain is essential to protect us from injury. Inflammatory pain can be beneficial in protecting damaged areas from further harm, but when chronic it can cause a major reduction in quality of life. Neuropathic pain serves no obvious protective function and is a severe handicap for those who suffer from it. It is also the most poorly understood mode of pain, and the most difficult to treat.Recent work in the applicant laboratory has found that a single ion channel, HCN2, which is expressed in the surface membranes of nociceptive (pain-sensitive) neurons, controls their rate of firing of action potentials in response to inflammatory mediators. Selective genetic deletion of HCN2 in a subset of nociceptors abolishes the heat sensitivity seen in inflammatory pain, and also abolishes neuropathic pain. This work showed for the first time that a single molecular entity underlies both forms of pain, and moreover it opens up possibilities for new treatments of pain by the development of selective blockers of HCN2. This last will be a particularly welcome development, as all current analgesics in common use have major side effects.The present application aims to build on this advance by extending our scientific understanding of the involvement of HCN2 in pain. We have identified a particular population of nociceptors where the expression of HCN2 is critical for pain. The method that we used to achieve the genetic deletion tells us that these neurons must express a voltage-dependent sodium channel, NaV1.8. What are the other electrophysiological and histological properties of these neurons that make them critical for pain? We will mark these neurons genetically and will isolate them for further study. In two further lines of enquiry we will investigate which inflammatory mediators are crucial for modulating HCN2. Our previous work has identified prostaglandin E2 as one such mediator, but we have strong indications that others are important, in particular for neuropathic pain. We will test candidate mediators in isolated neurons, and we will elucidate their intracellular signalling pathways. We will then test inhibitors of these mediators, or of their signalling pathways, in in vivo experiments. This work may open up therapeutic possibilities for controlling pain by attacking the modulation of HCN2 rather than directly blocking the channel.A fourth area will investigate how the expression of HCN2 in neurons, and its expression in the neuronal membrane itself, may be modulated in a more long-term way by inflammatory mediators of the growth factor family, such as NGF and GDNF. There is evidence that these factors are released following nerve injury, and they may be important in the long-term maintenance of neuropathic pain.The work outlined above will put our understanding of the role of HCN2 in pain ona firm basis. There are two other members of the HCN ion channel family, HCN3 and HCN4, which remain to be investigated (previous work in our group has already ruled out HCN1 as a major contributor to pain). In the final phase of the project we will construct animals with genetic deletions of these two ion channels and will examine the effect of the deletion on pain. This work will tell us in a straightforward fashion whether either channel plays an important role in either inflammatory or neuropathic pain.