Role of HCN channels in somatic sensation and pain

神经细胞沿其轴突的长度通过动作电位或跨细胞膜电压的瞬时逆转（去极化）进行通信。当感觉刺激冲击到皮肤表面时，为了被检测到，它必须在支配体表接触点的感觉神经或神经元中引起动作电位。如果刺激是持续的，那么它通常会引发一系列动作电位，其频率编码刺激的强度，频率越高表明刺激越强烈。在一个动作电位序列中，膜电位会返回到之前的负水平（再极化），为了引起下一个动作电位序列，膜电压必须再次去极化到阈值。因此，调节动作电位去极化速率的过程是决定动作电位频率的关键因素，因此也决定了刺激的感知强度。这一比率的一个重要决定因素是刺激力度，但它不是唯一的决定因素。在许多感觉神经元中，重极化打开一个向内电流，然后帮助去极化达到下一个动作电位的阈值。这个电流，超极化激活的内向电流，简称Ih，是本次拨款申请的主题。有趣的是，它可以被许多促进疼痛感觉的介质增强，因此它可能在痛觉过敏，或损伤后的疼痛增强，以及神经性疼痛中很重要，神经性疼痛是一种异常的疼痛状态，其特征是持续的疼痛和对中度触觉和热刺激的过敏。神经性疼痛尚未得到很好的理解，目前可用的药物治疗效果也很差。这种情况通常是终生的，并导致患者的生活质量大幅下降。氢离子通道由四种不同亚基HCN1-4的不同组合组成。在此应用之前的初步证据表明，这些亚基的表达存在分离，快速的HCN1亚基在感应光触摸的大神经元中表达，而较慢的HCN2亚基在感应各种疼痛刺激的小神经元中表达。为什么会这样？一个可能的原因是HCN2被各种已知的增强疼痛的介质增强，而HCN1不受影响。因此，这种分离至少可以部分解释损伤后疼痛增加的原因。我们的目标是更多地了解哪些亚基在哪些类型的感觉神经元中表达，以及它们的行为如何被炎症介质调节。其他实验室的工作也有证据表明，Ih与神经性疼痛有关，但哪个亚基在这里是重要的，以及它如何增强神经性疼痛尚不清楚。我们将通过使用每个HCN亚基被基因删除（敲除）的小鼠来解决这些问题和其他问题。我们将使用一系列技术来研究这些老鼠，并将它们与野生型的同伴进行比较。一项主要的技术将是记录神经元的电反应，无论是在细胞培养中，它们的行为更容易被研究，还是在皮肤中分离的神经元原位制备中，其优点是神经元处于自然环境中。此外，我们将研究野生型和HCN基因敲除小鼠对轻度疼痛刺激的反应行为，当疼痛开始时，它们不会退缩。这些研究将促进我们对HCN亚基在疼痛中的作用的理解，如果特定的亚基在疼痛的某些方面（例如神经性疼痛）具有关键作用，那么这项工作将刺激针对特异性阻断这些亚基的新型药物的开发。

Nerve cells communicate along the length of their axons by means of action potentials, or transient reversals (depolarisations) of the voltage across the cell membrane. When a sensory stimulus impinges on the skin surface it must, in order to be detected, elicit action potentials in the sensory nerve, or neurone, innervating the body surface at the point of contact. If the stimulus is sustained then it will often elicit a train of action potentials whose frequency encodes the intensity of the stimulus, with higher frequencies signalling a more intense stimulus. Each action potential in a train is followed by a return of the membrane potential to its former negative level (a repolarisation), and in order to elicit the next action potential in the train the membrane voltage must be depolarised again to threshold. The processes which mediate the rate of this depolarisation between action potentials is thus a crucial determinant of the action potential frequency and therefore of the perceived intensity of the stimulus. One important determinant of this rate is the strength of the stimulus, but it is not the only one. In many sensory neurones repolarisation switches on an inward current which then aids the depolarisation to threshold of the next action potential. This current, the hyperpolarisation-activated inward current, or Ih, is the subject of this grant application. Ih is interesting because it can be enhanced by many mediators which promote a sensation of pain, and it therefore may be important in hyperalgesia, or the enhanced pain which follows injury, and in neuropathic pain, an anomalous pain state characterised by ongoing pain and hypersensitivity to even moderate tactile and thermal stimuli. Neuropathic pain is not well understood and is poorly treated by currently available drugs. The condition is often life-long and causes a substantial reduction in quality of life for those who suffer from it. Ih ion channels are made up from various combinations of four different subunits, HCN1-4. Preliminary evidence leading up to this application has shown that there is a segregation in expression of these subunits, with the fast HCN1 subunit expressed in large neurones sensing light touch, and the slower HCN2 in small neurones, most of which sense painful stimuli of various kinds. Why is this? A possible reason is that HCN2 is enhanced by various mediators known to enhance pain, while HCN1 is unaffected. Thus this segregation may provide at least a partial explanation for the increase in pain following injury. We aim to find out more about which subunits are expressed in which types of sensory neurones, and how their behaviour is modulated by inflammatory mediators. There is also evidence from work in other labs that Ih is involved in neuropathic pain, but which subunit is important here and how it enhances neuropathic pain is unknown. We will tackle these and other questions by the use of mice in which each HCN subunits has been genetically deleted (knocked out). We will use a range of techniques to study these mice and to compare them with their wild-type littermates. One major technique will be to record the electrical responses from neurones, both in cell culture, where their behaviour can more readily be investigated, and in an isolated preparation of neurones in situ in skin, which has the advantage that the neurones are in their natural environment. In addition, we will study the behaviour of wild-type and HCN knockout mice in response to a mild painful stimulus, from which they are free with withdraw when it begins to hurt. These studies will advance our understanding of the role of HCN subunits in pain, and if particular subunits have crucial roles in some aspects of pain (e.g. in neuropathic pain) the work will act as a stimulus to the development of novel drugs aimed at specifically blocking those subunits.