Modulation of thermo-TRP ion channel activity by phosphorylation and trafficking to the membrane

在大多数感觉系统中，恒定刺激的感知强度随着暴露时间的推移而降低。这个过程，被称为适应，是至关重要的，使动物能够在广泛的刺激强度存在于我们的自然环境。疼痛的感觉是不同的，然而/疼痛不会减少，而是随着时间的推移而增加，在一个称为敏化的过程中，这确保了潜在的破坏性刺激不会被忽视。虽然敏化对于生物体的生存至关重要，但它也负责增强慢性疾病的疼痛，如关节炎和背痛，其中疼痛是不可避免的，并且是个人痛苦和经济损失的主要原因。我们开始了解敏化过程的细胞和分子基础，这要归功于在单个疼痛敏感神经元（称为伤害感受器）水平及其负责感知疼痛刺激的分子组分水平上的工作。这项工作很重要，因为它满足了我们对疼痛的自然好奇心，这是一个我们都熟悉的基本而重要的过程。它也有潜在的好处，了解其他类似的信号通路，并提供基础知识，可用于未来的药物开发，以抑制疼痛。这项工作的主要目的是研究如何敏化增强热激活离子通道的功能。这些离子通道是更广泛的TRP家族的成员，在加热或冷却时打开，因此允许电流以离子的形式流入神经细胞和其他细胞。通过打开，这些离子通道改变细胞内的膜电压/例如，在神经细胞的情况下，名为TRPV 1的热敏离子通道的打开使内部电压变得更正，这引发了神经冲动，进而将热痛的感觉传递到更高的中心。以前的工作，大部分是在申请人实验室进行的，已经表明TRPV 1的敏化是由特定氨基酸的磷酸化引起的，并且有两种独立的机制在起作用：细胞膜中已经存在的离子通道对热的敏感性增强，更多的离子通道插入膜中。我们将更详细地研究这些过程的分子机制。哪些酶（称为激酶）进行磷酸化，TRPV 1上的哪些磷酸化位点对增强敏感性或促进通道插入很重要？支架蛋白AKAP 79似乎将两种这样的激酶保持在一个大分子复合物中，这提高了直接向TRPV 1发出信号的直接性。我们能否删除或拮抗这种分子，从而消除致敏作用？TRPV 1被运输到细胞膜并从细胞膜上去除的分子机制是什么？我们将在几个系统中研究这些问题：在人工表达TRPV 1等蛋白质的细胞中;在真实的伤害感受神经元中;以及在经过基因工程改造以表达改变水平的分子（如AKAP 79）的完整动物中。最后，TRPV 1的研究在多大程度上推广到其他热TRP？这项工作将提高我们的理解疼痛和其他感觉介导的热TRP家族成员的离子通道。这些信息将为开发新型镇痛药提供基础。从更广泛的意义上说，我们的研究将提高我们对离子通道如何进出细胞表面膜的理解，以及一旦它们在膜中，它们的敏感性是如何调节的。

In most sensory systems the perceived intensity of a constant stimulus decreases with time of exposure. This process, known as adaptation, is vital in enabling an animal to operate over the wide range of stimulus intensities present in our natural environment. The sensation of pain is different, however / pain does not decrease but instead increases with time, in a process known as sensitisation, which ensures that a potentially damaging stimulus is not ignored. While sensitisation is vital for the survival of the organism, it is also responsible for enhancing pain in chronic conditions such as arthritis and back pain, where the pain is inescapable, and is a major cause of personal suffering and economic loss. We are beginning to understand the cellular and molecular basis of the process of sensitisation, thanks to work at the level of single pain-sensitive neurons (called nociceptors) and at the level of their molecular components responsible for sensing painful stimuli. The work is important in terms of satisfying our natural curiosity about pain, a fundamental and important process with which we are all familiar. It also has potential benefits for the understanding of other similar signalling pathways, and to provide the underpinning knowledge which can be used in the future development of drugs to suppress pain. The main aim of this work will be to study how sensitisation enhances the function of thermally-activated ion channels. These ion channels, members of the wider TRP family, open in response to heating or cooling and so allow electrical current, in the form of ions, to flow into nerve cells and other cells. By opening, these ion channels alter the membrane voltage inside the cell / for instance, in the case of a nerve cell, opening of a heat-sensitive ion channel named TRPV1 makes the internal voltage more positive, and this initiates nerve impulses which in turn communicate the sensation of heat pain to higher centres. Previous work, much of it in the applicant lab, has shown that sensitisation of TRPV1 is caused by phosphorylation of particular amino acids, and that two separate mechanisms are at work: the sensitivity to heat of ion channels already in the cell membrane is enhanced, and more ion channels are inserted into the membrane. We will investigate the molecular mechanisms of these processes in more detail. Which enzymes (called kinases) carry out the phosphorylation, and which phosphorylated sites on TRPV1 are important for enhancing sensitivity or for promoting channel insertion? A scaffolding protein, AKAP79, seems to hold two such kinases into a macro-molecular complex, which improves the immediacy of signalling direct to TRPV1. Can we delete or antagonise this molecule and so abolish sensitisation? What is the molecular mechanism by which TRPV1 is trafficked to the membrane and removed from the membrane? We will investigate these questions in several systems: in cells where proteins such as TRPV1 have been artificially expressed; in real nociceptive neurons; and in intact animals genetically engineered to express altered levels of molecules such as AKAP79. And finally, how far do studies of TRPV1 generalise to the other thermo-TRPs? This work will enhance our understanding of pain and other sensations mediated by members of the thermo TRP family on ion channels. This information will provide the basis for the development of novel analgesics. In a wider sense our studies will enhance our understanding of how ion channels are trafficked to and from the cell surface membrane, and of how their sensitivity is modulated once they are in the membrane.