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.

Technical Abstract:
The membrane ion channel TRPV1 is activated by heat, low pH and capsaicin (the active extract of chilli peppers), and is important in the detection of pain caused by heat or by acid produced in ischaemia or inflammation. Six putative members of the thermo TRP family of ion channels have now been identified, covering the physiologically relevant range of temperatures from painful cold to noxious heat. Activation of TRPV1 is potentiated by a number of pro-inflammatory factors, amongst them bradykinin and nerve growth factor (NGF), thus causing sensitisation and enhancing pain. We have found in recent studies that the scaffolding protein AKAP79 is critical in promoting phosphorylation, and hence sensitisation, by maintaining the serine/threonine kinases PKA and PKC in a macromolecular complex with TRPV1. Recent evidence also shows that two separate mechanisms cause sensitisation of TRPV1: potentiation of activation of channels in the surface membrane by stimuli such as heat or capsaicin; and trafficking of channels to the membrane from an intracellular vesicular pool. The aim of the study will be to learn more about the molecular mechanisms of sensitisation of TRPV1, and to extend our findings to other members of the thermo TRP family of ion channels. We will work on expression systems, on intact neurons and, in order to demonstrate the in vivo relevance of our findings, we will repeat certain critical experiments on genetically modified animals. We will delineate binding sites for AKAP79 on TRPV1 and other thermo TRP channels and will examine the effect on sensitisation of antagonising binding, or of deleting AKAP79 in an intact animal. We will examine how phosphorylation causes sensitization of thermo TRPs and how it promotes trafficking to the membrane. Finally, we will seek to understand the molecular mechanisms controlling insertion of thermo TRPs into the membrane, and of retrieval from it by endocytosis.