Probing G protein coupled receptor signaling networks in trigeminal nociception

Two of the most common causes of pain are toothache and headache, but scientists don?t really understand how nerves collect and communicate ?painful? information from the mouth, face and brain. If they did, it would open the door to new methods for pain relief. Our research is centered on the question of how we sense tooth- and headache, how this sensation is altered in inflammation and how can we stop such pains. To tackle these intricate questions we are going to use an array of cutting-edge technological tools and approaches available through Leeds University and through our collaborators at the University of Texas Health Science Center at San Antonio which hosts one of the best dental schools in USA. These tools will include electrical recordings from living nerve cells, imaging (to look deep inside cells) and molecular biology. For example, we will use a latest generation of an instrument called an ?optical fluorescent microscope? (the Europe?s first machine of this kind is now being assembled in our lab at Leeds) which can see in such fine detail that we will be able to make videos of the individual molecules that transmit pain to the brain. Ultimately this work should lead to brand new ways to understand and treat pains which not only cause incredible discomfort, but cost the Health Services and society billions of pounds.

In our group we are strong advocates of Public Engagement in Science. The general public are incredibly interested in biomedical research (not least because they pay for it), and we have to get more involved in communicating our basic research to non-scientific audience. As an ongoing activity, we have recently obtained a collaborative Wellcome ?People Award? with the Glasgow-based artist Jim Pattison to develop an exhibition to show how signalling proteins in nerve cells work, and how technological changes have altered the way how we represent these. We also plan to have a lab ?Open Day? for schoolchildren and interested parents once a year; we envisage that they will be extremely interested in seeing cutting edge technology applied to pain research. Our institution considers public engagement highly important, and we have been shortlisted for the Beacons of Public Engagement , the new #8m initiative oh HEFCE, Research Councils and the Wellcome Trust, which aims to promote excellence in public engagement.

We seek to obtain a mechanistic understanding of the networking and cross-talk between G protein-coupled receptors (GPCR) and their associated voltage-gated ion channels in trigeminal (TG) nociceptors and subsequently test this framework in a behavioral model. Pain in the TG system is of particular importance because it includes both tooth and headache pain (such as migraine). We focus on two pivotal ion channels, which are principal players in determining neuronal output: voltage gated Ca2+ channels (VGCC) and M-type K+ channels (KCNQ, Kv7). We will test how these ion channels are regulated by G protein-coupled inflammatory mediators and opioid receptors, how this regulation affects excitability and neuropeptide release from TG nociceptors and, finally, what is the role this receptor-channel network in pain signalling. There are three specific aims. 1) To characterize the Gq/11-coupled receptor profile of TG sensory neurons. We will use an optical probe for phospholipase C (PLC) activation (the main output of Gq or G11-coupled receptor triggering) to detect real-time functional responses of TG neurons to different Gq/11-coupled receptor agonists. The imaging data will be complemented by immunocytochemistry. 2) To probe the interaction between Gq/11 and Go/i-coupled signaling systems in TG: does stimulation of Gq/11-coupled receptors affect modulation of VGCC by delta-opioid receptors (DOR)? Do DORs activate PLC and modulate M current? In patch clamp experiments we will test if Gq/11-coupled inflammatory mediators (e.g. bradykinin) facilitate DOR-induced modulation of VGCC by induction of trafficking and enhancement of DOR receptors. 3) To establish the functional link between GPCR modulation of ion channels and pain. We will study effects of modulation of VGCC and M channels on excitability (current clamp) and nociceptive peptide release (confocal and TIRF imaging) from TG neurons. Importantly, we will take our experiments to the whole animal by testing effects of ion channel modulation on nocifensive behavior in rats using a novel pain model. Overall, we believe that the synthesis of these powerful and complementary approaches will help us to reveal the complex signaling network that links inflammatory agents, GPCRs and ion channels in regulating pain transmission.