Probing G protein coupled receptor signaling networks in trigeminal nociception

Lead Research Organisation: University of Leeds
Department Name: Institute of Membrane & Systems Biology

Abstract

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.

Technical Summary

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.

Publications

10 25 50

publication icon
Elies J (2014) Hydrogen sulfide inhibits Cav3.2 T-type Ca2+ channels. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

publication icon
Linley JE (2010) Understanding inflammatory pain: ion channels contributing to acute and chronic nociception. in Pflugers Archiv : European journal of physiology

publication icon
Linley JE (2008) Inhibition of M current in sensory neurons by exogenous proteases: a signaling pathway mediating inflammatory nociception. in The Journal of neuroscience : the official journal of the Society for Neuroscience

publication icon
Linley JE (2012) M channel enhancers and physiological M channel block. in The Journal of physiology

 
Description China Partnering Award: Emerging Approaches to Intracellular Signaling
Amount £30,612 (GBP)
Funding ID BB/R02104X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 07/2022
 
Description International Joint Project
Amount £12,000 (GBP)
Funding ID JP080524 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2009 
End 09/2011
 
Description International Partnering Awards
Amount £30,000 (GBP)
Funding ID BB/R02104X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 06/2022
 
Description Project Grant
Amount £622,032 (GBP)
Funding ID BB/R02104X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2017 
End 09/2020
 
Description Project grant
Amount £652,223 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2011 
End 12/2014
 
Title Heterologous expression of genes in cultured sensory neurons 
Description We have developed a method for successful transfection of cultured mammalian sensory neurons 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2013 
Provided To Others? Yes  
Impact The chapter describing this method developed by us is part of the book Ion Channels. Methods and Protocols, 2nd edition (N Gamper, Editor) Humana Press 2013 
 
Title Robotic Patch-Clamp technique 
Description A method for robotic multiwell planar patch-clamp for native and primary mammalian cells has been published in journal Nature Protocols (PMID: 19197268). 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact These protocols developed by researchers at Leeds University become avilable for scientific comunity. 
URL http://europepmc.org/abstract/MED/19197268
 
Title pHluorin-delta-opioid-receptor 
Description A pH-sensitive pHluorin-DOR rpobe to study trafficking of delta-opioid receptors 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The publication is pending 
 
Description Ion channels in peripheral pain pathways 
Organisation Hebei Medical University
Department Pharmacology
Country China 
Sector Academic/University 
PI Contribution This collaboration is based on my appointment as an Ajunct Professor of Pharmacology at Hebei Medical University. I have 2 PhD students and Co-PI there and we are investigating role of M channels and other ion channels in pain
Collaborator Contribution Partner provides administrative and financial support for this activity
Impact This is a multidisciplinary collaboration which involves Medicinal Chemistry, Pharmacology, Biophysics and neuroscience. 1 Jin, X., Shah, S., Liu, Y., Zhang, H., Lees, M., Fu, Z., Lippiat, J.D., Beech, D.J., Sivaprasadarao, A., Baldwin, S.A., Zhang, H., Gamper, N. (2013) Activation of the Cl- Channel ANO1 by Localized Calcium Signals in Nociceptive Sensory Neurons Requires Coupling with the IP3 Receptor. Sci Signal. 6(290):ra73. 2 Linley, J.E., Pettinger, L., Huang, D., Gamper, N. (2011) M Channel Enhancers and Physiological M channel Block. J Physiol. 590, 793-807. 3 Liu, B., Linley, J.E., Du, X., Zhang, X., Ooi, L., Zhang, H., Gamper, N. (2010) The Acute Nociceptive Signals Induced by Bradykinin in Rat Sensory Neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl- channels. J. Clin. Invest. 120, 1240-52.
Start Year 2011
 
Description Optical methods for studying neuropeptide release from sensory neurons 
Organisation Peking University
Department Laboratory of Cellular Biophysics
Country China 
Sector Academic/University 
PI Contribution This collaboration was initiated by me.
Collaborator Contribution A new pH-sensitive, genetically-encoded optical probe for imaging neuropeptide release from sensory neurons has been developed. The probe is now in use in my laboratory.
Impact This is a collaboration with the research group in china (Peking University). We received a small funds from Leeds University to sponsor a 3-months visit of the PhD student from China to my lab. The visit was very successful and there is a publication being prepared.
Start Year 2009
 
Description Study of the mechanisms of perypheral sensory neuron excitability induced by bradykinin 
Organisation Hebei Medical University
Country China 
Sector Academic/University 
PI Contribution I am the initiator of this collaboration and my lab provided preliminary data and resources for the application for the Internationa Joint Project Grant to Royal Society and National Science Foundation of China
Collaborator Contribution This is a collaboration sponsored by Royal society and National Science Foundation of China. I'm expecting short-term visitors from the laboratory of my collaborators in China to undertake research in my lab.
Impact This collaboration resulted in a paper published in the Journal of Clinical Investigation (see the 'Publications' section).
Start Year 2008
 
Description Supperesolution imaging 
Organisation University of Texas
Department Health Science Center at San Antonio
Country United States 
Sector Academic/University 
PI Contribution I am providing the tissue samples and consumables
Collaborator Contribution Time allocated at their NIKON STORM microscope; a postdoc in UTHSCSA actually performs the imaging
Impact No outputs yet, a collaboration started in October 2014
Start Year 2014
 
Description Cafe Scientific Public lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact ~40 people (general public) attended an evening lecture on pain.

The event was very lively, the audience expressed high level of engagement; lots of interest and questions after the talk.
Year(s) Of Engagement Activity 2013
 
Description Press Release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release covering our publication in Proc Natl Acad Sci USA has been issued by the University of Leeds in May 2012.

The press release has been picked up by the media worldwide
Year(s) Of Engagement Activity 2012
 
Description Press release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact The press release about our discovery of the inflammatory pain mechanism has been issued by the University of Leeds; it has been picked up by the Yorkshire Post, Science Daily and by many medical on-line resources worldwide.

I have received direct enquiry from the general public (phone calls) about the our results and possible implications
Year(s) Of Engagement Activity 2010
 
Description Society for Neuroscience 2009's media materials 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Our latest findings on the role of voltage-gated K+ channels in the perypheral somatosensory system has been accepted into the Society for Neuroscience 2009's media materials and are included into the SFN "Hot Topics" book and online media room.

Public dissemination of our findings
Year(s) Of Engagement Activity 2009