Regulation of M-type K+ channel expression in sensory neurones as a novel mechanism contributing to chronic pain states

Lead Research Organisation: University of Leeds
Department Name: Research & Innovation Services

Abstract

Chronic pain constitutes an enormous health problem. It brings physical and psychological distress to the sufferers and their carers and puts significant pressure on the health care system. It is especially true because many types of chronic pains are particularly difficult to treat, most of the conventional analgesics are either ineffective or have serious side-effects. One of the common primary causes of many chronic pain conditions is an uncontrolled activity of specific peripheral nerves which normally are silent and only become active in response to potentially damaging stimulation. This pathological activity of damage-sensing nerves (peripheral sensitisation) may last for months or years (e.g. in arthritis or neuropathic pain) causing unrestrained pain. Unfortunately, despite of much effort, the mechanisms underlying peripheral sensitisation are poorly understood. Recent work in the laboratories of the applicants has lead up to the accumulation of a ?critical mass? of evidence towards the discovery of what may become one of the key molecular mechanism contributing to the chronic pain. Our findings revealed that nerve injury triggers long-lasing down-regulation of specific group of genes that encode family of proteins called ?M-type potassium channels? that control activity of peripheral nerves. Importantly, we identified another protein called ?REST?, which is able to specifically interact with and regulate expression of the M-type potassium channel genes. Our hypothesis (supported by our recently published and yet unpublished data) is that nerve injury or inflammation triggers the expression of REST which, in turn, suppresses the expression of M-type potassium channels in peripheral sensory nerves thus increasing their spontaneous activity. The aim of this proposal is to perform comprehensive and rigorous investigation of this novel mechanism. We will use known animal models for neuropathic pain in combination with an array of cutting edge genetic, molecular, biochemical and electrophysiological approaches to fully characterise the changes in the expression patterns of the M-type potassium channel genes induced by nerve injury. We will further probe the mechanism responsible for control of M-channel expression and function in sensory nerves with particular attention to the newly discovered role of REST. Our work has the potential to identify new targets and strategies that may be used to develop better medicines for the treatment and management of chronic pain.

Technical Summary

Excitation of damage-sensing (nociceptive) neurons underlies nociceptive sensory transmission which signals the occurrence or threat of body damage. This pathway is crucial for avoiding harm, however, with the nerve injury or inflammation, electrical activity of nociceptors often persists beyond the duration of the stimulus and causes persistent pain. We seek to investigate the physiological and genetic mechanisms underlying this increased excitability of nociceptive neurons (peripheral sensitization). Our preliminary experiments revealed dramatic down-regulation of the gene encoding a subunit of M-type potassium channel (KCNQ2) in dorsal root ganglia (DRG) neurons of rats developing a neuropathic pain condition. Our published data show that KCNQ2 expression can be suppressed by the transcription factor REST and as a consequence of the decrease in M channel expression the excitability of in DRG neurons is increased. We have shown the relevance of this mechanism in response to inflammatory insult; interestingly, our preliminary data suggest that REST is also up-regulated under neuropathic pain conditions. These findings put forward the hypothesis for a novel mechanism contributing to peripheral sensitization: transcriptional suppression of M channels. We seek to investigate this novel mechanism. The proposal has three specific aims. I) To characterise long-term changes in the expression of Kv7 channels in peripheral sensory neurons after nerve injury. We will provide a comprehensive characterization of the changes in the expression of individual M channel subunits in dorsal root ganglia (DRG) neurons following the experimentally-induced nerve injury. II) To determine the role of transcriptional repressor REST in the development of chronic pain. We will generate a transgenic mouse line with tamoxifen-inducible knock-out of REST specifically in sensory neurones (RESTloxP/loxP/AdvCreER-T2 mice); we will characterize the effects of REST knockdown in vivo on the development of neuropathic pain. III) To determine the contribution of the suppression of the KCNQ genes by REST to the development of peripheral sensitization. We will determine how the KCNQ-REST interaction contributes to the development of neuropathic pain; this will include an investigation of the contribution of M channel downregulation to the excitability of neuropathic DRG neurones and a study on the efficacy of M channel openers in neuropathic injury. To answer these questions we have developed a strategy incorporating genetic, biochemical, electrophysiological and behavioural methods. The results of this proposal will reveal a novel mechanism of peripheral sensitization, provide potential new targets for the pain management industry and generate new transgenic mice available for use in other pain-related research.

Publications

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Boycott HE (2013) Carbon monoxide inhibition of Cav3.2 T-type Ca2+ channels reveals tonic modulation by thioredoxin. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

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Gamper Nikita (Ed) (2013) Ion Channels. Methods and Protocols

 
Description Functional expression of M channel subunits in peripheral somatosensory system
Amount £130,431 (GBP)
Funding ID 1947288 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2021
 
Title Ganglionic slice recording 
Description Method for patch clamp recording from acute slices of dorsal root ganglion 
Type Of Material Technology assay or reagent 
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 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 Computational model of mammalian nociceptive neuron 
Description Computational model of mammalian nociceptive neurons for modelling of the action potential propagation through the t-junction. 
Type Of Material Computer model/algorithm 
Year Produced 2014 
Provided To Others? Yes  
Impact The model can be used for prediction of drug action in peripheral somatosensory system 
 
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 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