Functional expression of M channel subunits in peripheral somatosensory system

Lead Research Organisation: University of Leeds
Department Name: School of Biomedical Sciences

Abstract

M-type K+ channels (KCNQ1-5) are non-inactivating K+ channels with a low activation threshold and slow kinetics, allowing them to control neuronal excitability. Loss of M channel function often leads to hyperexcitability disorders (e.g. epilepsy, deafness, pain). We and others have identified functional KCNQ subunits in 'pain' sensory neurons (nociceptors) and shown that 1) M channel inhibition in nociceptive nerves increases excitability and causes pain-like behavior in rats; 2) the inflammatory mediator bradykinin inhibits M channels in sensory neurons, an effect that contributes to inflammatory pain; 3) neuropathic injury causes downregulation of M channel subunit KCNQ2 in dorsal root ganglia (DRG) and spinal cord, and this contributes to neuropathic pain development.

M channel enhancers ('openers') produce analgesia which is largely mediated by their action within peripheral nerves. Accordingly, a number of analgesic drug discovery projects worldwide are focused on novel M channel openers. Yet, two issues hamper the progress in this area: a) various KCNQ subunits are expressed in the nervous and cardiovascular systems and other tissues, making broad-spectrum M channel openers prone to side effects; b) the subunit composition of M channels in peripheral nerves is not firmly established. Thus, in order to develop specific M-channel-targeting analgesics it is imperative to i) define the molecular identity of M channels in nociceptors; ii) develop subunit-selective M channel openers. The goal of this project is to comprehensively address point i); we have four specific aims.

Aim I. Correlative electrophysiological and transcriptome analysis of KCNQ channel expression in rat DRG neurons. We will electrophysiologically characterize DRG neurons for M current amplitude and kinetics and for sensory modality. After each recording, single DRG neurons will be collected and shipped to Lilly for single-cell transcriptome profiling. Successful pilot work has been already performed using rat (prepared in Gamper's lab) and human (from Anabios) DRG neurons in which electrophysiological responses to capsaicin in individual neurons were correlated with TRPV1 (and other DRG markers) transcript abundance.

Aim II. In situ analysis of KCNQ subunit expression in DRG. To circumvent possible neuronal remodelling in culture, distribution of KCNQ subunits will be studied using in situ hybridization and immunolabeling in DRG slices.

Aim III. Pharmacological profiling. We will use subunit-selective M channel openers (provided by Lilly) to seek pharmacological correlations with results obtained under Aims I-II.

Aim IV. We will test analgesic efficacy and side-effect severity (e.g. motor coordination, sedation) of selected selective M channel openers compared to a broad-spectrum opener, retigabine, using inflammatory/neuropathic pain models.

This project will help to rationalize the development of analgesics based on the mechanism of M current potentiation.

Publications

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Title Behavioural assessment 
Description Hypersensitivity testing was used for assessing changes in painful response. This was done using Hargreaves for thermal sensitivity Von Frey for mechanical sensitivity Nocifensive behaviour observations to assess acute pain. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? Yes  
Impact These tests have been used for many studies in the pain field 
 
Title Patch clamp electrophysiology 
Description Using electrodes to record electrical current from inside cells both endogenously and in cell lines. This allows measuring of both ion currents flowing through cells and of the changes in voltages of the cell in response to different currents. The technique allows us to measure changes in response to drug application too. 
Type Of Material Physiological assessment or outcome measure 
Provided To Others? Yes  
Impact All ion channel biology and most neuroscience and muscle physiology research uses some variation of this technique. 
 
Description Functional expression of M channel subunits in the peripheral somatosensory system 
Organisation Eli Lilly & Company Ltd
Country United Kingdom 
Sector Private 
PI Contribution We are completing in vitro experiments to characterise a confidential mouse line.
Collaborator Contribution completing experiments and providing animals
Impact Confidential
Start Year 2017
 
Description Poster at Kv7 symposium 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Symposium in Naples, dedicated to Kv7 channels. I presented a poster of my research here, won a n award and got to meet numerous giants in the field.
Year(s) Of Engagement Activity 2019
URL https://kv7channels2019naples.org