Functional expression of M channel subunits in peripheral somatosensory system

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.