John E. Linley

Neurological perspectives on voltage-gated sodium channels.

The activity of voltage-gated sodium channels has long been linked to disorders of neuronal excitability such as epilepsy and chronic pain. Recent genetic studies have now expanded the role of sodium channels in health and disease, to include autism, migraine, multiple sclerosis, cancer as well as muscle and immune system disorders. Transgenic mouse models have proved useful in understanding the physiological role of individual sodium channels, and there has been significant progress in the development of subtype selective inhibitors of sodium channels. This review will outline the functions and roles of specific sodium channels in electrical signalling and disease, focusing on neurological aspects. We also discuss recent advances in the development of selective sodium channel inhibitors.

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

Bradykinin (BK) is an inflammatory mediator and one of the most potent endogenous pain-inducing substances. When released at sites of tissue damage or inflammation, or applied exogenously, BK produces acute spontaneous pain and causes hyperalgesia (increased sensitivity to potentially painful stimuli). The mechanisms underlying spontaneous pain induced by BK are poorly understood. Here we report that in small nociceptive neurons from rat dorsal root ganglia, BK, acting through its B2 receptors, PLC, and release of calcium from intracellular stores, robustly inhibits M-type K+ channels and opens Ca2+-activated Cl- channels (CaCCs) encoded by Tmem16a (also known as Ano1). Summation of these two effects accounted for the depolarization and increase in AP firing induced by BK in DRG neurons. Local injection of inhibitors of CaCC and specific M-channel openers both strongly attenuated the nociceptive effect of local injections of BK in rats. These results provide a framework for understanding spontaneous inflammatory pain and may suggest new drug targets for treatment of such pain.

TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

Summary Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.

A role for Piezo2 in EPAC1-dependent mechanical allodynia

Aberrant mechanosensation has an important role in different pain states. Here we show that Epac1 (cyclic AMP sensor) potentiation of Piezo2-mediated mechanotransduction contributes to mechanical allodynia. Dorsal root ganglia Epac1 mRNA levels increase during neuropathic pain, and nerve damage-induced allodynia is reduced in Epac1−/− mice. The Epac-selective cAMP analogue 8-pCPT sensitizes mechanically evoked currents in sensory neurons. Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton. In vivo, 8-pCPT induces long-lasting allodynia that is prevented by the knockdown of Epac1 and attenuated by mouse Piezo2 knockdown. Piezo2 knockdown also enhanced thresholds for light touch. Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres. These data indicate that the Epac1–Piezo2 axis has a role in the development of mechanical allodynia during neuropathic pain.

Inhibition of M Current in Sensory Neurons by Exogenous Proteases: A Signaling Pathway Mediating Inflammatory Nociception

Inflammatory pain is thought to be mediated in part through the action of inflammatory mediators on membrane receptors of peripheral nerve terminals, however, the downstream signaling events which lead to pain are poorly understood. In this study we investigated the nociceptive pathways induced by activation of protease-activated receptor 2 (PAR-2) in damage-sensing (nociceptive) neurons from rat dorsal root ganglion (DRG). We found that activation of PAR-2 in these cells strongly inhibited M-type potassium currents (conducted by Kv7 potassium channels). Such inhibition caused depolarization of the neuronal resting membrane potential leading, ultimately, to nociception. Consistent with this mechanism, injection of the specific M channel blocker XE991 into rat paw induced nociception in a concentration-dependent manner. Injection of a PAR-2 agonist peptide also induced nociception but coinjection of XE991 and the PAR-2 agonist did not result in summation of nociception, suggesting that the action of both agents may share a similar mechanism. We also studied the signaling pathway of M current inhibition by PAR-2 using patch-clamp and fluorescence imaging of DRG neurons. These experiments revealed that the PAR-2 effect was mediated by phospholipase C (PLC). Further experiments demonstrated that M current inhibition required concurrent rises in cytosolic Ca2+ concentration and depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP2). We propose that PLC- and Ca2+/PIP2-mediated inhibition of M current in sensory neurons may represent one of the general mechanisms underlying pain produced by inflammatory mediators, and may therefore open up a new therapeutic window for treatment of this major clinical problem.

Understanding inflammatory pain: ion channels contributing to acute and chronic nociception.

Inflammatory pain results from the increased excitability of peripheral nociceptive sensory fibres produced by the action of inflammatory mediators. This excitatory effect, in turn, is a result of the altered activity of ion channels within affected sensory fibres. This review will consider the molecular consequences of inflammation within the peripheral nerves with particular focus on the effects of different inflammatory mediators on the ion channels in sensory neurons. We will discuss the main signalling pathways triggered in neurons by inflammatory mediators; the ionic mechanisms underlying inflammatory hyperalgesia and spontaneous inflammatory pain and finally will briefly consider ion channels underlying pain in chronic inflammation.

Reactive oxygen species are second messengers of neurokinin signaling in peripheral sensory neurons

Substance P (SP) is a prominent neuromodulator, which is produced and released by peripheral damage-sensing (nociceptive) neurons; these neurons also express SP receptors. However, the mechanisms of peripheral SP signaling are poorly understood. We report a signaling pathway of SP in nociceptive neurons: Acting predominantly through NK1 receptors and Gi/o proteins, SP stimulates increased release of reactive oxygen species from the mitochondrial electron transport chain. Reactive oxygen species, functioning as second messengers, induce oxidative modification and augment M-type potassium channels, thereby suppressing excitability. This signaling cascade requires activation of phospholipase C but is largely uncoupled from the inositol 1,4,5-trisphosphate sensitive Ca2+ stores. In rats SP causes sensitization of TRPV1 and produces thermal hyperalgesia. However, the lack of coupling between SP signaling and inositol 1,4,5-trisphosphate sensitive Ca2+ stores, together with the augmenting effect on M channels, renders the SP pathway ineffective to excite nociceptors acutely and produce spontaneous pain. Our study describes a mechanism for neurokinin signaling in sensory neurons and provides evidence that spontaneous pain and hyperalgesia can have distinct underlying mechanisms within a single nociceptive neuron.

Transcriptional Control of KCNQ Channel Genes and the Regulation of Neuronal Excitability

Regulation of the resting membrane potential and the repolarization of neurons are important in regulating neuronal excitability. The potassium channel subunits Kv7.2 and Kv7.3 play a key role in stabilizing neuronal activity. Mutations in KCNQ2 and KCNQ3, the genes encoding Kv7.2 and Kv7.3, cause a neonatal form of epilepsy, and activators of these channels have been identified as novel antiepileptics and analgesics. Despite the observations that regulation of these subunits has profound effects on neuronal function, almost nothing is known about the mechanisms responsible for controlling appropriate expression levels. Here we identify two mechanisms responsible for regulating KCNQ2 and KCNQ3 mRNA levels. We show that the transcription factor Sp1 activates expression of both KCNQ2 and KCNQ3, whereas the transcriptional repressor REST (repressor element 1-silencing transcription factor) represses expression of both of these genes. Furthermore, we show that transcriptional regulation of KCNQ genes is mirrored by the correlated changes in M-current density and excitability of native sensory neurons. We propose that these mechanisms are important in the control of excitability of neurons and may have implications in seizure activity and pain.

M channel enhancers and physiological M channel block.

Non‐technical summary  Specific membrane proteins called ‘M channels’ control the excitability of peripheral pain‐sensing nerves. In certain disease conditions (e.g. inflammation), M channels become inhibited, and this contributes to the increased excitability of these nerves and, ultimately, to pain sensation. Recently, chemical compounds that enhance M channel activity have been discovered and were suggested as prospective analgesics. However, it was previously unknown whether these M channel enhancers could augment the activity of M channels that are inhibited in inflammatory conditions. We tested four compounds that possess M channel enhancer activity in various conditions mimicking inflammation. Our conclusions suggest that while the overall effect of the enhancers is reduced when M channels are inhibited, the remaining enhancement is sufficient to ‘recover’ M channel activity from inflammation‐induced inhibition. Our results support pharmacological targeting of M channels in peripheral nerves as a strategy against inflammatory pain.

Altered cryptal expression of luminal potassium (BK) channels in ulcerative colitis.

Decreased sodium (Na+), chloride (Cl−), and water absorption, and increased potassium (K+) secretion, contribute to the pathogenesis of diarrhoea in ulcerative colitis. The cellular abnormalities underlying decreased Na+ and Cl− absorption are becoming clearer, but the mechanism of increased K+ secretion is unknown. Human colon is normally a K+ secretory epithelium, making it likely that K+ channels are expressed in the luminal (apical) membrane. Based on the assumption that these K+ channels resembled the high conductance luminal K+ (BK) channels previously identified in rat colon, we used molecular and patch clamp recording techniques to evaluate BK channel expression in normal and inflamed human colon, and the distribution and characteristics of these channels in normal colon. In normal colon, BK channel α‐subunit protein was immunolocalized to surface cells and upper crypt cells. By contrast, in ulcerative colitis, although BK channel α‐subunit protein expression was unchanged in surface cells, it extended along the entire crypt irrespective of whether the disease was active or quiescent. BK channel α‐subunit protein and mRNA expression (evaluated by western blotting and real‐time PCR, respectively) were similar in the normal ascending and sigmoid colon. Of the four possible β‐subunits (β1–4), the β1‐ and β3‐subunits were dominant. Voltage‐dependent, barium‐inhibitable, luminal K+ channels with a unitary conductance of 214 pS were identified at low abundance in the luminal membrane of surface cells around the openings of sigmoid colonic crypts. We conclude that increased faecal K+ losses in ulcerative colitis, and possibly other diseases associated with altered colonic K+ transport, may reflect wider expression of luminal BK channels along the crypt axis. Copyright