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 G i/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 Ca 2+ 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 Ca 2+ 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.G protein coupled receptors | inflammatory pain | intracellular signaling | KCNQ | M current E xcitation of peripheral terminals of sensory neurons is a primary event in somatosensation, including pain. A large proportion of sensory neurons (mostly TRPV1 + damage-sensing or "nociceptive" neurons) produce neuropeptides such as substance P (SP), which are released in response to nociceptive stimulation both at spinal cord synapses and in the periphery (1). In the spinal cord SP acts as an excitatory neurotransmitter or cotransmitter (2, 3), whereas peripheral SP release is thought to underlie the inflammatory response known as "neurogenic inflammation" (4). Many peripheral nociceptors also express receptors for SP [neurokinin receptors (NKR) 1-3 (NK1-3)]; this expression may suggest paracrine or autocrine actions of this peptide. However, the nature of such actions is controversial, and the molecular events triggered by NKR in peripheral nociceptors are not well established. The NKR traditionally are classed as G q/11 -coupled G protein-coupled receptors (GPCR) that activate phospholipase C (PLC), with subsequent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and release of inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG) (5, 6). Common downstream steps of G q/11 signaling include IP 3 -mediated release of Ca 2+ from intracellular stores and DAG-mediated activation of PKC. However, it was hitherto unknown whether NKR in peripheral sensory neurons couple fully to this signaling cascade, because a lack of coupling between NKR and Ca 2+ release in dorsal root ganglia (DRG) neurons has been noted (ref. 7 and 8, but cf. ref. 9). Nevertheless, it is accepted that peripherally act...

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.Abstract M-type (Kv7, KCNQ) K + channels control the resting membrane potential of many neurons, including peripheral nociceptive sensory neurons. Several M channel enhancers were suggested as prospective analgesics, and targeting M channels specifically in peripheral nociceptors is a plausible strategy for peripheral analgesia. However, receptor-induced inhibition of M channels in nociceptors is often observed in inflammation and may contribute to inflammatory pain. Such inhibition is predominantly mediated by phospholipase C. We investigated four M channel enhancers (retigabine, flupirtine, zinc pyrithione and H 2 O 2 ) for their ability to overcome M channel inhibition via two phospholipase C-mediated mechanisms, namely depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and a rise in intracellular Ca 2+ (an action mediated by calmodulin). Data from overexpressed Kv7.2/Kv7.3 heteromers and native M currents in dorsal root ganglion neurons suggest the following conclusions. (i) All enhancers had a dual effect on M channel activity, a negative shift in voltage dependence and an increase of the maximal current at saturating voltages. The enhancers differed in their efficacy to produce these effects. (ii) Both PIP 2 depletion and Ca 2+ /calmodulin strongly reduced the M current amplitude; however, at voltages near the threshold for M channel activation (−60 mV) all enhancers were able to restore M channel activity to a control level or above, while at saturating voltages the effects were more variable. (iii) Receptor-mediated inhibition of M current in nociceptive dorsal root ganglion neurons did not reduce the efficacy of retigabine or flupirtine to hyperpolarize the resting membrane potential. In conclusion, we show that all four M channel enhancers tested could overcome both PIP 2 and Ca 2+ -calmodulin-induced inhibition of Kv7.2/7.3 at voltages close to the threshold for action potential firing (−60 mV) but generally had reduced efficacy at a saturating voltag...

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T-type Ca(2+) channels play diverse roles in tissues such as sensory neurons, vascular smooth muscle, and cancers, where increased expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1) is often found. Here, we report regulation of T-type Ca(2+) channels by carbon monoxide (CO) a HO-1 by-product. CO (applied as CORM-2) caused a concentration-dependent, poorly reversible inhibition of all T-type channel isoforms (Cav3.1-3.3, IC50 ∼3 μM) expressed in HEK293 cells, and native T-type channels in NG108-15 cells and primary rat sensory neurons. No recognized CO-sensitive signaling pathway could account for the CO inhibition of Cav3.2. Instead, CO sensitivity was mediated by an extracellular redox-sensitive site, which was also highly sensitive to thioredoxin (Trx). Trx depletion (using auranofin, 2-5 μM) reduced Cav3.2 currents and their CO sensitivity by >50% but increased sensitivity to dithiothreitol ∼3-fold. By contrast, Cav3.1 and Cav3.3 channels, and their sensitivity to CO, were unaffected in identical experiments. Our data propose a novel signaling pathway in which Trx acts as a tonic, endogenous regulator of Cav3.2 channels, while HO-1-derived CO disrupts this regulation, causing channel inhibition. CO modulation of T-type channels has widespread implications for diverse physiological and pathophysiological mechanisms, such as excitability, contractility, and proliferation.

We have identified a new signaling role for nitric oxide (NO) in neurons from the trigeminal ganglia (TG). We show that in rat sensory neurons from the TG the NO donor, S-nitroso-N-acetyl-DL-penicillamine, inhibited M-current. This inhibitory effect was blocked by NO scavenging, while inhibition of NO synthases increased M-current, suggesting that tonic NO levels inhibit M-current in TG neurons. Moreover NO increased neuronal excitability and calcitonin gene-related peptide (CGRP) release and these effects could be prevented by perturbing M-channel function. First, NO-induced depolarization was prevented by pre-application of the M-channel blocker XE991 and second, NO-induced increase in CGRP release was prevented by incubation with the M-channel opener retigabine. We investigated the mechanism of the effects of NO on M-channels and identified a site of action of NO to be the redox modulatory site at the triplet of cysteines within the cytosolic linker between transmembrane domains 2 and 3, which is also a site of oxidative modification of M-channels by reactive oxygen species (ROS). NO and oxidative modifications have opposing effects on M-current, suggesting that a tightly controlled local redox and NO environment will exert fine control over M-channel activity and thus neuronal excitability. Together our data have identified a dynamic redox sensor within neuronal M-channels, which mediates reciprocal regulation of channel activity by NO and ROS. This sensor may play an important role in mediating excitatory effects of NO in such trigeminal disorders as headache and migraine.