||Masami Kimura, Hiroaki Shiokawa, Yuji Karashima, Makoto Sumie, Sumio Hoka, Ken Yamaura, Antinociceptive effect of selective G protein-gated inwardly rectifying K+ channel agonist ML297 in the rat spinal cord, PloS one, 10.1371/journal.pone.0239094, 15, 9, e0239094, 2020.09, The G protein-gated inwardly rectifying K+ (GIRK) channels play important signaling roles in the central and peripheral nervous systems. However, the role of GIRK channel activation in pain signaling remains unknown mainly due to the lack of potent and selective GIRK channel activators until recently. The present study was designed to determine the effects and mechanisms of ML297, a selective GIRK1/2 activator, on nociception in the spinal cord by using behavioral studies and whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurons. Rats were prepared for chronic lumber catheterization and intrathecal administration of ML297. The nociceptive flexion reflex was tested using an analgesy-meter, and the influence on motor performance was assessed using an accelerating rotarod. We also investigated pre- and post-synaptic actions of ML297 in spinal cord preparations by whole-cell patch-clamp recordings. Intrathecal administration of ML297 increased the mechanical nociceptive threshold without impairing motor function. In voltage-clamp mode of patch-clamp recordings, bath application of ML297 induced outward currents in a dose-dependent manner. The ML297-induced currents demonstrated specific equilibrium potential like other families of potassium channels. At high concentration, ML297 depressed miniature excitatory postsynaptic currents (mEPSCs) but not their amplitude. The ML297-induced outward currents and suppression of mEPSCs were not inhibited by naloxone, a μ-opioid receptor antagonist. These results demonstrated that intrathecal ML297 showed the antinociceptive effect, which was mediated through direct activation of pre- and post-synaptic GIRK channels. Selective GIRK channel activation is a promising strategy for the development of new agents against chronic pain and opioid tolerance..
||Yuji Karashima, Jean Prenen, Karel Talavera, Annelies Janssens, Thomas Voets, Bernd Nilius, Agonist-induced changes in Ca2+ permeation through the nociceptor cation channel TRPA1, Biophysical Journal, 10.1016/j.bpj.2009.11.007, 98, 5, 773-783, 2010.03, The Ca2+-permeable cation channel TRPA1 acts as an ionotropic receptor for various pungent compounds and as a noxious cold sensor in sensory neurons. It is unclear what proportion of the TRPA1-mediated current is carried by Ca2+ ions and how the permeation pathway changes during stimulation. Here, based on the relative permeability of the nonstimulated channel to cations of different size, we estimated a pore diameter of ∼11 Å. Combined patch-clamp and Fura-2 fluorescence recordings revealed that with 2 mM extracellular Ca2+, and at a membrane potential of -80 mV, ∼17% of the inward TRPA1 current is carried by Ca2+. Stimulation with mustard oil evoked an apparent dilatation of the pore of 3 Å and an increase in divalent cation selectivity and fractional Ca2+ current. Mutations in the putative pore that reduced the divalent permeability and fractional Ca2+ current also prevented mustard-oil-induced increases in Ca2+ permeation. It is interesting that fractional Ca2+ currents for wild-type and mutant TRPA1 were consistently higher than values predicted based on biionic reversal potentials using the Goldman-Hodgkin-Katz equation, suggesting that binding of Ca2+ in the pore hinders monovalent cation permeation. We conclude that the pore of TRPA1 is dynamic and supports a surprisingly large Ca2+ influx..
||Karel Talavera, Maarten Gees, Yuji Karashima, Víctor M. Meseguer, Jeroen A.J. Vanoirbeek, Nils Damann, Wouter Everaerts, Melissa Benoit, Annelies Janssens, Rudi Vennekens, Félix Viana, Benoit Nemery, Bernd Nilius, Thomas Voets, Nicotine activates the chemosensory cation channel TRPA1, Nature Neuroscience, 10.1038/nn.2379, 12, 10, 1293-1299, 2009.10, Topical application of nicotine, as used in nicotine replacement therapies, causes irritation of the mucosa and skin. This reaction has been attributed to activation of nicotinic acetylcholine receptors (nAChRs) in chemosensory neurons. In contrast with this view, we found that the chemosensory cation channel transient receptor potential A1 (TRPA1) is crucially involved in nicotine-induced irritation. We found that micromolar concentrations of nicotine activated heterologously expressed mouse and human TRPA1. Nicotine acted in a membrane-delimited manner, stabilizing the open state(s) and destabilizing the closed state(s) of the channel. In the presence of the general nAChR blocker hexamethonium, nociceptive neurons showed nicotine-induced responses that were strongly reduced in TRPA1-deficient mice. Finally, TRPA1 mediated the mouse airway constriction reflex to nasal instillation of nicotine. The identification of TRPA1 as a nicotine target suggests that existing models of nicotine-induced irritation should be revised and may facilitate the development of smoking cessation therapies with less adverse effects..
||Yuji Karashima, Karel Talavera, Wouter Everaerts, Annelies Janssens, Kelvin Y. Kwan, Rudi Vennekens, Bernd Nilius, Thomas Voets, TRPA1 acts as a cold sensor in vitro and in vivo, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0808487106, 106, 4, 1273-1278, 2009.01, TRPA1 functions as an excitatory ionotropic receptor in sensory neurons. It was originally described as a noxious cold-activated channel, but its cold sensitivity has been disputed in later studies, and the contribution of TRPA1 to thermosensing is currently a matter of strong debate. Here, we provide several lines of evidence to establish that TRPA1 acts as a cold sensor in vitro and in vivo. First, we demonstrate that heterologously expressed TRPA1 is activated by cold in a Ca2+-independent and Ca2+ store-independent manner; temperature-dependent gating of TRPA1 is mechanistically analogous to that of other temperature-sensitive TRP channels, and it is preserved after treatment with the TRPA1 agonist mustard oil. Second, we identify and characterize a specific subset of cold-sensitive trigeminal ganglion neurons that is absent in TRPA1-deficient mice. Finally, cold plate and tail-flick experiments reveal TRPA1-dependent, cold-induced nociceptive behavior in mice. We conclude that TRPA1 acts as a major sensor for noxious cold..
||Yuji Karashima, Jean Prenen, Victor Meseguer, Grzegorz Owsianik, Thomas Voets, Bernd Nilius, Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators, Pflugers Archiv European Journal of Physiology, 10.1007/s00424-008-0493-6, 457, 1, 77-89, 2008.10, The transient receptor potential channel of the ankyrin-binding repeat subfamily, TRPA1, is a Ca2+-permeable non-selective cation channel that depolarizes the plasma membrane and causes Ca2+ influx. A typical feature of TRPA1 is its rapid desensitization following activation by agonists such as mustard oil (MO), cinnamaldehyde, and a high intracellular Ca2+ concentration. In whole-cell recordings on Chinese hamster ovary (CHO) cells expressing TRPA1, desensitization was delayed when phosphatidylinositol 4,5-biphosphate (PIP2) was supplemented via the patch pipette, whereas the PIP2 scavenger neomycin accelerated desensitization. Preincubation with the PI-4 kinase inhibitor wortmannin reduced both constitutive TRPA1 channels activity and the response to MO. Run down was also accelerated by high intracellular Mg2+ concentrations, whereas chelating intracellular Mg2+ with 10 mM ethylenedinitrilotetraacetic acid (EDTA) increased the basal channel activity. In inside-out patches, we observed a rapid run down of TRPA1 activity, which could be prevented by application of diC8-PIP2 or 2 mM Mg-ATP but not Na2-ATP to the cytosolic side of the excised patches. In isolated trigeminal ganglion neurons, preincubation with wortmannin resulted in inhibition of endogenous TRPA1 activation by MO. Taken together, our data indicate that PIP2 modulates TRPA1, albeit to a lesser extent than other known PIP 2-dependent TRP channels, and that tools modifying the plasma membrane PIP2 content often have direct effects on this channel..
||Victor Meseguer, Yuji Karashima, Karel Talavera, Dieter D'Hoedt, Tansy Donovan-Rodríguez, Felix Viana, Bernd Nilius, Thomas Voets, Transient receptor potential channels in sensory neurons are targets of the antimycotic agent clotrimazole, Journal of Neuroscience, 10.1523/JNEUROSCI.4772-07.2008, 28, 3, 576-586, 2008.01, Clotrimazole (CLT) is a widely used drug for the topical treatment of yeast infections of skin, vagina, and mouth. Common side effects of topical CLT application include irritation and burning pain of the skin and mucous membranes. Here, we provide evidence that transient receptor potential (TRP) channels in primary sensory neurons underlie these unwanted effects of CLT. We found that clinically relevant CLT concentrations activate heterologously expressed TRPV1 and TRPA1, two TRP channels that act as receptors of irritant chemical and/or thermal stimuli in nociceptive neurons. In line herewith, CLT stimulated a subset of capsaicin-sensitive and mustard oil-sensitive trigeminal neurons, and evoked nocifensive behavior and thermal hypersensitivity with intraplantar injection in mice. Notably, CLT-induced pain behavior was suppressed by the TRPV1-antagonist BCTC [(N-(-4-tertiarybutylphenyl)-4-(3- cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide)] and absent in TRPV1-deficient mice. In addition, CLT inhibited the cold and menthol receptor TRPM8, and blocked menthol-induced responses in capsaicin- and mustard oil-insensitive trigeminal neurons. The concentration for 50% inhibition (IC50) of inward TRPM8 current was ∼200 nM, making CLT the most potent known TRPM8 antagonist and a useful tool to discriminate between TRPM8- and TRPA1-mediated responses. Together, our results identify TRP channels in sensory neurons as molecular targets of CLT, and offer means to develop novel CLT preparations with fewer unwanted sensory side effects..
||Yuji Karashima, Nils Damann, Jean Prenen, Karel Talavera, Andrei Segal, Thomas Voets, Bernd Nilius, Bimodal action of menthol on the transient receptor potential channel TRPA1, Journal of Neuroscience, 10.1523/JNEUROSCI.2221-07.2007, 27, 37, 9874-9884, 2007.09, TRPA1 is a calcium-permeable nonselective cation transient receptor potential (TRP) channel that functions as an excitatory ionotropic receptor in nociceptive neurons. TRPA1 is robustly activated by pungent substances in mustard oil, cinnamon, and garlic and mediates the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate a bimodal sensitivity of TRPA1 to menthol, a widely used cooling agent and known activator of the related cold receptor TRPM8. In whole-cell and single-channel recordings of heterologously expressed TRPA1, submicromolar to low-micromolar concentrations of menthol cause channel activation, whereas higher concentrations lead to a reversible channel block. In addition, we provide evidence for TRPA1-mediated menthol responses in mustard oil-sensitive trigeminal ganglion neurons. Our data indicate that TRPA1 is a highly sensitive menthol receptor that very likely contributes to the diverse psychophysical sensations after topical application of menthol to the skin or mucous membranes of the oral and nasal cavities..
||Yuji Karashima, Masahiro Oike, Shosuke Takahashi, Yushi Ito, Propofol prevents endothelial dysfunction induced by glucose overload, British Journal of Pharmacology, 10.1038/sj.bjp.0704912, 137, 5, 683-691, 2002.11, 1. Surgical operations often induce acute hyperglycemia, which is known to affect endothelial functions. In this study, we examined the effects of propofol, a commonly used general anaesthetic, on bovine aortic endothelial cell (BAEC) dysfunction induced by glucose overload. 2. D-glucose overload (23 mM) induced an accumulation of superoxide anion (O2-), assessed by MCLA chemiluminescence, to a similar extent as that generated by 233 μU ml-1 xanthine oxidase (XO) and 100 μM xanthine. Propofol inhibited this accumulation with an IC50 of 0.21 μM, whereas much higher concentrations of propofol were required to scavenge O2- generated by 250 μU ml-1 XO and 100 μM xanthine (IC50: 13.5 μM). 3. D-glucose overload attenuated ATP-induced NO production which was detected using diaminofluorescence-2 (DAF-2). The inhibition was reversed by propofol with an EC50 of 0.60 μM. In contrast, inhibitions caused by xanthine/XO were not altered by propofol (1 μM). 4. D-glucose overload suppressed ATP-induced Ca2+ oscillations and capacitative Ca2+ entry (CCE), which were both restored by superoxide dismutase, indicating that O2- was responsible. Propofol restored these attenuated Ca2+ oscillations and CCE with EC50 of 0.31 and 1.0 μM, respectively. 5. D-glucose overload (23 mM) increased the intracellular glucose concentration 4 fold, compared with cells exposed to 5.75 mM glucose, and 1 μM propofol reduced this increase to 2.8 fold. 6. We conclude from these results that anaesthetic concentrations of propofol prevent the impairment of Ca2+-dependent NO production in BAEC induced by glucose overload. This effect is mainly due to the reduction of O2- accumulation, and involves, at least in part, the inhibition of cellular glucose uptake..