Damage-sensing nociceptors in the skin provide an indispensable protective function thanks to their specialized ability to detect and transmit hot temperatures that would block or inflict irreversible damage in other mammalian neurons. Here we show that the exceptional capacity of skin C-fiber nociceptors to encode noxiously hot temperatures depends on two tetrodotoxin (TTX)-resistant sodium channel α-subunits: NaV1.8 and NaV1.9. We demonstrate that NaV1.9, which is commonly considered an amplifier of subthreshold depolarizations at 20°C, undergoes a large gain of function when temperatures rise to the pain threshold. We also show that this gain of function renders NaV1.9 capable of generating action potentials with a clear inflection point and positive overshoot. In the skin, heat-resistant nociceptors appear as two distinct types with unique and possibly specialized features: one is blocked by TTX and relies on NaV1.9, and the second type is insensitive to TTX and composed of both NaV1.8 and NaV1.9. Independent of rapidly gated TTX-sensitive NaV channels that form the action potential at pain threshold, NaV1.8 is required in all heat-resistant nociceptors to encode temperatures higher than ∼46°C, whereas NaV1.9 is crucial for shaping the action potential upstroke and keeping the NaV1.8 voltage threshold within reach.
- MeSH
- akční potenciály MeSH
- buněčné linie MeSH
- kůže MeSH
- metoda terčíkového zámku MeSH
- molekulární evoluce MeSH
- myši inbrední C57BL MeSH
- napětově řízený sodíkový kanál typ 10 metabolismus MeSH
- napětově řízený sodíkový kanál typ 11 metabolismus MeSH
- nociceptory metabolismus MeSH
- práh bolesti MeSH
- techniky in vitro MeSH
- vysoká teplota * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ciguatoxins (CTXs) are marine toxins that cause ciguatera fish poisoning, a debilitating disease dominated by sensory and neurological disturbances that include cold allodynia and various painful symptoms as well as long-lasting pruritus. Although CTXs are known as the most potent mammalian sodium channel activator toxins, the etiology of many of its neurosensory symptoms remains unresolved. We recently described that local application of 1 nM Pacific Ciguatoxin-1 (P-CTX-1) into the skin of human subjects induces a long-lasting, painful axon reflex flare and that CTXs are particularly effective in releasing calcitonin-gene related peptide (CGRP) from nerve terminals. In this study, we used mouse and rat skin preparations and enzyme-linked immunosorbent assays (ELISA) to study the molecular mechanism by which P-CTX-1 induces CGRP release. We show that P-CTX-1 induces CGRP release more effectively in mouse as compared to rat skin, exhibiting EC50 concentrations in the low nanomolar range. P-CTX-1-induced CGRP release from skin is dependent on extracellular calcium and sodium, but independent from the activation of various thermosensory transient receptor potential (TRP) ion channels. In contrast, lidocaine and tetrodotoxin (TTX) reduce CGRP release by 53-75%, with the remaining fraction involving L-type and T-type voltage-gated calcium channels (VGCC). Using transgenic mice, we revealed that the TTX-resistant voltage-gated sodium channel (VGSC) NaV1.9, but not NaV1.8 or NaV1.7 alone and the combined activation of the TTX-sensitive VGSC subtypes NaV1.7 and NaV1.1 carry the largest part of the P-CTX-1-caused CGRP release of 42% and 34%, respectively. Given the contribution of CGRP to nociceptive and itch sensing pathways, our findings contribute to a better understanding of sensory symptoms of acute and chronic ciguatera that may help in the identification of potential therapeutics.
- MeSH
- ciguatera metabolismus MeSH
- ciguatoxiny chemie farmakologie MeSH
- ELISA MeSH
- hyperalgezie chemicky indukované MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- lidokain farmakologie MeSH
- membránové potenciály účinky léků MeSH
- mořské toxiny farmakologie MeSH
- myši transgenní MeSH
- myši MeSH
- napětím řízený sodíkový kanál, typ 1 účinky léků MeSH
- napětově řízený sodíkový kanál typ 11 účinky léků MeSH
- napěťově řízený sodíkový kanál, typ 9 účinky léků MeSH
- peptid spojený s genem pro kalcitonin účinky léků MeSH
- receptory peptidu se vztahem ke genu kalcitoninu účinky léků MeSH
- tetrodotoxin farmakologie MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Crotalphine is a structural analogue to a novel analgesic peptide that was first identified in the crude venom from the South American rattlesnake Crotalus durissus terrificus. Although crotalphine's analgesic effect is well established, its direct mechanism of action remains unresolved. The aim of the present study was to investigate the effect of crotalphine on ion channels in peripheral pain pathways. We found that picomolar concentrations of crotalphine selectively activate heterologously expressed and native TRPA1 ion channels. TRPA1 activation by crotalphine required intact N-terminal cysteine residues and was followed by strong and long-lasting desensitization of the channel. Homologous desensitization of recombinant TRPA1 and heterologous desensitization in cultured dorsal root ganglia neurons was observed. Likewise, crotalphine acted on peptidergic TRPA1-expressing nerve endings ex vivo as demonstrated by suppression of calcitonin gene-related peptide release from the trachea and in vivo by inhibition of chemically induced and inflammatory hypersensitivity in mice. The crotalphine-mediated desensitizing effect was abolished by the TRPA1 blocker HC030031 and absent in TRPA1-deficient mice. Taken together, these results suggest that crotalphine is the first peptide to mediate antinociception selectively and at subnanomolar concentrations by targeting TRPA1 ion channels.
- MeSH
- akční potenciály účinky léků MeSH
- analgetika farmakologie terapeutické užití MeSH
- bradykinin toxicita MeSH
- HEK293 buňky MeSH
- hyperalgezie chemicky indukované farmakoterapie etiologie MeSH
- kationtové kanály TRP antagonisté a inhibitory genetika metabolismus MeSH
- kationtový kanál TRPA1 MeSH
- kultivované buňky MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- mutace genetika MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- neurony účinky léků metabolismus MeSH
- peptidy farmakologie terapeutické užití MeSH
- spinální ganglia cytologie MeSH
- vápník metabolismus MeSH
- zánět komplikace MeSH
- zvířata MeSH
- zymosan toxicita MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Currently available behavioral assays to quantify normal cold sensitivity, cold hypersensitivity and cold hyperalgesia in mice have betimes created conflicting results in the literature. Some only capture a limited spectrum of thermal experiences, others are prone to experimenter bias or are not sensitive enough to detect the contribution of ion channels to cold sensing because in mice smaller alterations in cold nociception do not manifest as frank behavioral changes. To overcome current limitations we have designed a novel device that is automated, provides a high degree of freedom, i.e. thermal choice, and eliminates experimenter bias. The device represents a thermal gradient assay designed as a circular running track. It allows discerning exploratory behavior from thermal selection behavior and provides increased accuracy by providing measured values in duplicate and by removing edge artifacts. Our custom-designed automated offline analysis by a blob detection algorithm is devoid of movement artifacts, removes light reflection artifacts and provides an internal quality control parameter which we validated. The assay delivers discrete information on a large range of parameters extracted from the occupancy of thermally defined zones such as preference temperature and skew of the distribution. We demonstrate that the assay allows increasingly accurate phenotyping of thermal sensitivity in transgenic mice by disclosing yet unrecognized details on the phenotypes of TRPM8-, TRPA1- and TRPM8/A1-deficient mice.
- Publikační typ
- časopisecké články MeSH
Gain-of-function (GOF) mutations in ion channels are rare events, which lead to increased agonist sensitivity or altered gating properties, and may render the channel constitutively active. Uncovering and following characterization of such mutants contribute substantially to the understanding of the molecular basis of ion channel functioning. Here we give an overview of some GOF mutants in polymodal ion channels specifically involved in transduction of painful stimuli--TRPV1 and TRPA1, which are scrutinized by scientists due to their important role in development of some pathological pain states. Remarkably, a substitution of single amino acid in the S4-S5 region of TRPA1 (N855S) has been recently associated with familial episodic pain syndrome. This mutation increases chemical sensitivity of TRPA1, but leaves the voltage sensitivity unchanged. On the other hand, mutations in the analogous region of TRPV1 (R557K and G563S) severely affect all aspects of channel activation and lead to spontaneous activity. Comparison of the effects induced by mutations in homologous positions in different TRP receptors (or more generally in other distantly related ion channels) may elucidate the gating mechanisms conserved during evolution.
- MeSH
- bolest patofyziologie MeSH
- kationtové kanály TRP chemie genetika metabolismus MeSH
- kationtové kanály TRPV chemie genetika metabolismus MeSH
- lidé MeSH
- mícha patofyziologie MeSH
- mutace genetika MeSH
- percepce bolesti MeSH
- proteiny nervové tkáně chemie genetika metabolismus MeSH
- vápníkové kanály chemie genetika metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Topically applied camphor elicits a sensation of cool, but nothing is known about how it affects cold temperature sensing. We found that camphor sensitizes a subpopulation of menthol-sensitive native cutaneous nociceptors in the mouse to cold, but desensitizes and partially blocks heterologously expressed TRPM8 (transient receptor potential cation channel subfamily M member 8). In contrast, camphor reduces potassium outward currents in cultured sensory neurons and, in cold nociceptors, the cold-sensitizing effects of camphor and menthol are additive. Using a membrane potential dye-based screening assay and heterologously expressed potassium channels, we found that the effects of camphor are mediated by inhibition of Kv7.2/3 channels subtypes that generate the M-current in neurons. In line with this finding, the specific M-current blocker XE991 reproduced the cold-sensitizing effect of camphor in nociceptors. However, the M-channel blocking effects of XE991 and camphor are not sufficient to initiate cold transduction but require a cold-activated inward current generated by TRPM8. The cold-sensitizing effects of XE991 and camphor are largest in high-threshold cold nociceptors. Low-threshold corneal cold thermoreceptors that express high levels of TRPM8 and lack potassium channels are not affected by camphor. We also found that menthol--like camphor--potently inhibits Kv7.2/3 channels. The apparent functional synergism arising from TRPM8 activation and M-current block can improve the effectiveness of topical coolants and cooling lotions, and may also enhance TRPM8-mediated analgesia.
- MeSH
- kafr farmakologie MeSH
- kationtové kanály TRPM genetika metabolismus MeSH
- menthol farmakologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nervová vlákna nemyelinizovaná účinky léků metabolismus MeSH
- nízká teplota MeSH
- nociceptory metabolismus fyziologie MeSH
- signální transdukce účinky léků fyziologie MeSH
- spinální ganglia účinky léků metabolismus MeSH
- termoreceptory metabolismus fyziologie MeSH
- vnímání teploty účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The recent discovery that camphor activates and strongly desensitizes the capsaicin-sensitive and noxious heat-sensitive channel transient receptor potential vanilloid subfamily member 1 (TRPV1) has provided new insights and opened up new research paths toward understanding why this naturally occurring monoterpene is widely used in human medicine for its local counter-irritant, antipruritic, and anesthetic properties. However, the molecular basis for camphor sensitivity remains mostly unknown. The authors attempt to explore the nature of the activation pathways evoked by camphor and narrow down a putative interaction site at TRPV1. METHODS: The authors transiently expressed wild-type or specifically mutated recombinant TRPV1 channels in human embryonic kidney cells HEK293T and recorded cation currents with the whole cell, patch clamp technique. To monitor changes in the spatial distribution of phosphatidylinositol 4,5-bisphosphate, they used fluorescence resonance energy transfer measurements from cells transfected with the fluorescent protein-tagged pleckstrin homology domains of phospholipase C. RESULTS: The results revealed that camphor modulates TRPV1 channel through the outer pore helix domain by affecting its overall gating equilibrium. In addition, camphor, which generally is known to decrease the fluidity of cell plasma membranes, may also regulate the activity of TRPV1 by inducing changes in the spatial distribution of phosphatidylinositol-4,5-bisphosphate on the inner leaflet of the plasma membrane. CONCLUSIONS: The findings of this study provide novel insights into the structural basis for the modulation of TRPV1 channel by camphor and may provide an explanation for the mechanism by which camphor modulates thermal sensation in vivo.
- MeSH
- gating iontového kanálu účinky léků fyziologie MeSH
- HEK293 buňky MeSH
- jaderný pór účinky léků metabolismus MeSH
- kafr chemie farmakologie MeSH
- kationtové kanály TRPV antagonisté a inhibitory chemie fyziologie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- terciární struktura proteinů účinky léků fyziologie MeSH
- vazebná místa účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Capsaicin and other vanilloids selectively excite and subsequently desensitize pain-conducting nerve fibers (nociceptors) and this process contributes to the analgesic (and thus therapeutically relevant) effects of these compounds. Such a desensitization process is triggered by the activation of the transient receptor potential vanilloid subtype 1 receptor channels (TRPV1) that open their cationic pores, permeable to sodium, potassium and calcium (Ca(2+)) ions. Depending on the duration of capsaicin exposure and the external calcium concentration, the Ca(2+) influx via TRPV1 channels desensitizes the channels themselves, which, from the cellular point of view, represents a feedback mechanism protecting the nociceptive neuron from toxic Ca(2+) overload. The 'acute desensitization' accounts for most of the reduction in responsiveness occurring within the first few (~20) seconds after the vanilloids are administered to the cell for the first time. Another form of desensitization is 'tachyphylaxis', which is a reduction in the response to repeated applications of vanilloid. The wealth of pathways following TRPV1 activation that lead to increased intracellular Ca(2+) levels and both forms of desensitization is huge and they might utilise just about every known type of signalling molecule. This review will not attempt to cover all historical aspects of research into all these processes. Instead, it will try to highlight some new challenging thoughts on the important phenomenon of TRPV1 desensitization and will focus on the putative mechanisms that are thought to account for the acute phase of this process.
- MeSH
- analgetika metabolismus farmakologie MeSH
- fosfolipasa C fosfoinositidové signalizace metabolismus MeSH
- fosforylace MeSH
- kapsaicin metabolismus farmakologie MeSH
- kationtové kanály TRPV agonisté metabolismus MeSH
- lidé MeSH
- nociceptory metabolismus MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Ankyrinový receptor typu 1 (TRPAl) je iontový kanál, jehož funkce se uplatňuje v přenosu bolestivých podnětů na primárních nociceptivních neuronech. Tento z nichž mnohé zprostředkovávají senzorické vjemy přicházející z okolního prostředí: algogenní, pálivé nebo chladivé látky nacházející se v různých rostlinách, bolestivé teplo nebo chlad. TRPAl kanál lze aktivovat řadou dráždivých látek, organosulfátů, obsažených v hořčici, křenu, česneku nebo cibuli, dráždivými aldehydy nacházejícími se ve výfukových a slzných plynech nebo v cigaretovém kouři. Tento iontový kanál je ale také )římo aktivován chladem, mediatory zánětu, endogenními produkty oxidativního stresu a překvapivě i psychoaktivními látkami, jako jsou fytokanabinoidy. Cílem tohoto příspěvku je informovat o současných poznatcích týkajících se strukturálních, funkčních a farmakologických vlastností TRPAl receptoru, zejména z hlediska možného fyziologického významu a případných souvislostí s periferními mechanizmy nocicepce, které byly v uplynulých letech studovány na oddělení buněčné neurofyziologie Fyziologického ústavu AV CR, v.v.i. (http.7/www2.biomed.cas. cz/d331/index.htm).
The ankyrin transient receptor potential subtype 1 (TRPA1) is an ion channel that is highly expressed in primary sensory neuron s with noci- ceptive properties. This channel belongs to a diverse collection of membrane proteins: transient receptor potential (TRP) chann el superfamily, some members of which mediate a variety of sensory qualities that are clearly related to pain: stinging, pricking, warmth, burn ing or cold. The TRPA1 channel can be activated by some organosulfur compounds contained in mustard oil, horse radish or garlic, environmental i rritants contained in air polution, cigarette smoke or tear gas, by cold, inflammatory mediators and, interestingly, also by some psycho active drugs such as phytocannabinoids. The purpose of this article is to give an overview of the pharmacological, structural and functional prop erties of the TRPA1 receptor with a focus on its potential physiological role in peripheral mechanisms of nociception that have been studied at the Department of Cellular Neurophysiology of the Institute of Physiology AS CR, v.v.i. (http://www2.biomed.cas.cz/d331/index_eng.htm).
- Klíčová slova
- ankyrinový receptor, chlad, termo-TRP receptory, ganglia zadních kořenů míšních, senzorická transdukce,
- MeSH
- bolest MeSH
- financování organizované MeSH
- gating iontového kanálu fyziologie MeSH
- hyperalgezie metabolismus MeSH
- kanabinoidy farmakologie metabolismus MeSH
- kationtové kanály TRP agonisté antagonisté a inhibitory fyziologie MeSH
- modely u zvířat MeSH
- neurony metabolismus MeSH
- nocicepce MeSH
- nociceptory fyziologie metabolismus účinky léků MeSH
- psychotropní léky farmakologie metabolismus MeSH
The past decade has seen a progressive unraveling of the molecular identities of receptors implicated in transduction of painful stimuli. Among the line-up of important ion channels involved in peripheral pain pathways, the family of the transient receptor potential (TRP) ion channels has attracted considerable attention and research work as a target class for drug discovery. One of these channels, the ankyrin transient receptor potential TRPA1, which expression is restricted to nociceptive neurons of peripheral ganglia, can be activated by isothiocyanates, pungent products from mustard oil and horse radish, phytocannabinoids, nicotin, environmental irritants contained in air polution, cigarette smoke and tear gas, but also by inflammatory mediators and endogenous products of oxidative stress. The purpose of this paper is to give an overview of the pharmacological, structural and functional properties of the TRPA1 channel in mammalian sensory system and to summarize recent evidence regarding its key properties that can be exploited for potential therapeutic advantage.
- Klíčová slova
- ankyrinový receptor, chlad, enzorická transdukce,
- MeSH
- ankyriny fyziologie genetika MeSH
- bolest MeSH
- financování organizované MeSH
- hyperalgezie etiologie farmakoterapie MeSH
- kationtové kanály TRP fyziologie chemie MeSH
- nocicepce MeSH
- nociceptory fyziologie účinky léků MeSH
- receptory Notch fyziologie chemie metabolismus MeSH
- Publikační typ
- přehledy MeSH
