The rationale for the topical application of capsaicin and other vanilloids in the treatment of pain is that such compounds selectively excite and subsequently desensitize nociceptive neurons. This desensitization is triggered by the activation of vanilloid receptors (TRPV1), which leads to an elevation in intracellular free Ca2+ levels. Depending on the vanilloid concentration and duration of exposure, the Ca2+ influx via TRPV1 desensitizes the channels themselves, which may represent not only a feedback mechanism protecting the cell from toxic Ca2+ overload, but also likely contributes to the analgesic effects of capsaicin. This review summarizes the current state of knowledge concerning the mechanisms that underlie the acute capsaicin-induced Ca2+-dependent desensitization of TRPV1 channels and explores to what extent they may contribute to capsaicin-induced analgesia. In view of the polymodal nature of TRPV1, we illustrate how the channels behave in their desensitized state when activated by other stimuli such as noxious heat or depolarizing voltages. We also show that the desensitized channel can be strongly reactivated by capsaicin at concentrations higher than those previously used to desensitize it. We provide a possible explanation for a high incidence of adverse effects of topical capsaicin and point to a need for more accurate clinical criteria for employing it as a reliable remedy.
- MeSH
- analgetika aplikace a dávkování MeSH
- analgezie * MeSH
- aplikace lokální MeSH
- baryum metabolismus MeSH
- bolest farmakoterapie MeSH
- gating iontového kanálu účinky léků MeSH
- kalmodulin metabolismus MeSH
- kapsaicin aplikace a dávkování MeSH
- kationtové kanály TRPV fyziologie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- membránové potenciály fyziologie MeSH
- molekulární modely MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Agonist-induced desensitization of the transient receptor potential vanilloid receptor-1 (TRPV1) is one of the key strategies that offer a way to alleviate neuropathic and inflammatory pain. This process is initiated by TRPV1 receptor activation and the subsequent entry of extracellular Ca(2+) through the channel into sensory neurones. One of the prominent mechanisms responsible for TRPV1 desensitization is dephosphorylation of the TRPV1 protein by the Ca(2+)/calmodulin-dependent enzyme, phosphatase 2B (calcineurin). Of several consensus phosphorylation sites identified so far, the most notable are two sites for Ca(2+)/calmodulin dependent kinase II (CaMKII) at which the dynamic equilibrium between the phosphorylated and dephosphorylated states presumably regulates agonist binding. We examined the mechanisms of acute Ca(2+)-dependent desensitization using whole-cell patch-clamp techniques in human embryonic kidney (HEK) 293T cells expressing the wild type or CaMKII phosphorylation site mutants of rat TRPV1. The nonphosphorylatable mutant S502A/T704I was capsaicin-insensitive but the S502A/T704A construct was fully functional, indicating a requirement for a specific residue at position 704. A point mutation at the nearby conserved residue R701 strongly affected the heat, capsaicin and pH-evoked currents. As this residue constitutes a stringent CaMKII consensus site but is also predicted to be involved in the interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), these data suggest that in addition to dephosphorylation, or as its consequence, a short C-terminal juxtamembrane segment adjacent to the transient receptor potential box composed of R701 and T704 might be involved in the decelerated gating kinetics of the desensitized TRPV1 channel.
- MeSH
- elektrická stimulace metody MeSH
- financování organizované MeSH
- fosfatidylinositol-4,5-difosfát farmakologie MeSH
- kapsaicin farmakologie MeSH
- kationtové kanály TRPV fyziologie genetika MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- membránové potenciály MeSH
- metoda terčíkového zámku metody MeSH
- mutace fyziologie MeSH
- proteinkinasa závislá na vápníku a kalmodulinu typ 2 metabolismus MeSH
- teplota MeSH
- transfekce MeSH
- transformované buněčné linie MeSH
- vápník MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
We have previously reported that the reducing agent dithiothreitol (DTT) strongly increases thermally induced activity of the transient receptor potential vanilloid receptor-1 (TRPV1) channel. Here, we show that exposure to oxidizing agents also enhances the heat-induced activation of TRPV1. The actions of sulfhydryl modifiers on heat-evoked whole-cell membrane currents were examined in TRPV1-transfected human embryonic kidney 293T cells. The sensitizing effects of the membrane-permeable oxidizing agents diamide (1 mM), chloramine-T (1 mM), and the copper-o-complex (100:400 microM) were not reversed by washout, consistent with the stable nature of covalently modified sulfhydryl groups. In contrast, the membrane-impermeable cysteine-specific oxidant 5,5'-dithio-bis-(2-nitrobenzoic acid) (0.5 mM) was ineffective. The alkylating agent N-ethylmaleimide (1 mM) strongly and irreversibly affected heat-evoked responses in a manner that depended on DTT pretreatment. Extracellular application of the membrane-impermeable reducing agent glutathione (10 mM) mimicked the effects of 10 mM DTT in potentiating the heat-induced and voltage-induced membrane currents. Using site-directed mutagenesis, we identified Cys621 as the residue responsible for the extracellular modulation of TRPV1 by reducing agents. These data suggest that the vanilloid receptor is targeted by redox-active substances that directly modulate channel activity at sites located extracellularly as well as within the cytoplasmic domains. The results obtained demonstrate that an optimal redox state is crucial for the proper functioning of the TRPV1 channel and both its reduced and oxidized states can result in an increase in responsiveness to thermal stimuli.
- MeSH
- buněčné linie MeSH
- diamid farmakologie MeSH
- dithiothreitol farmakologie MeSH
- ethylmaleimid farmakologie MeSH
- financování organizované MeSH
- kapsaicin farmakologie MeSH
- kationtové kanály TRPV fyziologie genetika MeSH
- krysa rodu rattus MeSH
- kyselina dithionitrobenzoová farmakologie MeSH
- lidé MeSH
- membránové potenciály účinky léků MeSH
- metoda terčíkového zámku MeSH
- missense mutace genetika MeSH
- mutace genetika MeSH
- mutantní proteiny fyziologie genetika MeSH
- oxidancia farmakologie MeSH
- peroxid vodíku farmakologie MeSH
- redukční činidla farmakologie MeSH
- sulfhydrylová reagencia farmakologie MeSH
- transfekce MeSH
- vysoká teplota MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- MeSH
- analgezie MeSH
- bolest terapie MeSH
- kapsaicin terapeutické užití MeSH
- lidé MeSH
- nociceptory klasifikace MeSH
- Check Tag
- lidé MeSH
Gadolinium is a recognized blocker of many types of cation channels, including several channels of the transient receptor potential (TRP) superfamily. In this study, we demonstrate that Gd(3+), in addition to its blocking effects, activates and potentiates the recombinant vanilloid receptor TRPV1 expressed in HEK293T cells. Whole-cell currents through TRPV1 were induced by Gd(3+) with a half-maximal activation achieved at 72 microM at +40 mV. Gd(3+), at concentrations up to 100 microM, lowered the threshold for heat activation and potentiated the currents induced by capsaicin (1 microM) and low extracellular pH (6). Higher concentrations of Gd(3+) (>300 microM) blocked the TRPV1 channel. Neutralizations of the two acidic residues, Glu600 and Glu648, which are the key residues conferring the proton-sensitivity to TRPV1, resulted in a loss of Gd(3+)-induced activation and/or a reduction in its potentiating effects. A trivalent nonlanthanide, Al(3+), that possesses much a smaller atomic mass than Gd(3+) blocked but did not activate or sensitize the TRPV1 channel. These findings indicate that Gd(3+) activates and potentiates the TRPV1 by neutralizing two specific proton-sensitive sites on the extracellular side of the pore-forming loop.
- MeSH
- buněčné linie MeSH
- DNA primery MeSH
- elektrofyziologie metody MeSH
- financování organizované MeSH
- gadolinium farmakologie MeSH
- genetické vektory MeSH
- iontové kanály fyziologie genetika účinky léků MeSH
- kationtové kanály TRPV MeSH
- ledviny MeSH
- lidé MeSH
- mutageneze cílená MeSH
- sekvence nukleotidů MeSH
- substituce aminokyselin MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
Skupina TRP (transient receptor potential) iontových kanálů je rozsáhlou třídou membránových receptorů, z nichž mnohé jsou aktivovány podněty přicházejícími z okolního prostředí: světlem, tlakem, teplem či chladem. Nedávná molekulární identifikace teplotně aktivovaného iontového kanálu TRPV1, který se uplatňuje v přenosu bolestivých podnětů na primárních nociceptivních neuronech, vedla k prudkému zvýšení zájmu fyziologů o další iontové kanály této skupiny a díky rozsáhlým a cíleným genomickým projektům naznačila existenci obecných molekulárních principů senzorické transdukce. Současné poznatky ukazují, že TRP kanály zajišťují důležité biologické funkce jako teplotní, mechanické, chuťové i feromonové vnímání. Studium mechanizmů, kterými jsou tyto iontové kanály otvírány působením různých podnětů, směřuje k odhalení příčiny některých onemocnění nervového systému a je nutnou podmínkou pro vyhledávání nových látek se specifickým působením. Cílem tohoto příspěvku je informovat o současných poznatcích, které byly získány o struktuře a polymodální funkci TRP receptorů v nervové soustavě, a naznačit cesty dalšího výzkumu v oblasti studia mechanizmů senzorické transdukce na buněčné a molekulární úrovni.
The TRP (transient receptor potential) group of ion channels comprises a large subset of membrane receptors, many of them are activated by environmental stimuli such as light, pressure, heat or cold. Identification of the vanilloid receptor TRPV1, an ion channel that can be activated by temperatures over 43°C and plays an important role in transduction of noxious stimuli in primary sensory neurons, has attracted recent attention of physiologists to other members of this group of channels. Owing to recent extensive and aimed genomic projects, strong evidence had been gained for the existence of general molecular mechanisms of sensory transduction. It is supposed that TRP channels are responsible for important biological functions as temperature, mechanical, taste and pheromone sensation. Better understanding of the mechanisms involved in gating of these ion channels by different stimuli may lead to identification of the cause of some nervous system diseases and is a neccessary requirement for searching new compounds with specific effects. The aim of this report is to focus on recent new insights concerning the structure and polymodal function of TRP receptors in nervous system and to indicate ways of further research of the mechanisms involved in sensory transduction at the cellular and molecular level.
- MeSH
- finanční podpora výzkumu jako téma MeSH
- lidé MeSH
- nervové receptory klasifikace MeSH
- nociceptory klasifikace MeSH
- senzorická ganglia fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Ligandem aktivované iontové kanály tvoří skupinu transmembránových proteinových struktur, které hrají klíčovou úlohu při vzniku a přenosu elektrických signálů. Pro zjištění mechanizmů, které se podílejí na vazbě ligandů, změnách pH a vysoké teploty vedoucích ke strukturálním změnám iontových kanálů, bylo v nedávné době využito nových technik. Cílem tohoto přehledu je sumarizovat komplexní mechanizmy podílející se na aktivaci glutamátových (NMDA, AMPA, kainátových) a vaniloidních (TRPV1) receptorů spojených s příslušnými iontovými kanály a rovněž shrnout výsledky studií provedených v nedávné době v Oddělení buněčné neurofyziologie Fyziologického ústavu AV ČR (http://www2.biomed.cas.cz /d331/index.html), které je součástí Centra neuropsychiatrických studií; http://www.pcp.lf3.cuni.cz/cns/.
Ligand-gated ion channels form a family of transmembrane proteins that play key role in generation and propagation of electrical signals. Recently, new techniques have become available for identification of the mechanisms involved in ligand binding, pH changes and elevated temperatures into structural rearrangements leading to channel gating. The aim of this article is to review the complexity of the gating mechanisms involved in the activation of glutamate (NMDA, AMPA, kainate) and vanilloid (TRPV1) receptor channels and summarize the recent results from studies performed in the Department of Cellular Neurophysiology of the Institute of Physiology AS CR (http://www2.biomed.cas.cz/d331/index.html) a member of the Centre of Neuropsychiatric Studies http://www.pcp.lf3.cuni.cz/cns/.
- MeSH
- finanční podpora výzkumu jako téma MeSH
- iontové kanály analýza antagonisté a inhibitory fyziologie MeSH
- membránové proteiny fyziologie MeSH
- neurofyziologie MeSH
- neurony fyziologie MeSH
- receptory N-methyl-D-aspartátu fyziologie MeSH
- synapse fyziologie MeSH
- teplota MeSH
- Publikační typ
- přehledy MeSH
- Publikační typ
- abstrakt z konference MeSH
