Alterations in the excitability of dorsal root ganglion (DRG) neurons are critical in the pathogenesis of acute and chronic pain. Neurotransmitter release from the terminals of DRG neurons is regulated by cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1), both activated by anandamide (AEA). In our experiments, the AEA precursor N-arachidonoylphosphatidylethanolamine (20:4-NAPE) was used to study the modulation of nociceptive DRG neurons excitability using K+-evoked Ca2+ transients. Intrathecal administration was used to evaluate in vivo effects. Application of 20:4-NAPE at lower concentrations (10 nM - 1 µM) decreased the excitability of DRG neurons, whereas the higher (10 µM) increased it. Both effects of 20:4-NAPE were blocked by the N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor LEI-401. Similarly, lower concentrations of externally applied AEA (1 nM - 10 nM) inhibited DRG neurons, whereas higher concentration (100 nM) did not change it. High AEA concentration (10 µM) evoked Ca2+ transients dependent on TRPV1 activation in separate experiments. Inhibition of the CB1 receptor by PF514273 (400 nM) prevented the 20:4-NAPE- and AEA-induced inhibition, whereas TRPV1 inhibition by SB366791 (1 µM) prevented the increased DRG neuron excitability. In behavioral tests, lower 20:4-NAPE concentration caused hyposensitivity, while higher evoked mechanical allodynia. Intrathecal LEI-401 prevented both in vivo effects of 20:4-NAPE. These results highlight anti- and pro-nociceptive effects of 20:4-NAPE mediated by CB1 and TRPV1 in concentration-dependent manner. Our study underscores the complexity of endocannabinoid signaling in pain transmission modulation and highlights 20:4-NAPE as a potential therapeutic target, offering new insights for developing analgesic strategies.

• Klíčová slova
• 20:4-NAPE, Anandamide, CB1, DRG neurons, NAPE-PLD, TRPV1,
• MeSH
• endokanabinoidy farmakologie   metabolismus   MeSH
• fosfatidylethanolaminy * farmakologie   MeSH
• fosfolipasa D * metabolismus   antagonisté a inhibitory   MeSH
• kationtové kanály TRPV metabolismus   MeSH
• krysa rodu Rattus MeSH
• kyseliny arachidonové * farmakologie   MeSH
• neurony * účinky léků   metabolismus   MeSH
• polynenasycené alkamidy farmakologie   MeSH
• potkani Sprague-Dawley MeSH
• receptor kanabinoidní CB1 metabolismus   MeSH
• spinální ganglia * účinky léků   metabolismus   cytologie   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• anandamide MeSH Prohlížeč
• endokanabinoidy MeSH
• fosfatidylethanolaminy * MeSH
• fosfolipasa D * MeSH
• kationtové kanály TRPV MeSH
• kyseliny arachidonové * MeSH
• polynenasycené alkamidy MeSH
• receptor kanabinoidní CB1 MeSH
• Trpv1 protein, rat MeSH Prohlížeč
• vápník MeSH

TRPV2 is a ligand-operated temperature sensor with poorly defined pharmacology. Here, we combine calcium imaging and patch-clamp electrophysiology with cryo-electron microscopy (cryo-EM) to explore how TRPV2 activity is modulated by the phytocannabinoid Δ9-tetrahydrocannabiorcol (C16) and by probenecid. C16 and probenecid act in concert to stimulate TRPV2 responses including histamine release from rat and human mast cells. Each ligand causes distinct conformational changes in TRPV2 as revealed by cryo-EM. Although the binding for probenecid remains elusive, C16 associates within the vanilloid pocket. As such, the C16 binding location is distinct from that of cannabidiol, partially overlapping with the binding site of the TRPV2 inhibitor piperlongumine. Taken together, we discover a new cannabinoid binding site in TRPV2 that is under the influence of allosteric control by probenecid. This molecular insight into ligand modulation enhances our understanding of TRPV2 in normal and pathophysiology.

• MeSH
• elektronová kryomikroskopie MeSH
• kanabidiol * farmakologie   MeSH
• kanabinoidy * farmakologie   MeSH
• kationtové kanály TRPV metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• ligandy MeSH
• probenecid farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• kanabidiol * MeSH
• kanabinoidy * MeSH
• kationtové kanály TRPV MeSH
• ligandy MeSH
• probenecid MeSH
• TRPV2 protein, human MeSH Prohlížeč
• Trpv2 protein, rat MeSH Prohlížeč

TRP channels sense temperatures ranging from noxious cold to noxious heat. Whether specialized TRP thermosensor modules exist and how they control channel pore gating is unknown. We studied purified human TRPA1 (hTRPA1) truncated proteins to gain insight into the temperature gating of hTRPA1. In patch-clamp bilayer recordings, ∆1-688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas ∆1-854 hTRPA1, also lacking the S1-S4 voltage sensing-like domain (VSLD), gained sensitivity to cold but lost its heat sensitivity. In hTRPA1 intrinsic tryptophan fluorescence studies, cold and heat evoked rearrangement of VSLD and the C-terminus domain distal to the transmembrane pore domain S5-S6 (CTD). In whole-cell electrophysiology experiments, replacement of the CTD located cysteines 1021 and 1025 with alanine modulated hTRPA1 cold responses. It is proposed that hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5-S6 pore region and the VSLD, respectively.

• MeSH
• alanin MeSH
• ankyrinová repetice * MeSH
• kationtový kanál TRPA1 genetika   metabolismus   MeSH
• lidé MeSH
• tryptofan MeSH
• vnímání teploty MeSH
• vysoká teplota * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• kationtový kanál TRPA1 MeSH
• TRPA1 protein, human MeSH Prohlížeč
• tryptofan MeSH

The development of painful paclitaxel-induced peripheral neuropathy (PIPN) represents a major dose-limiting side effect of paclitaxel chemotherapy. Here we report a promising effect of duvelisib (Copiktra), a novel FDA-approved PI3Kδ/γ isoform-specific inhibitor, in preventing paclitaxel-induced pain-like behavior and pronociceptive signaling in DRGs and spinal cord dorsal horn (SCDH) in rat and mouse model of PIPN. Duvelisib blocked the development of mechanical hyperalgesia in both males and females. Moreover, duvelisib prevented paclitaxel-induced sensitization of TRPV1 receptors, and increased PI3K/Akt signaling in small-diameter DRG neurons and an increase of CD68+ cells within DRGs. Specific optogenetic stimulation of inhibitory neurons combined with patch-clamp recording revealed that duvelisib inhibited paclitaxel-induced weakening of inhibitory, mainly glycinergic control on SCDH excitatory neurons. Enhanced excitatory and reduced inhibitory neurotransmission in the SCDH following PIPN was also alleviated by duvelisib application. In summary, duvelisib showed a promising ability to prevent neuropathic pain in PIPN. The potential use of our findings in human medicine may be augmented by the fact that duvelisib is an FDA-approved drug with known side effects.SIGNIFICANCE STATEMENT We show that duvelisib, a novel FDA-approved PI3Kδ/γ isoform-specific inhibitor, prevents the development of paclitaxel-induced pain-like behavior in males and females and prevents pronociceptive signaling in DRGs and spinal cord dorsal horn in rat and mouse model of paclitaxel-induced peripheral neuropathy.

• Klíčová slova
• PI3K, TRPV1, dorsal horn, glycine, neuropathy, pain,
• MeSH
• bolest MeSH
• fosfatidylinositol-3-kinasy MeSH
• fytogenní protinádorové látky * farmakologie   MeSH
• hyperalgezie chemicky indukované   farmakoterapie   prevence a kontrola   MeSH
• isochinoliny MeSH
• krysa rodu Rattus MeSH
• myši MeSH
• nemoci periferního nervového systému MeSH
• neuralgie * chemicky indukované   farmakoterapie   prevence a kontrola   MeSH
• paclitaxel škodlivé účinky   MeSH
• puriny MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• duvelisib MeSH Prohlížeč
• fytogenní protinádorové látky * MeSH
• isochinoliny MeSH
• paclitaxel MeSH
• puriny MeSH

Numerous physiological functions rely on distinguishing temperature through temperature-sensitive transient receptor potential channels (thermo-TRPs). Although the function of thermo-TRPs has been studied extensively, structural determination of their heat- and cold-activated states has remained a challenge. Here, we present cryo-EM structures of the nanodisc-reconstituted wild-type mouse TRPV3 in three distinct conformations: closed, heat-activated sensitized and open states. The heat-induced transformations of TRPV3 are accompanied by changes in the secondary structure of the S2-S3 linker and the N and C termini and represent a conformational wave that links these parts of the protein to a lipid occupying the vanilloid binding site. State-dependent differences in the behavior of bound lipids suggest their active role in thermo-TRP temperature-dependent gating. Our structural data, supported by physiological recordings and molecular dynamics simulations, provide an insight for understanding the molecular mechanism of temperature sensing.

• MeSH
• buněčné linie MeSH
• elektronová kryomikroskopie MeSH
• gating iontového kanálu MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPV metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• lipidy chemie   MeSH
• myši MeSH
• nízká teplota MeSH
• termodynamika MeSH
• vazba proteinů fyziologie   MeSH
• vnímání teploty fyziologie   MeSH
• vysoká teplota MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• kationtové kanály TRPV MeSH
• lipidy MeSH
• Trpv3 protein, mouse MeSH Prohlížeč

Our understanding of the general principles of the polymodal regulation of transient receptor potential (TRP) ion channels has grown impressively in recent years as a result of intense efforts in protein structure determination by cryo-electron microscopy. In particular, the high-resolution structures of various TRP channels captured in different conformations, a number of them determined in a membrane mimetic environment, have yielded valuable insights into their architecture, gating properties and the sites of their interactions with annular and regulatory lipids. The correct repertoire of these channels is, however, organized by supramolecular complexes that involve the localization of signaling proteins to sites of action, ensuring the specificity and speed of signal transduction events. As such, TRP ankyrin 1 (TRPA1), a major player involved in various pain conditions, localizes into cholesterol-rich sensory membrane microdomains, physically interacts with calmodulin, associates with the scaffolding A-kinase anchoring protein (AKAP) and forms functional complexes with the related TRPV1 channel. This perspective will contextualize the recent biochemical and functional studies with emerging structural data with the aim of enabling a more thorough interpretation of the results, which may ultimately help to understand the roles of TRPA1 under various physiological and pathophysiological pain conditions. We demonstrate that an alteration to the putative lipid-binding site containing a residue polymorphism associated with human asthma affects the cold sensitivity of TRPA1. Moreover, we present evidence that TRPA1 can interact with AKAP to prime the channel for opening. The structural bases underlying these interactions remain unclear and are definitely worth the attention of future studies.

• Klíčová slova
• A-kinase anchoring protein, TRP channel, TRPA1, calmodulin, transient receptor potential,
• Publikační typ
• časopisecké články MeSH

Transient receptor potential ankyrin 1 channel (TRPA1) serves as a key sensor for reactive electrophilic compounds across all species. Its sensitivity to temperature, however, differs among species, a variability that has been attributed to an evolutionary divergence. Mouse TRPA1 was implicated in noxious cold detection but was later also identified as one of the prime noxious heat sensors. Moreover, human TRPA1, originally considered to be temperature-insensitive, turned out to act as an intrinsic bidirectional thermosensor that is capable of sensing both cold and heat. Using electrophysiology and modeling, we compare the properties of human and mouse TRPA1, and we demonstrate that both orthologues are activated by heat, and their kinetically distinct components of voltage-dependent gating are differentially modulated by heat and cold. Furthermore, we show that both orthologues can be strongly activated by cold after the concurrent application of voltage and heat. We propose an allosteric mechanism that could account for the variability in TRPA1 temperature responsiveness.

• Klíčová slova
• TRP channel, ankyrin receptor subtype 1, noxious cold, noxious heat, thermoTRP, transient receptor potential,
• MeSH
• biologické modely MeSH
• druhová specificita MeSH
• elektrofyziologie metody   MeSH
• HEK293 buňky MeSH
• kationtový kanál TRPA1 metabolismus   MeSH
• lidé MeSH
• myši MeSH
• napětím ovládané aniontové kanály metabolismus   fyziologie   MeSH
• nízká teplota MeSH
• sekvence aminokyselin MeSH
• vysoká teplota MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kationtový kanál TRPA1 MeSH
• napětím ovládané aniontové kanály MeSH
• TRPA1 protein, human MeSH Prohlížeč
• Trpa1 protein, mouse MeSH Prohlížeč

The vanilloid transient receptor potential channel TRPV3 is a putative molecular thermosensor widely considered to be involved in cutaneous sensation, skin homeostasis, nociception, and pruritus. Repeated stimulation of TRPV3 by high temperatures above 50 °C progressively increases its responses and shifts the activation threshold to physiological temperatures. This use-dependence does not occur in the related heat-sensitive TRPV1 channel in which responses decrease, and the activation threshold is retained above 40 °C during activations. By combining structure-based mutagenesis, electrophysiology, and molecular modeling, we showed that chimeric replacement of the residues from the TRPV3 cytoplasmic inter-subunit interface (N251-E257) with the homologous residues of TRPV1 resulted in channels that, similarly to TRPV1, exhibited a lowered thermal threshold, were sensitized, and failed to close completely after intense stimulation. Crosslinking of this interface by the engineered disulfide bridge between substituted cysteines F259C and V385C (or, to a lesser extent, Y382C) locked the channel in an open state. On the other hand, mutation of a single residue within this region (E736) resulted in heat resistant channels. We propose that alterations in the cytoplasmic inter-subunit interface produce shifts in the channel gating equilibrium and that this domain is critical for the use-dependence of the heat sensitivity of TRPV3.

• Klíčová slova
• ankyrin repeat, nociception, noxious heat, transient receptor potential, transient receptor potential vanilloid 1 (TRPV1),
• MeSH
• cytoplazma metabolismus   MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPV chemie   genetika   metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• simulace molekulární dynamiky MeSH
• vysoká teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kationtové kanály TRPV MeSH
• podjednotky proteinů MeSH
• TRPV3 protein, human MeSH Prohlížeč

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
• Názvy látek
• napětově řízený sodíkový kanál typ 10 MeSH
• napětově řízený sodíkový kanál typ 11 MeSH
• Scn10a protein, mouse MeSH Prohlížeč
• Scn11a protein, mouse MeSH Prohlížeč

Exposure to repetitive low-frequency electromagnetic field (LF-EMF) shows promise as a non-invasive approach to treat various sensory and neurological disorders. Despite considerable progress in the development of modern stimulation devices, there is a limited understanding of the mechanisms underlying their biological effects and potential targets at the cellular level. A significant impact of electromagnetic field on voltage-gated calcium channels and downstream signalling pathways has been convincingly demonstrated in many distinct cell types. However, evidence for clear effects on primary sensory neurons that particularly may be responsible for the analgesic actions of LF-EMF is still lacking. Here, we used F11 cells derived from dorsal root ganglia neurons as an in vitro model of peripheral sensory neurons and three different protocols of high-induction magnetic stimulation to determine the effects on chemical responsiveness and spontaneous activity. We show that short-term (<180 sec.) exposure of F11 cells to LF-EMF reduces calcium transients in response to bradykinin, a potent pain-producing inflammatory agent formed at sites of injury. Moreover, we characterize an immediate and reversible potentiating effect of LF-EMF on neuronal spontaneous activity. Our results provide new evidence that electromagnetic field may directly modulate the activity of sensory neurons and highlight the potential of sensory neuron-derived cell line as a tool for studying the underlying mechanisms at the cellular and molecular level.

• Klíčová slova
• bradykinin receptor, electromagnetic field, ion channel, primary sensory neuron, transient receptor potential channel,
• MeSH
• bradykinin farmakologie   MeSH
• buněčné linie MeSH
• elektromagnetická pole * MeSH
• kationtový kanál TRPA1 metabolismus   MeSH
• lidé MeSH
• nervové receptory účinky léků   metabolismus   MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bradykinin MeSH
• kationtový kanál TRPA1 MeSH
• TRPA1 protein, human MeSH Prohlížeč
• vápník MeSH