Conservation of functional and pharmacological properties in the distantly related temperature sensors TRVP1 and TRPM8

Members of the transient receptor potential (TRP) superfamily of ion channels have now been defined as molecular transducers capable of reproducing the spectrum of temperature sensation exhibited by mammals. Because of their pivotal role in sensory transduction, many of these channels represent good targets for drug discovery. With a view to gaining further insight into the functional and pharmacological properties of these channels, we have used the whole-cell patch-clamp technique to study the human cold-sensitive menthol receptor transient receptor potential melastatin 8 (TRPM8) and compared its behavior with that of its distant relative, the heat-sensitive capsaicin-gated transient receptor potential vanilloid 1 (TRPV1). It is remarkable to find that TRPM8, in addition to its behavior as an outwardly rectifying, nonselective cation channel, shares many functional and pharmacological properties with TRPV1. TRPM8 exhibits prominent time- and voltage-dependent behavior, a property that may underlie the conserved rectification or gating mechanisms exhibited by these channels. We also show that TRPM8 is modulated by ethanol but unlike TRPV1 is insensitive to extracellular acidification. There is also significant overlap in the antagonist pharmacology of these channels with many TRPV1 antagonists such as capsazepine, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide (BCTC), (2R)-4-(3-chloro-2-pyridinyl)-2-methyl-N-[4-(trifluoromethyl)phenyl]-1-piperazinecarboxamide (CTPC), and N-(2-bromophenyl)-N'-{2-[ethyl(3-methylphenyl)amino]ethyl}-urea (SB-452533) exhibiting similar activity at TRPM8. Overall, the degree of pharmacological overlap between TRPV1 and TRPM8 has implications for the interpretation of studies conducted with these ligands to date and highlights a clear challenge for the design of selective TRP channel antagonists. Our finding that N-(3-methoxyphenyl)-4-chlorocinnamide (SB-366791), at least, represents an apparently selective antagonist for TRPV1 suggests that this goal is attainable.     

Production of a specific extracellular inhibitor of TRPM3 channels

Background and purpose:

Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family.

Experimental approach:

A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca²⁺ measurement.

Key results:

Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5–10 min. Ca²⁺ entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response.

Conclusions and implications:

The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers.     

Nociception and TRP Channels

Noxious thermal, mechanical, or chemical stimuli evoke pain through excitation of the peripheral terminals called nociceptor, and many kinds of ionotropic and metabotropic receptors are involved in this process. Capsaicin receptor TRPV1 is a nociceptor-specific ion channel that serves as the molecular target of capsaicin. TRPV1 can be activated not only by capsaicin but also by noxious heat (with a thermal threshold >43 degrees C) or protons (acidification), all of which are known to cause pain in vivo. Studies using TRPV1-deficient mice have shown that TRPV1 is essential for selective modalities of pain sensation and for thermal hyperalgesia. One mechanism underlying inflammatory pain which is initiated by tissue damage/inflammation and characterized by hypersensitivity is sensitization of TRPV1. In addition to TRPV1, there are five thermosensitive ion channels in mammals, all of which belong to the TRP (transient receptor potential) super family. These include TRPV2, TRPV3, TRPV4, TRPM8 and TRPA1. These channels exhibit distinct thermal activation thresholds (> 52 degrees C for TRPV2, > approximately 34-38 degrees C for TRPV3, > approximately 27-35 degrees C for TRPV4, < approximately 25-28 degrees C for TRPM8 and < 17 degrees C for TRPA1) and are expressed in primary sensory neurons as well as other tissues. Some of the thermosensitive TRP channels are likely to be involved in thermal nociception, since their activation thresholds are within the noxious range of temperatures.     

Design,Synthesis, and Biological Evaluation of 1,5‐Diaryl‐1,2,4‐triazole Derivatives as Selective Cyclooxygenase‐2 Inhibitors

A series of 1,5‐diaryl‐1,2,4‐triazole derivatives were synthesized and evaluated as cyclooxygenase‐2 (COX‐2) inhibitors. The results of the preliminary biological assays in vivo showed that eight compounds 5b , 6b , 6c , 7c , 8b , 8d , 9c , and 9d have potent anti‐inflammatory activity (P < 0.01), while compounds 6b , 6c , and 9c exhibit marked potency. Compound 6c was then selected for further investigation. In the COX inhibition assay in vitro, compound 6c was identified as a potent and selective inhibitor of COX‐2 (COX‐2 IC₅₀ = 0.37 µM; SI = 0.018), being equipotent to celecoxib (COX‐2 IC₅₀ = 0.26 µM; SI = 0.015). In a rat carrageenan‐induced paw edema assay, 6c exhibited moderate anti‐inflammatory activity (35% inhibition of inflammation) at 2 h after administration of 15 mg/kg as an oral dose. A docking study also revealed that compound 6c binds in the active site of COX‐2 in a similar mode to that of the known selective COX‐2 inhibitor SC‐558.     

Identification of herbal components as TRPA1 agonists and TRPM8 antagonists

Transient receptor potential (TRP) channels are non-selective cation channels that are implicated in analgesia, bowel motility, wound healing, thermoregulation, vasodilation and voiding dysfunction. Many natural products have been reported to affect the activity of TRP channels. We hypothesize that numerous traditional herbal medicines (THMs) might exert their pharmacological activity through modulating the activity of TRP channels. The present study aimed to evaluate the effects of flavonoid aglycones and their glycosides, which are the main components of many THMs, on the TRP channel subtypes. A Ca²⁺ influx assay was performed using recombinant human TRPA1, TRPV1, TRPV4 and TRPM8 cell lines. Our findings showed that flavonoid aglycones and glycycoumarin activated TRPA1. In particular, isoflavone and chalcone compounds displayed potent TRPA1 agonistic activity. Furthermore, flavone aglycones showed concomitant potent TRPM8 inhibiting activity. Indeed, flavone, isoflavone aglycones, non-prenylated chalcones and glycycoumarin were found to be TRPM8 inhibitors. Hence, flavonoid aglycones metabolized by lactase-phlorizin hydrolase and β-glucosidase in the small intestine or gut microbiota of the large intestine could generate TRPA1 agonists and TRPM8 antagonists.

     

Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7

BACKGROUND AND PURPOSE

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells.

EXPERIMENTAL APPROACH

We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments.

KEY RESULTS

TRPM7 currents were inhibited by modulators of small conductance Ca²⁺-activated K⁺ channels (K_Ca2.1–2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC₅₀ 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg²⁺-dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

This study reveals a tight overlap in the pharmacological profiles of TRPM7 and K_Ca2.1–2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.     

Inhibition of temperature-sensitive TRPV3 channel by two natural isochlorogenic acid isomers for alleviation of dermatitis and chronic pruritus

Genetic gain-of-function mutations of warm temperature-sensitive transient receptor potential vanilloid 3 (TRPV3) channel cause Olmsted syndrome characterized by severe itching and keratoderma, indicating that pharmacological inhibition of TRPV3 may hold promise for therapy of chronic pruritus and skin diseases. However, currently available TRPV3 tool inhibitors are either nonselective or less potent, thus impeding the validation of TRPV3 as therapeutic target. Using whole-cell patch-clamp and single-channel recordings, we report the identification of two natural dicaffeoylquinic acid isomers isochlorogenic acid A (IAA) and isochlorogenic acid B (IAB) that selectively inhibit TRPV3 currents with IC₅₀ values of 2.7 ± 1.3 and 0.9 ± 0.3 μmol/L, respectively, and reduce the channel open probability to 3.7 ± 1.2% and 3.2 ± 1.1% from 26.9 ± 5.5%, respectively. In vivo evaluation confirms that both IAA and IAB significantly reverse the ear swelling of dermatitis and chronic pruritus. Furthermore, the isomer IAB is able to rescue the keratinocyte death induced by TRPV3 agonist carvacrol. Molecular docking combined with site-directed mutations reveals two residues T636 and F666 critical for the binding of the two isomers. Taken together, our identification of isochlorogenic acids A and B that act as specific TRPV3 channel inhibitors and gating modifiers not only provides an essential pharmacological tool for further investigation of the channel pharmacology and pathology, but also holds developmental potential for treatment of dermatitis and chronic pruritus.     

Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3

BACKGROUND AND PURPOSE

Fenamates are N-phenyl-substituted anthranilic acid derivatives clinically used as non-steroid anti-inflammatory drugs in pain treatment. Reports describing fenamates as tools to interfere with cellular volume regulation attracted our attention based on our interest in the role of the volume-modulated transient receptor potential (TRP) channels TRPM3 and TRPV4.

EXPERIMENTAL APPROACH

Firstly, we measured the blocking potencies and selectivities of fenamates on TRPM3 and TRPV4 as well as TRPC6 and TRPM2 by Ca²⁺ imaging in the heterologous HEK293 cell system. Secondly, we further investigated the effects of mefenamic acid on cytosolic Ca²⁺ and on the membrane voltage in single HEK293 cells that exogenously express TRPM3. Thirdly, in insulin-secreting INS-1E cells, which endogenously express TRPM3, we validated the effect of mefenamic acid on cytosolic Ca²⁺ and insulin secretion.

KEY RESULTS

We identified and characterized mefenamic acid as a selective and potent TRPM3 blocker, whereas other fenamate structures non-selectively blocked TRPM3, TRPV4, TRPC6 and TRPM2.

CONCLUSIONS AND IMPLICATIONS

This study reveals that mefenamic acid selectively inhibits TRPM3-mediated calcium entry. This selectivity was further confirmed using insulin-secreting cells. K_ATP channel-dependent increases in cytosolic Ca²⁺ and insulin secretion were not blocked by mefenamic acid, but the selective stimulation of TRPM3-dependent Ca²⁺ entry and insulin secretion induced by pregnenolone sulphate were inhibited. However, the physiological regulator of TRPM3 in insulin-secreting cells remains to be elucidated, as well as the conditions under which the inhibition of TRPM3 can impair pancreatic β-cell function. Our results strongly suggest mefenamic acid is the most selective fenamate to interfere with TRPM3 function.     

Synthesis and Anticholinergic Activity of 4‐hydroxycoumarin Derivatives Containing Substituted Benzyl‐1,2,3‐triazole Moiety

A series of 4‐hydroxycoumarin‐derived compounds 8a‐p containing N‐benzyl‐1,2,3‐triazole motif were designed as AChE inhibitors. The title compounds were obtained conveniently using multicomponent click reaction. The in vitro anticholinesterase evaluation of synthesized compounds against AChE and BuChE showed that some of them are potent and selective inhibitors of AChE. Among them, 2‐chlorobenzyl derivative 8k showed the most potent activity against AChE (IC₅₀ = 0.18 μm ). Its activity was also superior to that of standard drug tacrine. The kinetic study and molecular docking simulation of the most potent compound 8k were also described.     

Triphenylphosphine oxide is a potent and selective inhibitor of the transient receptor potential melastatin-5 ion channel

Transient receptor potential melastatin-5 (TRPM5) is a calcium-gated monovalent cation channel expressed in highly specialized cells of the taste bud and gastrointestinal tract, as well as in pancreatic β-cells. Well established as a critical signaling protein for G protein-coupled receptor-mediated taste pathways, TRPM5 also has recently been implicated as a regulator of incretin and insulin secretion. To date, no inhibitors of practical use have been described that could facilitate investigation of TRPM5 functions in taste or secretion of metabolic hormones. Using recombinant TRPM5-expressing cells in a fluorescence imaging plate reader-based membrane potential assay, we identified triphenylphosphine oxide (TPPO) as a selective and potent inhibitor of TRPM5. TPPO inhibited both human (IC???=?12?μM) and murine TRPM5 (IC???=?30?μM) heterologously expressed in HEK293 cells, but had no effect (up to 100?μM) on the membrane potential responses of TRPA1, TRPV1, or TRPM4b. TPPO also inhibited a calcium-gated TRPM5-dependent conductance in taste cells isolated from the tongues of transgenic TRPM5(+/)? mice. In contrast, TPP had no effect on TRPM5 responses, indicating a strict requirement of the oxygen atom for activity. Sixteen additional TPPO derivatives also inhibited TRPM5 but none more potently than TPPO. Structure-activity relationship of tested compounds was used for molecular modeling-based analysis to clarify the positive and negative structural contributions to the potency of TPPO and its derivatives. TPPO is the most potent TRPM5 inhibitor described to date and is the first demonstrated to exhibit selectivity over other channels.     

Design,synthesis, and evaluation of the anticancer activity of 2‐amino‐aryl‐7‐aryl‐benzoxazole compounds

A series of 2‐amino‐aryl‐7‐aryl‐benzoxazole derivatives have been designed, synthesized, and evaluated as anticancer agents. Fourteen of the compounds exhibited cytotoxic effects toward human A549 lung cancer cells. We found 12l was the most potent with an EC₅₀ of 0.4 μm , equivalent to the anticancer drug doxorubicin, but had low selectivity following cross screening in monkey kidney Vero cells. Eight of the most potent or most selective compounds were further profiled in additional cell lines (MCF7, NCI‐H187, and KB) to better understand their cytotoxic activity. Only compound 12l had a measurable EC₅₀ in a single cell line (3.3 μm in the KB cell line). Taken together, this data suggest the series as a whole display specific cytotoxicity toward A549 cells. Cheminformatics searches pointed to JAK2 as a possible target. A subset of compounds assayed at this target showed IC₅₀s ranging from 10 to 0.08 μm ; however, no clear correlation between JAK2 potency and A549 cytotoxicity was observed.     

瞬时受体电位8型的功能结构及其化学调节剂的研究进展

瞬时受体电位8型(transient receptor potential melastatin-related 8,TRPM8)是一种非选择性钙离子通道,其功能异常或缺失会导致多种疾病。TRPM8参与生物体的体温调节、肿瘤、炎症、疼痛和哮喘等生理及病理活动,特别是在肿瘤治疗过程中,TRPM8常被用作治疗的潜在靶点。大部分化学调节剂是通过影响细胞内Ca²⁺浓度平衡来激活TRPM8通道。本文对TRPM8通道的结构、功能及其对不同疾病的调节作用等进行归纳及分析,为TRPM8作为临床治疗及新药开发靶点提供借鉴。     

Pharmacological comparison of novel synthetic fenamate analogues with econazole and 2-APB on the inhibition of TRPM2 channels

BACKGROUND AND PURPOSE

Fenamate analogues, econazole and 2-aminoethoxydiphenyl borate (2-APB) are inhibitors of transient receptor potential melastatin 2 (TRPM2) channels and are used as research tools. However, these compounds have different chemical structures and therapeutic applications. Here we have investigated the pharmacological profile of TRPM2 channels by application of newly synthesized fenamate analogues and the existing channel blockers.

EXPERIMENTAL APPROACH

Human TRPM2 channels in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells and the expression was induced by tetracycline. Whole cell currents were recorded by patch-clamp techniques. Ca²⁺ influx or release was monitored by fluorometry.

KEY RESULTS

Flufenamic acid (FFA), mefenamic acid (MFA) and niflumic acid (NFA) concentration-dependently inhibited TRPM2 current with potency order FFA > MFA = NFA. Modification of the 2-phenylamino ring by substitution of the trifluoromethyl group in FFA with –CH₃, –F, –CF₃, –OCH₃, –OCH₂CH₃, –COOH, and –NO₂ at various positions, reduced channel blocking potency. The conservative substitution of 3-CF₃ in FFA by –CH₃ (3-MFA), however, gave the most potent fenamate analogue with an IC₅₀ of 76 µM, comparable to that of FFA, but unlike FFA, had no effect on Ca²⁺ release. 3-MFA and FFA inhibited the channel intracellularly. Econazole and 2-APB showed non-selectivity by altering cytosolic Ca²⁺ movement. Econazole also evoked a non-selective current.

CONCLUSION AND IMPLICATIONS

The fenamate analogue 3-MFA was more selective than other TRPM2 channel blockers. FFA, 2-APB and econazole should be used with caution as TRPM2 channel blockers, as these compounds can interfere with intracellular Ca²⁺ movement.     

Activation of TRPA1 channels by the fatty acid amide hydrolase inhibitor 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597)

As a member of the transient receptor potential (TRP) ion channel superfamily, the ligand-gated ion channel TRPA1 has been implicated in nociceptive function and pain states. The endogenous ligands that activate TRPA1 remain unknown. However, various agonists have been identified, including environmental irritants (e.g., acrolein) and ingredients of pungent natural products [e.g., allyl isothiocyanate (ITC), cinnamaldehyde, allicin, and gingerol]. In general, these agents are either highly reactive, nonselective, or not potent or efficacious, significantly limiting their utilities in the study of TRPA1 channel properties and biological functions. In a search for novel TRPA1 agonists, we identified 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597), a potent and systemically active inhibitor of fatty acid amide hydrolase (FAAH). This enzyme is responsible for anandamide degradation and therefore has been pursued as an antinociceptive and antiepileptic drug target. Using Ca(2+) influx assays and patch-clamp techniques, we demonstrated that URB597 could activate heterologously expressed human and rat TRPA1 channels, whereas two other FAAH inhibitors (i.e., URB532 and Compound 7) had no effect. When applied to inside-out membrane patches expressing rat TRPA1, URB597 elicited single-channel activities with a unitary conductance of 40 pS. Furthermore, URB597 activated TRPA1 channels endogenously expressed in a population of rat dorsal root ganglion neurons that also responded to ITC. In contrast to its effect on TRPA1, URB597 inhibited TRPM8 and had no effects on TRPV1 or TRPV4. Thus, we conclude that URB597 is a novel agonist of TRPA1 and probably activates the channel through a direct gating mechanism.     

The role of natural products in the ligand deorphanization of TRP channels

The ligand deorphanization of TRP channels has a tremendous potential for biomedical and nutritional research, and this review highlights the role that natural products have played in the identification of ligands for these targets and their establishment as viable candidates for drug discovery. Specific ligands have so far been discovered only for some thermoTRPs, like TRPV1, TRPV3, TRPV4, TRPM8 and TRPA1, and the lack of selective pharmacology has been a major drawback for unraveling the biological role of TRPs. While genetic approaches (transgenic animal models) have partially compensate for the lack of ligands, the universal expression of TRPs in living systems and the success achieved with TRPV1 suggest that a systematic investigation of the natural products pool might alleviate this shortage, fostering adoption by small molecules within this class of still largely orphan biological targets.     

Advances in modulating thermosensory TRP channels

Introduction: Thermosensory channels are a subfamily of the transient receptor potential (TRP) channel family that are activated by changes in the environmental temperature. These channels, known as thermoTRPs, cover the entire spectrum of temperatures, from noxious cold (< 15°C) to injurious heat (> 42°C). In addition, dysfunction of these channels contributes to the thermal hypersensitivity that accompanies painful conditions. Moreover, because of their wide tissue and cellular distribution, thermoTRPs are also involved in the pathophysiology of several diseases, from inflammation to cancer. Areas covered: Although the number of thermoTRPs is increasing with the identification of novel members such as TRPM3, we will cover the recent advances in the pharmacology of the classical thermosensory channels, namely TRPV1, TRPV2, TRPV3, TRPV4, TRPM8 and TRPA1. This review will focus on the therapeutic progress carried out for all these channels and will highlight the tenet that TRPV1, TRPM8 and TRPA1 are the most exploited channels, and that the interest on TRPV3 and TRPV4 is growing with the first TRPV3 antagonist that moves into Phase-II clinical trials. In contrast, the pharmacology of TRPV2 is yet in its infancy. Expert opinion: Despite the tremendous academic and industrial investment to develop therapeutic modulators of thermoTRPs, it apparently seems that we are still far from the first successful product, although hope is maintained high for all compounds currently in clinical trials. A major concern has been the appearance of side effects. A better knowledge of the thermosensory protein networks (signal-plexes), along with the application of system biology approaches may provide novel strategies to modulate thermoTRPs activity with improved therapeutic index. A case in point is TRPV1, where acting on interacting proteins is providing new therapeutic opportunities.     

化疗引起的神经痛治疗靶点温度选择性TRP通道的研究进展

作为钙离子渗透性的瞬时受体电位(TRP),5种通道(TRPV1~4和TRPM2)被不同的高温激活,两种通道(TRPV1和TRPV8)被低温激活。越来越多的证据表明,TRPA1和TRPM8拮抗剂可预防顺铂、奥沙利铂和紫杉醇诱导的线粒体氧化应激、炎症、冷痛和痛觉过敏。TRPV1在顺铂引起的感觉神经元热痛觉和机械异常中有应答。TRPA1、TRPM8和TRPV2蛋白表达水平主要通过这些治疗方法在背根(DRG)和三叉神经节中增加。主要总结了5种温度调节TRP通道(TRPA1、TRPM8、TRPV1、TRPV2和TRPV4)。     

Design and optimization of benzimidazole-containing transient receptor potential melastatin 8 (TRPM8) antagonists

Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that is thermoresponsive to cool to cold temperatures (8-28 °C) and also may be activated by chemical agonists such as menthol and icilin. Antagonism of TRPM8 activation is currently under investigation for the treatment of painful conditions related to cold, such as cold allodynia and cold hyperalgesia. The design, synthesis, and optimization of a class of selective TRPM8 antagonists based on a benzimidazole scaffold is described, leading to the identification of compounds that exhibited potent antagonism of TRPM8 in cell-based functional assays for human, rat, and canine TRPM8 channels. Numerous compounds in the series demonstrated excellent in vivo activity in the TRPM8-selective "wet-dog shakes" (WDS) pharmacodynamic model and in the rat chronic constriction injury (CCI)-induced model of neuropathic pain. Taken together, the present results suggest that the in vivo antagonism of TRPM8 constitutes a viable new strategy for treating a variety of disorders associated with cold hypersensitivity, including certain types of neuropathic pain.     

The M-channel blocker linopirdine is an agonist of the capsaicin receptor TRPV1

Linopirdine is a well known blocker of voltage-gated potassium channels from the Kv7 (or KCNQ) family that generate the so called M current in mammalian neurons. Kv7 subunits are also expressed in pain-sensing neurons in dorsal root ganglia, in which they modulate neuronal excitability. In this study we demonstrate that linopirdine acts as an agonist of TRPV1 (transient receptor potential vanilloid type 1), another ion channel expressed in nociceptors and involved in pain signaling. Linopirdine induces increases in intracellular calcium concentration in human embryonic kidney 293 (HEK293) cells expressing TRPV1, but not TRPA1 and TRPM8 or in wild-type HEK293 cells. Linopirdine also activates an inward current in TRPV1-expressing HEK293 cells that is almost completely blocked by the selective TRPV1 antagonist capsazepine. At low concentrations linopirdine sensitizes both recombinant and native TRPV1 channels to heat, in a manner that is not prevented by the Kv7-channel opener flupirtine. Taken together, these results indicate that linopirdine exerts an excitatory action on mammalian nociceptors not only through inhibition of the M current but also through activation of the capsaicin receptor TRPV1.