Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR‐32 human neuroblastoma cells

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non‐selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR‐32 undergoes a remarkable differentiation in response to treatment with 5‐bromo‐2‐deoxyuridine. The cells acquire a neuronal morphology with increased expression of N‐type voltage gated calcium channels and neurotransmitters. Here we show using RT‐PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR‐32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl‐isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC‐030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR‐32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR‐32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression. J. Cell. Physiol. 221: 67–74, 2009. © 2009 Wiley‐Liss, Inc     

Functional Expression of TRPM8 and TRPA1 Channels in Rat Odontoblasts

Odontoblasts produce dentin during development, throughout life, and in response to pathological conditions by sensing stimulation of exposed dentin. The functional properties and localization patterns of transient receptor potential (TRP) melastatin subfamily member 8 (TRPM8) and ankyrin subfamily member 1 (TRPA1) channels in odontoblasts remain to be clarified. We investigated the localization and the pharmacological, biophysical, and mechano-sensitive properties of TRPM8 and TRPA1 channels in rat odontoblasts. Menthol and icilin increased the intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Icilin-, WS3-, or WS12-induced [Ca²⁺]_i increases were inhibited by capsazepine or 5-benzyloxytriptamine. The increase in [Ca²⁺]_i elicited by allyl isothiocyanate (AITC) was inhibited by HC030031. WS12 and AITC exerted a desensitizing effect on [Ca²⁺]_i increase. Low-temperature stimuli elicited [Ca²⁺]_i increases that are sensitive to both 5-benzyloxytriptamine and HC030031. Hypotonic stimulation-induced membrane stretch increased [Ca²⁺]_i; HC030031 but not 5-benzyloxytriptamine inhibited the effect. The results suggest that TRPM8 channels in rat odontoblasts play a role in detecting low-temperature stimulation of the dentin surface and that TRPA1 channels are involved in sensing membrane stretching and low-temperature stimulation. The results also indicate that odontoblasts act as mechanical and thermal receptor cells, detecting the stimulation of exposed dentin to drive multiple cellular functions, such as sensory transduction.     

Expression of the transient receptor potential channels TRPV1, TRPA1 and TRPM8 in mouse trigeminal primary afferent neurons innervating the dura

ABSTRACT: BACKGROUND: Migraine and other headache disorders affect a large percentage of the population and cause debilitating pain. Activation and sensitization of the trigeminal primary afferent neurons innervating the dura and cerebral vessels is a crucial step in the "headache circuit". Many dural afferent neurons respond to algesic and inflammatory agents. Given the clear role of the transient receptor potential (TRP) family of channels in both sensing chemical stimulants and mediating inflammatory pain, we investigated the expression of TRP channels in dural afferent neurons. METHODS: We used two fluorescent tracers to retrogradely label dural afferent neurons in adult mice and quantified the abundance of peptidergic and non-peptidergic neuron populations using calcitonin gene-related peptide immunoreactivity (CGRP-ir) and isolectin B4 (IB4) binding as markers, respectively. Using immunohistochemistry, we compared the expression of TRPV1 and TRPA1 channels in dural afferent neurons with the expression in total trigeminal ganglion (TG) neurons. To examine the distribution of TRPM8 channels, we labeled dural afferent neurons in mice expressing farnesylated enhanced green fluorescent protein (EGFPf) from a TRPM8 locus. We used nearest-neighbor measurement to predict the spatial association between dural afferent neurons and neurons expressing TRPA1 or TRPM8 channels in the TG.Results and conclusionsWe report that the size of dural afferent neurons is significantly larger than that of total TG neurons and facial skin afferents. Approximately 40% of dural afferent neurons exhibit IB4 binding. Surprisingly, the percentage of dural afferent neurons containing CGRP-ir is significantly lower than those of total TG neurons and facial skin afferents. Both TRPV1 and TRPA1 channels are expressed in dural afferent neurons. Furthermore, nearest-neighbor measurement indicates that TRPA1-expressing neurons are clustered around a subset of dural afferent neurons. Interestingly, TRPM8-expressing neurons are virtually absent in the dural afferent population, nor do these neurons cluster around dural afferent neurons. Taken together, our results suggest that TRPV1 and TRPA1 but not TRPM8 channels likely contribute to the excitation of dural afferent neurons and the subsequent activation of the headache circuit. These results provide an anatomical basis for understanding further the functional significance of TRP channels in headache pathophysiology.     

The Contribution of TRPM8 and TRPA1 Channels to Cold Allodynia and Neuropathic Pain

Cold allodynia is a common feature of neuropathic pain however the underlying mechanisms of this enhanced sensitivity to cold are not known. Recently the transient receptor potential (TRP) channels TRPM8 and TRPA1 have been identified and proposed to be molecular sensors for cold. Here we have investigated the expression of TRPM8 and TRPA1 mRNA in the dorsal root ganglia (DRG) and examined the cold sensitivity of peripheral sensory neurons in the chronic construction injury (CCI) model of neuropathic pain in mice.In behavioral experiments, chronic constriction injury (CCI) of the sciatic nerve induced a hypersensitivity to both cold and the TRPM8 agonist menthol that developed 2 days post injury and remained stable for at least 2 weeks. Using quantitative RT-PCR and in situ hybridization we examined the expression of TRPM8 and TRPA1 in DRG. Both channels displayed significantly reduced expression levels after injury with no change in their distribution pattern in identified neuronal subpopulations. Furthermore, in calcium imaging experiments, we detected no alterations in the number of cold or menthol responsive neurons in the DRG, or in the functional properties of cold transduction following injury. Intriguingly however, responses to the TRPA1 agonist mustard oil were strongly reduced.Our results indicate that injured sensory neurons do not develop abnormal cold sensitivity after chronic constriction injury and that alterations in the expression of TRPM8 and TRPA1 are unlikely to contribute directly to the pathogenesis of cold allodynia in this neuropathic pain model.     

Human TRPM8 and TRPA1 pain channels,including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor

TRPM8 (transient receptor potential M8) and TRPA1 (transient receptor potential A1) are cold-temperature-sensitive nociceptors expressed in sensory neurons but their behaviour in neuronal cells is poorly understood. Therefore DNA expression constructs containing human TRPM8 or TRPA1 cDNAs were transfected into HEK (human embryonic kidney cells)-293 or SH-SY5Y neuroblastoma cells and G418 resistant clones analysed for effects of agonists and antagonists on intracellular Ca²⁺ levels. Approximately 51% of HEK-293 and 12% of SH-SY5Y cell clones expressed the transfected TRP channel. TRPM8 and TRPA1 assays were inhibited by probenecid, indicating the need to avoid this agent in TRP channel studies. A double-residue mutation in ICL-1 (intracellular loop-1) of TRPM8 (SV762,763EL, mimicking serine phosphorylation) or one in the C-terminal tail region (FK1045,1046AG, a lysine knockout) retained sensitivity to agonists (WS 12, menthol) and antagonist {AMTB [N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide]}. SNP (single nucleotide polymorphism) variants in TRPA1 ICL-1 (R797T, S804N) and TRPA1 fusion protein containing C-terminal (His)₁₀ retained sensitivity to agonists (cinnamaldehyde, allyl-isothiocyanate, carvacrol, eugenol) and antagonists (HC-030031, A967079). One SNP variant, 797T, possessed increased sensitivity to agonists. TRPA1 became repressed in SH-SY5Y clones but was rapidly rescued by Src-family inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Conversely, TRPM8 in SH-SY5Y cells was inhibited by PP2. Further studies utilizing SH-SY5Y may identify structural features of TRPA1 and TRPM8 involved in conferring differential post-translational regulation.     

3-Ylidenephthalides as a new class of transient receptor potential channel TRPA1 and TRPM8 modulators

Following the recent identification of the naturally occurring 3-ylidene-4,5-dihydrophthalide ligustilide and its oxidation product dehydroligustilide as novel TRPA1 modulators, a series of seventeen 3-ylidenephthalides was synthesized and tested on TRPA1 and TRPM8 channels. Most of these compounds acted as strong modulators of the two channel types with EC₅₀ and/or IC₅₀ values distinctly lower than those of the reference compounds.     

The Outer Pore and Selectivity Filter of TRPA1

TRPA1 (transient-receptor-potential-related ion channel with ankyrin domains) is a direct receptor or indirect effector for a wide variety of nociceptive signals, and thus is a compelling target for development of analgesic pharmaceuticals such as channel blockers. Recently, the structure of TRPA1 was reported, providing insights into channel assembly and pore architecture. Here we report whole-cell and single-channel current recordings of wild-type human TRPA1 as well as TRPA1 bearing point mutations of key charged residues in the outer pore. These measurements demonstrate that the glutamate at position 920 plays an important role in collecting cations into the mouth of the pore, by changing the effective surface potential by ~16 mV, while acidic residues further out have little effect on permeation. Electrophysiology experiments also confirm that the aspartate residue at position 915 represents a constriction site of the TRPA1 pore and is critical in controlling ion permeation.     

Biophysical properties of menthol-activated cold receptor TRPM8 channels

The temperature-sensitive transient receptor potential channel, TRPM8, was recently cloned and found to be activated by cold and menthol. Whole-cell recordings show that TRPM8 is permeable to multiple cations and exhibits a strong outward rectification. Here, we examine the mechanism underlying menthol-evoked current rectification of TRPM8 transiently expressed in tsA-201 cells at room temperature ( approximately 25 degrees C). Whole-cell currents (ruptured, bath: Na(+), K(+), Ca(2+), or Ba(2+); pipette: KCl) exhibited a strong outward rectification in the presence of menthol, consistent with previous studies. The outward K(+) current was reduced in the presence of external Ca(2+) or Ba(2+). Single-channel recordings (cell-attached) showed that menthol induced brief channel openings with two conducting states in the voltage range between -80 and +60mV. The small current (i(S)) conducted both monovalent and divalent ions, and the large one (i(L)) predominantly monovalent ions. The i-V plot for Ca(2+) was weakly outward rectifying, whereas those for monovalent ions were linear. The i(S) may result in the divalent ion-induced reduction of the whole-cell outward current. The open probability (P(o)) in all ion conditions tested was low at negative voltages and increased with depolarization, accounting for the small inward currents observed at the whole-cell level. In conclusion, our results indicate that menthol induced steep outward rectification of TRPM8 results from the voltage-dependent open channel probability and the permeating ion-dependent modulation of the unitary channel conductance.     

Effects of activation of skin ion channels TRPM8, TRPV1, and TRPA1 on the immune response. Comparison with effects of cold and heat exposure

The effects of pharmacological stimulation of skin ion channels TRPA1, TRPM8, TRPV1 on the immune response are presented. These effects are compared with the effects of different types of temperature exposures - skin cooling, deep cooling, and deep heating. This analysis allows us to clear the differences in the influence on the immune response of thermosensitive ion channels localized in the skin; (2) whether the changes in the immune response under temperature exposures are due to these thermosensitive ion channels. Experiments were performed on Wistar rats. For stimulation of TRPM8 ion channel, an application to the skin of 1% menthol was used, for TRPA1 - 0.04% allylisotiocianate, and for TRPV1 - capsaicin in a concentration of 0.001.The antigen binding in the spleen was two-times stimulated by activation of the cold-sensitive ion channel TRPM8 and much weaker by activation of warm-sensitive TRPV1 (by 15%), and another cold-sensitive ion channel TRPA1 (by 40%). Only the stimulation of TRPA1 significantly (by 140%) increased antibody formation in the spleen, while TRPM8 had practically no effect on this process, and activation of TRPV1 significantly (by 60%) inhibited antibody formation. Stimulation of the TRPM8 ion channel significantly (by 60%) reduced the level of IgG in the blood, which is believed to control of infectious diseases.The obtained results show that pharmacological activation of the skin TRPA1, TRPM8, TRPV1 ion channels can differently affect the immune system. At the epicenter of changes there were the antigen binding and antibody formation in the spleen, as well as the level of IgG in the blood. Exactly stimulation of the TRPM8 ion channel determines the changes in the immune response when only the skin is cooling, while at deep body heating, the changes in the immune response are mostly determined by the activation of the skin TRPV1 ion channel.     

Nocistatin sensitizes TRPA1 channels in peripheral sensory neurons

The ability of sensory neurons to detect potentially harmful stimuli relies on specialized molecular signal detectors such as transient receptor potential (TRP) A1 ion channels. TRPA1 is critically implicated in vertebrate nociception and different pain states. Furthermore, TRPA1 channels are subject to extensive modulation and regulation - processes which consequently affect nociceptive signaling. Here we show that the neuropeptide Nocistatin sensitizes TRPA1-dependent calcium influx upon application of the TRPA1 agonist mustard oil (MO) in cultured sensory neurons of dorsal root ganglia (DRG). Interestingly, TRPV1-mediated cellular calcium responses are unaffected by Nocistatin. Furthermore, Nocistatin-induced TRPA1-sensitization is likely independent of the Nocistatin binding partner 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) as assessed by siRNA-mediated knockdown in DRG cultures. In conclusion, we uncovered the sensitization of TRPA1 by Nocistatin, which may represent a novel mechanism how Nocistatin can modulate pain.     

Intragastric administration of allyl isothiocyanate increases carbohydrate oxidation via TRPV1 but not TRPA1 in mice

The transient receptor potential (TRP) channel family is composed of a wide variety of cation-permeable channels activated polymodally by various stimuli and is implicated in a variety of cellular functions. Recent investigations have revealed that activation of TRP channels is involved not only in nociception and thermosensation but also in thermoregulation and energy metabolism. We investigated the effect of intragastric administration of TRP channel agonists on changes in energy substrate utilization of mice. Intragastric administration of allyl isothiocyanate (AITC; a typical TRPA1 agonist) markedly increased carbohydrate oxidation but did not affect oxygen consumption. To examine whether TRP channels mediate this increase in carbohydrate oxidation, we used TRPA1 and TRPV1 knockout (KO) mice. Intragastric administration of AITC increased carbohydrate oxidation in TRPA1 KO mice but not in TRPV1 KO mice. Furthermore, AITC dose-dependently increased intracellular calcium ion concentration in cells expressing TRPV1. These findings suggest that AITC might activate TRPV1 and that AITC increased carbohydrate oxidation via TRPV1.     

Voltage- and cold-dependent gating of single TRPM8 ion channels

Transient receptor potential (TRP) channels play critical roles in cell signaling by coupling various environmental factors to changes in membrane potential that modulate calcium influx. TRP channels are typically activated in a polymodal manner, thus integrating multiple stimuli. Although much progress has been made, the underlying mechanisms of TRP channel activation are largely unknown. The TRPM8 cation channel has been extensively investigated as a major neuronal cold sensor but is also activated by voltage, calcium store depletion, and some lipids as well as by compounds that produce cooling sensations, such as menthol or icilin. Several models of TRPM8 activation have been proposed to explain the interaction between these diverse stimuli. However, a kinetic scheme is not yet available that can describe the detailed single-channel kinetics to gain further insight into the underlying gating mechanism. To work toward this goal, we investigated voltage-dependent single-channel gating in cell-attached patches at two different temperatures (20 and 30 °C) using HEK293 cells stably expressing TRPM8. Both membrane depolarization and cooling increased channel open probability (P(o)) mainly by decreasing the duration of closed intervals, with a smaller increase in the duration of open intervals. Maximum likelihood analysis of dwell times at both temperatures indicated gating in a minimum of five closed and two open states, and global fitting over a wide range of voltages identified a seven-state model that described the voltage dependence of P(o), the single-channel kinetics, and the response of whole-cell currents to voltage ramps and steps. The major action of depolarization and cooling was to accelerate forward transitions between the same two sets of adjacent closed states. The seven-state model provides a general mechanism to account for TRPM8 activation by membrane depolarization at two temperatures and can serve as a starting point for further investigations of multimodal TRP activation.     

Rapid demethylation of the IFN-gamma gene occurs in memory but not naive CD8 T cells

DNA methylation is an epigenetic mechanism of gene regulation. We have determined that specific modifications in DNA methylation at the IFN-gamma locus occur during memory CD8 T cell differentiation in vivo. Expression of the antiviral cytokine IFN-gamma in CD8 T cells is highly developmental stage specific. Most naive cells must divide before they express IFN-gamma, while memory cells vigorously express IFN-gamma before cell division. Ag-specific CD8 T cells were obtained during viral infection of mice and examined directly ex vivo. Naive cells had an IFN-gamma locus with extensive methylation at three specific CpG sites. An inhibitor of methylation increased the amount of IFN-gamma in naive cells, indicating that methylation contributes to the slow and meager production of IFN-gamma. Effectors were unmethylated and produced large amounts of IFN-gamma. Interestingly, while memory cells were also able to produce large amounts of IFN-gamma, the gene was partially methylated at the three CpG sites. Within 5 h of antigenic stimulation, however, the gene was rapidly demethylated in memory cells. This was independent of DNA synthesis and cell division, suggesting a yet unidentified demethylase. Rapid demethylation of the IFN-gamma promoter by an enzymatic factor only in memory cells would be a novel mechanism of differential gene regulation. This differentiation stage-specific mechanism reflects a basic immunologic principle: naive cells need to expand before becoming an effective defense factor, whereas memory cells with already increased precursor frequency can rapidly mount effector functions to eliminate reinfecting pathogens in a strictly Ag-dependent fashion.     

Gating and calcium-sensing mechanisms of TRPA1 channels revealed

Novel structures of the human TRPA1 channel were determined in the presence of the agonist iodoacetamide and the antagonist A-967079, to reveal the open and closed states of the channel, respectively. The structures further revealed the location of Ca²⁺ modulatory site that is likely conserved among several TRP subgroups.     

TRPA1 channels: novel targets of 1,4-dihydropyridines

Transient receptor potential type A1 (TRPA1) channels are cation permeable channels activated by irritant chemicals and pungent natural compounds. Their location in peptidergic sensory terminals innervating the skin and blood vessels makes them important effectors of vasodilator responses of neural origin. 1,4-dihydropyridines are a class of L-type calcium channel antagonists commonly used in the treatment of hypertension and ischemic heart disease. Here we show that four different 1,4-dihydropyridines (nifedipine, nimodipine, nicardipine and nitrendipine), and the structurally related L-type calcium channel agonist BayK8644, exert powerful excitatory effects on TRPA1 channels. The activation does not depend on elevated Ca2+ levels and cross-desensitizes with that produced by other TRPA1 agonists. The activation produced by nifedipine was reduced by camphor and the selective TRPA1 antagonist HC03001. In a subclass of mouse nociceptors expressing TRPA1 channels, assessed by responses to the TRPA1 agonist mustard oil, nifedipine also produced large elevations in [Ca2+](i). These responses were fully abrogated in TRPA1(-/-) mice. These findings identify TRPA1 channels as a new molecular target for the 1,4-dihydropyridine class of calcium channel modulators.     

Transient receptor potential melastatin 8 (TRPM8) channels are involved in body temperature regulation

ABSTRACT: BACKGROUND: Transient receptor potential cation channel subfamily M member 8 (TRPM8) is activated by cold temperature in vitro and has been demonstrated to act as a 'cold temperature sensor' in vivo. Although it is known that agonists of this 'cold temperature sensor', such as menthol and icilin, cause a transient increase in body temperature (Tb), it is not known if TRPM8 plays a role in Tb regulation. Since TRPM8 has been considered as a potential target for chronic pain therapeutics, we have investigated the role of TRPM8 in Tb regulation. RESULTS: We characterized five chemically distinct compounds (AMG0635, AMG2850, AMG8788, AMG9678, and Compound 496) as potent and selective antagonists of TRPM8 and tested their effects on Tb in rats and mice implanted with radiotelemetry probes. All five antagonists used in the study caused a transient decrease in Tb (maximum decrease of 0.98degreesC). Since thermoregulation is a homeostatic process that maintains Tb about 37degreesC, we further evaluated whether repeated administration of an antagonist attenuated the decrease in Tb. Indeed, repeated daily administration of AMG9678 for four consecutive days showed a reduction in the magnitude of the Tb decrease Day 2 onwards. CONCLUSIONS: The data reported here demonstrate that TRPM8 channels play a role in Tb regulation. Further, a reduction of magnitude in Tb decrease after repeated dosing of an antagonist suggests that TRPM8's role in Tb maintenance may not pose an issue for developing TRPM8 antagonists as therapeutics.     

Near-membrane dynamics and capture of TRPM8 channels within transient confinement domains

Background

The cold and menthol receptor, TRPM8, is a non-selective cation channel expressed in a subset of peripheral neurons that is responsible for neuronal detection of environmental cold stimuli. It was previously shown that members of the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process. Single particle tracking (SPT) is a useful tool for probing the organization and dynamics of protein constituents in the plasma membrane.

Methodology/Principal Findings

We used SPT to study the receptor dynamics and describe membrane/near-membrane behavior of particles containing TRPM8-EGFP in transfected HEK-293T and F-11 cells. Cells were imaged using total internal reflection fluorescence (TIRF) microscopy and the 2D and 3D trajectories of TRPM8 molecules were calculated by analyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel, most TRPM8 particles move in network covering the PM, periodically lingering for 2–8 s in confined microdomains of about 800 nm radius. Removing cholesterol with methyl-beta-cyclodextrin (MβCD) stabilizes TRPM8 motion in the PM and is correlated with larger TRPM8 current amplitude that results from an increase in the number of available channels without a change in open probability.

Conclusions/Significance

These results reveal a novel mechanism for regulating TRPM8 channel activity, and suggest that PM dynamics may play an important role in controlling electrical activity in cold-sensitive neurons.     

TRPA1-like channels enhance glycinergic transmission in medullary dorsal horn neurons

The effect of icilin, a potent agonist of transient receptor potential ankyrin 1 (TRPA1) and TRPM8, on glycinergic transmission was examined in mechanically isolated rat medullary dorsal horn neurons by use of the conventional whole-cell patch-clamp technique. Icilin increased the frequency of glycinergic spontaneous miniature inhibitory post-synaptic currents (mIPSCs) in a dose-dependent manner. Either allyl isothiocyanate(AITC) or cinnamaldehyde, other TRPA1 agonists, also increased mIPSC frequency, but the extent of facilitation induced by AITC or cinnamaldehyde was less than that induced by icilin. However, menthol, a TRPM8 agonist, had no facilitatory effect on glycinergic mIPSCs. The icilin-induced increase in mIPSC frequency was significantly inhibited by either HC030031, a selective TRPA1 antagonist, or ruthenium red, a non-selective transient receptor potential channel blocker. Icilin failed to increase glycinergic mIPSC frequency in the absence of extracellular Ca(2+), suggesting that the icilin-induced increase in mIPSC frequency is mediated by the Ca(2+) influx from the extracellular space. In contrast, icilin still increased mIPSC frequency either in the Na(+) -free external solution or in the presence of Cd(2+), a general voltage-dependent Ca(2+) channel blocker. The present results suggest that icilin acts on pre-synaptic TRPA1-like ion channels, which are permeable to Ca(2+), to enhance glycinergic transmission onto medullary dorsal horn neurons. The TRPA1-like channel-mediated enhancement of glycinergic transmission in medullary dorsal horn neurons would contribute to the regulation of pain information from the peripheral tissues.     

Antipsychotics inhibit TREK but not TRAAK channels

Schizophrenia is a chronic mental illness affecting 0.4% of the population. Existing antipsychotic drugs are mainly used to treat positive symptoms such as hallucinations but have only poor effects on negative symptoms such as cognitive deficits or depression. TREK and TRAAK channels are two P domain background potassium channels activated by polyunsaturated fatty acids and mechanical stress. TREK but not TRAAK channels are regulated by Gs- and Gq-coupled pathways. The inactivation of the TREK-1 but not the TRAAK channel in mice results in a depression-resistant phenotype. In addition, it has been shown that antidepressants such as fluoxetine or paroxetine directly inhibit TREK channel activity. Here we show that different antipsychotic drugs directly inhibit TREK currents with IC(50) values of approximately 1 to approximately 20 microM. No effect is seen on TRAAK channel activity. We conclude that TREK channels might be involved in the therapeutic action of antipsychotics or in their secondary effects. Furthermore, TREK channels could play a role in the pathophysiology of psychiatric disorders such as depression and schizophrenia.