TRPV1 Ion Channel Research Articles

Conditional permeabilization of the P. falciparum plasma membrane in infected cells links cation influx to reduced membrane integrity.

The intracellular human malaria parasite, Plasmodium falciparum, uses the PfATP4 cation pump to maintain Na+ and H+ homeostasis in parasite cytosol. PfATP4 is the target of advanced antimalarial leads, which produce many poorly understood metabolic disturbances within infected erythrocytes. Here, we expressed the mammalian ligand-gated TRPV1 ion channel at the parasite plasma membrane to study ion regulation and examine the effects of cation leak. TRPV1 expression was well-tolerated, consistent with negligible ion flux through the nonactivated channel. TRPV1 ligands produced rapid parasite death in the transfectant line at their activating concentrations, but were harmless to the wild-type parent. Activation triggered cholesterol redistribution at the parasite plasma membrane, reproducing effects of PfATP4 inhibitors and directly implicating cation dysregulation in this process. In contrast to predictions, TRPV1 activation in low Na+ media accentuated parasite killing but a PfATP4 inhibitor had unchanged efficacy. Selection of a ligand-resistant mutant revealed a previously uncharacterized G683V mutation in TRPV1 that occludes the lower channel gate, implicating reduced permeability as a mechanism for parasite resistance to antimalarials targeting ion homeostasis. Our findings provide key insights into malaria parasite ion regulation and will guide mechanism-of-action studies for advanced antimalarial leads that act at the host-pathogen interface.

Non-inositol 1,4,5-trisphosphate (IP3) receptor IP3-binding proteins

Conventionally, myo-D-inositol 1, 4,5-trisphosphate (IP3) is thought to exert its second messenger effects through the gating of IP3R Ca2+ release channels, located in Ca2+-storage organelles like the endoplasmic reticulum. However, there is considerable indirect evidence to support the concept that IP3 might interact with other, non-IP3R proteins within cells. To explore this possibility further, the Protein Data Bank was searched using the term “IP3”. This resulted in the retrieval of 203 protein structures, the majority of which were members of the IP3R/ryanodine receptor superfamily of channels. Only 49 of these structures were complexed with IP3. These were inspected for their ability to interact with the carbon-1 phosphate of IP3, since this is the least accessible phosphate group of its precursor, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This reduced the number of structures retrieved to 35, of which 9 were IP3Rs. The remaining 26 structures represent a diverse range of proteins, including inositol-lipid metabolizing enzymes, signal transducers, PH domain containing proteins, cytoskeletal anchor proteins, the TRPV4 ion channel, a retroviral Gag protein and fibroblast growth factor 2. Such proteins may impact on IP3 signalling and its effects on cell-biology. This represents an area open for exploration in the field of IP3 signalling.

The Ca2+ channel TRPV4 is dispensable for Ca2+ influx and cell volume regulation during hypotonic stress response in human keratinocyte cell lines

Cell swelling as a result of hypotonic stress is counteracted in mammalian cells by a process called regulatory volume decrease (RVD). We have recently discovered that RVD of human keratinocytes requires the LRRC8 volume-regulated anion channel (VRAC) and that Ca2+ exerts a modulatory function on RVD. However, the ion channel that is responsible for Ca2+ influx remains unknown. We investigated in this study whether the Ca2+-permeable TRPV4 ion channel, which functions as cell volume sensor in many cell types, may be involved in cell volume regulation during hypotonic stress response of human keratinocytes. We interfered with TRPV4 function in two human keratinocyte cell lines (HaCaT and NHEK-E6/E7) by using two TRPV4-specific inhibitors (RN1734 and GSK2193874), and by creating a CRISPR/Cas9-mediated genetic TRPV4−/− knockout in HaCaT cells. We employed electrophysiological patch clamp analysis, fluorescence-based Ca2+ imaging and cell volume measurements to determine the functional importance of TRPV4. We could show that both hypotonic stress and direct activation of TRPV4 by the specific agonist GSK1016790A triggered intracellular Ca2+ response. Strikingly, the Ca2+ increase upon hypotonic stress was neither affected by genetic knockout of TRPV4 in HaCaT cells nor by pharmacological inhibition of TRPV4 in both keratinocyte cell lines. Accordingly, hypotonicity-induced cell swelling, downstream activation of VRAC currents as well as subsequent RVD were unaffected both in TRPV4 inhibitor-treated keratinocytes and in HaCaT-TRPV4−/− cells. In summary, our study shows that keratinocytes do not require TRPV4 for coping with hypotonic stress, which implies the involvement of other, yet unidentified Ca2+ channels.

Endogenous Inflammatory Mediators Produced by Injury Activate TRPV1 and TRPA1 Nociceptors to Induce Sexually Dimorphic Cold Pain That Is Dependent on TRPM8 and GFRα3.

The detection of environmental temperatures is critical for survival, yet inappropriate responses to thermal stimuli can have a negative impact on overall health. The physiological effect of cold is distinct among somatosensory modalities in that it is soothing and analgesic, but also agonizing in the context of tissue damage. Inflammatory mediators produced during injury activate nociceptors to release neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P, inducing neurogenic inflammation, which further exasperates pain. Many inflammatory mediators induce sensitization to heat and mechanical stimuli but, conversely, inhibit cold responsiveness, and the identity of molecules inducing cold pain peripherally is enigmatic, as are the cellular and molecular mechanisms altering cold sensitivity. Here, we asked whether inflammatory mediators that induce neurogenic inflammation via the nociceptive ion channels TRPV1 (vanilloid subfamily of transient receptor potential channel) and TRPA1 (transient receptor potential ankyrin 1) lead to cold pain in mice. Specifically, we tested cold sensitivity in mice after intraplantar injection of lysophosphatidic acid or 4-hydroxy-2-nonenal, finding that each induces cold pain that is dependent on the cold-gated channel transient receptor potential melastatin 8 (TRPM8). Inhibition of CGRP, substance P, or toll-like receptor 4 (TLR4) signaling attenuates this phenotype, and each neuropeptide produces TRPM8-dependent cold pain directly. Further, the inhibition of CGRP or TLR4 signaling alleviates cold allodynia differentially by sex. Last, cold pain induced by both inflammatory mediators and neuropeptides requires TRPM8, as well as the neurotrophin artemin and its receptor GDNF receptor α3 (GFRα3). These results are consistent with artemin-induced cold allodynia requiring TRPM8, demonstrating that neurogenic inflammation alters cold sensitivity via localized artemin release that induces cold pain via GFRα3 and TRPM8.SIGNIFICANCE STATEMENT The cellular and molecular mechanisms that generate pain are complex with a diverse array of pain-producing molecules generated during injury that act to sensitize peripheral sensory neurons, thereby inducing pain. Here we identify a specific neuroinflammatory pathway involving the ion channel TRPM8 (transient receptor potential cation channel subfamily M member 8) and the neurotrophin receptor GFRα3 (GDNF receptor α3) that leads to cold pain, providing select targets for potential therapies for this pain modality.

Molecular Mechanisms of Neurogenic Inflammation of the Skin.

The skin, including the hypodermis, is the largest body organ and is in constant contact with the environment. Neurogenic inflammation is the result of the activity of nerve endings and mediators (neuropeptides secreted by nerve endings in the development of the inflammatory reaction in the skin), as well as interactions with other cells such as keratinocytes, Langerhans cells, endothelial cells and mast cells. The activation of TRPV-ion channels results in an increase in calcitonin gene-related peptide (CGRP) and substance P, induces the release of other pro-inflammatory mediators and contributes to the maintenance of cutaneous neurogenic inflammation (CNI) in diseases such as psoriasis, atopic dermatitis, prurigo and rosacea. Immune cells present in the skin (mononuclear cells, dendritic cells and mast cells) also express TRPV1, and their activation directly affects their function. The activation of TRPV1 channels mediates communication between sensory nerve endings and skin immune cells, increasing the release of inflammatory mediators (cytokines and neuropeptides). Understanding the molecular mechanisms underlying the generation, activation and modulation of neuropeptide and neurotransmitter receptors in cutaneous cells can aid in the development of effective treatments for inflammatory skin disorders.

Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes.

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human-induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype (WT). There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations up-regulated several anterior HOX genes and down-regulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment up-regulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.

Energetics and structural determinations of TRPV1 activation by vanilloids.

TRPV1 ion channel is a prototypical nociceptor. Vanilloids with similar structures, such as capsaicin, resiniferatoxin (RTX), and capsazepine, exhibit highly variable gating effects, from being a potent agonist to being an antagonist. To understand the structural and mechanistic differences in gating between these vanilloids, we first isolated intermediates states with varying number of bound RTX, using concatemers of wildtype rat TRPV1 subunits (RTX binds irreversibly) and Y511A mutant subunits (RTX binds reversibly). Equilibrium measurements from these concatemers revealed that binding of RTX to each subunit contributes 1.8 kcal/mol towards stabilizing the open state. Replacing RTX with capsaicin (whose binding to wildtype or mutant subunit is reversible) revealed that the Y511A mutation reduced capsaicin binding affinity by two orders of magnitude; capsaicin exerts a similar stabilization energy as RTX. Finally, we compared the energetic differences between RTX and two derivatives (5’-iRTX and 6’-iRTX), in which a one-atom difference makes the analogous molecules exhibit completely different gating effects on TRPV1: RTX is a full agonist, 6’-iRTX is a partial agonist, and 5’iRTX is an antagonist. We found that compared with RTX, 6’-iRTX only makes a small contribution (0.3 kcal/mol) to stabilize the open state, while 5’-iRTX contributes 1.2 kcal/mol to stabilize the close state. Combining energetic information with computational modeling and structural details of ligand-channel interactions allows a better understanding of TRPV1 activation and nociception.

Delivery of nonbiologically-compatible membrane protein constructs to mammalian cells for functional characterization.

A hallmark of integral membrane protein biogenesis is the various cellular pathways that identify and tightly regulate protein fates. These quality control mechanisms identify misfolded from well-folded membrane proteins and target them for degradation or trafficking to the appropriate cellular membrane. A current limitation in membrane protein biophysics is that many engineered membrane proteins used in structural, molecular, and mechanistic studies; including mutations, truncations, and chimeras, do not satisfy cellular quality control requirements, limiting their ability for cellular characterization. Our recently developed membrane delivery method enables recombinantly expressed and purified membrane proteins to be delivered back to cells for functional and other characterization. Here we extend these methods to characterize the delivery and evaluate the function of a minimal TRPV1 ion channel by whole-cell patch-clamp electrophysiology and other techniques. This minimal construct is ∼35% of the full-length human TRPV1 receptor and does not satisfy cellular quality control, and is thereby excluded from trafficking to the plasma membrane. Delivery of minimal TRPV1 directly to the plasma membrane enables its characterization and shows that it retains polymodal activation by heat, protons, and capsaicin, analogously to the full-length TRPV1 protein. These outcomes are validated with other methods such as electron microscopy and NMR data, enabling a bridge between cellular and biophysical studies by bypassing cellular quality control and trafficking pathways.

Structural Mechanism of Ionic Conductivity of the TRPV1 Channel

The so-called “hydrophobic gating” is widely discussed as a putative mechanism to control water and ion conduction via ion channels. This effect can occur in narrow areas of the channels pore lined by non-polar residues. In the closed state of the channel, such regions may spontaneously transit to a dehydrated state to block water and ions transport without full pore occlusion. In the open state, the hydrophobic gate is wide enough to provide sustainable hydration and conduction. Apparently, the transport through the open hydrophobic gate may by facilitated by some polar residues that assist polar/charged substances to overcome the energy barrier created by nonpolar environment. In this work, we investigated the behavior of Na+ ions and their hydration shells in the open pore of the rat TRPV1 ion channel by molecular dynamics simulations. We show that polar protein groups coordinate water molecules in such a way as to restore the hydration shell of ions in the hydrophobic gate that ensures ion transport through the gate in a fully hydrated state.

Protective and Therapeutic Role of Ginkgo Biloba Extract Through TRPV1 Channels in an in Vitro Alzheimer's Disease Model.

The effect of Ginkgo biloba (GB) on mitochondria-dependent TRPV1 ion channels in neuroblastoma cells was investigated by creating an Alzheimer's disease (AD) model. Okadaic acid was applied on SH-SY5Y cells to create an AD model. After cellular differentiation, the study was organized with the seven main groups, examining the effect of GB on calcium depended TRPV1 channels in neuroblastoma cells AD, has been established in vitro. The higher Ca2+ concentration was detected in the GB+AD, AD and AD+GB groups when compared with the control (p<0.001). The Ca2+ level was lower in GB+AD and AD+GB groups than in the AD group (p<0.001). Also, cytosolic Ca2+ concentration was lower in the GB+AD than in the AD+GB group (p<0.05), the apoptosis and intracellular reactive oxygen species (ROS) values were higher in the GB+AD, AD and AD+GB groups than in the control (p<0.001). The apoptosis and intracellular ROS values were higher in AD group than in the GB+AD and AD+GB group (p<0.001) and the apoptosis level was higher in AD+GB group than GB+AD group (p<0.001) and the mitochondrial depolarization, caspase 3 and caspase 9 levels were higher in the GB+AD, AD and AD+GB groups when compared to the control group (p<0.001). Also, the values were lower in the GB+AD group, AD group and AD+GB groups when compared with the GB+AD+capsazepine group, AD+capsazepine group and AD+GB+capsazepine respectively (p<0.001). These results show us that GB has a protective effect besides its therapeutic effect in Alzheimer's disease via TRPV1 channel.

Bioactive cell niche mediating uniform thermal stimulus for BMSC neural differentiation through TRPV1 channel activation.

As one of the physical stimulus tools to target neuromodulation-related biological entities, mild thermal stimulus has attracted increasing attention in unraveling neural differentiation processing. However, thermal stimulus for neural behavior regulation has been relatively unexplored due to the challenge in finding a good method of exerting thermal stimulus. Considering the distance-dependent temperature preservation efficiency and the native importance of a bioactive matrix, we herein put forward the design of a photothermal hydrogel by immobilizing photothermal dopamine (DA) in hyaluronic acid (HA) chains. Benefitting from the minuscule disaccharide repeat unit size (≈1 nm) of HA used for the DA grafting, and the additional adhesion capacity of the DA for recruiting cells, a uniformly close distance from heating source to cells is realized. Therefore, we successfully established a near-infrared light initiated photothermal stimulus platform, with full bioactivity and high thermal manipulation efficiency. After extensive characterization, we proved that the thermal activation, from matrix to cells, triggered TRPV1 ion channel opening and Ca2+ influx, which finally promoted neural differentiation of bone marrow mesenchymal stem cells (BMSCs). This work broadens the possibilities of polymeric photothermal materials, and is of great significance for remotely manipulating neural and other cellular machinery for stem cell therapeutics in tissue engineering.

How should we define a nociceptor in the gut-brain axis?

In the past few years, there has been extraordinary interest in how the gut communicates with the brain. This is because substantial and gathering data has emerged to suggest that sensory nerve pathways between the gut and brain may contribute much more widely in heath and disease, than was originally presumed. In the skin, the different types of sensory nerve endings have been thoroughly characterized, including the morphology of different nerve endings and the sensory modalities they encode. This knowledge is lacking for most types of visceral afferents, particularly spinal afferents that innervate abdominal organs, like the gut. In fact, only recently have the nerve endings of spinal afferents in any visceral organ been identified. What is clear is that spinal afferents play the major role in pain perception from the gut to the brain. Perhaps surprisingly, the majority of spinal afferent nerve endings in the gut express the ion channel TRPV1, which is often considered to be a marker of "nociceptive" neurons. And, a majority of gut-projecting spinal afferent neurons expressing TRPV1 are activated at low thresholds, in the "normal" physiological range, well below the normal threshold for detection of painful sensations. This introduces a major conundrum regarding visceral nociception. How should we define a "nociceptor" in the gut? We discuss the notion that nociception from the gut wall maybe a process encrypted into multiple different morphological types of spinal afferent nerve ending, rather than a single class of sensory ending, like free-endings, suggested to underlie nociception in skin.

Magnetic Systems for Regenerative Medicine

Magnetic Systems for Regenerative Medicine

Optimized method for the recombinant production of a sea anemone’s peptide

The production of the analgesic peptide HCRG21 for medical use is restricted by a number of limitations in the technology. The optimal biotechnological method scalable to industrial has been developed based on the production of a fusion protein containing a special leader, His-tag, and Smt3 sequence upstream in the peptide sequence. The resulting peptide shared its inhibitory activity to TRPV1 ion channel identical to the published early.

Thermal Nociception of Ionic Skin: TRPV1 Ion Channel-Inspired Heat-Activated Dynamic Ionic Liquid

The artificial reproduction of the tactile sensory function of natural skin is crucial for intelligent sensing, human-computer interaction, and medical health. Thermal nociception is an essential human tactile function to avoid noxious thermal stimuli, which depends on the specific heat-activation of the TRPV1 ion channel. Inspired by the TRPV1, a dynamic ionic liquid with heat-activation characteristics is designed and prepared, which can be activated at 45 °C, which is near the physiological noxious temperature, accompanied by a steep rise in electrical response signals. Its electrical behavior can be deemed to be the extreme version of temperature sensation similar to the natural thermal nociceptor. The heat-activation mechanism is confirmed as a feasible strategy to regulate the thermal response behavior of ions, and this reported dynamic ionic liquid has an unprecedented intrinsic temperature response sensitivity of up to 156.79%/°C. In consideration of the similarity between the heat-activated dynamic ionic liquid and the TRPV1 ion channel in terms of heat-activation characteristics, electrical output signal, and ultrathermal sensitivity, an all-liquid ionic skin with the ability of thermal nociception is further fabricated, which shows considerable potential to assist patients with tactile desensitization to avoid noxious thermal stimuli.

Site-Specific Fluorescent Labeling of the Cysteine-Rich Toxin, DkTx, for TRPV1 Ion Channel Imaging and Membrane Binding Studies.

Peptide toxins secreted by venomous animals bind to mammalian ion channel proteins and modulate their function. The high specificity of these toxins for their target ion channels enables them to serve as powerful tools for ion channel biology. Toxins labeled with fluorescent dyes are employed for the cellular imaging of channels and also for studying toxin-channel and toxin-membrane interactions. Several of these toxins are cysteine-rich, rendering the production of properly folded fluorescently labeled toxins technically challenging. Herein, we evaluate a variety of site-specific protein bioconjugation approaches for producing fluorescently labeled double-knot toxin (DkTx), a potent TRPV1 ion channel agonist that contains an uncommonly large number of cysteines (12 out of a total of 75 amino acids present in the protein). We find that popular cysteine-mediated bioconjugation approaches are unsuccessful as the introduction of a non-native cysteine residue for thiol modification leads to the formation of misfolded toxin species. Moreover, N-terminal aldehyde-mediated bioconjugation approaches are also not suitable as the resultant labeled toxin lacks activity. In contrast to these approaches, C-terminal bioconjugation of DkTx via the sortase bioconjugation technology yields functionally active fluorescently labeled DkTx. We employ this labeled toxin for imaging rat TRPV1 heterologously expressed in Xenopus laevis oocytes, as well as for performing membrane binding studies on giant unilamellar vesicles composed of different lipid compositions. Our studies set the stage for using fluorescent DkTx as a tool for TRPV1 biology and provide an informative blueprint for labeling cysteine-rich proteins.

Role of TRPV1 ion channel in cervical squamous cell carcinoma genesis.

The transient receptor potential (TRP) family is a widely expressed superfamily of ion channels that regulate intracellular Ca2+ homeostasis and signal transduction. Abnormal expression of TRPV1 is closely related to malignant tumors of the female reproductive system such as breast, ovarian, cervical and endometrial cancers. In this study, we found a significant reduction of TRPV1 expression in cervical squamous cell carcinoma and this expression is inversely association with the risk of cervical squamous cell carcinoma. Furthermore, TRPV1 is involved in cell differentiation, iron death, inflammatory response, and metabolic regulation in cervical squamous cell carcinoma. Meanwhile TRPV1 is positively correlated with T cells and negatively associated with macrophages, indicating that TRPV is associated with tumor cell immunity. Therefore, TRPV1 may be a potential marker of cervical cancer and a promising anti-cancer drug candidate.

Etv4 regulates nociception by controlling peptidergic sensory neuron development and peripheral tissue innervation.

The perception of noxious environmental stimuli by nociceptive sensory neurons is an essential mechanism for the prevention of tissue damage. Etv4 is a transcriptional factor expressed in most nociceptors in dorsal root ganglia (DRG) during the embryonic development. However, its physiological role remains unclear. Here, we show that Etv4 ablation results in defects in the development of the peripheral peptidergic projections in vivo, and in deficits in axonal elongation and growth cone morphology in cultured sensory neurons in response to NGF. From a mechanistic point of view, our findings reveal that NGF regulates Etv4-dependent gene expression of molecules involved in extracellular matrix (ECM) remodeling. Etv4-null mice were less sensitive to noxious heat stimuli and chemical pain, and this behavioral phenotype correlates with a significant reduction in the expression of the pain-transducing ion channel TRPV1 in mutant mice. Together, our data demonstrate that Etv4 is required for the correct innervation and function of peptidergic sensory neurons, regulating a transcriptional program that involves molecules associated with axonal growth and pain transduction.

Magnetothermal Modulation of Calcium‐Dependent Nerve Growth

AbstractNerve injuries are common, and the available treatments including invasive surgeries do not guarantee complete regeneration of the injured nerves and restoration of function. Despite the ability of peripheral nerves to regenerate, the slow rate of axonal growth hampers the functional recovery. Development of new approaches to discover underlying mechanisms that may accelerate axonal growth is needed to overcome these limitations and augment available treatments of nerve injury. In addition to chemical factors, recent studies suggested the use of optogenetics and electrical stimulation to promote axonal growth. The underlying mechanisms of these approaches, however, require further investigation. Furthermore, their application relies on invasive hardware, which may not be compatible with injured nerves under significant mechanical deformation. Here, it is shown that thermal activation of a heat sensitive ion channel TRPV1 promotes axonal growth in a calcium‐dependent manner. By leveraging heat dissipation of magnetic nanoparticles in alternating magnetic fields, the calcium influx through TRPV1 channels endogenously expressed in dorsal root ganglion explants is triggered remotely. The accelerated axonal growth through elongation of neurofilaments and increased Schwann cell migration following magnetothermal stimulation is observed. These findings suggest future applications of magnetothermal modulation of axonal growth as a minimally invasive approach to accelerate nerve regeneration.

Activation of serotonin receptor 2 by glucosylsphingosine can be enhanced by TRPA1 but not TRPV1: Implication of a novel glucosylsphingosine-mediated itch pathway

Glucosylsphingosine (GS) is an endogenous sphingolipid that specifically accumulates in the skin of patients with atopic dermatitis (AD). Notably, it was recently found that GS can induce itch sensation by activating serotonin receptor 2A and TRPV4 ion channels. However, it is still uncertain whether other molecules are involved in GS-induced itch sensation. Therefore, by using the calcium imaging technique, we investigated whether serotonin receptor 2 – specifically 2A and 2B – can interact with TRPV1 and TRPA1, because these are representative ion channels in the transmission of itch. As a result, it was found that GS did not activate TRPV1 or TRPA1 per se. Moreover, cells expressing both serotonin receptor 2 and TRPV1 did not show any changes in calcium responses. However, enhanced calcium responses were observed in cells expressing serotonin receptor 2 and TRPA1, suggesting a possible interaction between these two molecules. Similar synergistic effects were also observed in cells expressing serotonin receptor 2 and TRPA1, but not TRPV1. Furthermore, a phospholipase C inhibitor (U73122) and a store-operated calcium entry blocker (SKF96365) significantly reduced GS-induced responses in cells expressing both serotonin receptor 2 and TRPA1, but not with pre-treatment with a Gβγ-complex blocker (gallein). Therefore, we propose a putative novel pathway for GS-induced itch sensation, such that serotonin receptor 2 could be coupled to TRPA1 but not TRPV1 in sensory neurons.