A diacylglycerol photoswitching protocol for studying TRPC channel functions in mammalian cells and tissue slices.

Small molecular probes designed for photopharmacology and opto-chemogenetics are rapidly gaining widespread recognition for investigations of transient receptor potential canonical (TRPC) channels. This protocol describes the use of three photoswitchable diacylglycerol analogs-PhoDAG-1, PhoDAG-3, and OptoDArG-for ultrarapid activation and deactivation of native TRPC2 channels in mouse vomeronasal sensory neurons and olfactory type B cells, as well as heterologously expressed human TRPC6 channels. Photoconversion can be achieved in mammalian tissue slices and enables all-optical stimulation and shutoff of TRPC channels. For complete details on the use and execution of this protocol, please refer to Leinders-Zufall et al. (2018).

TRP Channels Expression Profile in Human End-Stage Heart Failure.

Objectives: Many studies indicate the involvement of transient receptor potential (TRP) channels in the development of heart hypertrophy. However, the data is often conflicted and has originated in animal models. Here, we provide systematic analysis of TRP channels expression in human failing myocardium. Methods and results: Left-ventricular tissue samples were isolated from explanted hearts of NYHA III-IV patients undergoing heart transplants (n = 43). Quantitative real-time PCR was performed to assess the mRNA levels of TRPC, TRPM and TRPV channels. Analysis of functional, clinical and biochemical data was used to confirm an end-stage heart failure diagnosis. Compared to myocardium samples from healthy donor hearts (n = 5), we detected a distinct increase in the expression of TRPC1, TRPC5, TRPM4 and TRPM7, and decreased expression of TRPC4 and TRPV2. These changes were not dependent on gender, clinical or biochemical parameters, nor functional parameters of the heart. We detected, however, a significant correlation of TRPC1 and MEF2c expression. Conclusions: The end-stage heart failure displays distinct expressional changes of TRP channels. Our findings provide a systematic description of TRP channel expression in human heart failure. The results highlight the complex interplay between TRP channels and the need for deeper analysis of early stages of hypertrophy and heart failure development.

Activation of mutated TRPA1 ion channel by resveratrol in human prostate cancer associated fibroblasts (CAF).

Previous studies showed the effects of resveratrol (RES) on several cancer cells, including prostate cancer (PCa) cell apoptosis without taking into consideration the impact of the tumor microenvironment (TME). The TME is composed of cancer cells, endothelial cells, blood cells, and cancer-associated fibroblasts (CAF), the main source of growth factors. The latter cells might modify in the TME the impact of RES on tumor cells via secreted factors. Recent data clearly show the impact of CAF on cancer cells apoptosis resistance via secreted factors. However, the effects of RES on PCa CAF have not been studied so far. We have investigated here for the first time the effects of RES on the physiology of PCa CAF in the context of TME. Using a prostate cancer CAF cell line and primary cultures of CAF from prostate cancers, we show that RES activates the N-terminal mutated Transient Receptor Potential Ankyrin 1 (TRPA1) channel leading to an increase in intracellular calcium concentration and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells. Interestingly, in the present work, we also show that when the prostate cancer cells were co-cultured with CAF, the RES-induced cancer cell apoptosis was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. The present work highlights CAF TRPA1 ion channels as a target for RES and the importance of the channel in the epithelial-stromal crosstalk in the TME leading to resistance to the RES-induced apoptosis.

TRPA1 receptor is upregulated in human oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory disease of unknown etiology with antigen-specific and non-specific mechanisms. Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel activated by noxious stimuli such as oxidative stress products evoking pain and release of proinflammatory mediators from sensory nerve endings culminating in neurogenic inflammation. Extraneuronal TRPA1s, for example, on immune cells possess yet unknown functions. SUBJECTS AND METHODS: We studied the buccal mRNA expression (qPCR) and protein localization (immunohistochemistry) of TRPA1 receptors and key OLP mediator transcripts in oral mucosa samples of healthy volunteers (n = 9), OLP patients (n = 43), and OLP-like hyperkeratotic patients (n = 12). RESULTS: We measured 27.7- and 25.5-fold TRPA1 mRNA increase in OLP and OLP-like hyperkeratotic patients compared to healthy controls. TRPA1 transcripts elevated 2.4-fold in hypertensive OLP but not in hyperkeratotic patients compared to counterparts, reduced by 1.6-fold by angiotensin-convertase inhibitor intake. TRPA1 messenger RNA was more coexpressed with transcripts of tumor necrosis factor α than with interferon γ. Keratinocytes, macrophages but not T cells expressed TRPA1. CONCLUSIONS: We provided evidence for the extraneuronal presence and upregulation of the proinflammatory TRPA1 receptor in buccal samples of patients with OLP. This may implicate the ion channel in the pathomechanism of OLP.

Localization and expression patterns of TRP channels in submandibular gland development.

OBJECTIVE: Expression of Transient receptor potential (TRP) channels: TRP canonical (TRPC)1, TRP vanilloid (TRPV)3, TRPV4 and TRP melastatin (TRPM)8 in adult rat salivary gland has recently been reported. The authors investigated expression of these TRP channels in the submandibular gland during early developmental stage in which the cell constitution is different, and discussed the function of TRP in the submandibular gland in early development. DESIGN: Using rat submandibular gland at embryonic days (E)18 and E20 and postnatal days (PN)0 and PN5 and PN28, expression of TRPV3, TRPV4, TRPC1 and TRPM8 was investigated using real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: All TRP channels were expressed in cells constituting the submandibular gland in early developmental stage, but an increase in the expression level at PN5 on RT-PCR was significant compared with those at E18, PN0 and PN28 in TRPC1 and TRPV4 channels, whereas an increase was observed but not significant in the others. On immunohistochemical staining at PN5, whereas strong reactions of anti-TRPM8 antibody, anti-TRPV3 and anti-TRPV4 antibodies were observed in cells which proliferated from a terminal portion of cells arranged tubular structure which previously constituted mostly the submandibular gland. CONCLUSION: It was clarified that TRP channels are expressed in the rat submandibular gland in early developmental stage although cells constituting the submandibular gland are different from those in adult animals, suggesting that these TRP channels are involved in cell differentiation in at PN5 into the adult submandibular gland during early development.

Transient receptor potential ankyrin 1 (TRPA1) is functionally expressed in primary human osteoarthritic chondrocytes.

BACKGROUND: Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, widely expressed in neuronal cells and involved in nociception and neurogenic inflammation. We showed recently that TRPA1 mediates cartilage degradation and joint pain in the MIA-model of osteoarthritis (OA) suggesting a hitherto unknown role for TRPA1 in OA. Therefore, we aimed to investigate whether TRPA1 is expressed and functional in human OA chondrocytes. METHODS: Expression of TRPA1 in primary human OA chondrocytes was assessed by qRT-PCR and Western blot. The functionality of the TRPA1 channel was assessed by Ca(2+)-influx measurements. Production of MMP-1, MMP-3, MMP-13, IL-6, and PGE2 subsequent to TRPA1 activation was measured by immunoassay. RESULTS: We show here for the first time that TRPA1 is expressed in primary human OA chondrocytes and its expression is increased following stimulation with inflammatory factors IL-1ß, IL-17, LPS, and resistin. Further, the TRPA1 channel was found to be functional, as stimulation with the TRPA1 agonist AITC caused an increase in Ca(2+) influx, which was attenuated by the TRPA1 antagonist HC-030031. Genetic depletion and pharmacological inhibition of TRPA1 downregulated the production of MMP-1, MMP-3, MMP-13, IL-6, and PGE2 in osteoarthritic chondrocytes and murine cartilage, respectively. CONCLUSIONS: The TRPA1 cation channel was found to be functionally expressed in primary human OA chondrocytes, which is an original finding. The presence and inflammatory and catabolic effects of TRPA1 in human OA chondrocytes propose a highly intriguing role for TRPA1 as a pathogenic factor and drug target in OA.

Data-driven Analysis of TRP Channels in Cancer: Linking Variation in Gene Expression to Clinical Significance.

BACKGROUND: Experimental evidence has suggested that transient receptor potential (TRP) channels play a crucial role in tumor biology. However, clinical relevance and significance of TRP channels in cancer remain largely unknown. MATERIALS AND METHODS: We applied a data-driven approach to dissect the expression landscape of 27 TRP channel genes in 14 types of human cancer using International Cancer Genome Consortium data. RESULTS: TRPM2 was found overexpressed in most tumors, whereas TRPM3 was broadly down-regulated. TRPV4 and TRPA1 were found up- and down-regulated respectively in a cancer type-specific manner. TRPC4 was found to be closely associated with incidence of head and neck cancer and poor survival of patients with kidney cancer. TRPM8 was identified as a new molecular marker for lung cancer diagnosis and TRPP1 for kidney cancer prognosis. CONCLUSION: Our data-driven approach demonstrates that the variation in the expression of TRP channel genes is manifested across various human cancer types and genes, for certain TRP channels have strong predictive diagnostic and prognostic potential.

Increased expression of three types of transient receptor potential channels (TRPA1, TRPV4 and TRPV3) in burn scars with post-burn pruritus.

Post-burn pruritus is a common distressing consequence of burn wounds. Empirical treatment often fails to have a satisfactory outcome on post-burn pruritus, as the mechanism of post-burn pruritus has not been fully elucidated. The aim of this study was to evaluate the manifestation of transient receptor potential (TRP) channels in post-burn pruritus. Fifty-one burn patients with (n=33) or without (n=18) pruritus were investigated, including skin biopsies. Not unexpectedly, the scarred body area was larger in the former group. In immunohistochemistry, TPRV3 was significantly elevated in the epidermis of burn scars with pruritus. Furthermore, real time- PCR showed that mRNA of TRPA1 and TRPV4 was increased in itching burn scars. Staining for substance P and CGRP did not differ between the 2 grouped, but the former neuropeptide was increased in burn scars. These results may help determine a specific therapeutic approach for post-burn pruritus.

Fatty acid composition of Drosophila photoreceptor light-sensitive microvilli.

Phototransduction, the mechanism underlying the electrical response to light in photoreceptor cells, has been thoroughly investigated in Drosophila melanogaster, an essential model in signal transduction research. These cells present a highly specialized photosensitive membrane consisting of thousands of microvilli forming a prominent structure termed a rhabdomere. These microvilli encompass the phototransduction proteins, most of which are transmembrane and exclusively rhabdomeric. Rhabdomere membrane lipids play a crucial role in the activation of the transient receptor potential ionic channels (TRP and TRPL) responsible for initiating the photoresponse. Despite its importance, rhabdomere lipid composition has not been established. We developed a novel preparation enriched in rhabdomere membranes to perform a thorough characterization of the lipidomics of Drosophila rhabdomeres. Isolated eyes (500) were homogenized and subjected to a differential centrifugation protocol that generates a fraction enriched in rhabdomere membrane. Lipids extracted from this preparation were identified and quantified by gas chromatography coupled to mass spectrometry. We found an abundance of low sterol esters (C16:0, C18:0), highly abundant and diverse triglycerides, free fatty acids, a moderate variety of mono and diacyglycerols (C:16:0, 18:0, C18:1) and abundant phospholipids (principally C18:2). This preparation opens a new avenue for investigating essential aspects of phototransduction.

The electrovomeronasogram: field potential recordings in the mouse vomeronasal organ.

Mammalian vomeronasal neurons (VSNs) located in the sensory epithelium of the vomeronasal organ (VNO) detect and transduce molecular cues emitted by other individuals and send this information to the olfactory forebrain. The initial steps in the detection of pheromones and other chemosignals by VSNs involve interaction of a ligand with a G protein-coupled receptor and downstream activation of the primary signal transduction cascade, which includes activation of ion channels located in microvilli and the dendritic tip of a VSN. The electrovomeronasogram (EVG) recording technique provides a sensitive means through which ligand-induced activation of populations of VSNs can be recorded from the epithelial surface using an intact, ex vivo preparation of the mouse VNO. We describe methodological aspects of this preparation and the EVG recording technique which, together with single-cell recordings, contributed significantly to our understanding of mammalian vomeronasal function, the identification of pheromonal ligands, and the analysis of mice with targeted deletions in specific signal transduction molecules such as Trpc2, Gαo, V1R, or V2R receptors.

Immunolocalization and distribution of functional temperature-sensitive TRP channels in salivary glands.

Transient receptor potential (TRP) cation channels are unique cellular sensors involved in multiple cellular functions. Their role in salivary secretion remains to be elucidated. The expression and localization of temperature-sensitive TRP channels in salivary (submandibular, sublingual and parotid) glands were analyzed by immunohistochemistry and quantitative real-time reverse transcription plus the polymerase chain reaction (RT-PCR). The effects of various TRP channel agonists on carbachol (CCh)-induced salivary secretion in the submandibular gland and on the intracellular Ca(2+) concentration ([Ca(2+)]i) in a submandibular epithelial cell line were also investigated. Immunohistochemistry revealed the expression of TRP-melastatin subfamily member 8 (TRPM8) and TRP-ankyrin subfamily member 1 (TRPA1) in myoepithelial, acinar and ductal cells in the sublingual, submandibular and parotid glands. In addition, TRP-vanilloid subfamily member 1 (TRPV1), TRPV3 and TRPV4 were also expressed in myoepithelial, acinar and ductal cells in all three types of gland. Quantitative real-time RT-PCR results demonstrated the mRNA expression of TRPV1, TRPV3, TRPV4, TRPM8 and TRPA1 in acinar and ductal cells in these salivary glands. Perfusion of the entire submandibular gland with the TRPV1 agonist capsaicin (1 µM) via the submandibular artery significantly increased CCh-induced salivation, whereas perfusion with TRPM8 and TRPA1 agonists (0.5 µM WS12 and 100 µM allyl isothiocyanate) decreased it. Application of agonists for each of the thermosensitive TRP channels increased [Ca(2+)]i in a submandibular epithelial cell line. These results indicate that temperature-sensitive TRP channels are localized and distributed in acinar, ductal and myoepithelial cells in salivary glands and that they play a functional role in the regulation and/or modulation of salivary secretion.

Novel therapeutic strategy to prevent chemotherapy-induced persistent sensory neuropathy by TRPA1 blockade.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe and painful adverse reaction of cancer treatment in patients that is little understood or treated. Cytotoxic drugs that cause CIPN exert their effects by increasing oxidative stress, which activates the ion channel TRPA1 expressed by nociceptors. In this study, we evaluated whether TRPA1 acted as a critical mediator of CIPN by bortezomib or oxaliplatin in a mouse model system. Bortezomib evoked a prolonged mechanical, cold, and selective chemical hypersensitivity (the latter against the TRPA1 agonist allyl isothiocyanate). This CIPN hypersensitivity phenotype that was stably established by bortezomib could be transiently reverted by systemic or local treatment with the TRPA1 antagonist HC-030031. A similar effect was produced by the oxidative stress scavenger α-lipoic acid. Notably, the CIPN phenotype was abolished completely in mice that were genetically deficient in TRPA1, highlighting its essential role. Administration of bortezomib or oxaliplatin, which also elicits TRPA1-dependent hypersensitivity, produced a rapid, transient increase in plasma of carboxy-methyl-lysine, a by-product of oxidative stress. Short-term systemic treatment with either HC-030031 or α-lipoic acid could completely prevent hypersensitivity if administered before the cytotoxic drug. Our findings highlight a key role for early activation/sensitization of TRPA1 by oxidative stress by-products in producing CIPN. Furthermore, they suggest prevention strategies for CIPN in patients through the use of early, short-term treatments with TRPA1 antagonists.

Transient receptor potential channels - emerging novel drug targets for the treatment of pain.

In mammals several members of the Transient Receptor Potential channel family (TRPs), expressed mainly in the sensory neurons and skin keratinocytes, are implicated in relevant physiological functions, including thermosensation,nociception and vision. Since the TRPV1-4, TRPA1 and TRPM8 channels from this family play a pivotal role in both the detection and possibly modulation of painful stimuli, they are regarded as a very promising target of novel analgesic drugs. A few agents acting at TRPs, such as capsaicin or menthol, have a long history of their application as analgesics,whereas others (e.g. SB705498, JTS653, JNJ17203212, AP18, A967079, Chembridge-5861528 or PBMC) are currently being evaluated both in animals and in humans. In this review we discuss pain physiology, as well as the pharmacological properties of the TRPs involved in pain detection as potential critical peripheral analgesic targets. We present one of the most relevant strategies in the search for novel analgesic drugs, namely the TRP channels and their ligands, both agonists and antagonists as potential novel therapeutics for inflammatory and neuropathic pain syndromes. The safety profile of these agents, in particular their impact on thermosensation, is also discussed below.

Fatty acid composition of Drosophila photoreceptor light-sensitive microvilli

Phototransduction, the mechanism underlying the electrical response to light in photoreceptor cells, has been thoroughly investigated in Drosophila melanogaster, an essential model in signal transduction research. These cells present a highly specialized photosensitive membrane consisting of thousands of microvilli forming a prominent structure termed a rhabdomere. These microvilli encompass the phototransduction proteins, most of which are transmembrane and exclusively rhabdomeric. Rhabdomere membrane lipids play a crucial role in the activation of the transient receptor potential ionic channels (TRP and TRPL) responsible for initiating the photoresponse. Despite its importance, rhabdomere lipid composition has not been established. We developed a novel preparation enriched in rhabdomere membranes to perform a thorough characterization of the lipidomics of Drosophila rhabdomeres. Isolated eyes (500) were homogenized and subjected to a differential centrifugation protocol that generates a fraction enriched in rhabdomere membrane. Lipids extracted from this preparation were identified and quantified by gas chromatography coupled to mass spectrometry. We found an abundance of low sterol esters (C16:0, C18:0), highly abundant and diverse triglycerides, free fatty acids, a moderate variety of mono and diacyglycerols (C:16:0, 18:0, C18:1) and abundant phospholipids (principally C18:2). This preparation opens a new avenue for investigating essential aspects of phototransduction.

Expression of transient receptor potential ankyrin 1 in human dental pulp.

INTRODUCTION: Transient receptor potential ankyrin 1 (TRPA1) is activated by noxious cold (<17°C) and contributes to cold and mechanical hypersensitivity after inflammation and nerve injury. METHODS: To investigate whether TRPA1 is involved in the mediation of nociception, including noxious cold and cold hypersensitivity in teeth, we examined the expression of TRPA1 and sodium channel Nav1.8 in human dental pulp using fluorescent and electron microscopic immunocytochemistry. RESULTS: TRPA1 was expressed in a large number of axons branching extensively in the peripheral pulp and in a few axons within the nerve bundles in the core of the coronal pulp and in the radicular pulp. Under electron microscopy, TRPA1 immunoreactivity was typically localized near the plasma membrane of unmyelinated axons in the peripheral pulp, suggesting that in these axons it may act as a functional receptor. The proportion of axons expressing TRPA1 in neurofilament 200-positive axons significantly increased in the painful pulp compared with the normal pulp. TRPA1 was also densely expressed in the processes and the cell body of odontoblasts. A large number of axons coexpressed TRPA1 and Nav1.8. CONCLUSIONS: These findings support the notion that TRPA1 is involved in the perception of noxious cold and cold hypersensitivity in human dental pulp and that TRPA1-mediated nociception is primarily mediated by axons and odontoblasts in the peripheral pulp.

Primary alcohols activate human TRPA1 channel in a carbon chain length-dependent manner.

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is mainly expressed in primary nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent compounds such as mustard oil and cinnamaldehyde, and intracellular alkalization. Here, we show that primary alcohols, which have been reported to cause skin, eye or nasal irritation, activate human TRPA1 (hTRPA1). We measured intracellular Ca(2+) changes in HEK293 cells expressing hTRPA1 induced by 1 mM primary alcohols. Higher alcohols (1-butanol to 1-octanol) showed Ca(2+) increases proportional to the carbon chain length. In whole-cell patch-clamp recordings, higher alcohols (1-hexanol to 1-octanol) activated hTRPA1 and the potency increased with the carbon chain length. Higher alcohols evoked single-channel opening of hTRPA1 in an inside-out configuration. In addition, cysteine at 665 in the N terminus and histidine at 983 in the C terminus were important for hTRPA1 activation by primary alcohols. Furthermore, straight-chain secondary alcohols increased intracellular Ca(2+) concentrations in HEK293 cells expressing hTRPA1, and both primary and secondary alcohols showed hTRPA1 activation activities that correlated highly with their octanol/water partition coefficients. On the other hand, mouse TRPA1 did not show a strong response to 1-hexanol or 1-octanol, nor did these alcohols evoke significant pain in mice. We conclude that primary and secondary alcohols activate hTRPA1 in a carbon chain length-dependent manner. TRPA1 could be a sensor of alcohols inducing skin, eye and nasal irritation in human.

Human dental pulp fibroblasts express the "cold-sensing" transient receptor potential channels TRPA1 and TRPM8.

INTRODUCTION: Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts. METHODS: Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca(2+) microfluorimetry. RESULTS: Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca(2+) microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca(2+)](i)) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists. CONCLUSIONS: Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth.

Odontoblast TRP channels and thermo/mechanical transmission.

Odontoblasts function as mechanosensory receptors because of the expression of mechanosensitive channels in these cells. However, it is unclear if odontoblasts direct the signal transmission evoked by heat/cold or osmotic changes. This study investigated the effects of heat/cold or osmotic changes on calcium signaling and the functional expression of the thermo/mechanosensitive transient receptor potential (TRP) channels in primary cultured mouse odontoblastic cells, with the use of RT-PCR, fluorometric calcium imaging, and electrophysiology. TRPV1, TRPV2, TRPV3, TRPV4, and TRPM3 mRNA was expressed, but TRPM8 and TRPA1 mRNA was not. The receptor-specific stimulation of TRPV1-3 (heat-sensing receptors) and TRPV4/ TRPM3 (mechanic receptors) caused increases in the intracellular calcium concentration. Moreover, the channel activities of TRPV1-4 and TRPM3 were confirmed by a whole-cell patch-clamp technique. These results suggest that primary cultured mouse odontoblasts express heat/mechanosensitive TRP channels and play a role in the underlying mechanisms of thermo/mechanosensitive sensory transmission.

Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine.

The functional activity of a number of ion channels is highly sensitive to large changes in temperature. Foremost among these are the thermosensing TRP channels which include cold- (TRPM8, TRPA1), warmth- (TRPV3, TRPV4), and heat-sensing (TRPV1, TRPV2) members. TRPV1, also known as the vanilloid receptor (VR1), is activated by ligands such as capsaicin, acidic pH, and heat (an increase in temperature to approximately 42 degrees C will lead to channel opening). Screening against the thermal gating of TRPV1 is generally performed using perfusion systems or water baths for temperature control, in conjunction with electrophysiology or Ca2 + influx readouts for direct functional assessment. These approaches are very useful, but have limited throughput or minimal thermo-temporal control. A standard real-time PCR machine with standard microplates allowed us to combine fluorescent Ca2 + detection with precise temperature manipulation to develop a homogeneous (Z' = 0.53), cell-based assay that uses temperature as the agonist. A temperature response curve of TRPV1 was obtained, which provided a T50 of 46.1 degrees C, and IC50 values against heat agonism were determined for known TRPV1 antagonists. Furthermore, we expanded this approach to a cold-activated ion channel, TRPM8. We developed and validated an analytical technique with broad applications for the study and screening of temperature-gated ion channels.