Calcium-permeable Non-selective Cation Channel Research Articles

Role of TRPM7 in cerebral ischaemia and hypoxia.

Transient receptor potential melastatin 7 (TRPM7) channel, a calcium-permeable non-selective divalent cation channel, is broadly expressed in various cells and tissues, including the brain. TRPM7 is thought to be coupled to the metabolic state and regulate calcium homeostasis in the cell. TRPM7 takes part in a wide range of cell biology processes that affect cell growth and proliferation, as well as in embryonic development and skeleton formation. TRPM7 plays a significant role in ischaemic and hypoxic brain injury and neuronal cell death. TRPM7, as a key non-glutamate mechanism of cerebral ischaemia, also triggers an intracellular ionic imbalance and neuronal cell death in ischaemia and hypoxia. We have reported that TRPM7 is expressed in neurons of the hippocampus and cortex and activation of TRPM7 induced ischaemic neuronal cell death; suppression of TRPM7 with virally mediated gene silencing using siRNA reduced ischaemic neuronal cell death and improved neurobehavioural outcomes in vivo. Recently, we also demonstrated that inhibition of TRPM7 using pharmacological means promoted neuronal outgrowth in vitro and provided neuroprotection against brain injury to hypoxia in vivo. Thus, we have shown the contributions of TRPM7 in many physiological and pathophysiological processes, including hypoxia and ischaemia.

Resiniferatoxin: The Evolution of the "Molecular Scalpel" for Chronic Pain Relief.

Control of chronic pain is frequently inadequate or can be associated with debilitating side effects. Ablation of certain nociceptive neurons, while retaining all other sensory modalities and motor function, represents a new therapeutic approach to controlling severe pain while avoiding off-target side effects. transient receptor potential cation channel subfamily V member 1 (TRPV1) is a calcium permeable nonselective cation channel expressed on the peripheral and central terminals of small-diameter sensory neurons. Highly selective chemoablation of TRPV1-containing peripheral nerve endings, or the entire TRPV1-expressing neuron itself, can be used to control chronic pain. Administration of the potent TRPV1 agonist resiniferatoxin (RTX) to neuronal perikarya or nerve terminals induces calcium cytotoxicity and selective lesioning of the TRPV1-expressing nociceptive primary afferent population. This selective neuroablation has been coined “molecular neurosurgery” and has the advantage of sparing motor, proprioceptive, and other somatosensory functions that are so important for coordinated movement, performing activities of daily living, and maintaining quality of life. This review examines the mechanisms and preclinical data underlying the therapeutic use of RTX and examples of such use for the management of chronic pain in clinical veterinary and human pain states.

TRPV4: Molecular Conductor of a Diverse Orchestra.

Transient receptor potential vanilloid type 4 (TRPV4) is a calcium-permeable nonselective cation channel, originally described in 2000 by research teams led by Schultz (Nat Cell Biol 2: 695-702, 2000) and Liedtke (Cell 103: 525-535, 2000). TRPV4 is now recognized as being a polymodal ionotropic receptor that is activated by a disparate array of stimuli, ranging from hypotonicity to heat and acidic pH. Importantly, this ion channel is constitutively expressed and capable of spontaneous activity in the absence of agonist stimulation, which suggests that it serves important physiological functions, as does its widespread dissemination throughout the body and its capacity to interact with other proteins. Not surprisingly, therefore, it has emerged more recently that TRPV4 fulfills a great number of important physiological roles and that various disease states are attributable to the absence, or abnormal functioning, of this ion channel. Here, we review the known characteristics of this ion channel's structure, localization and function, including its activators, and examine its functional importance in health and disease.

Activation of TRPV4 Regulates Respiration through Indirect Activation of Bronchopulmonary Sensory Neurons.

Transient receptor potential vanilloid receptor 4 (TRPV4) is a calcium-permeable non-selective cation channel implicated in numerous physiological and pathological functions. This study aimed to investigate the effect of TRPV4 activation on respiration and to explore the potential involvement of bronchopulmonary sensory neurons. Potent TRPV4 agonist GSK1016790A was injected into right atrium in anesthetized spontaneously breathing rats and the changes in breathing were measured. Patch-clamp recording was performed to investigate the effect of GSK1016790A or another TRPV4 activator 4α-PDD on cultured rat vagal bronchopulmonary sensory neurons. Immunohistochemistry was carried out to determine the TRPV4-expressing cells in lung slices obtained from TRPV4-EGFP mice. Our results showed, that right-atrial injection of GSK1016790A evoked a slow-developing, long-lasting rapid shallow breathing in anesthetized rats. Activation of TRPV4 also significantly potentiated capsaicin-evoked chemoreflex responses. The alteration in ventilation induced by GSK1016790A was abolished by cutting or perineural capsaicin treatment of both vagi, indicating the involvement of bronchopulmonary afferent neurons. The stimulating and sensitizing effects of GSK1016790A were abolished by a selective TRPV4 antagonist GSK2193874 and also by inhibiting cyclooxygenase with indomethacin. Surprising, GSK1016790A or 4α-PDD did not activate isolated bronchopulmonary sensory neurons, nor did they modulate capsaicin-induced inward currents in these neurons. Furthermore, TRPV4 expression was found in alveolar macrophages, alveolar epithelial, and vascular endothelial cells. Collectively, our results suggest that GSK1016790A regulates the respiration through an indirect activation of bronchopulmonary sensory neurons, likely via its stimulation of other TRPV4-expressing cells in the lungs and airways.

Intrathecal resiniferatoxin in a dog model: efficacy in bone cancer pain.

Resiniferatoxin (RTX) is the most potent among all known endogenous and synthetic agonists for the transient receptor potential vanilloid 1 (TRPV1) receptor, which is a calcium-permeable nonselective cation channel, expressed on the peripheral and central terminals of small-diameter sensory neurons. Prolonged calcium influx induced by RTX causes cytotoxicity and death of only those sensory neurons that express the TRPV1 ion channel leading to selective targeting and permanent deletion of the TRPV1-expressing C-fiber neuronal cell bodies in the dorsal root ganglia. The goal of this project was to provide preclinical efficacy data, that intrathecal RTX could provide effective pain relief and improve function in dogs with bone cancer without significant long-term side effects. In a single-blind, controlled study, 72 companion dogs with bone cancer pain were randomized to standard of care analgesic therapy alone (control, n = 36) or 1.2 μg/kg intrathecal RTX in addition to standard of care analgesic therapy (treated, n = 36). Significantly more dogs in the control group (78%) required unblinding and adjustment in analgesic protocol or euthanasia within 6 weeks of randomization, than dogs that were treated with RTX (50%; P < 0.03); and overall, dogs in the control group required unblinding significantly sooner than dogs that had been treated with RTX (P < 0.02). The analgesic effect was documented in these dogs without any evidence of development of deafferentation pain syndrome that can be seen with neurolytic therapies.

TRPV4 Agonist GSK1016790A Regulates Respiration through Indirect Activation of Bronchopulmonary Sensory Neurons

Transient receptor potential vanilloid receptor 4 (TRPV4) is a calcium-permeable non-selective cation channel that has recently been implicated in numerous physiological functions. In this study, we investigate the ventilatory effect of a novel and potent TRPV4 agonist GSK1016790A and further explore the potential involvement of bronchopulmonary sensory neurons. Our results show: 1) in anesthetized rats, right-atrial injection of GSK1016790A evokes a slow-developing, long-lasting rapid shallow breathing; it also significantly potentiates capsaicin-evoked chemoreflexes; 2) the alteration in ventilation induced by GSK1016790A is prevented by cutting or perineural capsaicin treatment of both vagi, indicating the involvement of bronchopulmonary afferent neuron activation; 3) stimulating and sensitizing effects of GSK1016790A are abolished by TRPV4 antagonist GSK2193874 and also by systemic infusion of COX inhibitor indomethacin; 4) in patch-clamp recordings, GSK1016790A does not activate isolated bronchopulmonary sensory neurons, nor does it modulate capsaicin-evoked TRPV1 current in these neurons; and 5) abundant TRPV4 expression is found in alveolar macrophages as well as in vascular endothelial and alveolar epithelial cells. Collectively, our results suggest that GSK1016790A regulates respiration through an indirect activation of bronchopulmonary sensory neurons, likely via its stimulation of other TRPV4 expressing cells in lungs and airways. (Funded by Medcen Community Health Foundation, Mercer University School of Medicine and NIGMS)

Identification of Significant Amino Acids in Multiple Transmembrane Domains of Human Transient Receptor Potential Ankyrin 1 (TRPA1) for Activation by Eudesmol, an Oxygenized Sesquiterpene in Hop Essential Oil

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is activated by various noxious or irritant substances in nature, including spicy compounds. Many TRPA1 chemical activators have been reported; however, only limited information is available regarding the amino acid residues that contribute to the activation by non-electrophilic activators, whereas activation mechanisms by electrophilic ligands have been well characterized. We used intracellular Ca(2+) measurements and whole-cell patch clamp recordings to show that eudesmol, an oxygenated sesquiterpene present at high concentrations in the essential oil of hop cultivar Hallertau Hersbrucker, could activate human TRPA1. Gradual activation of inward currents with outward rectification by eudesmol was observed in human embryonic kidney-derived 293 cells expressing human TRPA1. This activation was completely blocked by a TRPA1-specific inhibitor, HC03-0031. We identified three critical amino acid residues in human TRPA1 in putative transmembrane domains 3, 4, and 5, namely threonine at 813, tyrosine at 840, and serine at 873, for activation by β-eudesmol in a systematic mutational study. Our results revealed a new TRPA1 activator in hop essential oil and provide a novel insight into mechanisms of human TRPA1 activation by non-electrophilic chemicals.

Functional Analysis of the Thermosensor TRPM3 in Intact Sensory Fibers Using the Skin-Nerve Assay

Transient receptor potential melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is selectively activated by the neurosteroid pregnenolone sulphate (PS). Previous in vitro and in vivo experiments demonstrated that TRPM3 functions as a heat sensor in the somatosensory system. Although behavioral assays in combination with single-cell recordings on isolated sensory neurons can yield important insight into the role of TRPM3 in thermosensation, the procedure results in the isolation of neuronal cell bodies, devoid of the sensory nerve endings. Moreover, DRG neurons in primary culture lack the context of adjacent cells (e.g. keratinocytes), which are known to influence thermal sensation by the somatosensory system. The skin-nerve preparation is an ex vivo technique that bridges the gap between in vitro cellular recordings and behavioral assays. With this technique, we evaluated the functional role of TRPM3 in C-fibers. Our results indicate functional expression of TRPM3 in sensory terminals of a subset of mechano-heat sensitive C-fibers. Other types of C-fibers (mechano-cold, high- and low-threshold mechano) were insensitive to TRPM3 agonists. These experiments indicate a role for TRPM3 in heat sensation by C-fibers in the somatosensory system.

Abstract 2722: Suppression of breast cancer cell migration by novel inhibitors that target transient receptor potential-melastatin-like 7 (Trpm7) kinase activity

Abstract TRPM7 (transient receptor potential melastatin 7) encodes a calcium permeable non-selective cation channel implicated in cell adhesion and magnesium homoeostasis. The TRPM7 gene encodes for a protein kinase whose activity is linked to the control of actomyosin contractility. TRPM7 mediates adhesion and migration of MDA-MB-231 breast cancer cells and recently has been reported to promote breast tumor cell metastasis. The lack of cell-permeable pharmacological inhibitors of the kinase domain represents a barrier to fully understanding kinase function. Herein, we describe the discovery of several compounds that target TRPM7 kinase activity and which were shown to decrease the binding of Myosin IIB to TRPM7 in HEK293 cells and MDA-MB-231 breast cancer cells transfected with pCMV6-TRPM7. Interestingly, two of the inhibitors potentially inhibited MDA-MB-231 breast cancer cells migration that have been reported to be regulated by TRPM7 kinase activity. Finally, magnesium starvation, which promotes TRPM7 kinase activity, induces phosphorylation of eEF2. Treatment of cells with several of the compounds decreased eEF2 phosphorylation under conditions of magnesium starvation, consistent with the notion that they inhibit TRPM7 kinase activity in cells. Citation Format: Tamer Saad Kaoud, Jihyun Park, Clint D.J. Tavares, Shreya Mitra, Micael Cano, Chun-Chia Tseng, Kevin N. Dalby. Suppression of breast cancer cell migration by novel inhibitors that target transient receptor potential-melastatin-like 7 (Trpm7) kinase activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2722. doi:10.1158/1538-7445.AM2014-2722

Activation of transient receptor potential vanilloid 1 decreases endothelial nitric oxide synthase phosphorylation at Thr497 by protein phosphatase 2B‐dependent dephosphorylation of protein kinase C (696.1)

We investigated the effects and underlying molecular mechanism of transient receptor potential vanilloid 1 (TRPV1), a calcium‐permeable non‐selective cation channel, on phosphorylation of endothelial nitric oxide synthase (eNOS) at threonine 497 (Thr497) in bovine aortic endothelial cells (BAECs) and in mice. In BAECs, treatment with the TRPV1 ligand evodiamine decreased the phosphorylation of eNOS at Thr497, protein kinase Cα (PKCα at Serine 657, and PKCβ2 at Serine 660. Evodiamine increased protein phosphatase 2B (PP2B) activity and promoted the formation of a PP2B‐PKC complex. Inhibition of TRPV1 activation, removal of extracellular calcium or pharmacological inhibition of PI3K/Akt/CaMKII/AMPK signaling pathway abolished the evodiamine‐induced alterations in phosphorylation of eNOS at Thr497, PKCα, PKCβ2 and PP2B activity, as well as the formation of a PP2B‐PKC complex. Inhibition of PP2B activation partially reduced the evodiamine‐induced NO bioavailability and tube formation in ECs and angiogenesis in mice. Moreover, evodiamine decreased the phosphorylation of eNOS at Thr497, PKCα, and PKCβ2 in apolipoprotein E (ApoE)‐deficient mouse aortas but not TRPV1‐deficient or ApoE/TRPV1 double‐knockout mice. TRPV1 activation in ECs may elicit a calcium‐dependent effect on PP2B‐PKC signaling, which leads to dephosphorylation of eNOS at Thr497 in ECs and in mice.Grant Funding Source: National Science Council, Taiwan

Novel TRPM3 Agonist - Single Compound Opens Multiple Ion Permeation Pathways

TRPM3 is a calcium-permeable nonselective cation channel in the melastatin (TRPM) subfamily of TRP channels. It represents a typical example of a polymodally gated TRP channel, in that it can be activated by chemical ligands such as nifedipine and the neurosteroid pregnenolone sulphate (PS), as well as by physical stimuli such as heat and membrane depolarization. Recently, we have found evidence for an alternative ion permeation pathway distinct from the central pore, which can be gated by combined application of PS and exogenous chemicals such as clotrimazole. This alternative ion permeation pathway is preserved following desensitization, blockade, mutagenesis and chemical modification of the central pore, and enables massive Na+ influx at negative voltages. By screening a compound library, we identified CIM021600 as potent agonist of TRPM3. The compound exhibited a marked specificity for TRPM3 and induced [Ca2+]I signals in somatosensory neurons. Intriguingly, single application of CIM021600 was able to activate both the central pore ion permeation pathway and the alternative ion permeation pathway. Since physiological functions of TRPM3 and the alternative ion permeation pathway of TRPM3 are still poorly defined, the identification of a potent and selective activator is expected to contribute to clarifying the role of TRPM3 in vivo.

The Cation Channel TRPV4 Regulates Epithelial Barrier Responses to Lipopolysaccharides

TRPV4 is a calcium-permeable non-selective cation channel belonging to the vanilloid subfamily of Transient Receptor Potential proteins. This channel has a rather ubiquitous expression in epithelial cells (skin, airways, endothelium and urothelium), and it is known to be sensitive to a wide variety of physical and chemical stimuli, including heat, arachidonic acid metabolites and synthetic α-phorbol derivatives. We have found that lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, activates TRPV4 in HEK293T cells, independently of the renowned TLR4 pathway. Interestingly, conical LPS from E.coli induced larger TRPV4 response than cylindrical LPS from S. minnesota. Considering the role of TRPV4 in mechanosensation we hypothesized that TRPV4 senses the disturbances induced by insertion of LPS in the plasma membrane. By measuring membrane fluidity using fluorescence probes, we found that conically-shaped LPS from E. coli, but not cylindrical LPS from S. minnesota, causes a membrane phase shift towards gel-like state. This suggests that LPS structural differences are important in its ability to induce mechanical alterations in the membrane. We also found that LPS activates native TRPV4 in the human bronchial epithelial cell line 16HBE. Acute stimulation of TRPV4 in monolayer cultures of 16HBE cells induced a rapid increase of the transepithelial electrical resistance. Additionally, LPS-mediated TRPV4 activation increased colocalization of ZO-1 and Occludin in the plasma membrane. Altogether, these data suggest that LPS induces a TRPV4-dependent reorganization of tight junction proteins, leading to an increase in barrier function. We propose that TRPV4 participates in the orchestration of the innate immune response to the challenge with bacterial endotoxins.

Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors.

BackgroundProlonged intracellular calcium elevation contributes to sensitization of nociceptors and chronic pain in inflammatory conditions. The underlying molecular mechanisms remain unknown but store-operated calcium entry (SOCE) components participate in calcium homeostasis, potentially playing a significant role in chronic pain pathologies. Most G protein-coupled receptors activated by inflammatory mediators trigger calcium-dependent signaling pathways and stimulate SOCE in primary afferents. The aim of the present study was to investigate the role of TRPC3, a calcium-permeable non-selective cation channel coupled to phospholipase C and highly expressed in DRG, as a link between activation of pro-inflammatory metabotropic receptors and SOCE in nociceptive pathways.ResultsUsing in situ hybridization, we determined that TRPC3 and TRPC1 constitute the major TRPC subunits expressed in adult rat DRG. TRPC3 was found localized exclusively in small and medium diameter sensory neurons. Heterologous overexpression of TRPC3 channel subunits in cultured primary DRG neurons evoked a significant increase of Gd3+-sensitive SOCE following thapsigargin-induced calcium store depletion. Conversely, using the same calcium add-back protocol, knockdown of endogenous TRPC3 with shRNA-mediated interference or pharmacological inhibition with the selective TRPC3 antagonist Pyr10 induced a substantial decrease of SOCE, indicating a significant role of TRPC3 in SOCE in DRG nociceptors. Activation of P2Y2 purinoceptors or PAR2 protease receptors triggered a strong increase in intracellular calcium in conditions of TRPC3 overexpression. Additionally, knockdown of native TRPC3 or its selective pharmacological blockade suppressed UTP- or PAR2 agonist-evoked calcium responses as well as sensitization of DRG neurons. These data show a robust link between activation of pro-inflammatory receptors and calcium homeostasis through TRPC3-containing channels operating both in receptor- and store-operated mode.ConclusionsOur findings highlight a major contribution of TRPC3 to neuronal calcium homeostasis in somatosensory pathways based on the unique ability of these cation channels to engage in both SOCE and receptor-operated calcium influx. This is the first evidence for TRPC3 as a SOCE component in DRG neurons. The flexible role of TRPC3 in calcium signaling as well as its functional coupling to pro-inflammatory metabotropic receptors involved in peripheral sensitization makes it a potential target for therapeutic strategies in chronic pain conditions.

Flavanones That Selectively Inhibit TRPM3 Attenuate Thermal Nociception In Vivo

Transient receptor potential melastatin 3 (TRPM3) is a calcium-permeable nonselective cation channel that is expressed in a subset of dorsal root (DRG) and trigeminal ganglia sensory neurons. TRPM3 can be activated by the neurosteroid pregnenolone sulfate (PregS) and heat. TRPM3⁻/⁻ mice display an impaired sensation of noxious heat and thermal hyperalgesia. We have previously shown that TRPM3 is blocked by the citrus fruit flavanones hesperetin, naringenin, and eriodictyol as well as by ononetin, a deoxybenzoin from Ononis spinosa. To further improve the tolerability, potency, and selectivity of TRPM3 blockers, we conducted a hit optimization procedure by rescreening a focused library that was composed of chemically related compounds. Within newly identified TRPM3 blockers, isosakuranetin and liquiritigenin displayed favorable properties with respect to their inhibitory potency and a selective mode of action. Isosakuranetin, a flavanone whose glycoside is contained in blood oranges and grapefruits, displayed an IC₅₀ of 50 nM and is to our knowledge the most potent inhibitor of TRPM3 identified so far. Both compounds exhibited a marked specificity for TRPM3 compared with other sensory TRP channels, and blocked PregS-induced intracellular free Ca²⁺ concentration signals and ionic currents in freshly isolated DRG neurons. Furthermore, isosakuranetin and previously identified hesperetin significantly reduced the sensitivity of mice to noxious heat and PregS-induced chemical pain. Because the physiologic functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo.

Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the “canonical TRP” family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

Abstract 4551: Identification of TRPM7 kinase inhibitors.

Abstract TRPM7 (transient receptor potential melastatin 7) encodes a calcium permeable non-selective cation channel implicated in cell adhesion and magnesium homoeostasis. The TRPM7 gene encodes for a protein kinase whose activity is linked to the control of actomyosin contractility. TRPM7 mediates adhesion and migration of MDA-MB-231 breast cancer cells and recently has been reported to promote breast tumor cell metastasis. The lack of cell-permeable pharmacological inhibitors of the kinase domain represent a barrier to fully understanding kinase function. Herein, we describe the discovery of several compounds that target TRPM7 kinase activity and which were shown to decrease the binding of Myosin IIB to TRPM7 in HEK293 cells transfected with pCMV6-TRPM7. Magnesium starvation, which promotes TRPM7 kinase activity, induces phosphorylation of eEF2. Treatment of cells with several of the compounds decreased eEF2 phosphorylation under conditions of magnesium starvation, consistent with the notion that they inhibit TRPM7 kinase activity in cells. Citation Format: Tamer S. Kaoud, Jihyun Park, Shreya Mitra, Clint D.J. Tavares, Anna Tseng, Kevin N. Dalby. Identification of TRPM7 kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4551. doi:10.1158/1538-7445.AM2013-4551

Citrus fruit and fabacea secondary metabolites potently and selectively block TRPM3

The melastatin-related transient receptor potential TRPM3 is a calcium-permeable nonselective cation channel that can be activated by the neurosteroid pregnenolone sulphate (PregS) and heat. TRPM3-deficient mice show an impaired perception of noxious heat. Hence, drugs inhibiting TRPM3 possibly get in focus of analgesic therapy. Fluorometric methods were used to identify novel TRPM3-blocking compounds and to characterize their potency and selectivity to block TRPM3 but not other sensory TRP channels. Biophysical properties of the block were assessed using electrophysiological methods. Single cell calcium measurements confirmed the block of endogenously expressed TRPM3 channels in rat and mouse dorsal root ganglion (DRG) neurones. By screening a compound library, we identified three natural compounds as potent blockers of TRPM3. Naringenin and hesperetin belong to the citrus fruit flavanones, and ononetin is a deoxybenzoin. Eriodictyol, a metabolite of naringenin and hesperetin, was still biologically active as a TRPM3 blocker. The compounds exhibited a marked specificity for recombinant TRPM3 and blocked PregS-induced [Ca(2+)]i signals in freshly isolated DRG neurones. The data indicate that citrus fruit flavonoids are potent and selective blockers of TRPM3. Their potencies ranged from upper nanomolar to lower micromolar concentrations. Since physiological functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

Optical Recording of Single-Channel TRPV1 Activity and Mobility in Isolated Dorsal Root Ganglion (DRG) Neurons and Cultured Mammalian Cells

TRPV1 is a calcium-permeable non-selective cation channel recognized for its sensitivity to heat, low pH, oxidation and numerous endogenous agonists. To date there is very little known about the activity of the individual channel in the intact cell. In order to explore TRPV1 in this context we employed total internal reflection fluorescence (TIRF) microscopy to observe the open states of the channel as a fluorescent “sparklet” at the site where calcium influx occurs. Preliminary investigations into the sparklet activity elicited by capsaicin in DRG neurons indicate that TRPV1 is free to move laterally in the plasma membrane while conducting calcium into the cytosol. Importantly, two-state fluorescent sparklets indicate that channels function independently in vivo. In HEK293T/17 cells transiently transfected with TRPV1-eGFP we simultaneously imaged mobile channels by their eGFP label and their capsaicin-activated sparklets. We implemented both the eGFP photobleaching subunit counting strategy as well as sparklet intensity analysis of TRPV1 sites. Consistent with results in DRG neurons, we found that individual channels, rather than assemblies of channels, were present and functional in the plasma membrane. Further analysis of the subset of mobile sparklets revealed that the mobility of TRPV1 sparklets steadily decreased with its open duration. Activity dependence of TRPV1 mobility may represent a new form of channel regulation in which its function becomes spatially compartmentalized.

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 1mM 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.

Functional interaction between AQP2 and TRPV4 in renal cells

We have previously demonstrated that renal cortical collecting duct cells (RCCD(1)), responded to hypotonic stress with a rapid activation of regulatory volume decrease (RVD) mechanisms. This process requires the presence of the water channel AQP2 and calcium influx, opening the question about the molecular identity of this calcium entry path. Since the calcium permeable nonselective cation channel TRPV4 plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types, the aim of this work was to test the hypothesis that the increase in intracellular calcium concentration and the subsequent rapid RVD, only observed in the presence of AQP2, could be due to a specific activation of TRPV4. We evaluated the expression and function of TRPV4 channels and their contribution to RVD in WT-RCCD(1) (not expressing aquaporins) and in AQP2-RCCD(1) (transfected with AQP2) cells. Our results demonstrated that both cell lines endogenously express functional TRPV4, however, a large activation of the channel by hypotonicity only occurs in cells that express AQP2. Blocking of TRPV4 by ruthenium red abolished calcium influx as well as RVD, identifying TRPV4 as a necessary component in volume regulation. Even more, this process is dependent on the translocation of TRPV4 to the plasma membrane. Our data provide evidence of a novel association between TRPV4 and AQP2 that is involved in the activation of TRPV4 by hypotonicity and regulation of cellular response to the osmotic stress, suggesting that both proteins are assembled in a signaling complex that responds to anisosmotic conditions.