TRPV5 and TRPV6 calcium channels in human T cells

The recent cloning of the special calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) has provided a molecular basis for studying previously unidentified calcium influx channels in electrically nonexcitable cells. In the present work using RT-PCR, we obtained the endogenous expression of mRNAs of genes trpv5 and trpv6 in lymphoblast leukemia Jurkat cells and in normal human T lymphocytes. Additionally, by immunoblotting, the presence of the channel-forming TRPV5 proteins has been shown both in the total lysate and in crude membrane fractions from Jurkat cells and normal T lymphocytes. The use of immunoprecipitation revealed TRPV6 proteins in Jurkat cells, whereas in normal T lymphocytes, this protein was not detected. The expression pattern and the selective Ca²⁺ permeation properties of TRPV5 and TRPV6 channels indicate the important role of these channels in Ca²⁺ homeostasis, as well as most likely in malignant transformation of blood cells.     

Structural insights into the gating mechanisms of TRPV channels

Transient Receptor Potential channels from the vanilloid subfamily (TRPV) are a group of cation channels modulated by a variety of endogenous stimuli as well as a range of natural and synthetic compounds. Their roles in human health make them of keen interest, particularly from a pharmacological perspective. However, despite this interest, the complexity of these channels has made it difficult to obtain high resolution structures until recently. With the cryo-EM resolution revolution, TRPV channel structural biology has blossomed to produce dozens of structures, covering every TRPV family member and a variety of approaches to examining channel modulation. Here, we review all currently available TRPV structures and the mechanistic insights into gating that they reveal.     

TRPV4 channels activity in bovine articular chondrocytes: Regulation by obesity-associated mediators

Turnover of the cartilage extracellular matrix depends exclusively on chondrocytes and varies in response to load and osmolarity fluctuations. Obesity can affect chondrocyte physiology; adipokines, insulin and proinflammatory cytokines levels are all altered in the obese and are related to matrix turnover impairments and thus to osteoarthritis. TRPV4, a mechanosensitive cation channel, is responsible for reacting to hypotonic variations. In this study, the presence and activity of TRPV4 channels in bovine chondrocytes were evaluated using the whole-cell patch-clamp technique and fluorescence measurements to perform characterisations of these channels and to determine intracellular calcium responses. The expression of TRPV4 was determined by RT-PCR. The TRPV4 regulation by hypotonic shock, insulin and adipokines were analysed. Hypoosmolarity induced a Gd³⁺-, ruthenium red-, and HC-067047-sensitive current, predominantly inward, an intracellular Ca²⁺ concentration increase and a membrane depolarisation. The current had a reversal potential of +28 ± 4 mV and exhibited preferential permeability to Ca²⁺; 4αPDD, a specific TRPV4 agonist, evoked the same response. TNFα, IL-1β, insulin, and, to a lesser degree, leptin and resistin attenuated the TRPV4-mediated effects; in contrast, adiponectin did not affect them. These results confirm the function of TRPV4 in bovine articular chondrocytes and its regulation by obesity-associated mediators.     

Formation of a physiological complex between TRPV2 and RGA protein promotes cell surface expression of TRPV2

The transient receptor potential, sub-family Vanilloid (TRPV)(2) cation channel is activated in response to extreme temperature elevations in sensory neurons. However, TRPV2 is widely expressed in tissues with no sensory function, including cells of the immune system. Regulation of GRC, the murine homolog of TRPV2 has been studied in insulinoma cells and myocytes. GRC is activated in response to certain growth factors and neuropeptides, via a mechanism that involves regulated access of the channel to the plasma membrane. This is likely to be an important primary control mechanism for TRPV2 outside the CNS. Here, we report that a regulated trafficking step controls the access of TRPV2 to the cell surface in mast cells. In mast cells, elevations in cytosolic cAMP are sufficient to drive plasma membrane localization of TRPV2. We have previously proposed that the recombinase gene activator protein (RGA), a four-transmembrane domain, intracellular protein, associates with TRPV2 during the biosynthesis and early trafficking of the channel. We use a polyclonal antibody to RGA to confirm the formation of a physiological complex between RGA and TRPV2. Finally, we show that over-expression of the RGA protein potentiates the basal surface localization of TRPV2. We propose that trafficking and activation mechanisms intersect for TRPV2, and that cAMP mobilizing stimuli may regulate TRPV2 localization in non-sensory cells. RGA participates in the control of TRPV2 surface levels, and co-expression of RGA may be a key component of experimental systems that seek to study TRPV2 physiology.     

Astrocytes express functional TRPV2 ion channels

Thermosensitive transient receptor potential (thermo TRP) channels are important for sensory transduction. Among them, TRPV2 has an interesting characteristic of being activated by very high temperature (>52 °C). In addition to the heat sensor function, TRPV2 also acts as a mechanosensor, an osomosensor and a lipid sensor. It has been reported that TRPV2 is expressed in heart, intestine, pancreas and sensory nerves. In the central nervous system, neuronal TRPV2 expression was reported, however, glial expression and the precise roles of TRPV2 have not been determined. To explore the functional expression of TRPV2 in astrocytes, the expression was determined by histological and physiological methods. Interestingly, TRPV2 expression was detected in plasma membrane of astrocytes, and the astrocytic TRPV2 was activated by very high temperature (>50 °C) consistent with the reported characteristic. We revealed that the astrocytic TRPV2 was also activated by lysophosphatidylcholine, a known endogenous lipid ligand for TRPV2, suggesting that astrocytic TRPV2 might regulate neuronal activities in response to lipid metabolism. Thus, for the first time we revealed that TRPV2 is functionally expressed in astrocytes in addition to neurons.     

PDMS微柱阵列型拓扑结构基底增强HepG2细胞TRPV1、TRPV4通道表达及功能响应性

制备了微柱名义直径为4μm或10μm,名义间距为4μm或7μm,名义高度为4μm的聚二甲基硅氧烷微柱阵列型拓扑结构基底,研究了HepG2细胞与拓扑结构基底复合后细胞瞬时受体电位通道TRPV1、TRPV4在基因和蛋白水平的表达及其功能响应性。细胞TRPV1和TRPV4在基因水平表达的评价采用定量PCR技术进行;TRPV1和TRPV4在蛋白水平的表达以免疫印迹和免疫荧光染色确认;TRPV1和TRPV4功能响应性的研究系以TRPV1和TRPV4激动剂辣椒素和4α-佛波醇-12,13-二葵酸酯刺激细胞,采用钙离子染料钙绿-1结合激光共聚焦显微技术记录钙内流动态过程,以钙内流荧光响应幅度及阳性响应比率进行评价。实验结果表明,在四种拓扑结构基底上细胞TRPV1和TRPV4的mRNA表达量均显著高于平面基底上相应值。免疫印迹实验证实了TRPV1和TRPV4在蛋白水平的表达,且拓扑结构基底上TRPV1和TRPV4免疫荧光染色强度较之平面基底相应值明显增高或趋于增高。在激动剂作用下,TRPV1介导的钙内流表现为快速去敏感化(25秒内)的瞬态内流,且拓扑结构基底上阳性响应细胞比例或相对荧光响应幅度较之平面基底相应值增高;而拓扑结构基底上细胞TRPV4阳性响应细胞比例和相对荧光响应幅度较之平面基底均全面明显升高。上述结果表明,TRPV介导的离子信号可能是基底拓扑结构优化HepG2细胞功能表型的重要信号机制。     

TRPV3 endogenously expressed in murine colonic epithelial cells is inhibited by the novel TRPV3 blocker 26E01

TRPV3 is a Ca²⁺-permeable cation channel, prominently expressed by keratinocytes where it contributes to maintaining the skin barrier, skin regeneration, and keratinocyte differentiation. However, much less is known about its physiological function in other tissues and there is still a need for identifying novel and efficient TRPV3 channel blockers. By screening a compound library, we identified 26E01 as a novel TRPV3 blocker. 26E01 blocks heterologously expressed TRPV3 channels overexpressed in HEK293 cells as assessed by fluorometric intracellular free Ca²⁺ assays (IC₅₀ = 8.6 μM) but does not affect TRPV1, TRPV2 or TRPV4 channels. Electrophysiological whole-cell recordings confirmed the reversible block of TRPV3 currents by 26E01, which was also effective in excised inside-out patches, hinting to a rather direct mode of action. 26E01 suppresses endogenous TRPV3 currents in the mouse 308 keratinocyte cell line and in the human DLD-1 colon carcinoma cell line (IC₅₀ = 12 μM). In sections of the gastrointestinal epithelium of mice, the expression of TRPV3 mRNA follows a gradient along the gastrointestinal tract, with the highest expression in the distal colon. 26E01 efficiently attenuates 2-aminoethoxydiphenyl borate-induced calcium influx in primary colonic epithelial cells isolated from the distal colon. As 26E01 neither shows toxic effects on DLD-1 cells at concentrations of up to 100 μM in MTT assays nor on mouse primary colonic crypts as assessed by calcein-AM/propidium iodide co-staining, it may serve as a useful tool to further study the physiological function of TRPV3 in various tissues.     

Positive modulation of the TMEM16B mediated currents by TRPV4 antagonist

Calcium-activated chloride channels (CaCCs) play important roles in many physiological processes and their malfunction is implicated in diverse pathologies such as cancer, asthma, and hypertension. TMEM16A and TMEM16B proteins are the structural components of the CaCCs. Recent studies in cell cultures and animal models have demonstrated that pharmacological inhibition of CaCCs could be helpful in the treatment of some diseases, however, there are few specific modulators of these channels. CaCCs and Transient Receptor Potential Vanilloid-4 (TRPV4) channels are co-expressed in some tissues where they functionally interact. TRPV4 is activated by different stimuli and forms a calcium permeable channel that is activated by GSK1016790A and antagonized by GSK2193874. Here we report that GSK2193874 enhances the chloride currents mediated by TMEM16B expressed in HEK cells at nanomolar concentrations and that GSK1016790A enhances native CaCCs of Xenopus oocytes. Thus, these compounds may be used as a tool for the study of CaCCs, TRPV4 and their interactions.     

TRPV4 is involved in irisin-induced endothelium-dependent vasodilation

Irisin, an exercise-induced myokine, induces conversion of white into brown adipocytes, promoting mitochondrial biogenesis and energy expenditure. Irisin has a vascular protective effect on endothelial function in animals, including humans. Defects in irisin signaling pathways result in endothelial dysfunction in obesity and diabetes. However, the mechanisms underlying the effects of irisin on endothelial function have not been elucidated. Transient receptor potential vanilloid subtype 4 (TRPV4) channels are one of the most important Ca²⁺-permeable cation channels in vascular endothelial cells. In this study, we hypothesized that irisin may induce endothelium-dependent vasodilation by activating Ca²⁺ influx into endothelial cells via TRPV4 channels. In primary cultured rat mesenteric artery endothelial cells, irisin caused an increase in [Ca²⁺]_i due to extracellular Ca²⁺ influx rather than release from Ca²⁺ stores. Moreover, irisin-induced increases in [Ca²⁺]_i were completely abolished by a TRPV4 inhibitor. In addition, irisin induced endothelium-dependent vasodilation of rat mesenteric arteries. However, irisin had no effect on endothelium-independent vasodilation. Furthermore, irisin-induced vasodilation was fully abolished in the presence of a TRPV4 inhibitor, indicating the involvement of TRPV4 channels in endothelium-dependent vasodilation. This study provides the first evidence that irisin-induced endothelium-dependent vasodilation is related to the stimulation of extracellular Ca²⁺ influx via TRPV4 channels in rat mesenteric arteries.     

A conserved gating element in TRPV6 channels

The Ca²⁺-selective tetrameric Transient Receptor Potential Vanilloid 6 (TRPV6) channel is an inwardly rectifying ion channel. The constitutive current endures Ca²⁺-induced inactivation as a result of the activation of phospholipase C followed depletion of phosphatidylinositol 4,5-bisphosphate, and calmodulin binding. Replacing a glycine residue within the cytosolic S4-S5 linker of the human TRPV6 protein, glycine 516, which is conserved in all TRP channel proteins, by a serine residue forces the channels into an open conformation thereby enhancing constitutive Ca²⁺ entry and preventing inactivation. Introduction of a second mutation (T621A) into TRPV6_G516S reduces constitutive activity and partially rescues the TRPV6 function. According to the recently revealed crystal structure of the rat TRPV6 the T621 is adjacent to the distal end of the transmembrane segment 6 (S6) within a short linker between S6 and the helix formed by the TRP domain. These results indicate that the S4-S5 linker and the S6-TRP-domain linker are critical constituents of TRPV6 channel gating and that disturbance of their sequences foster constitutive Ca²⁺ entry.     

Jellyfish and other cnidarian envenomations cause pain by affecting TRPV1 channels

Cnidarian envenomations cause a burning-pain sensation of which the underlying mechanisms are unknown. Activation of TRPV1, a non-selective cation channel expressed in nociceptive neurons, leads to cell depolarisation and pain. Here, we show in vitro and in vivo evidence for desensitization-dependent TRPV1 activation in cnidarian envenomations. Cnidarian venom induced a nociceptive reactivity, comparable to capsaicin, in laboratory rats, which could be reduced by the selective TRPV1 antagonist, BCTC. These findings are the first to explain at least part of the symptomology of cnidarian envenomations and provide insights into the design of more effective treatments for this global public health problem.     

Activation of TRPV4 channels reduces migration of immortalized neuroendocrine cells

Calcium is a universal signal, and its capacity to encode intracellular messages via spatial, temporal and amplitude characteristics allows it to participate in most cellular events. In a specific context, calcium plays a pivotal role in migration, although its role has not been elucidated fully. By using immortalized gonadotropin-releasing hormone-secreting neurons (GN11), we have now investigated the role of TRPV4, a member of the vanilloid family of Ca(2+) channels, in neuronal migration. Our results show that TRPV4 channels are present and functional in GN11 cells and their localization is polarized and enriched in lamellipodial structures. TRPV4 activation leads to a retraction of the lamellipodia and to a decrease in migratory behaviour; moreover cells migrate slower and in a more random manner. We therefore provide evidence for a new regulation of gonadotropin-releasing hormone neurons and a new role for calcium at the leading edge of migratory cells.     

TRPV1 研究的新亮点：TRPV1 剪接变体

TRPV1是一种非选择性阳离子通道蛋白,可被伤害性热刺激、辣椒素和氢离子等所激活。由于TRPV1在痛觉传导(尤其是炎症情况下的痛觉传导)中起重要作用,所以TRPV1的研究对临床治疗有十分重要的意义,研究也越来越深入。因为TRPV1可被多种刺激所激活,人们推论其有多个剪接变体(splice variant),不久,即证实了此设想。本文对迄今为止发现的TRPV1剪接变体做一简单综述。     

Human TRPV5 and TRPV6: Key players in cadmium and zinc toxicity

TRPV5 and TRPV6 are two major calcium transport pathways in the human body maintaining calcium homeostasis. TRPV5 is mainly expressed in the distal convoluted and connecting tubule where it is the major, regulated pathway for calcium reabsorption. TRPV6 serves as an important calcium entry pathway in the duodenum and the placenta. Previously, we showed that human TRPV6 (hTRPV6) transports several heavy metals. In this study we tested whether human TRPV5 (hTRPV5) also transports cadmium and zinc, and whether hTRPV5 together with hTRPV6 are involved in cadmium and zinc toxicity. The hTRPV5 mRNA and protein were expressed in HEK293 cells transiently transfected with pTagRFP-C1-hTRPV5. The overexpression of the hTRPV5 protein at the plasma membrane was revealed by cell surface biotinylation and immunofluorescence techniques. We observed that both cadmium and zinc permeate hTRPV5 in ion imaging experiments using Fura-2 or Newport Green DCF. Our results were further confirmed using whole-cell patch clamp technique. Transient overexpression of hTRPV5 or hTRPV6 sensitized cells to cadmium and zinc. Toxicity curves of cadmium and zinc were also shifted in hTRPV6 expressing HEK293 cells clones. Our results suggest that TRPV5 and TRPV6 are crucial gates controlling cadmium and zinc levels in the human body especially under low calcium dietary conditions, when these channels are maximally upregulated.     

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave k_on = 13 x 10⁶ M^?1s^?1 and k_off = 1609 sec^?1 with K_D = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a K_D = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.     

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave k_on = 13 x 10⁶ M⁻¹s⁻¹ and k_off = 1609 sec⁻¹ with K_D = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a K_D = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.     

Chroman and tetrahydroquinoline ureas as potent TRPV1 antagonists

Novel chroman and tetrahydroquinoline ureas were synthesized and evaluated for their activity as TRPV1 antagonists. It was found that aryl substituents on the 7- or 8-position of both bicyclic scaffolds imparted the best in vitro potency at TRPV1. The most potent chroman ureas were assessed in chronic and acute pain models, and compounds with the ability to cross the blood-brain barrier were shown to be highly efficacious. The tetrahydroquinoline ureas were found to be potent CYP3A4 inhibitors, but replacement of bulky substituents at the nitrogen atom of the tetrahydroisoquinoline moiety with small groups such as methyl can minimize the inhibition.