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。     

The Distribution of TRPV1 and TRPV2 in the Rat Pharynx

Immunohistochemistry for two nociceptive transducers, the transient receptor potential cation channel subfamily V members 1 (TRPV1) and 2 (TRPV2), was performed on the pharynx and its adjacent regions. TRPV1-immunoreactivity (IR) was detected in nerve fibers beneath and within the epithelium and/or taste bud-like structure. In the pharynx, these nerve fibers were abundant in the naso-oral part and at the border region of naso-oral and laryngeal parts. They were also numerous on the laryngeal side of the epiglottis and in the soft palate. TRPV2-IR was expressed by dendritic cells in the pharynx and epiglottis, as well as in the root of the tongue and soft palate. These cells were located in the epithelium and lamina propria. TRPV2-immunoreactive (IR) dendritic cells were numerous in the naso-oral part of the pharynx, epiglottis, and tongue. Abundance of TRPV2-IR dendritic processes usually obscured the presence of TRPV2-IR nerve fibers in these portions. However, some TRPV2-IR nerve fibers could be observed in the epithelium of the soft palate. Retrograde tracing method also revealed that sensory neurons which innervate the pharynx or soft palate were abundant in the jugular–petrosal ganglion complex and relatively rare in the nodose ganglion. In the jugular–petrosal ganglion complex, TRPV1- and TRPV2-IR were expressed by one-third of pharyngeal and soft palate neurons. TRPV2-IR was also detected in 11.5 % pharyngeal and 30.9 % soft palate neurons in the complex. Coexpression of TRPV1 and CGRP was frequent among pharyngeal and soft palate neurons. The present study suggests that TRPV1- and TRPV2-IR jugular–petrosal neurons may be associated with the regulation of the swallowing reflex.     

2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3

The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation.     

Single Residues in the Outer Pore of TRPV1 and TRPV3 Have Temperature-Dependent Conformations

Thermosensation is mediated by ion channels that are highly temperature-sensitive. Several members of the family of transient receptor potential (TRP) ion channels are activated by cold or hot temperatures and have been shown to function as temperature sensors in vivo. The molecular mechanism of temperature-sensitivity of these ion channels is not understood. A number of domains or even single amino acids that regulate temperature-sensitivity have been identified in several TRP channels. However, it is unclear what precise conformational changes occur upon temperature activation. Here, we used the cysteine accessibility method to probe temperature-dependent conformations of single amino acids in TRP channels. We screened over 50 amino acids in the predicted outer pore domains of the heat-activated ion channels TRPV1 and TRPV3. In both ion channels we found residues that have temperature-dependent accessibilities to the extracellular solvent. The identified residues are located within the second predicted extracellular pore loop. These residues are identical or proximal to residues that were shown to be specifically required for temperature-activation, but not chemical activation. Our data precisely locate conformational changes upon temperature-activation within the outer pore domain. Collectively, this suggests that these specific residues and the second predicted pore loop in general are crucial for the temperature-activation mechanism of these heat-activated thermoTRPs.     

Structural analyses of the ankyrin repeat domain of TRPV6 and related TRPV ion channels

Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.     

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.     

Differential Regulation of TRPV1, TRPV3, and TRPV4 Sensitivity through a Conserved Binding Site on the Ankyrin Repeat Domain

Transient receptor potential vanilloid (TRPV) channels, which include the thermosensitive TRPV1–V4, have large cytoplasmic regions flanking the transmembrane domain, including an N-terminal ankyrin repeat domain. We show that a multiligand binding site for ATP and calmodulin previously identified in the TRPV1 ankyrin repeat domain is conserved in TRPV3 and TRPV4, but not TRPV2. Accordingly, TRPV2 is insensitive to intracellular ATP, while, as previously observed with TRPV1, a sensitizing effect of ATP on TRPV4 required an intact binding site. In contrast, ATP reduced TRPV3 sensitivity and potentiation by repeated agonist stimulations. Thus, ATP and calmodulin, acting through this conserved binding site, are key players in generating the different sensitivity and adaptation profiles of TRPV1, TRPV3, and TRPV4. Our results suggest that competing interactions of ATP and calmodulin influence channel sensitivity to fluctuations in calcium concentration and perhaps even metabolic state. Different feedback mechanisms likely arose because of the different physiological stimuli or temperature thresholds of these channels.     

Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6

The molecular assembly of the epithelial Ca(2+) channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85-100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immuno blotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co-expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd(2+) sensitivity and voltage-dependent gating. Immuno precipitations using membrane fractions from oocytes co-expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca(2+) channels that varied with respect to Ca(2+)-dependent inactivation, Ba(2+) selectivity and pharmacological block. Thus, Ca(2+)-transporting epithelia co-expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca(2+) transport kinetics.     

PACSINs bind to the TRPV4 cation channel. PACSIN 3 modulates the subcellular localization of TRPV4

TRPV4 is a cation channel that responds to a variety of stimuli including mechanical forces, temperature, and ligand binding. We set out to identify TRPV4-interacting proteins by performing yeast two-hybrid screens, and we isolated with the avian TRPV4 amino terminus the chicken orthologues of mammalian PACSINs 1 and 3. The PACSINs are a protein family consisting of three members that have been implicated in synaptic vesicular membrane trafficking and regulation of dynamin-mediated endocytotic processes. In biochemical interaction assays we found that all three murine PACSIN isoforms can bind to the amino terminus of rodent TRPV4. No member of the PACSIN protein family was able to biochemically interact with TRPV1 and TRPV2. Co-expression of PACSIN 3, but not PACSINs 1 and 2, shifted the ratio of plasma membrane-associated versus cytosolic TRPV4 toward an apparent increase of plasma membrane-associated TRPV4 protein. A similar shift was also observable when we blocked dynamin-mediated endocytotic processes, suggesting that PACSIN 3 specifically affects the endocytosis of TRPV4, thereby modulating the subcellular localization of the ion channel. Mutational analysis shows that the interaction of the two proteins requires both a TRPV4-specific proline-rich domain upstream of the ankyrin repeats of the channel and the carboxyl-terminal Src homology 3 domain of PACSIN 3. Such a functional interaction could be important in cell types that show distribution of both proteins to the same subcellular regions such as renal tubule cells where the proteins are associated with the luminal plasma membrane.     

TRPV4-mediated channelopathies

Transient Receptor Potential Vanilloid sub type 4 (TRPV4) is a member of non-selective cation channel which is important for sensation of several physical and chemical stimuli and also involved in multiple physiological functions. Recently it gained immense medical and clinical interest as several independent studies have demonstrated that mutations in the TRPV4 gene can results in genetic disorders like Brachyolmia, Charcot-Marie-Tooth disease type 2C, Spinal Muscular Atrophy and Hereditary Motor and Sensory Neuropathy type 2. Close analysis of the data obtained from these naturally occurring as well as other TRPV4 mutants suggest that it is not the altered channel activity of these mutants per se, but the involvement and interaction of other factors that seem to modulate oligomerization, trafficking and degradation of TRPV4 channels. Also, these factors can either enhance or reduce the activity of TRPV4. In addition, there are some potential signaling events that can also be involved in these genetic disorders. In this review, we analyzed how and what extent certain cellular and molecular functions like oligomerization, surface expression, ubiquitination and functional interactions might be affected by these mutations.     

TRPV3 and TRPV4 mediate warmth-evoked currents in primary mouse keratinocytes

Recently, a family of temperature-activated ion channels has been identified in mammalian and nonmammalian species that appear to contribute to thermosensation. Two of these proteins, TRPV3 and TRPV4, are ion channels activated by modest increases in ambient temperature. Localization studies have indicated that both proteins, in addition to being expressed in sensory neurons, are also expressed in skin keratinocytes. These and other findings have suggested that keratinocytes might act in concert with sensory neurons to perceive our thermal environment. In this study, we demonstrate that primary keratinocytes isolated from mouse skin exhibit two distinct heat-evoked current responses to mild increases in ambient temperature. The more common of these response types bears considerable similarity to responses mediated by recombinant TRPV4, is absent in mice lacking this ion channel, and is restored upon TRPV4 reintroduction. The second, rarer response strongly resembles those mediated by recombinant TRPV3. Together, these findings demonstrate that keratinocytes can indeed act as thermosensory cells and that they do so via at least two distinct transduction mechanisms.     

Characterization of calmodulin binding domains in TRPV2 and TRPV5 C-tails

The transient receptor potential channels TRPV2 and TRPV5 belong to the vanilloid TRP subfamily. TRPV2 is highly similar to TRPV1 and shares many common properties with it. TRPV5 (and also its homolog TRPV6) is a rather distinct member of the TRPV subfamily. It is distant for being strictly Ca²⁺-selective and features quite different properties from the rest of the TRPV subfamily. It is known that TRP channels are regulated by calmodulin in a calcium-dependent manner. In our study we identified a calmodulin binding site on the C-termini of TRPV2 (654–683) and TRPV5 (587–616) corresponding to the consensus CaM binding motif 1-5-10. The R679 and K681 single mutants of TRPV2 caused a 50% decrease in binding affinity and a double mutation of K661/K664 of the same peptide lowered the binding affinity by up to 75%. A double mutation of R606/K607 and triple mutation of R594/R606/R610 in TRPV5 C-terminal peptide resulted in the total loss of binding affinity to calmodulin. These results demonstrate that the TRPV2 C-tail and TRPV5 C-tail contain calmodulin binding sites and that the basic residues are strongly involved in TRP channel binding to calmodulin.     

TRPV1 and TRPV4 Play Pivotal Roles in Delayed Onset Muscle Soreness

Unaccustomed strenuous exercise that includes lengthening contraction (LC) often causes tenderness and movement related pain after some delay (delayed-onset muscle soreness, DOMS). We previously demonstrated that nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are up-regulated in exercised muscle through up-regulation of cyclooxygenase (COX)-2, and they sensitized nociceptors resulting in mechanical hyperalgesia. There is also a study showing that transient receptor potential (TRP) ion channels are involved in DOMS. Here we examined whether and how TRPV1 and/or TRPV4 are involved in DOMS. We firstly evaluated a method to measure the mechanical withdrawal threshold of the deep tissues in wild-type (WT) mice with a modified Randall-Selitto apparatus. WT, TRPV1−/− and TRPV4−/− mice were then subjected to LC. Another group of mice received injection of murine NGF-2.5S or GDNF to the lateral gastrocnemius (LGC) muscle. Before and after these treatments the mechanical withdrawal threshold of LGC was evaluated. The change in expression of NGF, GDNF and COX-2 mRNA in the muscle was examined using real-time RT-PCR. In WT mice, mechanical hyperalgesia was observed 6–24 h after LC and 1–24 h after NGF and GDNF injection. LC induced mechanical hyperalgesia neither in TRPV1−/− nor in TRPV4−/− mice. NGF injection induced mechanical hyperalgesia in WT and TRPV4−/− mice but not in TRPV1−/− mice. GDNF injection induced mechanical hyperalgesia in WT but neither in TRPV1−/− nor in TRPV4−/− mice. Expression of NGF and COX-2 mRNA was significantly increased 3 h after LC in all genotypes. However, GDNF mRNA did not increase in TRPV4−/− mice. These results suggest that TRPV1 contributes to DOMS downstream (possibly at nociceptors) of NGF and GDNF, while TRPV4 is located downstream of GDNF and possibly also in the process of GDNF up-regulation.     

Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H₁ receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H₁ receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.     

Phosphoinositide 3-kinase binds to TRPV1 and mediates NGF-stimulated TRPV1 trafficking to the plasma membrane

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85beta subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85beta coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.     

Consequences of activating the calcium-permeable ion channel TRPV1 in breast cancer cells with regulated TRPV1 expression

Increased expression of specific calcium channels in some cancers and the role of calcium signaling in proliferation and invasion have led to studies assessing calcium channel inhibitors as potential therapies for some cancers. The use of channel activators to promote death of cancer cells has been suggested, but the risk of activators promoting cancer cell proliferation and the importance of the degree of channel over-expression is unclear. We developed an MCF-7 breast cancer cell line with inducible TRPV1 overexpression and assessed the role of TRPV1 levels on cell death mediated by the TRPV1 activator capsaicin and the potential for submaximal activation to promote proliferation. The TRPV1 level was a determinant of cell death induced by capsaicin. A concentration response curve with varying TRPV1 expression levels identified the minimum level of TRPV1 required for capsaicin induced cell death. At no level of TRPV1 over-expression or capsaicin concentration did TRPV1 activation enhance proliferation. Cell death induced by capsaicin was necrotic and associated with up-regulation of c-Fos and RIP3. These studies suggest that activators of specific calcium channels may be an effective way to induce necrosis and that this approach may not always be associated with enhancement of cancer cell proliferation.     

瞬时受体电位香草醛亚家族1(TRPV1)的生物学功能

瞬时受体电位香草醛亚家族1 (TRPV1)又称辣椒素受体(VR1),是一类可被辣椒素、热(>43℃)、酸(pH<6.0)所激活的配体门控性非选择性阳离子通道,对Ca²⁺有高度通透性。早期研究发现TRPV1主要分布在神经系统并介导瘙痒及痛觉反应,近些年研究表明其在非神经细胞如肥大细胞、膀胱上皮细胞、单核细胞、皮肤角化上皮细胞、胰岛细胞等中也广泛分布,在代谢性疾病、消化、呼吸和心血管系统疾病、皮肤病及肿瘤等疾病的发生发展中均发挥了重要作用。本文介绍了TRPV1的分布、结构特征及其功能研究的最新进展,并重点综述了TRPV1介导的瘙痒和疼痛信号通路及以TRPV1为靶点的中草药研究进展,以期为以TRPV1为潜在治疗靶点相关疾病的中西医防治提供理论指导。