Transient receptor potential M2 (TRPM2) ion channel is a non-selective cationic channel that can permeate calcium ions, and plays an important role in neuroinflammation, ischemic reperfusion brain injury, neurodegenerative disease, neuropathic pain, epilepsy and other neurological diseases. In ischemic reperfusion brain injury, TRPM2 mediates neuronal death by modulating the different subunits of glutamate N-methyl-D-aspartic acid receptor in response to calcium/zinc signal. In Alzheimer's disease, TRPM2 is activated by reactive oxygen species generated by β-amyloid peptide to form a malignant positive feedback loop that induces neuronal death and is involved in the pathological process of glial cells by promoting inflammatory response and oxidative stress. In epilepsy, the TRPM2-knockout alleviates epilepsy induced neuronal degeneration by inhibiting autophagy and apoptosis related proteins. The roles of TRPM2 channel in the pathogenesis of various central nervous system diseases and its potential drug development and clinical application prospects are summarized in this review.
Amyloid beta-Peptides/metabolism* ; Humans ; Neurodegenerative Diseases ; Neuroglia ; TRPM Cation Channels/genetics* ; Transient Receptor Potential Channels

OBJECTIVES: To investigate the role of transient receptor potential cation channel subfamily M member 2 (TRPM2) in hepatic ischemia-reperfusion injury of mouse (HIRI) and the possible mechanisms. METHODS: Sixty adult male C57BL/6 mice were randomly divided into 4 groups: a sham group (S group), a HIRI model group (M group), a TRPM2 adenovirus interference vector group (T group), and a TRPM2 adenovirus control vector group (C group) (=15 in each group). The liver tissues of mice before perfusion were obtained. The efficiency of adenovirus infection was detected by fluorescence microscopy, and the silencing efficiency of adenovirus against TRPM2 was detected by real-time PCR.The abdominal aorta blood and liver tissues were collected from mice at 2, 4 and 8 h after reperfusion. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of mice were detected. Hepatic pathological changes were examined by hematoxylin-eosin (HE) staining. The protein expression of TRPM2 and Rac family small GTPase 1 (RAC1) in liver tissues was detected by Western blotting. Changes of malondialdehyde (MDA), superoxide dismutase (SOD) and myeloperoxidase (MPO) activities in liver tissues were detected by enzyme-linked immunosorbent assay. RESULTS: A strong signal of green fluorescence was observed in the liver tissues of mice in the T and C groups compared to the S or M group. Compared with the S, M or C group, the expression of TRPM2 mRNA in liver tissue in the T group was significantly down-regulated (all <0.05). The morphology of hepatocytes was normal in the S group under light microscope.Hepatic sinus dilatation, congestion, hepatocyte degeneration, central necrosis of lobule, and massive inflammatory granulocyte infiltration were observed in the M and C group, respectively. The degree of hepatocyte damage in the T group was significantly reduced compared with that in the M and C group, respectively. Compared with the S group, the serum ALT and AST activities in the M, T and C groups were significantly increased at 2, 4 and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum ALT and AST activities in the T group were significantly lower in serum of mice at 2, 4, and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum SOD activity in the T group was significantly increased at 2, 4, and 8 h after reperfusion (all <0.05), while the serum MDA and MPO activities were significantly decreased (all <0.05). The protein expression of TRPM2 and RAC1 in liver tissues in the T group were significantly lower than those in the M and C groups at 2, 4 and 8 h after reperfusion (all <0.05). CONCLUSIONS Pretreatment with TRPM2 adenovirus interference vector can effectively silence TRPM2 gene expression in liver tissues of mice and attenuate HIRI, which may be related to inhibiting oxidative stress and reducing the expression of RAC1 protein.
Alanine Transaminase ; Animals ; Aspartate Aminotransferases ; Liver ; Male ; Mice ; Mice, Inbred C57BL ; Neuropeptides ; Rats, Sprague-Dawley ; Reperfusion Injury ; TRPM Cation Channels ; genetics ; Transient Receptor Potential Channels ; rac1 GTP-Binding Protein

OBJECTIVE: To observe the effect of Miao medicinal acupuncture therapy on transient receptor potential vanilloid (TRPV) channel in knee joint synovial tissue of the rabbits with knee osteoarthritis (KOA) model and to explore the mechanism of Miao medicinal acupuncture therapy in treatment of KOA. METHODS: Of 34 New Zealand male rabbits, 6 rabbits were selected randomly as the normal group. KOA model was established in the rest rabbits by injecting a mixture of papain and L-cysteine in right knee joints. The 24 successfully modeled rabbits were randomized into a model group, a Miao medicinal acupuncture therapy group, a dermal needle group and a smearing group, 6 rabbits in each one. In the Miao medicinal acupuncture therapy group, Miao medicinal acupuncture therapy was adopted, in which, the roller type of dermal needle was used on the surface of right knee joint [a rectangle shape formed by "Xuehai" (SP 10), "Liangqiu" (ST 34), "Yanglingquan" (GB 34) and "Yinlingquan" (SP 9)], rolling in a " shape, on which, Miao medicinal solution was smeared in advance. In the dermal needle group, the rolling stimulation was exerted on the right the right knee joint surface with the roller type of dermal needle. In the smearing group, Miao medicinal solution was smeared on the right knee joint surface. The intervention was given once every two days, 3 times weekly and the intervention was exerted consecutively for 4 weeks. Successively, on day 1, 21, 28, 35, 42 and 49 of experiment, paw withdrawal threshold (von Frey threshold) after mechanical stimulation was detected in the rabbits. HE staining was adopted to observe the histomorphological changes of the right knee joint cartilage in the rabbits. ELISA was used to determine the contents of interleukin-1 (IL-1β) and tumor necrosis factor-α (TNF-α) in the right knee synovial fluid. Western blot method and real-time PCR were used to determine the relative expressions of protein and mRNA of TRPV1 and TRPV4 in knee synovial tissue of the rabbits. RESULTS: Compared with the normal group, on day 49 of experiment, von Frey threshold was reduced significantly in the rabbits of the model group ( CONCLUSION Miao medicinal acupuncture therapy plays a role in treatment of KOA probably through inhibiting the expressions of IL-1β and TNF-α of knee synovial fluid and down-regulating the expressions of protein and mRNA of TRPV1 and TRPV4 in knee synovial tissue.
Acupuncture Therapy ; Animals ; Knee Joint ; Male ; Osteoarthritis, Knee/therapy* ; Rabbits ; Synovial Fluid ; Transient Receptor Potential Channels

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.
Amino Acids ; Binding Sites ; Calcium ; Cytosol ; Glutamic Acid ; Myocytes, Smooth Muscle ; Permeability ; Polyamines ; Receptors, Muscarinic ; Spermine ; Transient Receptor Potential Channels

Pruritus (itching) is classically defined as an unpleasant cutaneous sensation that leads to scratching behavior. Although the scientific criteria of classification for pruritic diseases are not clear, it can be divided as acute or chronic by duration of symptoms. In this study, we investigated whether skin injury caused by chemical (contact hypersensitivity, CHS) or physical (skin-scratching stimulation, SSS) stimuli causes initial pruritus and analyzed gene expression profiles systemically to determine how changes in skin gene expression in the affected area are related to itching. In both CHS and SSS, we ranked the Gene Ontology Biological Process terms that are generally associated with changes. The factors associated with upregulation were keratinization, inflammatory response and neutrophil chemotaxis. The Kyoto Encyclopedia of Genes and Genomes pathway shows the difference of immune system, cell growth and death, signaling molecules and interactions, and signal transduction pathways. Il1a , Il1b and Il22 were upregulated in the CHS, and Tnf, Tnfrsf1b, Il1b, Il1r1 and Il6 were upregulated in the SSS. Trpc1 channel genes were observed in representative itching-related candidate genes. By comparing and analyzing RNA-sequencing data obtained from the skin tissue of each animal model in these characteristic stages, it is possible to find useful diagnostic markers for the treatment of itching, to diagnose itching causes and to apply customized treatment.
Animals ; Biological Processes ; Chemotaxis ; Classification ; Cytokines ; Dermatitis, Contact* ; Gene Expression ; Gene Ontology ; Genome ; Hypersensitivity ; Immune System ; Interleukin-6 ; Mice* ; Models, Animal ; Neutrophils ; Pruritus* ; RNA* ; Sensation ; Sequence Analysis, RNA* ; Signal Transduction ; Skin* ; Transcriptome ; Transient Receptor Potential Channels ; Up-Regulation ; Wound Healing

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model. Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(HO),calcium-sensitive receptor agonist gadolinium trichloride(GdCl),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of HO.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without HO.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot. Results After treatment with HO,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1β(P= 0.036)were significantly reduced.Cells in the HO group and HO+GdCl group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 μmol/L of HO concentration were similar.HO-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of HO.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the HO +siRNA treatment group and the HO treatment group.Conclusion CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.
Animals ; Cathepsin B ; antagonists & inhibitors ; metabolism ; Cell Line ; Gene Silencing ; Hydrogen Peroxide ; Inflammasomes ; metabolism ; Interleukin-1beta ; Mice ; Microglia ; NLR Family, Pyrin Domain-Containing 3 Protein ; metabolism ; Oxidative Stress ; Pyrin Domain ; Transient Receptor Potential Channels ; metabolism

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when coexpressed with CaM and CaMΔN. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ΔEF-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.
Binding Sites ; Calcium ; Calcium Signaling ; Calmodulin ; Ion Channels ; Ions ; Patch-Clamp Techniques ; Polycystic Kidney Diseases ; Transient Receptor Potential Channels

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) depletion. When PI(4,5)P₂ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²⁺ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²⁺ due to Ca²⁺ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P₂ depletion.
Calcium ; Cytoplasm ; Endoplasmic Reticulum ; GTP-Binding Proteins ; HEK293 Cells ; Inositol ; Phosphatidylinositol 4,5-Diphosphate ; Phospholipases ; Phosphotransferases ; Protein Kinase C ; Transient Receptor Potential Channels ; Type C Phospholipases

Depending on the intracellular buffering of calcium by chelation, zinc has the following two apparent effects on neuronal excitability: enhancement or reduction. Zinc increased tonic activity in the depolarized state when neurons were intracellularly dialyzed with EGTA but attenuated the neuronal activity when BAPTA was used as an intracellular calcium buffer. This suggests that neuronal excitability can be modulated by zinc, depending on the internal calcium buffering capacity. In this study, we elucidated the mechanisms of zinc-mediated alterations in neuronal excitability and determined the effect of calcium-related channels on zinc-mediated alterations in excitability. The zinc-induced augmentation of firing activity was mediated via the inhibition of small-conductance calcium-activated potassium (SK) channels with not only the contribution of voltage-gated L-type calcium channels (VGCCs) and ryanodine receptors (RyRs), but also through the activation of VGCCs via melastatin-like transient receptor potential channels. We suggest that zinc modulates the dopaminergic neuronal activity by regulating not only SK channels as calcium sensors, but also VGCCs or RyRs as calcium sources. Our results suggest that the cytosolic calcium-buffering capacity can tightly regulate zinc-induced neuronal firing patterns and that local calcium-signaling domains can determine the physiological and pathological state of synaptic activity in the dopaminergic system.
Animals ; Calcium ; Calcium Channels, L-Type ; Cytosol ; Dopaminergic Neurons ; Egtazic Acid ; Electrophysiology ; Fires ; Neurons ; Potassium ; Rats ; Ryanodine Receptor Calcium Release Channel ; Transient Receptor Potential Channels ; Zinc

Epidermal barrier formation and the maintenance of barrier homeostasis are essential to protect us from the external environments and organisms. Moreover, impaired keratinocytes differentiation and dysfunctional skin barrier can be the primary causes or aggravating factors for many inflammatory skin diseases including atopic dermatitis and psoriasis. Therefore, understanding the regulation mechanisms of keratinocytes differentiation and skin barrier homeostasis is important to understand many skin diseases and establish an effective treatment strategy. Calcium ions (Ca²⁺) and their concentration gradient in the epidermis are essential in regulating many skin functions, including keratinocyte differentiation, skin barrier formation, and permeability barrier homeostasis. Recent studies have suggested that the intracellular Ca²⁺ stores such as the endoplasmic reticulum (ER) are the major components that form the epidermal calcium gradient and the ER calcium homeostasis is crucial for regulating keratinocytes differentiation, intercellular junction formation, antimicrobial barrier, and permeability barrier homeostasis. Thus, both Ca²⁺ release from intracellular stores, such as the ER and Ca²⁺ influx mechanisms are important in skin barrier. In addition, growing evidences identified the functional existence and the role of many types of calcium channels which mediate calcium flux in keratinocytes. In this review, the origin of epidermal calcium gradient and their role in the formation and regulation of skin barrier are focused. We also focus on the role of ER calcium homeostasis in skin barrier. Furthermore, the distribution and role of epidermal calcium channels, including transient receptor potential channels, store-operated calcium entry channel Orai1, and voltage-gated calcium channels in skin barrier are discussed.
Calcium Channels ; Calcium* ; Dermatitis, Atopic ; Endoplasmic Reticulum ; Epidermis ; Homeostasis ; Intercellular Junctions ; Ions ; Keratinocytes ; Permeability ; Psoriasis ; Skin Diseases ; Skin* ; Transient Receptor Potential Channels