Molecular and structural basis of the dual regulation of the polycystin-2 ion channel by small-molecule ligands.

Mutations in the PKD2 gene, which encodes the polycystin-2 (PC2, also called TRPP2) protein, lead to autosomal dominant polycystic kidney disease (ADPKD). As a member of the transient receptor potential (TRP) channel superfamily, PC2 functions as a non-selective cation channel. The activation and regulation of the PC2 channel are largely unknown, and direct binding of small-molecule ligands to this channel has not been reported. In this work, we found that most known small-molecule agonists of the mucolipin TRP (TRPML) channels inhibit the activity of the PC2_F604P, a gain-of-function mutant of the PC2 channel. However, two of them, ML-SA1 and SF-51, have dual regulatory effects, with low concentration further activating PC2_F604P, and high concentration leading to inactivation of the channel. With two cryo-electron microscopy (cryo-EM) structures, a molecular docking model, and mutagenesis results, we identified two distinct binding sites of ML-SA1 in PC2_F604P that are responsible for activation and inactivation, respectively. These results provide structural and functional insights into how ligands regulate PC2 channel function through unusual mechanisms and may help design compounds that are more efficient and specific in regulating the PC2 channel and potentially also for ADPKD treatment.

LIPUS regulates the progression of knee osteoarthritis in mice through primary cilia-mediated TRPV4 channels.

Osteoarthritis (OA) is a common disease in middle-aged and elderly people. An imbalance in calcium ion homeostasis will contribute to chondrocyte apoptosis and ultimately lead to the progression of OA. Transient receptor potential channel 4 (TRPV4) is involved in the regulation of intracellular calcium homeostasis. TRPV4 is expressed in primary cilia, which can sense mechanical stimuli from outside the cell, and its abnormal expression is closely related to the development of OA. Low-intensity pulsed ultrasound (LIPUS) can alleviate chondrocyte apoptosis while the exact mechanism is unclear. In this project, with the aim of revealing the mechanism of action of LIPUS, we proposed to use OA chondrocytes and animal models, LIPUS intervention, inhibition of primary cilia, use TRPV4 inhibitors or TRPV4 agonist, and use Immunofluorescence (IF), Immunohistochemistry (IHC), Western Blot (WB), Quantitative Real-time PCR (QP) to detect the expression of cartilage synthetic matrix and endoplasmic reticulum stress markers. The results revealed that LIPUS altered primary cilia expression, promoted synthetic matrix metabolism in articular chondrocytes and was associated with primary cilia. In addition, LIPUS exerted a active effect on OA by activating TRPV4, inducing calcium inward flow, and facilitating the entry of NF-κB into the nucleus to regulate synthetic matrix gene transcription. Inhibition of TRPV4 altered primary cilia expression in response to LIPUS stimulation, and knockdown of primary cilia similarly inhibited TRPV4 function. These results suggest that LIPUS mediates TRPV4 channels through primary cilia to regulate the process of knee osteoarthritis in mice.

Klotho Stabilizes the Podocyte Actin Cytoskeleton in Idiopathic Membranous Nephropathy through Regulating the TRPC6/CatL Pathway.

INTRODUCTION: The aim of this study was to explore the renoprotective effects of Klotho on podocyte injury mediated by complement activation and autoantibodies in idiopathic membranous nephropathy (IMN). METHODS: Rat passive Heymann nephritis (PHN) was induced as an IMN model. Urine protein levels, serum biochemistry, kidney histology, and podocyte marker levels were assessed. In vitro, sublytic podocyte injury was induced by C5b-9. The expression of Klotho, transient receptor potential channel 6 (TRPC6), and cathepsin L (CatL); its substrate synaptopodin; and the intracellular Ca2+ concentration were detected via immunofluorescence. RhoA/ROCK pathway activity was measured by an activity quantitative detection kit, and the protein expression of phosphorylated-LIMK1 (p-LIMK1) and p-cofilin in podocytes was detected via Western blotting. Klotho knockdown and overexpression were performed to evaluate its role in regulating the TRPC6/CatL pathway. RESULTS: PHN rats exhibited proteinuria, podocyte foot process effacement, decreased Klotho and Synaptopodin levels, and increased TRPC6 and CatL expression. The RhoA/ROCK pathway was activated by the increased phosphorylation of LIMK1 and cofilin. Similar changes were observed in C5b-9-injured podocytes. Klotho knockdown exacerbated podocyte injury, while Klotho overexpression partially ameliorated podocyte injury. CONCLUSION: Klotho may protect against podocyte injury in IMN patients by inhibiting the TRPC6/CatL pathway. Klotho is a potential target for reducing proteinuria in IMN patients.

Joint CB1 and NGF Receptor Activation Suppresses TRPM8 Activation in Etoposide-Resistant Retinoblastoma Cells.

In childhood, retinoblastoma (RB) is the most common primary tumor in the eye. Long term therapeutic management with etoposide of this life-threatening condition may have diminishing effectiveness since RB cells can develop cytostatic resistance to this drug. To determine whether changes in receptor-mediated control of Ca2+ signaling are associated with resistance development, fluorescence calcium imaging, semi-quantitative RT-qPCR analyses, and trypan blue dye exclusion staining patterns are compared in WERI-ETOR (etoposide-insensitive) and WERI-Rb1 (etoposide-sensitive) cells. The cannabinoid receptor agonist 1 (CNR1) WIN55,212-2 (40 µM), or the transient receptor potential melastatin 8 (TRPM8) agonist icilin (40 µM) elicit similar large Ca2+ transients in both cell line types. On the other hand, NGF (100 ng/mL) induces larger rises in WERI-ETOR cells than in WERI-Rb1 cells, and its lethality is larger in WERI-Rb1 cells than in WERI-ETOR cells. NGF and WIN55,212-2 induced additive Ca2+ transients in both cell types. However, following pretreatment with both NGF and WIN55,212-2, TRPM8 gene expression declines and icilin-induced Ca2+ transients are completely blocked only in WERI-ETOR cells. Furthermore, CNR1 gene expression levels are larger in WERI-ETOR cells than those in WERI-Rb1 cells. Therefore, the development of etoposide insensitivity may be associated with rises in CNR1 gene expression, which in turn suppress TRPM8 gene expression through crosstalk.

Roles of Thermosensitive Transient Receptor Channels TRPV1 and TRPM8 in Paclitaxel-Induced Peripheral Neuropathic Pain.

Paclitaxel, a microtubule-stabilizing chemotherapy drug, can cause severe paclitaxel-induced peripheral neuropathic pain (PIPNP). The roles of transient receptor potential (TRP) ion channel vanilloid 1 (TRPV1, a nociceptor and heat sensor) and melastatin 8 (TRPM8, a cold sensor) in PIPNP remain controversial. In this study, Western blotting, immunofluorescence staining, and calcium imaging revealed that the expression and functional activity of TRPV1 were upregulated in rat dorsal root ganglion (DRG) neurons in PIPNP. Behavioral assessments using the von Frey and brush tests demonstrated that mechanical hyperalgesia in PIPNP was significantly inhibited by intraperitoneal or intrathecal administration of the TRPV1 antagonist capsazepine, indicating that TRPV1 played a key role in PIPNP. Conversely, the expression of TRPM8 protein decreased and its channel activity was reduced in DRG neurons. Furthermore, activation of TRPM8 via topical application of menthol or intrathecal injection of WS-12 attenuated the mechanical pain. Mechanistically, the TRPV1 activity triggered by capsaicin (a TRPV1 agonist) was reduced after menthol application in cultured DRG neurons, especially in the paclitaxel-treated group. These findings showed that upregulation of TRPV1 and inhibition of TRPM8 are involved in the generation of PIPNP, and they suggested that inhibition of TRPV1 function in DRG neurons via activation of TRPM8 might underlie the analgesic effects of menthol.

Roles of metabotropic glutamate receptor 5 in low [Mg2+]o-induced interictal epileptiform activity in rat hippocampal slices.

Group I metabotropic glutamate receptors (mGluRs) modulate postsynaptic neuronal excitability and epileptogenesis. We investigated roles of group I mGluRs on low extracellular Mg2+ concentration ([Mg2+]o)-induced epileptiform activity and neuronal cell death in the CA1 regions of isolated rat hippocampal slices without the entorhinal cortex using extracellular recording and propidium iodide staining. Exposure to Mg2+-free artificial cerebrospinal fluid can induce interictal epileptiform activity in the CA1 regions of rat hippocampal slices. MPEP, a mGluR 5 antagonist, significantly inhibited the spike firing of the low [Mg2+]o-induced epileptiform activity, whereas LY367385, a mGluR1 antagonist, did not. DHPG, a group 1 mGluR agonist, significantly increased the spike firing of the epileptiform activity. U73122, a PLC inhibitor, inhibited the spike firing. Thapsigargin, an ER Ca2+-ATPase antagonist, significantly inhibited the spike firing and amplitude of the epileptiform activity. Both the IP3 receptor antagonist 2-APB and the ryanodine receptor antagonist dantrolene significantly inhibited the spike firing. The PKC inhibitors such as chelerythrine and GF109203X, significantly increased the spike firing. Flufenamic acid, a relatively specific TRPC 1, 4, 5 channel antagonist, significantly inhibited the spike firing, whereas SKF96365, a relatively non-specific TRPC channel antagonist, did not. MPEP significantly decreased low [Mg2+]o DMEM-induced neuronal cell death in the CA1 regions, but LY367385 did not. We suggest that mGluR 5 is involved in low [Mg2+]oinduced interictal epileptiform activity in the CA1 regions of rat hippocampal slices through PLC, release of Ca2+ from intracellular stores and PKC and TRPC channels, which could be involved in neuronal cell death.

Transient Receptor Potential Channel Vanilloid 1 Contributes to Facial Mechanical Hypersensitivity in a Mouse Model of Atopic Asthma.

Sensitive skin, a common pathophysiological feature of allergic diseases, is defined as an unpleasant sensation in response to stimuli that normally should not provoke such sensations. However, the relationship between allergic inflammation and hypersensitive skin in the trigeminal system remains to be elucidated. To explore whether bronchial allergic inflammation affects facial skin and primary sensory neurons, we used an ovalbumin (OVA)-induced asthma mouse model. Significant mechanical hypersensitivity was observed in the facial skin of mice with pulmonary inflammation induced by OVA sensitization compared to mice treated with adjuvant or vehicle as controls. The skin of OVA-treated mice showed an increased number of nerve fibers, especially rich intraepithelial nerves, compared to controls. Transient receptor potential channel vanilloid 1 (TRPV1)-immunoreactive nerves were enriched in the skin of OVA-treated mice. Moreover, epithelial TRPV1 expression was higher in OVA-treated mice than in controls. Trigeminal ganglia of OVA-treated mice displayed larger numbers of activated microglia/macrophages and satellite glia. In addition, more TRPV1 immunoreactive neurons were found in the trigeminal ganglia of OVA-treated mice than in controls. Mechanical hypersensitivity was suppressed in OVA-treated Trpv1-deficient mice, while topical skin application of a TRPV1 antagonist before behavioral testing reduced the reaction induced by mechanical stimulation. Our findings reveal that mice with allergic inflammation of the bronchi had mechanical hypersensitivity in the facial skin that may have resulted from TRPV1-mediated neuronal plasticity and glial activation in the trigeminal ganglion.

Anti-Inflammatory Effects of Serotonin Receptor and Transient Receptor Potential Channel Ligands in Human Small Intestinal Epithelial Cells.

Intestinal inflammation and dysbiosis can lead to inflammatory bowel diseases (IBD) and systemic inflammation, affecting multiple organs. Developing novel anti-inflammatory therapeutics is crucial for preventing IBD progression. Serotonin receptor type 2A (5-HT2A) ligands, including psilocybin (Psi), 4-Acetoxy-N,N-dimethyltryptamine (4-AcO-DMT), and ketanserin (Ket), along with transient receptor potential (TRP) channel ligands like capsaicin (Cap), curcumin (Cur), and eugenol (Eug), show promise as anti-inflammatory agents. In this study, we investigated the cytotoxic and anti-inflammatory effects of Psi, 4-AcO-DMT, Ket, Cap, Cur, and Eug on human small intestinal epithelial cells (HSEIC). HSEIC were exposed to tumor necrosis factor (TNF)-α and interferon (IFN)-γ for 24 h to induce an inflammatory response, followed by treatment with each compound at varying doses (0-800 µM) for 24 to 96 h. The cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and protein expression by Western blot (WB) analysis. As single treatments, Psi (40 µM), Cur (0.5 µM), and Eug (50 µM) significantly reduced COX-2 levels without cytotoxic effects. When combined, Psi (40 µM) and Cur (0.5 µM) exhibited synergy, resulting in a substantial decrease in COX-2 protein levels (-28× fold change), although the reduction in IL-6 was less pronounced (-1.6× fold change). Psi (20 µM) and Eug (25 µM) demonstrated the most favorable outcomes, with significant decreases in COX-2 (-19× fold change) and IL-6 (-10× fold change) protein levels. Moreover, the combination of Psi and Eug did not induce cytotoxic effects in vitro at any tested doses. This study is the first to explore the anti-inflammatory potential of psilocybin and 4-AcO-DMT in the intestines while highlighting the potential for synergy between the 5-HT2A and TRP channel ligands, specifically Psi and Eug, in alleviating the TNF-α/IFN-γ-induced inflammatory response in HSEIC. Further investigations should evaluate if the Psi and Eug combination has the therapeutic potential to treat IBD in vivo.

Transient receptor potential channel 6 knockdown prevents high glucose-induced Müller cell pyroptosis.

BACKGROUND: Transient receptor potential channel 6 (TRPC6) is reported to be involved in the pathogenesis of diabetic complications, but its role in diabetic retinopathy (DR) remains unknown. The aim of our study was to determine the role and mechanism of TRPC6 in DR. METHODS: High glucose was used to construct a DR cell model using rat retinal Müller cells (rMC-1). Intracellular Ca2+, reactive oxygen species (ROS) and cell pyroptosis were evaluated by flow cytometry. Protein levels of NLRP3, pro-caspase-1, active caspase-1, gasdermin D (GSDMD), GSDMD-N, TRPC6 and H3K27ac were detected by Western blot. mRNA levels of EP300 and TRPC6 were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Levels of IL-1ß and IL-18 were estimated by enzyme linked immunosorbent assay (ELISA). The interaction between EP300 and TRPC6 was validated by a chromatin immunoprecipitation assay. RESULTS: The knockdown of TRPC6 reduced inflammation and cell pyroptosis in HG induced rMC-1 cells, whereas overexpression of TRPC6 had the opposite effects. The inhibition of ROS and NLRP3 reversed TRPC6-mediated cell pyroptosis in the DR cell model. In addition, EP300 increased the expression of H3K27ac and TRPC6 to promote cell pyroptosis, which was suppressed by the knockdown of TRPC6. CONCLUSIONS: Our study revealed a novel EP300/H3K27ac/TRPC6 signaling pathway that may contribute to HG induced Müller cell pyroptosis. TRPC6 played a novel role in Müller cell pyroptosis triggered by HG, and may be a potential target for DR treatment in the future.

Cancer-nerve interplay in cancer progression and cancer-induced bone pain.

INTRODUCTION: Cancer-induced bone pain (CIBP) is one of the most common and debilitating complications associated with bone metastasis. Although our understanding of the precise mechanism is limited, it has been known that bone is densely innervated, and that CIBP is elicited as a consequence of increased neurogenesis, reprogramming, and axonogenesis in conjunction with sensitization and excitation of sensory nerves (SNs) in response to the noxious stimuli that are derived from the tumor microenvironment developed in bone. Recent studies have shown that the sensitized and excited nerves innervating the tumor establish intimate communications with cancer cells by releasing various tumor-stimulating factors for tumor progression. APPROACHES: In this review, the role of the interactions of cancer cells and SNs in bone in the pathophysiology of CIBP will be discussed with a special focus on the role of the noxious acidic tumor microenvironment, considering that bone is in nature hypoxic, which facilitates the generation of acidic conditions by cancer. Subsequently, the role of SNs in the regulation of cancer progression in the bone will be discussed together with our recent experimental findings. CONCLUSION: It is suggested that SNs may be a newly-recognized important component of the bone microenvironment that contribute to not only in the pathophysiology of CIBP but also cancer progression in bone and dissemination from bone. Suppression of the activity of bone-innervating SNs, thus, may provide unique opportunities in the treatment of cancer progression and dissemination, as well as CIBP.

Molecular Mechanisms, Diagnosis and Treatments in Digestive Malignancy.

In this Special Issue, "Molecular Mechanisms, Diagnosis and Treatments in Digestive Malignancy", of the International Journal of Molecular Sciences, a total of 10 impactful articles have been published [...].

L-Carnitine Suppresses Transient Receptor Potential Vanilloid Type 1 Activation in Human Corneal Epithelial Cells.

Tear film hyperosmolarity induces dry eye syndrome (DES) through transient receptor potential vanilloid type 1 (TRPV1) activation. L-carnitine is a viable therapeutic agent since it protects against this hypertonicity-induced response. Here, we investigated whether L-carnitine inhibits TRPV1 activation by blocking heat- or capsaicin-induced increases in Ca2+ influx or hyperosmotic stress-induced cell volume shrinkage in a human corneal epithelial cell line (HCE-T). Single-cell fluorescence imaging of calcein/AM-loaded cells or fura-2/AM-labeled cells was used to evaluate cell volume changes and intracellular calcium levels, respectively. Planar patch-clamp technique was used to measure whole-cell currents. TRPV1 activation via either capsaicin (20 µmol/L), hyperosmolarity (≈450 mosmol/L) or an increase in ambient bath temperature to 43 °C induced intracellular calcium transients and augmented whole-cell currents, whereas hypertonicity induced cell volume shrinkage. In contrast, either capsazepine (10 µmol/L) or L-carnitine (1-3 mmol/L) reduced all these responses. Taken together, L-carnitine and capsazepine suppress hypertonicity-induced TRPV1 activation by blocking cell volume shrinkage.

TRPML3 enhances drug resistance in non-small cell lung cancer cells by promoting Ca2+-mediated lysosomal trafficking.

Lysosomes are emerging as versatile signaling hubs that mediate numerous cellular processes, including the development of drug resistance in cancer cells. Transient receptor potential mucolipin 3 (TRPML3), an endolysosomal Ca2+-permeable channel, is implicated in regulating lysosomal trafficking during endocytosis and autophagy. However, the role of TRPML3 in cancer progression remains unclear. In this study, we focused on identifying key molecules that modulate exosomal release triggered by lysosomal exocytosis during the development of gefitinib resistance in non-small cell lung cancer (NSCLC). We found that the basal release of exosomes and lysosomal exocytosis is higher in the gefitinib-resistant NSCLC cell line HCC827/GR than in the gefitinib-sensitive NSCLC cell line HCC827. Notably, exosomal release and lysosomal exocytosis were associated with an increase in TRPML3 expression. Lysosomal Ca2+ release via TRPML3 was triggered by the gefitinib-mediated elevation of lysosomal pH. Furthermore, TRPML3 deficiency enhanced the gefitinib-mediated increase in sub-G0 cell population, reduction of cell proliferation, and poly (ADP-ribose) polymerase cleavage. These data demonstrated that TRPML3 is a promising modulator of drug resistance. By sensing the elevation of lysosomal pH, it mediates lysosomal Ca2+ release, lysosomal trafficking and exocytosis, and exosomal release. Taken together, our study is the first to report the autonomous defense mechanism developed in NSCLC cells against the small-molecule tyrosine kinase inhibitor gefitinib, leading to acquired drug resistance.

How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP.

BACKGROUND: For many cell types, directional locomotion depends on their maintaining filopodia at the leading edge. Filopodia lack any Ca2+-binding structural protein but respond to store-operated Ca2+ entry (SOCE). METHODS: SOCE was induced by first replacing the medium with Ca2+-free salt solution with cyclopiazonic acid (CPA). This lowers Ca2+ in the ER and causes stromal interacting molecule (STIM) to be translocated to the cell surface. After this priming step, CPA was washed out, and Ca2+ influx restored by addition of extracellular Ca2+. Intracellular Ca2+ levels were measured by calcium orange fluorescence. Regulatory mechanisms were identified by pharmacological treatments. Proteins mediating SOCE were localized by immunofluorescence and analyzed after image processing. RESULTS: Depletion of the ER Ca2+ increased filopodia prevalence briefly, followed by a spontaneous decline that was blocked by inhibitors of endocytosis. Intracellular Ca2+ increased continuously for ~ 50 min. STIM and a transient receptor potential canonical (TRPC) protein were found in separate compartments, but an aquaporin unrelated to SOCE was present in both. STIM1- and TRPC1-bearing vesicles were trafficked on microtubules. During depletion, STIM1 migrated to the surface where it coincided with Orai in punctae, as expected. TRPC1 was partially colocalized with Vamp2, a rapidly releasable pool marker, and with phospholipases (PLCs). TRPC1 retreated to internal compartments during ER depletion. Replenishment of extracellular Ca2+ altered the STIM1 distribution, which came to resemble that of untreated cells. Vamp2 and TRPC1 underwent exocytosis and became homogeneously distributed on the cell surface. This was accompanied by an increased prevalence of filopodia, which was blocked by inhibitors of TRPC1/4/5 and endocytosis. CONCLUSIONS: Because the media were devoid of ligands that activate receptors during depletion and Ca2+ replenishment, we could attribute filopodia extension to SOCE. We propose that the Orai current stimulates exocytosis of TRPC-bearing vesicles, and that Ca2+ influx through TRPC inhibits PLC activity. This allows regeneration of the substrate, phosphatidylinositol 4,5 bisphosphate (PIP2), a platform for assembling proteins, e. g. Enabled and IRSp53. TRPC contact with PLC is required but is broken by TRPC dissemination. This explains how STIM1 regulates the cell's ability to orient itself in response to attractive or repulsive cues. Video Abstract.

Drug repurposing for the treatment of COVID-19.

Coronavirus disease 2019 (COVID-19) remains prevalent worldwide since its onset was confirmed in Wuhan, China in 2019. Vaccines against the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have shown a preventive effect against the onset and severity of COVID-19, and social and economic activities are gradually recovering. However, the presence of vaccine-resistant variants has been reported, and the development of therapeutic agents for patients with severe COVID-19 and related sequelae remains urgent. Drug repurposing, also called drug repositioning or eco-pharma, is the strategy of using previously approved and safe drugs for a therapeutic indication that is different from their original indication. The risk of severe COVID-19 and mortality increases with advancing age, cardiovascular disease, hypertension, diabetes, and cancer. We have reported three protein-protein interactions that are related to heart failure, and recently identified that one mechanism increases the risk of SARS-CoV-2 infection in mammalian cells. This review outlines the global efforts and outcomes of drug repurposing research for the treatment of severe COVID-19. It also discusses our recent finding of a new protein-protein interaction that is common to COVID-19 aggravation and heart failure.

Aberrant TRPC1 expression reflects stromal cervical invasion, lymphovascular invasion, elevated FIGO stage, and poor survival in resectable endometrial carcinoma patients.

BACKGROUND: Transient receptor potential channel 1 (TRPC1) promotes tumor growth and metastasis in endometrial carcinoma (EC) cell lines, whereas its clinical role in EC management remains unclear. Therefore, this study aimed to investigate the association of TRPC1 protein expression with the clinical features and survival of EC patients, then was further validated by TRPC1 mRNA measurement and data from The Human Protein Atlas. METHODS: TRPC1 protein expression in tumor tissues and normal endometria of 176 resectable EC patients was determined using immunohistochemistry. Besides, TRPC1 mRNA expression of partial patients (n = 80) was detected using RT-qPCR. Additionally, survival data from The Human Protein Atlas (derived from The Cancer Genome Atlas [TCGA]) was analyzed. RESULTS: TRPC1 protein expression was up-regulated in tumor tissue compared with normal endometrium (p < 0.001). Up-regulated TRPC1 protein expression was associated with stromal cervical invasion (p = 0.044), lymphovascular invasion (p = 0.032), and increased federation of gynecology and obstetrics (FIGO) stage (p = 0.005). Tumor TRPC1 protein high was linked with shortened accumulating disease-free survival (DFS) (p = 0.009) and overall survival (OS) (p = 0.026), which were also confirmed by multivariate Cox's regression analysis (both p < 0.050). Further, TRPC1 mRNA validation disclosed that TRPC1 mRNA high was related to shortened accumulating DFS (p = 0.038) and exhibited a correlating trend with declined OS (lacked statistical significance) (p = 0.162). Meanwhile, survival analysis on the data from The Human Protein Atlas (derived from TCGA) also exhibited that TRPC1 mRNA high was correlated with reduced accumulating OS (p < 0.001). CONCLUSION: Our findings support TRPC1 as a prognostic biomarker in resectable EC patients.

Dysregulated transient receptor potential channel 1 expression and its correlation with clinical features and survival profile in surgical non-small-cell lung cancer patients.

BACKGROUND: Transient receptor potential channel 1 (TRPC1) facilitates the tumor growth, metastasis, and chemoresistance in a series of neoplasms, while its correlation with clinical features and survival profile in NSCLC patients remains elusive. Hence, this study aimed to explore this topic. METHODS: Totally, 192 NSCLC patients were enrolled. Protein and mRNA expression of TRPC1 in carcinoma tissue and para-carcinoma tissue were evaluated by immunohistochemistry (IHC) assay and reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, respectively. RESULTS: Immunohistochemistry score and mRNA expression of TRPC1 were higher in carcinoma tissue compared with para-carcinoma tissue (both p < 0.001). Besides, increased TRPC1 IHC score (p = 0.004) and elevated TRPC1 mRNA overexpression (p = 0.016) were linked with occurrence of LYN metastasis; meanwhile, increased TRPC1 IHC score (p = 0.015) and raised TRPC1 mRNA expression (p = 0.009) were also linked with advanced TNM stage, whereas TRPC1 IHC score and TRPC1 mRNA expression were not correlated with other clinical features (all p > 0.05). Additionally, TRPC1 protein high (p = 0.007) and TRPC1 mRNA high (p = 0.015) were correlated with poor disease-free survival (DFS) but not correlated with overall survival (OS). Moreover, multivariate Cox's proportional hazards regression analysis showed that high TRPC1 protein expression (p = 0.046) and advanced TNM stage (p < 0.001) were independently correlated with poor DFS. However, TRPC1 protein and mRNA expression were not linked with OS (both p > 0.05), while poor differentiation (p = 0.003) and advanced TNM stage (p < 0.001) were independently associated with worse OS. CONCLUSIONS: TRPC1 is unregulated in NSCLC tissue with its overexpression relating to the occurrence of LYN metastasis and worse DFS in NSCLC patients.

cADPR Does Not Activate TRPM2.

cADPR is a second messenger that releases Ca2+ from intracellular stores via the ryanodine receptor. Over more than 15 years, it has been controversially discussed whether cADPR also contributes to the activation of the nucleotide-gated cation channel TRPM2. While some groups have observed activation of TRPM2 by cADPR alone or in synergy with ADPR, sometimes only at 37 °C, others have argued that this is due to the contamination of cADPR by ADPR. The identification of a novel nucleotide-binding site in the N-terminus of TRPM2 that binds ADPR in a horseshoe-like conformation resembling cADPR as well as the cADPR antagonist 8-Br-cADPR, and another report that demonstrates activation of TRPM2 by binding of cADPR to the NUDT9H domain raised the question again and led us to revisit the topic. Here we show that (i) the N-terminal MHR1/2 domain and the C-terminal NUDT9H domain are required for activation of human TRPM2 by ADPR and 2'-deoxy-ADPR (2dADPR), (ii) that pure cADPR does not activate TRPM2 under a variety of conditions that have previously been shown to result in channel activation, (iii) the cADPR antagonist 8-Br-cADPR also inhibits activation of TRPM2 by ADPR, and (iv) cADPR does not bind to the MHR1/2 domain of TRPM2 while ADPR does.

Transient Receptor Potential Channels and Itch.

Transient Receptor Potential (TRP) channels are multifunctional sensory molecules that are abundant in the skin and are involved in the sensory pathways of itch, pain, and inflammation. In this review article, we explore the complex physiology of different TRP channels, their role in modulating itch sensation, and their contributions to the pathophysiology of acute and chronic itch conditions. We also cover small molecule and topical TRP channel agents that are emerging as potential anti-pruritic treatments; some of which have shown great promise, with a few treatments advancing into clinical trials-namely, TRPV1, TRPV3, TRPA1, and TRPM8 targets. Lastly, we touch on possible ethnic differences in TRP channel genetic polymorphisms and how this may affect treatment response to TRP channel targets. Further controlled studies on the safety and efficacy of these emerging treatments is needed before clinical use.

TRPC6, a therapeutic target for pulmonary hypertension.

Idiopathic pulmonary arterial hypertension (PAH) is a fatal and progressive disease. Sustained vasoconstriction due to pulmonary arterial smooth muscle cell (PASMC) contraction and concentric arterial remodeling due partially to PASMC proliferation are the major causes for increased pulmonary vascular resistance and increased pulmonary arterial pressure in patients with precapillary pulmonary hypertension (PH) including PAH and PH due to respiratory diseases or hypoxemia. We and others observed upregulation of TRPC6 channels in PASMCs from patients with PAH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in PASMC triggers PASMC contraction and vasoconstriction, while Ca2+-dependent activation of PI3K/AKT/mTOR pathway is a pivotal signaling cascade for cell proliferation and gene expression. Despite evidence supporting a pathological role of TRPC6, no selective and orally bioavailable TRPC6 antagonist has yet been developed and tested for treatment of PAH or PH. In this study, we sought to investigate whether block of receptor-operated Ca2+ channels using a nonselective blocker of cation channels, 2-aminoethyl diphenylborinate (2-APB, administered intraperitoneally) and a selective blocker of TRPC6, BI-749327 (administered orally) can reverse established PH in mice. The results from the study show that intrapulmonary application of 2-APB (40 µM) or BI-749327 (3-10 µM) significantly and reversibly inhibited acute alveolar hypoxia-induced pulmonary vasoconstriction. Intraperitoneal injection of 2-APB (1 mg/kg per day) significantly attenuated the development of PH and partially reversed established PH in mice. Oral gavage of BI-749327 (30 mg/kg, every day, for 2 wk) reversed established PH by ∼50% via regression of pulmonary vascular remodeling. Furthermore, 2-APB and BI-749327 both significantly inhibited PDGF- and serum-mediated phosphorylation of AKT and mTOR in PASMC. In summary, the receptor-operated and mechanosensitive TRPC6 channel is a good target for developing novel treatment for PAH/PH. BI-749327, a selective TRPC6 blocker, is potentially a novel and effective drug for treating PAH and PH due to respiratory diseases or hypoxemia.