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Transient receptor potential canonical (TRPC) channels allow the intracellular entry of Ca 2+ and play important roles in several physio-pathological processes. In this study, we constructed transgenic mice expressing porcine TRPC1 (Tg-pTRPC1) to verify the effects of TRPC1 on skeletal muscle growth and elucidate the underlying mechanism. Porcine TRPC1 increased the muscle mass, fiber cross-sectional area, and exercise endurance of mice and accelerated muscle repair and regeneration. TRPC1 overexpression enhanced β-catenin expression and promoted myogenesis, which was partly reversed by inhibitors of β-catenin. TRPC1 facilitated the accumulation of intracellular Ca 2+ and nuclear translocation of the NFATC2/NFATC2IP complex involved in the Wnt/Ca 2+ pathway, promoting muscle growth. Paired related homeobox 1 (Prrx1) promoted the expression of TRPC1, NFATC2, and NFATC2IP that participate in the regulation of muscle growth. Taken together, our findings indicate that porcine TRPC1 promoted by Prrx1 could regulate muscle development through activating the canonical Wnt/β-catenin and non-canonical Wnt/Ca 2+ pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aims: Transient receptor potential canonical 5 (TRPC5) channels are redox-sensitive cation-permeable channels involved in temperature and mechanical sensation. Increased expression and over-activation of these channels has been implicated in several central nervous system disorders such as epilepsy, depression, traumatic brain injury, anxiety, Huntington's disease and stroke. TRPC5 channel activation causes increased calcium influx which in turn activates numerous downstream signalling pathways involved in the pathophysiology of neurological disorders. Therefore, we hypothesized that pharmacological blockade and knockdown of TRPC5 channels could attenuate the behavioural deficits and molecular changes seen in CNS disease models such as MPTP/MPP + induced Parkinson's disease (PD)., Materials and Methods: In the present study, PD was induced after bilateral intranigral infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the Sprague Dawley rats. Additionally, SH-SY5Y neurons were exposed to 1-methyl-4-phenylpyridinium (MPP + ) to further determine the role of TRPC5 channels in PD., Key Findings: We used clemizole hydrochloride, a potent TRPC5 channel blocker, to reverse the behavioural deficits, molecular changes and biochemical parameters in MPTP/MPP + -induced PD. Furthermore, knockdown of TRPC5 expression using siRNA also closely phenocopies these effects. We further observed restoration of tyrosine hydroxylase levels and improved mitochondrial health following clemizole treatment and TRPC5 knockdown. These changes were accompanied by diminished calcium influx, reduced levels of reactive oxygen species and decreased apoptotic signalling in the PD models., Significance: These findings collectively suggest that increased expression of TRPC5 channels is a potential risk factor for PD and opens a new therapeutic window for the development of pharmacological agents targeting neurodegeneration and PD., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objectives: Studies in certain cardiac hypertrophy models suggested the individual role of soluble epoxide hydrolase (sEH) and canonical transient receptor potential 3 (TRPC3) channels, however, whether they jointly mediate hypertrophic process remains unexplored. Hyperhomocysteinemia promotes cardiac hypertrophy while the involvement of sEH and TRPC3 channels remains unknown. This study aimed to explore the role of, and interrelation between sEH and TRPC3 channels in homocysteine-induced cardiac hypertrophy., Methods: Rats were fed methionine-enriched diet to induce hyperhomocysteinemia. H9c2 cells and neonatal rat cardiomyocytes were incubated with homocysteine. Cardiac hypertrophy was evaluated by echocardiography, histological examination, immunofluorescence imaging, and expressions of hypertrophic markers. Epoxyeicosatrienoic acids (EETs) were determined by ELISA. TRPC3 current was recorded by patch-clamp. Gene promotor activity was measured using dual-luciferase reporter assay., Results: Inhibition of sEH by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) reduced ventricular mass, lowered the expression of hypertrophic markers, decreased interstitial collagen deposition, and improved cardiac function in hyperhomocysteinemic rats, associated with restoration of EETs levels in myocardium. TPPU or knockdown of sEH suppressed TRPC3 transcription and translation as well as TRPC3 current that were enhanced by homocysteine. Exogenous 11,12-EET inhibited homocysteine-induced TRPC3 expression and cellular hypertrophy. Silencing C/EBPβ attenuated, while overexpressing C/EBPβ promoted homocysteine-induced hypertrophy and expressions of sEH and TRPC3, resulting respectively from inhibition or activation of sEH and TRPC3 gene promoters., Conclusions: sEH and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy. Homocysteine transcriptionally activates sEH and TRPC3 genes through a common regulatory element C/EBPβ. sEH activation leads to an upregulation of TRPC3 channels via a 11,12-EET-dependent manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Recent structural studies have shown that the carboxyl-terminus of many TRP channels, including TRPC3, are folded into a horizontal rib helix that is connected to the vertical pole helix, which play roles in inter-structural interactions and multimerization. In a previous work we identified I807 located in the pole helix with a role in regulation of TRPC3 by STIM1 (Lee et al., 2014, Liu et al., 2022). To further determine the role of the pole helix in TRPC3 function, here we identified key hydrophobic residues in the pole helix that form tight tunnel-like structure and used mutations to probe their role in TRPC3 regulation by Ca 2+ and Calmodulin. Our findings suggest that the hydrophobic starch formed by the I807-L818 residues has several roles, it modulates gating of TRPC3 by Ca 2+ , affects channel selectivity and the channel Ca 2+ permeability. Mutations of I807, I811, L814 and L818 all attenuated the Ca 2+ -dependent inactivation (CDI) of TRPC3, with I807 having the most prominent effect. The extent of modulation of the CDI depended on the degree of hydrophobicity of I807. Moreover, the TRPC3(I807S) mutant showed altered channel monovalent ion selectivity and increased Ca 2+ permeability, without affecting the channel permeability to Mg 2+ and Ba 2+ and without changing the pore diameter. The CDI of TRPC3 was reduced by an inactive calmodulin mutant and by a pharmacological inhibitor of calmodulin, which was eliminated by the I807S mutation. Notably, deletion of STIM1 caused similar alteration of TRPC3 properties. Taken together, these findings reveal a role of the pole helix in CDI, in addition to its potential role in channel multimerization that required gating of TRPC3 by STIM1. Since all TRPC and most TRP channels have pole helix structures, our findings raise the possibility that the pole helix may have similar roles in all the TRP family., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The canonical transient receptor potential 6 gene, TRPC6, has been implicated as a putative risk gene for chemotherapy-induced congestive heart failure, but knowledge of specific risk variants is lacking. Following our genome-wide association study and subsequent fine-mapping, a rare missense mutant of TRPC6 N338S, was identified in a breast cancer patient who received anthracycline-containing chemotherapy regiments and developed congestive heart failure. However, the function of N338S mutant has not been examined. Using intracellular Ca 2+ imaging, patch clamp recording and molecular docking techniques, we assessed the function of N338S mutant heterologously expressed in HEK293 cells and HL-1 cardiac cells. We found that expression of TRPC6 N338S significantly increased intracellular Ca 2+ levels ([Ca 2+ ] i ) and current densities in response to 50 μM 1-oleoyl 2-acetyl-sn-glycerol (OAG), an activator of TRPC6 channels, compared to those of TRPC6 WT. A 24-h pretreatment with 0.5 μM doxorubicin (DOX) further potentiated the OAG effects on TRPC6 N338S current densities and [Ca 2+ ] i , and these effects were abolished by 1 μM BI-749327, a highly selective TRPC6 inhibitor. Moreover, DOX treatment significantly upregulated the mRNA and protein expressions of TRPC6 N338S, compared to those of TRPC6 WT. Molecular docking and dynamics simulation showed that OAG binds to the pocket constituted by the pore-helix, S5 and S6 domains of TRPC6. However, the N338S mutation strengthened the interaction with OAG, therefore stabilizing the OAG-TRPC6 N338S complex and enhancing OAG binding affinity. Our results indicate that TRPC6 N338S is a gain-of-function mutant that may contribute to DOX-induced cardiotoxicity by increasing Ca 2+ influx and [Ca 2+ ] i in cardiomyocytes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the works reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Depression has a growing impact on public health. Accumulating evidence supports an association between depression and increased immune system activity. IL-10 is a key cytokine that inhibits excessive inflammatory responses and is related to the anti-inflammatory and protective functions of the central nervous system (CNS). Cx3cr1 CreER IL-10 -/- mice were used in our study. We aimed to identify the role of IL-10 in microglia in depression and anxiety-like behavior. We performed a series of behavioral tests on the mice; the Cx3cr1 CreER IL-10 -/- male mice showed depression- and anxiety-like behavior compared with the littermates. The expression of transient receptor potential canonical 5 (TRPC5) decreased in both the medial prefrontal cortex (mPFC) and amygdala regions. The cytokines IL-1β and IL-6 increased, and IL-10 was decreased by western blotting. The knockout mice showed different trends in the effects of synaptic proteins. In the mPFC, IL-10 knockout induced a decrease in NR2B and synaptophysin; in the amygdala region, there was a significant increase in NR2B and PSD95. IL-10 knockout from microglia induced a decrease in GAD67 and parvalbumin (Pv) in the mPFC, but not in the amygdala. Our results showed enhanced depression and anxiety-like behavior in the Cx3cr1 CreER IL-10 -/- mice, which could be related to an imbalance in local excitatory and inhibitory transmission, as well as neuroinflammation in the mPFC and amygdala. This imbalance was associated with increased local inflammation. Although many studies have demonstrated the role of TRPC channels in emotional responses, our study showed that TRPC was not involved in this process in Cx3cr1 CreER IL-10 -/- mice., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aims: Brain-derived neurotrophic factor (BDNF) is vital in the pathogenesis of mechanical allodynia with a paucity of reports available regarding diabetic neuropathy pain (DNP). Herein we identified the involvement of BDNF in driving mechanical allodynia in DNP rats via the activation of transient receptor potential canonical 6 (TRPC6) channel., Materials and Methods: The DNP rat model was established via streptozotocin (STZ) injection, and allodynia was assessed by paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL). The expression profiles of BDNF and TRPC6 in dorsal root ganglia (DRG) and spinal cord were illustrated by immunofluorescence and Western blotting. Intrathecal administration of K252a or TrkB-Fc was performed to inhibit BNDF/TrkB expression, and respective injection of GsMTX-4, BTP2 and TRPC6 antisense oligodeoxynucleotides (TRPC6-AS) was likewise conducted to inhibit TRPC6 expression in DNP rats. Calcium influx in DRG was monitored by calcium imaging., Key Findings: The time-dependent increase of BDNF and TRPC6 expression in DRG and spinal cord was observed since the 7th post-STZ day, correlated with the development of mechanical allodynia in DNP rats. Intrathecal administration of K252a, TrkB-Fc, GsMTX-4 and BTP2 prevented mechanical allodynia in DNP rats. Pre-treatment of TRPC6-AS reversed the BDNF-induced pain-like responses in DNP rats rather than the naïve rats. In addition, the TRPC6-AS reversed BDNF-induced increase of calcium influx in DRG neurons in DNP rats., Significance: The intrathecal inhibition of TRPC6 alleviated the BDNF-induced mechanical allodynia in DNP rat model. This finding may validate the application of TRPC6 antagonists as interesting strategy for DNP management., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Metastasis is frequently occurred in end-stage GC. Nevertheless, the initiation and progression of metastasis in GC remains unclear. The transient receptor potential canonical (TRPC) has been confirmed to be crucial for metastasis in many kinds of tumors, including GC. However, the molecular mechanisms regulating TRPC1 is unclear. Therefore, we investigated the role and mechanisms of TRPC1 in GC metastasis. We first evaluated the role of TRPC1 in GC by searching the public database, and tested the expression of TRPC1 in 50 paired GC tissues by qRT-PCR and IHC assays. Then, we generated BGC-823-shTRPC1 cells and MKN-45-TRPC1 cells to investigate the effects of TRPC1 on metastasis in vitro. For the mechanism study, we applied luciferase reporter assay, RNA pull-down assay, as well as RIP assay to validate the interation of ciRS-7, miR-135a-5p and TRPC1 in GC cells. This study, we showed that TRPC1 exacerbate EMT in gastric cancer via ciRS-7/miR-135a-5p/TRPC1 axis, and target TRPC1 could be beneficial for end-stage GC patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aims: Idiopathic scoliosis is a common deformity of the spine that has an especially high incidence rate in adolescents. Some studies have demonstrated a close relationship between idiopathic scoliosis and melatonin deficiency. Our team's previous research showed that melatonin can inhibit the proliferation of osteoblasts, but the mechanism remains unclear. This study aimed to determine the mechanism by which melatonin inhibits the proliferation of osteoblasts., Main Methods: Cell viability experiment, DNA fragment detection and alkaline phosphatase (ALP) activity assays were performed to determine the effects of melatonin on the proliferation, apoptosis and differentiation of osteoblasts. We used immunofluorescence to detect the expression of STIM1 in melatonin-treated osteoblasts. STIM1 interference was achieved using a specific siRNA, and a TRPC inhibitor was used to block the influx of Ca 2+ . The mRNA expression was determined by RT-qPCR, and protein levels were measured by Western blot., Key Findings: In this study, we found that melatonin inhibited the proliferation, differentiation and apoptosis of osteoblasts in a concentration-dependent manner. Additional studies showed that melatonin elevated cytosolic calcium levels by upregulation of STIM1, leading to osteoblast apoptosis via the mitochondrial pathway. Finally, we demonstrated that the STIM1-mediated increase in cytosolic calcium levels induced apoptosis through the ERK pathway., Significance: Melatonin induces mitochondrial apoptosis in osteoblasts by regulating the STIM1/cytosolic calcium elevation/ERK pathway. These basic findings provide a basis for further clinical studies on melatonin as a drug therapeutic for idiopathic scoliosis., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protein S-palmitoylation, the covalent lipid modification of the side chain of Cys residues with the 16‑carbon fatty acid palmitate, is the most common acylation, and it enhances the membrane stability of ion channels. This post-translational modification (PTM) determines a functional mechanism of ion channel life cycle from maturation and membrane trafficking to localization. Especially, neurodevelopment is regulated by balancing the level of synaptic protein palmitoylation/depalmitoylation. Recently, we revealed the pathological role of the transient receptor potential canonical type 5 (TRPC5) channel in striatal neuronal loss during Huntington's disease (HD), which is abnormally activated by oxidative stress. Here, we report a mechanism of TRPC5 palmitoylation at a conserved cysteine residue, that is critical for intrinsic channel activity. Furthermore, we identified the therapeutic effect of TRPC5 depalmitoylation by enhancing the TRPC5 membrane instability on HD striatal cells in order to lower TRPC5 toxicity. Collectively, these findings suggest that controlling S-palmitoylation of the TRPC5 channel as a potential risk factor can modulate TRPC5 channel expression and activity, providing new insights into a therapeutic strategy for neurodegenerative diseases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Long-term high salt intake leads to cardiac hypertrophy, but the mechanism remains elusive. Transient receptor potential channel, canonical 3(TRPC3), located in mitochondria, regulates mitochondrial calcium and reactive oxygen species(ROS) production. Herein, we investigated whether TRPC3 participates in high salt-induced cardiac hypertrophy by impairing cardiac mitochondrial function. High salt treatment increased the expression of mitochondrial TRPC3 in cardiomyocytes, accompanied by enhanced mitochondrial calcium uptake and elevated ROS production. Inhibition of TRPC3 significantly reduced high salt-induced ROS generation, promoted ATP production by stimulating oxidative phosphorylation, and increased enzyme activity in mitochondria in cardiomyocytes. Additionally, TRPC3 deficiency inhibited high salt-induced cardiac hypertrophy in vivo. A long-term high salt diet increased cardiac mitochondrial TRPC3 expression, elevated expression of cardiac hypertrophic markers atrial natriuretic peptide (ANP),brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) and decreased ATP production and mitochondrial complex I and II enzyme activity in a TRPC3-dependent manner. TRPC3 deficiency antagonises high salt diet-mediated cardiac hypertrophy by ameliorating TRPC3-mediated cardiac mitochondrial dysfunction. TRPC3 may therefore represent a novel target for preventing high salt-induced cardiac damage., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Our recent study demonstrated that the nonselective cation current mediated by the transient receptor potential canonical 1 (TRPC1) channel is activated by endothelin-1 (ET-1) in human atrial myocytes; however, the related signal molecules involved are unknown., Objective: The purpose of this study was to investigate how the TRPC1 channel is regulated by ET-1 and whether it is upregulated in human atria from patients with atrial fibrillation (AF)., Methods: Whole-cell patch technique and molecular biology techniques were used in the study., Results: The ET-1-evoked TRPC1 current was inhibited by the ET-1 type A (ET A ) receptor antagonist BQ123 and the ET-1 type B (ET B ) receptor antagonist BQ788 as well as the protein kinase C inhibitor chelerythrine. ET A receptor-mediated TRPC1 channel activity was selectively inhibited by the phosphoinositide-3-kinase inhibitor wortmannin, while ET B receptor-mediated TRPC1 activity was inhibited by the phospholipase C inhibitor U73122. The messenger RNAs and proteins of the TRPC1 channel and ET A receptor, but not the ET B receptor, were significantly upregulated in atria from patients with AF. The basal TRPC1 current increased in AF myocytes, and the response to ET-1 was greater in AF myocytes than in sinus rhythm myocytes. ET-1 induced a delayed repolarization in 20% of AF myocytes., Conclusion: These results demonstrate for the first time that TRPC1 activation by ET-1 is mediated by protein kinase C through the distinct phospholipids pathways phosphoinositide-3-kinase and phospholipase C and that the TRPC1 channel and ET A receptor are upregulated in AF atria, which are likely involved in atrial electrical remodeling in patients with AF., (Copyright © 2019 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Mutation of a single residue within the recently identified lipid (diacylglycerol) recognition window of TRPC3 (G652A) was found to abolish channel activation via endogenous lipid mediators while retaining sensitivity to the non-lipid activator GSK1702934A (abb. GSK). The mechanism of this change in chemical sensing by TRPC3 was analysed by whole-cell and single channel electrophysiology as well as Ca 2+ imaging. Currents initiated by GSK or the structural (benzimidazole) analog BI-2 were significantly larger in cells expressing the G652A mutant as compared to wild type (WT) channels. Whole cell patch-clamp experiments revealed that enhanced sensitivity to benzimidazoles was not due to augmented potency but reflected enhanced efficacy of benzimidazoles. Single channel analysis demonstrated that neither unitary conductance nor I-V characteristics were altered by the G652A mutation, precluding altered pore architecture as the basis of enhanced efficacy. These experiments uncovered a distinct gating pattern of BI-2-activated G652A mutant channels, featuring a unique, long-lived open state. Moreover, G652A mutant channels lacked PLC/diacylglycerol mediated cross-desensitization for GSK activation as typically observed for TRPC3. Lack of desensitization in G652A channels enabled large GSK/BI-2-induced Ca 2+ signals in conditions that fully desensitized TRPC3 WT channels. We demonstrate that the lipid-recognition window of TRPC3 determines both sensitivity to lipid mediators and chemical gating by benzimidazoles. TRPC3 mutations within this lipid interaction site are suggested as a basis for chemogenetic targeting of TRPC3-signaling., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Gain-of-function mutations in TRPC6 cause familial focal segmental glomerulosclerosis, and TRPC6 is upregulated in glomerular diseases including diabetic kidney disease. We studied the effect of systemic TRPC6 knockout in the Akita model of type 1 diabetes. Knockout of TRPC6 inhibited albuminuria in Akita mice at 12 and 16 weeks of age, but this difference disappeared by 20 weeks. Knockout of TRPC6 also reduced tubular injury in Akita mice; however, mesangial expansion was significantly increased. Hyperglycemia and blood pressure were similar between TRPC6 knockout and wild-type Akita mice, but knockout mice were more insulin resistant. In cultured podocytes, knockout of TRPC6 inhibited expression of the calcium/calcineurin responsive gene insulin receptor substrate 2 and decreased insulin responsiveness. Insulin resistance is reported to promote diabetic kidney disease independent of blood glucose levels. While the mechanisms are not fully understood, insulin activates both Akt2 and ERK, which inhibits apoptosis signal regulated kinase 1 (ASK1)-p38-induced apoptosis. In cultured podocytes, hyperglycemia stimulated p38 signaling and induced apoptosis, which was reduced by insulin and ASK1 inhibition and enhanced by Akt or ERK inhibition. Glomerular p38 signaling was increased in TRPC6 knockout Akita mice and was associated with enhanced expression of the p38 gene target cyclooxygenase 2. These data suggest that knockout of TRPC6 in Akita mice promotes insulin resistance and exacerbates glomerular disease independent of hyperglycemia., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Store-operated Ca 2+ entry (SOCE) is a functionally relevant mechanism for Ca 2+ influx present in electrically excitable and non-excitable cells. Regulation of Ca 2+ entry through store-operated channels is essential to maintain an appropriate intracellular Ca 2+ homeostasis and prevent cell damage. Calcium-release activated channels exhibit Ca 2+ -dependent inactivation mediated by two temporally separated mechanisms: fast Ca 2+ -dependent inactivation takes effect in the order of milliseconds and involves the interaction of Ca 2+ with residues in the channel pore while slow Ca 2+ -dependent inactivation (SCDI) develops over tens of seconds, requires a global rise in [Ca 2+ ] cyt and is a mechanism regulated by mitochondria. Recent studies have provided evidence that the protein SARAF (SOCE-associated regulatory factor) is involved in the mechanism underlying SCDI of Orai1. SARAF is an endoplasmic reticulum (ER) membrane protein that associates with STIM1 and translocate to plasma membrane-ER junctions in a STIM1-dependent manner upon store depletion to modulate SOCE. SCDI mediated by SARAF depends on the location of the STIM1-Orai1 complex within a PI(4,5)P 2 -rich microdomain. SARAF also interacts with Orai1 and TRPC1 in cells endogenously expressing STIM1 and cells with a low STIM1 expression and modulates channel function. This review focuses on the modulation by SARAF of SOCE and other forms of Ca 2+ influx mediated by Orai1 and TRPC1 in order to provide spatio-temporally regulated Ca 2+ signals., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background and Aims: Recent in vitro studies have showed that in macrophages, deletion of the non-selective Ca 2+ -permeable channel TRPC3 impairs expression of the osteogenic protein BMP-2. The pathophysiological relevance of this effect in atherosclerotic plaque calcification remains to be determined., Methods: We used Ldlr -/- mice with macrophage-specific loss of TRPC3 (MacTrpc3 -/- /Ldlr -/- ) to examine the effect of macrophage Trpc3 on plaque calcification and osteogenic features in advanced atherosclerosis., Results: After 25 weeks on high fat diet, aortic root plaques in MacTrpc3 -/- /Ldlr -/- mice showed reduced size, lipid and macrophage content compared to controls. Plaque calcification was decreased in MacTrpc3 -/- /Ldlr -/- mice, and this was accompanied by marked reduction in BMP-2, Runx-2 and phospho-SMAD1/5 contents within macrophage-rich areas. Expression of Bmp-2 and Runx-2 was also reduced in bone marrow-derived macrophages from MacTrpc3 -/- /Ldlr -/- mice., Conclusions: These findings show that, in advanced atherosclerosis, selective deletion of TRPC3 in macrophages favors plaque regression and impairs the activity of a novel macrophage-associated, BMP-2-dependent mechanism of calcification., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Classic lesion and physiology experiments identified the hypothalamic preoptic area as a pivotal region in the regulation of temperature homeostasis. The preoptic area can sense changes in local temperature, receives information about ambient temperature, contributes to fever, and can affect thermoregulation in response to several biologic signals. Electrophysiologic studies indicate that these actions are mediated by a neuronal circuitry that comprises temperature-sensitive as well as temperature-insensitive neurons. Little is known on the molecules that may be required for central thermosensation and much of the efforts towards their identification was done for warm-sensitive neurons. Here we summarize the current knowledge on the subject as well as what the search for these molecules revealed about warm-sensitive neurons., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Transient receptor potential (TRP) channels play important functional roles in the signal transduction machinery of hormone-secreting cells and have recently been implicated in reproductive physiology. While expression studies have demonstrated TRP channel expression at all levels of the hypothalamic-pituitary-gonadal (hpg) axis, functional details about TRP channel action at the level of the individual cells controlling reproduction are just beginning to emerge. Canonical TRP (TRPC) channels are prominently expressed in the reproductive center of the neuroendocrine brain, i.e. in kisspeptin and gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin neurons are depolarized by leptin via activation of TRPC channels and kisspeptin depolarizes GnRH neurons through TRPC4 activation. Recent studies have functionally identified TRPC channels also in gonadotrope cells in the anterior pituitary gland, which secrete gonadotropins in response to GnRH and thus regulate gonadal function. TRP channel expression in these cells exhibits remarkable plasticity and depends on the hormonal status of the animal. Subsequent functional analyses have demonstrated that TRPC5 in gonadotropes contributes to depolarization of the plasma membrane upon GnRH stimulation and increases the intracellular Ca 2+ concentration via its own Ca 2+ permeability and via the activation of voltage-gated Ca 2+ channels. However, conditional gene targeting experiments will be needed to unambiguously dissect the physiological role of TRPC channels in the different cell types of the reproductive axis in vivo., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Thyroid hormones play a crucial role in midbrain dopaminergic (DA) neuron development. However, the underlying molecular mechanisms remain largely unknown. In this study, we revealed that thyroid hormone treatment evokes significant calcium entry through canonical transient receptor potential (TRPC) channels in ventral midbrain neural stem cells and this calcium signaling is essential for thyroid hormone-dependent DA neuronal differentiation. We also found that TRPC1 is the dominant TRPC channel expressed in ventral midbrain neural stem cells which responds to thyroid hormone. In addition, thyroid hormone increases TRPC1 expression through its receptor alpha 1 during DA neuron differentiation, and, importantly, produces calcium signals by activating TRPC1 channels. In vivo and in vitro gene silencing experiments indicate that TRPC1-mediated calcium signaling is required for thyroid hormone-dependent DA neuronal differentiation. Finally, we confirmed that the activation of OTX2, a determinant of DA neuron development and the expression of which is induced by thyroid hormone, is dependent on TRPC1-mediated calcium signaling. These data revealed the molecular mechanisms of how thyroid hormone regulates DA neuron development from ventral midbrain neural stem cells, particularly endowing a novel physiological relevance to TRPC1 channels., (Copyright © 2017. Published by Elsevier B.V.)

Increasing circulating Ca 2+ levels within the normal range has been reported to positively correlate with the incidence of fatal cardiovascular diseases (CVDs). However, limited studies have been able to delineate the potential mechanism(s) linking circulating Ca 2+ to CVD. In this study, we exposed primary human umbilical vein endothelial cells (HUVECs) and human umbilical vein cell line (EA.hy926) to different extracellular Ca 2+ to mimic the physiological state. Our data revealed that increasing extracellular Ca 2+ significantly enhanced susceptibility to tumor necrosis factor (TNF)-alpha-stimulated vascular cell adhesion molecule (VCAM)-1 expression and monocytes adhesion. Knocking-down VCAM-1 by siRNA abolished calcium-induced monocytes adhesion on HUVECs. Follow up mechanistic investigations identified that extracellular Ca 2+ -increased calcium influx contributed to the activation of VCAM-1. This was mediated via upregulation of transient receptor potential channel (TRPC)1 in a nuclear factor (NF)κB-dependent manner. Most importantly, we found that a novel TRPC1-regulated extracellular signal-regulated kinase 1/2 (ERK1/2) pathway exclusively contributed to calcium-induced NFκB activation. This study provided direct evidence that increasing extracellular Ca 2+ enhanced TNF-alpha-induced VCAM-1 activation and monocytes adhesion. Moreover, we identified a novel TRPC1/ERK1/2/NFκB signaling pathway mediating VCAM-1 activation and monocyte adhesion in this pathological process. Our studies indicate that blood calcium levels should be strictly monitored to help prevent CVD, and that TRPC1 might act as a potential target for the treatment and prevention against increased circulating calcium-enhanced CVDs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The transient receptor potential canonical channel 5 (TRPC5) is a Ca 2+ -permeable ion channel, which is predominantly expressed in the brain. TRPC5-deficient mice exhibit a reduced innate fear response and impaired motor control. In addition, outgrowth of hippocampal and cerebellar neurons is retarded by TRPC5. However, pharmacological evidence of TRPC5 function on cellular or organismic levels is sparse. Thus, there is still a need for identifying novel and efficient TRPC5 channel modulators. We, therefore, screened compound libraries and identified the glucocorticoid methylprednisolone and N-[3-(adamantan-2-yloxy)propyl]-3-(6-methyl-1,1-dioxo-2H-1λ 6 ,2,4-benzothiadiazin-3-yl)propanamide (BTD) as novel TRPC5 activators. Comparisons with closely related chemical structures from the same libraries indicate important substructures for compound efficacy. Methylprednisolone activates TRPC5 heterologously expressed in HEK293 cells with an EC 50 of 12μM, while BTD-induced half-maximal activation is achieved with 5-fold lower concentrations, both in Ca 2+ assays (EC 50 =1.4μM) and in electrophysiological whole cell patch clamp recordings (EC 50 =1.3 μM). The activation resulting from both compounds is long lasting, reversible and sensitive to clemizole, a recently established TRPC5 inhibitor. No influence of BTD on homotetrameric members of the remaining TRPC family was observed. On the main sensory TRP channels (TRPA1, TRPV1, TRPM3, TRPM8) BTD exerts only minor activity. Furthermore, BTD can activate heteromeric channel complexes consisting of TRPC5 and its closest relatives TRPC1 or TRPC4, suggesting a high selectivity of BTD for channel complexes bearing at least one TRPC5 subunit., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The human TRP protein family comprises a family of 27 cation channels with diverse permeation and gating properties. The common theme is that they are very important regulators of intracellular Ca 2+ signaling in diverse cell types, either by providing a Ca 2+ influx pathway, or by depolarising the membrane potential, which on one hand triggers the activation of voltage-gated Ca 2+ channels, and on the other limits the driving force for Ca 2+ entry. Here we focus on the role of these TRP channels in vascular smooth muscle and cardiac striated muscle. We give an overview of highlights from the recent literature, and highlight the important and diverse roles of TRP channels in the pathophysiology of the cardiovascular system. The discovery of the superfamily of Transient Receptor Potential (TRP) channels has significantly enhanced our knowledge of multiple signal transduction mechanisms in cardiac muscle and vascular smooth muscle cells (VSMC). In recent years, multiple studies have provided evidence for the involvement of these channels, not only in the regulation of contraction, but also in cell proliferation and remodeling in pathological conditions. The mammalian family of TRP cation channels is composed by 28 genes which can be divided into 6 subfamilies groups based on sequence similarity: TRPC (Canonical), TRPM (Melastatin), TRPML (Mucolipins), TRPV (Vanilloid), TRPP (Policystin) and TRPA (Ankyrin-rich protein). Functional TRP channels are believed to form four-unit complexes in the plasma, each of them expressed with six transmembrane domain and intracellular N and C termini. Here we review the current knowledge on the expression of TRP channels in both muscle types, and discuss their functional properties and role in physiological and pathophysiological processes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Present study unravels the involvement of different calcium signaling pathways in oxytocin-induced contractions in myometrium of non-pregnant and pregnant buffaloes during early and mid-pregnancy stages. Uteri of pregnant animals were more sensitive than of non-pregnant buffaloes. Phasic contractions and frequency of contraction significantly increased with advancement of pregnancy, while tonic contractions non-significantly and amplitude significantly decreased from six months pregnancy onward. Oxytocin produced concentration-dependent-contraction on isolated myometrial strips of pregnant and non-pregnant buffaloes and the dose response curves (DRCs) of oxytocin were significantly (P < 0.05) shifted to right in the presence of nifedipine (1 μM), in Ca 2+ -free Ringer Locke solution (RLS), ruthenium red (30 μM), ruthenium red + nifedipine, cyclopiazonic acid (CPA; Ca 2+ free RLS as well as RLS), CPA (10 μM)+nifedipine, U-73122 (1 μM) + nifedipine and SKF96365 (25 μM) on uteri of non-pregnant and pregnant (early and mid) animals. The DRCs were also significantly shifted towards right in the presence of Y-27632 (10 μM), GF109203X (5 μM) and Pyr3 (10 μM) on uteri of non-pregnant and early pregnancy stage buffaloes while only in the presence of U-73122 (1 μM) on uteri of mid-pregnancy stage buffaloes. Our finding suggest that and L-type Ca 2+ channels, IP3-RyR-gated, and store-operated calcium channels including transient receptor potential channel (TRPC) pathways play significant role in mediating oxytocin-induced contractions in myometrium of pregnant and non-pregnant buffaloes. SERCA plays major role only during early-pregnancy while functional role of protein kinase C (PKC), Rho-kinase and TRPC3 pathways decreased and role of G-protein coupled receptor-phospholipase C (GPCR-PLC) pathway increased with advancement of pregnancy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fibrosis is an exaggerated form of tissue repair that occurs with serious damage or repetitive injury and ultimately leads to organ failure due to the excessive scarring. Increased calcium ion entry through the TRPC6 channel has been associated with the pathogenesis of heart and glomerular diseases, but its role in renal interstitial fibrosis is unknown. We studied this by deletion of Trpc6 in mice and found it decreased unilateral ureteral obstruction-induced interstitial fibrosis and blunted increased mRNA expression of fibrosis-related genes in the ureteral obstructed kidney relative to that in the kidney of wild-type mice. Administration of BTP2, a pyrazol derivative known to inhibit function of several TRPC channels, also ameliorated obstruction-induced renal fibrosis and gene expression in wild-type mice. BTP2 inhibited carbachol-activated TRPC3 and TRPC6 channel activities in HEK293 cells. Ureteral obstruction caused over a 10-fold increase in mRNA expression for TRPC3 as well as TRPC6 in the kidneys of obstructed relative to the sham-operated mice. The magnitude of protection against obstruction-induced fibrosis in Trpc3 and Trpc6 double knockout mice was not different from that in Trpc6 knockout mice. Klotho, a membrane and soluble protein predominantly produced in the kidney, is known to confer protection against renal fibrosis. Administration of soluble klotho significantly reduced obstruction-induced renal fibrosis in wild-type mice, but not in Trpc6 knockout mice, indicating that klotho and TRPC6 inhibition act in the same pathway to protect against obstruction-induced renal fibrosis. Thus klotho and TRPC6 may be pharmacologic targets for treating renal fibrosis., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Airway hyperresponsiveness (AHR), airway remodeling and inflammation are the fundamental pathological alterations that occur in asthma. Transient receptor potential canonical 3 (TRPC3) has been implicated in diverse functions of airway smooth muscle cells (ASMCs) in asthma. However, the underlying mechanisms remain incompletely understood. We investigated the mRNA and protein expression of TRPC3 in ASMCs from normal and OVA-sensitized mouse. And the effects of inhibition or knockdown of TRPC3 with Ethyl-1- (4- (2,3,3-trichloroacrylamide) phenyl) -5 - (trifluoromethyl) -1H -pyrazole -4-carboxylate (Pyr3) and lentiviral shRNA on OVA-sensitized mouse AHR, airway remodeling, circulating inflammatory cytokines, cell proliferation and migration. We found that TRPC3 mRNA and protein expression levels were significantly increased in ASMCs from OVA-sensitized mouse. Inhibiting TRPC3 with continuous subcutaneous administration of Pyr3 decreased enhanced pause (Penh) of OVA-sensitized mouse. Meanwhile, both Pyr3 and lentiviral shRNA treatment of ASMCs in OVA-sensitized mouse significantly decreased their proliferation and migration. These results suggest that TRPC3 plays a critical role in asthma and represents a promising new target for asthma treatment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Pulmonary fibrosis (PF) is a chronic progressive lung disease without effective medical treatment options leading to respiratory failure and death within 3-5years of diagnosis. The pathological process of PF is driven by aberrant wound-healing involving fibroblasts and myofibroblasts differentiated by secreted profibrotic transforming growth factor β (TGF-β1). Classical transient receptor potential 6 (TRPC6), a Na + - and Ca 2+ -permeable cation channel, is able to promote myofibroblast conversion of primary rat cardiac and human dermal fibroblasts and TRPC6-deficiency impaired wound healing after injury. To study a potential role of TRPC6 in the development of PF we analyzed lung function, gene and protein expression in wild-type (WT) and TRPC6-deficient (TRPC6-/-) lungs utilizing a bleomycin-induced PF-model. Fibrotic WT-mice showed a significant higher death rate while bleomycin-treated TRPC6-deficient mice were partly protected from fibrosis as a consequence of a lower production of collagen and an almost normal function of the respiratory system (reduced resistance and elastance compared to fibrotic WT-mice). On a molecular level TGF-β1 induced TRPC6 up-regulation, increased Ca 2+ influx and nuclear NFAT localization in WT primary murine lung fibroblasts (PMLFs) resulting in higher stress fiber formation and accelerated contraction rates as compared to treated TRPC6-deficient fibroblasts. Therefore, we conclude that TRPC6 is an important determinant for TGF-β1-induced myofibroblast differentiation during fibrosis and specific channel inhibitors might be beneficial in a future treatment of PF., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aberrant transforming growth factor β1 (TGFβ1) signaling plays a crucial role in the pathogenesis of vascular fibrosis. On the other hand, deregulated transient receptor potential canonical 6 (TRPC6) channel expression shows impaired vascular physiology and wound healing. However, it has little been known about the functional association between TGFβ1 and TRPC6 in vascular smooth muscle cells (VSMCs). In this study, we analyzed the microarray data obtained from TGFβ1-treated A7r5 VSMCs. We found that TGFβ1 specifically elevates the expression level of TRPC6 mainly through Smad-dependent canonical pathway. The siRNA against TRPC6 abolished TGFβ1-induced molecular and cellular phenotype changes, including myosin light chain phosphorylation, actin stress fiber formation, and cell migration. These results demonstrate that TRPC6 is an important component of TGFβ1 signaling pathway in VSMCs. Therefore, our findings provide a basis for future investigation aimed at developing novel therapeutic strategies for treatment of vascular fibrosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Spinal transient receptor potential ankyrin 1 (TRPA1) channel is associated with various pain hypersensitivity conditions. Spinally, TRPA1 is expressed by central terminals of nociceptive nerve fibers and astrocytes. Among potential endogenous agonists of TRPA1 is H 2 O 2 generated by d-amino acid oxidase (DAAO) in astrocytes. Here we studied whether prolonged block of the spinal TRPA1 or astrocytes starting at time of injury attenuates development and/or maintenance of neuropathic hypersensitivity. Additionally, TRPA1 and DAAO mRNA were determined in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH). Experiments were performed in rats with spared nerve injury (SNI) and chronic intrathecal catheter. Drugs were administered twice daily for the first seven injury days or only once seven days after injury. Mechanical hypersensitivity was assessed with monofilaments. Acute and prolonged treatment with Chembridge-5861528 (a TRPA1 antagonist), carbenoxolone (an inhibitor of activated astrocytes), or gabapentin (a comparison drug) attenuated tactile allodynia-like responses evoked by low (2g) stimulus. However, antihypersensitivity effect of these compounds was short of significance at a high (15g) stimulus intensity. No preemptive effects were observed. In healthy controls, carbenoxolone failed to prevent hypersensitivity induced by spinal cinnamaldehyde, a TRPA1 agonist. TRPA1 and DAAO mRNA in the DRG but not SDH were slightly increased in SNI, independent of drug treatment. The results indicate that prolonged peri-injury block of spinal TRPA1 or inhibition of spinal astrocyte activation attenuates maintenance but not development of mechanical (tactile allodynia-like) hypersensitivity after nerve injury., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The epithelium lining the oviduct is critical for early reproductive events, many of which are mediated via intracellular calcium ions. Despite this, little is known about the regulation of calcium homeostasis in the oviductal epithelium. Epithelial transient receptor potential channels (TRPCs) modulate calcium flux in other tissues, and their expression and functional regulation have therefore been examined using the bovine oviduct as a model for the human. The effects of FSH, LH, 17β-estradiol, and progesterone on TRPCs expression and intracellular calcium flux were determined. Transient receptor potential channels 1, 2, 3, 4, and 6 were expressed in the bovine reproductive tract, and their gene expression varied throughout the estrous cycle. In more detailed studies undertaken on TRPC1 and 6, we show that protein expression varied through the estrus cycle; specifically, 17β-estradiol, FSH, and LH individually and in combination upregulated TRPC1 and 6 expression in cultured bovine oviduct epithelial cells although progesterone antagonized these effects. Functional studies showed changes in calcium mobilization in bovine oviduct epithelial cells were dependent on TRPCs. In conclusion, TRPC1, 2, 3, 4, and 6 are present in the epithelium lining the bovine oviduct, and TRPC1 and 6 vary through the estrous cycle suggesting an important role in early reproductive function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Transendothelial migration (TEM) of leukocytes is the step in leukocyte emigration in which the leukocyte actually leaves the blood vessel to carry out its role in the inflammatory response. It is therefore, arguably the most critical step in emigration. This review focuses on two of the many aspects of this process that have seen important recent developments. The adhesion molecules, PECAM (CD31) and CD99 that regulate two major steps in TEM, do so by regulating specific signals. PECAM initiates the signaling pathway responsible for the calcium flux that is required for TEM. Calcium enters through the cation channel TRPC6 and recruits the first wave of trafficking of membrane from the lateral border recycling compartment (LBRC). CD99 signals through soluble adenylate cyclase to activate protein kinase A to recruit a second wave of LBRC trafficking. Another process that is critical for TEM is transient removal of VE-cadherin from the site of TEM. However, the local signaling pathways that are responsible for this appear to be different from those that open the junctions to increase vascular permeability., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Individuals with TRPC6 mutations have variable phenotypes, ranging from healthy carrier to focal segmental glomerulosclerosis (FSGS) leading to renal failure. Here, we describe a family where six members had a novel TRPC6 p.R68W (c.202C>T) mutation, two of whom had renal failure from FSGS, and one had proteinuria. One healthy carrier donated a kidney to her sister. Both donor and recipient had no proteinuria at 20 years posttransplant. Two synonymous NPHS1 polymorphisms, rs2285450 (c.294C>T) and rs437168 (c.2289C>T) segregated with renal failure in this family. These variants had higher allele frequencies in 97 unrelated patients with nephrotic syndrome or FSGS compared to 224 controls. Using patch-clamp experiments in HEK293 and podocytes, we showed that the p.R68W mutation increased TRPC6 current amplitudes, which may be explained by enhanced TRPC6 surface expression. Additionally, while wild-type nephrin suppressed TRPC6 currents, this ability was lost in the presence of NPHS1 c.294C>T polymorphism. When cells were transfected according to combined TRPC6 and NPHS1 genotypes in the family, those representing the donor had lower TRPC6 currents than cells representing the recipient, suggesting that interactions between TRPC6 and NPHS1 variants could possibly account for the variable penetrance of TRPC6 mutations and the absence of recurrence in the graft., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Changes in cytosolic Ca(2+) concentration ([Ca(2+)]c) regulate granule secretion in different cell types. Thrombin activates PAR1 and PAR4 receptors and promotes release of Ca(2+) from distinct intracellular stores, which, in turn, activates store-operated Ca(2+) entry (SOCE). A crucial step during platelet function is the release of physiological agonists stored in secretory granules to the extracellular compartment during activation. We aim to study the role of Ca(2+) mobilization from the extracellular compartment or from different intracellular stores in platelet granule secretion. By using flow cytometry, we have found that α- and δ-granules are secreted in thrombin-stimulated platelets in the absence of extracellular Ca(2+), and in a concentration-dependent manner. Our findings show that thrombin-stimulated granule secretion depends on Ca(2+) mobilization from intracellular stores. Analysis of the kinetics of granule secretion reveals that platelet stimulation with thrombin results in rapid release of α-granules which precedes the secretion of δ-granules. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 573-586 of TRPC6, inhibited thrombin-evoked δ-granule exocytosis. Our results indicate that the mechanisms underlying thrombin-induced α- and δ-granule secretion show differences in dependency on Ca(2+) mobilization., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mitsugumin 29 (MG29) is related to the fatigue and aging processes of skeletal muscle. To examine the roles of MG29 in conjunction with its binding protein, the canonical-type transient receptor potential cation channel 3 (TRPC3), in skeletal muscle, the binding region of MG29 to TRPC3 was studied along with the functional relevance of the binding in mouse primary skeletal myotubes using co-immunoprecipitation assays and Ca(2+) imaging experiments. The N-terminus and the I-II loop of MG29 constitute the binding region for TRPC3. The myotubes that expressed the MG29 mutant missing the entire TRPC3-binding region showed a disrupted binding between endogenous MG29 and TRPC3 and a reduction in Ca(2+) transients in response to membrane depolarization without affecting ryanodine receptor 1 activity, the resting cytosolic Ca(2+) level, and the amount of releasable Ca(2+) from the sarcoplasmic reticulum. Among the proteins mediating Ca(2+) movements in skeletal muscle, TRPC4 expression was significantly decreased by the MG29 mutant. Therefore, MG29 could be a new factor for regulating Ca(2+) transients during skeletal muscle contraction possibly via a correlation with TRPC3 and TRPC4., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Increasing evidence suggests that ischemia and hypoxia serve important functions in the development of renal diseases. However, the underlying mechanism of ischemic injury has not been fully understood. In this study, we found that renal ischemia-reperfusion injury induced podocyte effacement and the upregulation of TRPC6 mRNA and protein expression. In in vitro experiments, oxygen glucose deprivation (OGD) treatment enhanced the expression of TRPC6 and TRPC6-dependent Ca(2+) influx. TRPC6 knockdown by siRNA interference attenuated the OGD-induced [Ca(2+)]i and actin assembly. OGD treatment also increased ROS production. Furthermore, inhibition of ROS activity by N-acetyl-l-cysteine (NAC) eliminated the OGD-induced increase in TRPC6 expression and Ca(2+) influx. H2O2 treatment, which results in oxidative stress, also increased TRPC6 expression and Ca(2+) influx. We conclude that TRPC6 upregulation is involved in Ca(2+) signaling and actin reorganization in podocytes after OGD. These findings provide new insight into the mechanisms underlying the cellular response of podocytes to ischemic injury., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Chemotherapy targeting anti-angiogenesis in tumors may have insufficient efficacy, but little is known about the underlying mechanisms. Here, we showed that the Ca(2+)-permeable channel, TrpC5, is highly expressed in human breast cancer after long-term chemotherapy drug-treatment. It mediates downstream hypoxia-inducible factor 1α accumulation in the nucleus, and then activates the transcription of vascular endothelial growth factor which promotes tumor angiogenesis, leading to a poor chemotherapeutic outcome. We verified this mechanism at both the cellular and xenograft levels. Moreover, in samples from patients, high TrpC5 expression was correlated with enhanced tumor vasculature after chemotherapy. Taken together, our research demonstrated the essential role of TrpC5 in tumor angiogenesis when facing the challenge of chemotherapy and presents a new potential target for overcoming the high vasculature of human breast cancer after chemotherapy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: The measurement of intracellular Ca(2+), cytosolic or stored in organelles, i.e., mitochondria, gave valuable data for numerous areas of research. In case of tumor cells, mitochondrial Ca(2+) levels play essential roles in apoptosis along with endoplasmic reticulum (ER) Ca(2+). In this study, we describe a Ca(2+) monitoring system that allows studying both adherent cells and tissues and discuss data obtained from hepatocellular carcinoma cells and rat thoracic aorta by using this system., Methods: For this purpose, two apparatus, one for adherent cells and the other for intact rat aorta, were designed and produced. With this system, changes in cytosolic Ca(2+) levels following store-operated calcium (SOC) entry induced by sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) blockers were recorded in different hepatocellular carcinoma cells. Furthermore, cytosolic and mitochondrial Ca(2+) levels were simultaneously measured in TRPC1-silenced Huh7 hepatocellular carcinoma cells. In addition, the effects of trifluoromethylphenylimidazole (TRIM) on cyclopiazonic acid (CPA)-, serotonin (5-HT)-, and phenylephrine (PE)-induced changes in isometric force and cytosolic Ca(2+) levels were determined simultaneously in rat thoracic aorta. The effects of aging on PE-induced responses were also investigated., Results: After SOC entry activation, cytosolic Ca(2+) levels were increased, as expected in all hepatocellular carcinoma cells. Mitochondrial Ca(2+) levels following CPA-induced ER depletion were significantly (p<.05) diminished in TRPC1-silenced Huh7 cells. In addition, TRIM partially inhibited both 5-HT-induced contractions and cytosolic Ca(2+) levels without affecting CPA and PE responses. PE-induced contractions and cytosolic Ca(2+) levels were similar in aorta from young and old (3 and 22 months, respectively) rats., Discussion: We confirmed that the system provides valuable data about intracellular Ca(2+) dynamics by allowing simultaneous measurements and sequential addition of compounds in adherent cells. The decrease in mitochondrial Ca(2+) loading following CPA-induced ER depletion in TRPC1-silenced Huh7 cells suggests a possible role of TRPC1 in hepatocellular carcinoma cell apoptosis. The system also enables the simultaneous measurement of isometric force and cytosolic Ca(2+) levels and promotes understanding vascular physiology and disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Saccharomyces cerevisiae cells respond to hypotonic stress (HTS) by a cytosolic calcium rise, either generated by an influx of calcium from extracellular medium, when calcium is available, or by a release from intracellular stores in scarcity of extracellular calcium. Calcium release from intracellular compartments is peculiarly inhibited by external calcium in a calcineurin-independent and Cch1-, but not Mid1-, driven manner. HTS-induced calcium release is also negatively regulated by the ER protein Cls2 and involves a poorly characterized protein, FLC2/YAL053W gene product, previously proposed to be required for FAD transport in the ER, albeit, due to its molecular features, it was also previously classified as an ion transporter. A computational analysis revealed that this gene and its three homologs in S. cerevisiae, together with previously identified Schizosaccharomyces pombe pkd2 and Neurospora crassa calcium-related spray protein, belong to a fungal branch of TRP-like ion transporters related to human mucolipin and polycystin 2 calcium transporters. Moreover, disruption of FLC2 gene confers severe sensitivity to Calcofluor white and hyper-activation of the cell wall integrity MAPK cascade, suggesting a role in cell wall maintenance as previously suggested for the fission yeast homolog. Perturbation in cytosolic resting calcium concentration and hyper-activation of calcineurin in exponentially growing cells suggest a role for this transporter in calcium homeostasis in yeast., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and thus contributes to many vasoproliferative retinopathies including retinopathy of prematurity. Based on the importance of canonical transient receptor potential (TRPC) channels in VEGF signaling, we firstly evaluated the expression of TRPC channels in mouse retina by reverse transcriptase-polymerase chain reaction. All seven TRPC channels were expressed in mouse retina. TRPC4 channels were chosen for further analysis based on their upregulation on hypoxic retina according to the GEO database under the identifier GSE19886. Interestingly, TRPC4 suppression by intravitreal injection of siRNA against mTRPC4 significantly inhibited retinal neovascularization. To further investigate the effect of TRPC4 suppression on neovascularization, human retina microvascular endothelial cells (HRMECs) that are responsible for initiating neovascularization in response to increased VEGF in OIR retina were transfected with siRNA against TRPC4. As we have expected, suppression of TRPC4 effectively inhibited VEGF-induced migration and tube formation as well. Further evaluation on VEGF signaling pathway by western blot analysis of signaling molecules discovered that VEGF-induced activation of ERK, p38 MAPK and AKT signaling pathways were inhibited by suppression of TRPC4. These findings suggest that suppression of TRPC4 could be an alternative therapeutic option for VEGF-induced retinal neovascularization., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A key role for podocytes in the pathogenesis of proteinuric renal diseases has been established. Angiotensin II causes depolarization and increased intracellular calcium concentration in podocytes; members of the cation TRPC channels family, particularly TRPC6, are proposed as proteins responsible for calcium flux. Angiotensin II evokes calcium transient through TRPC channels and mutations in the gene encoding the TRPC6 channel result in the development of focal segmental glomerulosclerosis. Here we examined the effects of angiotensin II on intracellular calcium ion levels and endogenous channels in intact podocytes of freshly isolated decapsulated mouse glomeruli. An ion channel with distinct TRPC6 properties was identified in wild-type, but was absent in TRPC6 knockout mice. Single-channel electrophysiological analysis found that angiotensin II acutely activated native TRPC-like channels in both podocytes of freshly isolated glomeruli and TRPC6 channels transiently overexpressed in CHO cells; the effect was mediated by changes in the channel open probability. Angiotensin II evoked intracellular calcium transients in the wild-type podocytes, which was blunted in TRPC6 knockout glomeruli. Pan-TRPC inhibitors gadolinium and SKF 96365 reduced the response in wild-type glomerular epithelial cells, whereas the transient in TRPC6 knockout animals was not affected. Thus, angiotensin II-dependent activation of TRPC6 channels in podocytes may have a significant role in the development of kidney diseases.

Aims: Although evidence suggest that TRPA1 mediates some effects of prostaglandins, it is not known whether TRPA1 contributes to the in vivo nociceptive effects of prostaglandin E2 (PGE2), a key mediator of inflammatory pain., Main Methods: To address this issue, the effect of the pharmacological blockade of TRPA1 or of its gene silencing on the hyperalgesia induced in the rat paw by PGE2 or its downstream signaling molecules, protein kinase A (PKA) or protein kinase C-epsilon (PKCε), was evaluated. TRPA1 expression on dorsal root ganglia cells was assessed by western blot., Key Findings: The pharmacological blockade of local TRPA1 by its selective antagonist, HC 030031 decreased and reversed PGE2-induced hyperalgesia. The TRPA1 gene silencing induced by intrathecal pre-treatment with antisense oligodeoxynucleotide blocked PGE2-induced hyperalgesia and strongly reduced TRPA1 expression in dorsal root ganglia cells (L5 and L6). PGE2 injection into the hind paw did not significantly increase TRPA1 expression in dorsal root ganglia cells. Treatment with either HC 030031 or antisense oligodeoxynucleotide significantly decreased the hyperalgesia induced by PKA or PKCε. Since both kinases are the major components of PGE2-induced intracellular signal transduction, the modulation of TRPA1 function by PGE2 may be downstream PKA and PKC-epsilon., Significance: These findings show that TRPA1 is essential to the in vivo nociceptive effects induced by one of the most important mediators of inflammatory pain, PGE2. This is one of the crucial findings necessary to support TRPA1 as a promising target for the development of future drugs to pain treatment and control., (Copyright © 2014. Published by Elsevier Inc.)

Slit diaphragm and podocyte damage is crucial in the pathogenesis of proteinuria in diabetic nephropathy (DNP). Gain-of-function mutations in TRPC6, a slit diaphragm-associated ion channel, cause glomerulosclerosis; TRPC6 expression is increased in acquired glomerular disease. Hyperglycemia and high intrarenal angiotensin II (AngII) levels could contribute to podocyte injury in DNP. We determined whether glucose regulates TRPC6 expression and TRPC6-mediated Ca(2+) influx into the podocyte and whether these effects are AngII dependent. High glucose levels increased TRPC6 mRNA and protein expression in cultured podocytes; however, TRPC1 and TRPC5 mRNA expression was unaltered. AngII and inducing podocyte injury also specifically increased TRPC6 expression. Angiotensin receptor blockade and inhibition of local AngII production through angiotensin-converting enzyme inhibition prevented glucose-mediated increased TRPC6 expression. In addition, high glucose concentration pretreatment enhanced Ca(2+) influx in podocytes, which was prevented by concomitant angiotensin receptor blockade application and TRPC6 knockdown. Studies with a TRPC6 luciferase promoter construct demonstrated a glucose concentration-dependent effect on TRPC6 promoter activity. In vivo, podocyte TRPC6 protein expression was increased in proteinuric streptozotocin-induced diabetic rats. These data suggest that glucose can activate a local renin-angiotensin system in the podocyte, leading to increased TRPC6 expression, which enhances TRPC6-mediated Ca(2+) influx. Regulation of TRPC6 expression could be an important factor in podocyte injury due to chronic hyperglycemia and the antiproteinuric effect of angiotensin receptor blockade or angiotensin-converting enzyme inhibition in DNP., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

In Alzheimer's disease (AD), astrocytes undergo complex morphological and functional changes that include early atrophy, reactive activation and Ca(2+) deregulation. Recently, we proposed a mechanism by which nanomolar Aβ42 deregulates mGluR5 and InsP3 receptors, the key elements of astrocytic Ca(2+) signalling toolkit. To evaluate the specificity of these changes, we have now investigated whether the effects of Aβ42 on Ca(2+) signalling machinery can be reproduced by pro-inflammatory agents (TNFα, IL-1β, LPS). Here we report that Aβ42 (100nM, 72h) significantly increased mRNA levels of mGluR5, InsP3R1 and InsP3R2, whereas pro-inflammatory agents reduced expression of these specific mRNAs. Furthermore, DHPG-induced Ca(2+) signals and store operated Ca(2+) entry (SOCE) were augmented in Aβ42-treated cells due to up-regulation of a set of Ca(2+) signalling-related genes including TRPC1 and TRPC4. Opposite changes were observed when astrocytes were treated with TNFα, IL-1β and LPS. Last, the effects observed on SOCE by treating wild-type astrocytes with Aβ42 were also identified in untreated astrocytes from 3×Tg-AD animals, suggesting a link to the AD pathology. Our results demonstrate that effects of Aβ42 on astrocytic Ca(2+) signalling differ from and may contrast to the effects of pro-inflammatory agents., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure with lumen-occluding neointimal and plexiform lesions. Activation of store-operated calcium entry channels promotes contraction and proliferation of lung vascular cells. TRPC4 is a ubiquitously expressed store-operated calcium entry channel, but its role in PAH is unknown. We tested the hypothesis that TRPC4 promotes pulmonary arterial constriction and occlusive remodeling, leading to right ventricular failure in severe PAH. Severe PAH was induced in Sprague-Dawley rats and in wild-type and TRPC4-knockout Fischer 344 rats by a single subcutaneous injection of SU5416 [SU (semaxanib)], followed by hypoxia exposure (Hx; 10% O2) for 3 weeks and then a return to normoxia (Nx; 21% O2) for 3 to 10 additional weeks (SU/Hx/Nx). Although rats of both backgrounds exhibited indistinguishable pulmonary hypertensive responses to SU/Hx/Nx, Fischer 344 rats died within 6 to 8 weeks. Normoxic and hypertensive TRPC4-knockout rats recorded hemodynamic parameters similar to those of their wild-type littermates. However, TRPC4 inactivation conferred a striking survival benefit, due in part to preservation of cardiac output. Histological grading of vascular lesions revealed a reduction in the density of severely occluded small pulmonary arteries and in the number of plexiform lesions in TRPC4-knockout rats. TRPC4 inactivation therefore provides a survival benefit in severe PAH, associated with a decrease in the magnitude of occlusive remodeling., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The importance of Ca(2+) signaling in astrocytes is undisputed but a potential role of Ca(2+) influx via L-channels in the brain in vivo is disputed, although expression of these channels in cultured astrocytes is recognized. This study shows that an increase in free cytosolic Ca(2+) concentration ([Ca(2+)]i) in astrocytes in primary cultures in response to an increased extracellular K(+) concentration (45mM) is inhibited not only by nifedipine (confirming previous observations) but also to a very large extent by ryanodine, inhibiting ryanodine receptor-mediated release of Ca(2+), known to occur in response to an elevation in [Ca(2+)]i. This means that the actual influx of Ca(2+) is modest, which may contribute to the difficulty in demonstrating L-channel-mediated Ca(2+) currents in astrocytes in intact brain tissue. Chronic treatment with any of the 3 conventional anti-bipolar drugs lithium, carbamazepine or valproic acid similarly causes a pronounced inhibition of K(+)-mediated increase in [Ca(2+)]i. This is shown to be due to an inhibition of capacitative Ca(2+) influx, reflected by decreased mRNA and protein expression of the 'transient receptor potential channel' (TRPC1), a constituent of store-operated channels (SOCEs). Literature data are cited (i) showing that depolarization-mediated Ca(2+) influx in response to an elevated extracellular K(+) concentration is important for generation of Ca(2+) oscillations and for the stimulatory effect of elevated K(+) concentrations in intact, non-cultured brain tissue, and (ii) that Ca(2+) channel activity is dependent upon availability of metabolic substrates, including glycogen. Finally, expression of mRNA for Cav1.3 is demonstrated in freshly separated astrocytes from normal brain., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-β1 (TGF-β1) on Ca2+ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca2+ concentration [Ca2+]i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca(2+)-mediated [Ca2+]i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca2+ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca2+ entry. KB-R7943, a reverse-mode Na+/Ca2+ exchanger (NCX) inhibitor, and/or replacement of Na+ with NMDG+ inhibited thapsigargin-, OAG- and Ang II-induced Ca2+ entry. Treatment with TGF-β1 increased thapsigargin-, OAG- and Ang II-induced Ca2+ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-β1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca2+ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca2+ influx pathways, which activate the reverse-mode NCX. TGF-β1 enhances the Ca2+ influx pathways requiring Ca2+ signals for its effect on fibroblast proliferation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Utilizing a novel molecular model of TRPC3, based on the voltage-gated sodium channel from Arcobacter butzleri (Na(V)AB) as template, we performed structure-guided mutagenesis experiments to identify amino acid residues involved in divalent permeation and gating. Substituted cysteine accessibility screening within the predicted selectivity filter uncovered amino acids 629-631 as the narrowest part of the permeation pathway with an estimated pore diameter of < 5.8Å. E630 was found to govern not only divalent permeability but also sensitivity of the channel to block by ruthenium red. Mutations in a hydrophobic cluster at the cytosolic termini of transmembrane segment 6, corresponding to the S6 bundle crossing structure in Na(V)AB, distorted channel gating. Removal of a large hydrophobic residue (I667A or I667E) generated channels with approximately 60% constitutive activity, suggesting I667 as part of the dynamic structure occluding the permeation path. Destabilization of the gate was associated with reduced Ca2+ permeability, altered cysteine cross-linking in the selectivity filter and promoted channel block by ruthenium red. Collectively, we present a structural model of the TRPC3 permeation pathway and localize the channel's selectivity filter and the occluding gate. Moreover, we provide evidence for allosteric coupling between the gate and the selectivity filter in TRPC3., (Copyright © 2013 The Authors. Published by Elsevier India Pvt Ltd. All rights reserved.)

Background: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy., Methods and Results: In cultured neonatal rat cardiomyocytes, a 48 h treatment with 10nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes., Conclusions: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Genetic screening paradigms for congenital and infantile nephrotic syndrome are well established; however, screening in adolescents has received only minor attention. To help rectify this, we analyzed an unselected adolescent cohort of the international PodoNet registry to develop a rational screening approach based on 227 patients with nonsyndromic steroid-resistant nephrotic syndrome aged 10-20 years. Of these, 21% had a positive family history. Autosomal dominant cases were screened for WT1, TRPC6, ACTN4, and INF2 mutations. All other patients had the NPHS2 gene screened, and WT1 was tested in sporadic cases. In addition, 40 sporadic cases had the entire coding region of INF2 tested. Of the autosomal recessive and the sporadic cases, 13 and 6%, respectively, were found to have podocin-associated nephrotic syndrome, and 56% of them were compound heterozygous for the nonneutral p.R229Q polymorphism. Four percent of the sporadic and 10% of the autosomal dominant cases had a mutation in WT1. Pathogenic INF2 mutations were found in 20% of the dominant but none of the sporadic cases. In a large cohort of adolescents including both familial and sporadic disease, NPHS2 mutations explained about 7% and WT1 4% of cases, whereas INF2 proved relevant only in autosomal dominant familial disease. Thus, screening of the entire coding sequence of NPHS2 and exons 8-9 of WT1 appears to be the most rational and cost-effective screening approach in sporadic juvenile steroid-resistant nephrotic syndrome.

Increasing evidence indicates that podocyte apoptosis is a key event in the development of diabetic nephrology. However, the underlying mechanism of this apoptosis remains poorly understood. In this study, we report that high levels of glucose enhanced the expression of TRPC6 and TRPC6-dependent Ca(2+) influx, but glucose levels did not affect TRPC1 and TRPC5 expression. TRPC6 knockdown by siRNA interference attenuated the observed increase in glucose-induced podocyte apoptosis. High glucose levels also increased the generation of ROS; inhibition of ROS activity by N-acetyl-l-cysteine attenuated the high glucose-induced increase in TRPC6 expression and Ca(2+) influx. Exogenous treatment with H2O2 mimicked the high glucose response, resulting in an increase in TRPC6 expression and Ca(2+) influx. Taken together, these data suggest that high glucose levels induce ROS, thereby mediating TRPC6 expression and Ca(2+) influx. Because RhoA activity is increased following TRPC6 activation, we investigated whether TRPC6 is involved in high glucose-induced apoptosis via the RhoA/ROCK pathway. We report that high glucose levels produced an increase in RhoA activity, and this effect was abolished by the knockdown of TRPC6. Moreover, inhibition of the RhoA/ROCK pathway by a ROCK inhibitor, Y27632, also attenuated high glucose-induced apoptosis. We conclude that TRPC6 is involved in high glucose-induced podocyte apoptosis through the RhoA/ROCK pathway., (Copyright © 2013 Elsevier Inc. All rights reserved.)