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An elevated level of circulating homocysteine (Hcy) has been regarded as an independent risk factor for cardiovascular disease; however, the clinical benefit of Hcy lowering-therapy is not satisfying. To explore potential unrevealed mechanisms, we investigated the roles of Ca2+ influx through TRPC channels and regulation by Hcy–copper complexes. Using primary cultured human aortic endothelial cells and HEK-293 T-REx cells with inducible TRPC gene expression, we found that Hcy increased the Ca2+ influx in vascular endothelial cells through the activation of TRPC4 and TRPC5. The activity of TRPC4 and TRPC5 was regulated by extracellular divalent copper (Cu2+) and Hcy. Hcy prevented channel activation by divalent copper, but monovalent copper (Cu+) had no effect on the TRPC channels. The glutamic acids (E542/E543) and the cysteine residue (C554) in the extracellular pore region of the TRPC4 channel mediated the effect of Hcy–copper complexes. The interaction of Hcy–copper significantly regulated endothelial proliferation, migration, and angiogenesis. Our results suggest that Hcy–copper complexes function as a new pair of endogenous regulators for TRPC channel activity. This finding gives a new understanding of the pathogenesis of hyperhomocysteinemia and may explain the unsatisfying clinical outcome of Hcy-lowering therapy and the potential benefit of copper-chelating therapy.

Human corneal epithelial cells (HCEC), which are found on the outermost layer of the human cornea, play a vital role in maintaining vision. Research over the years has shown that various transient receptor potential channels (TRPs) are expressed on the surface of many ocular cells including the HCEC. With their intracellular and extracellular domains, TRPs are capable of converting external stimuli into signal transduction pathways within the cell. These channels play a significant role in Ca2+ regulation and mediate numerous intracellular processes by controlling the flow of Ca2+ ions across the cell membrane. Interestingly, studies have shown that growth factors can influence Ca2+ regulation via interaction with certain TRPs. As epidermal growth factor (EGF) in particular has been shown to promote healing of damaged corneal epithelial tissue, a closer study of the effect of EGF on HCEC would be useful. This study aims to investigate the influence EGF on Ca2+ regulation in cultured HCEC and the involvement of TRPs in this process. Fluorescence Ca2+ imaging of SV-40 transfected HCEC yielded the following results: Application of 50 ng/ml EGF caused a significant increase in intracellular Ca2+ concentration. This increase was significantly suppressed in the presence of the TRP canonical (TRPC) channel antagonist SKF 96365 (10–20 μM). When a Ca2+-free measuring solution was used, no increase in intracellular Ca2+ concentration was observed upon application of 50 ng/ml EGF. Patchclamp measurement of whole-cell currents revealed an increase in inward and outward currents through the cell membrane upon application of 50 ng/ml EGF. This EGFinduced increase in currents was suppressed in the presence of 20 μM SKF 96365. Taken together, this study demonstrates the involvement of TRPC channels in EGFinduced Ca2+ increase in HCEC. The results also suggest that this effect is due to EGFR-associated activation of TRPC channels on the cell membrane rather than the depletion of intracellular Ca2+ stores., Humane Hornhautepithelzellen (HCEC), die sich auf der äußersten Schicht der menschlichen Hornhaut befinden, spielen eine wichtige Rolle bei der Aufrechterhaltung des normalen Sehvermögens. Die Forschung im Laufe der Jahre zeigte, dass unterschiedliche Transient-Receptor-Potential-Kanäle (TRPs) in Zellen der Augenoberfläche einschließlich der HCEC exprimiert sind. Mit ihrer intra- und extrazellulären Domäne können TRPs externe Stimuli in Signaltransduktionswege innerhalb der Zelle umwandeln. Diese Kanäle spielen eine wichtige Rolle bei Ca2+- Regulation und vermitteln zahlreiche intrazelluläre Prozesse, indem sie den Fluss von Ca2+-Ionen durch die Zellmembran steuern. Interessanterweise zeigten Studien, dass Wachstumsfaktoren die Ca2+-Regulation durch Wechselwirkung mit bestimmten TRPs beeinflussen können. Da insbesondere der epidermale Wachstumsfaktor (EGF) gezeigt hat, dass er die Heilung von geschädigtem Hornhautepithelgewebe fördert, wäre eine nähere Untersuchung der Wirkung von EGF auf HCEC nützlich. Ziel dieser Studie ist es, den Einfluss von EGF auf die Ca2+-Regulation in kultivierten HCEC und die Beteiligung von TRPs an diesem Prozess zu untersuchen. Die Fluoreszenz-Ca2+- Image-Messungen von SV-40-transfizierten HCEC ergaben folgende Ergebnisse: Die extrazelluläre Applikation von 50 ng/ml EGF führte zu einem signifikanten Anstieg der intrazellulären Ca2+-Konzentration. Dieser Anstieg wurde in Gegenwart des kanonischen TRP (TRPC)-Kanalantagonisten SKF 96365 (10–20 μM) deutlich unterdrückt. Bei Verwendung einer Ca2+-freien Messlösung löste 50 ng/ml EGF keinen Anstieg der intrazellulären Ca2+-Konzentration aus. Patch-Clamp Messungen von Ganzzellströmen ergaben einen Anstieg der Ein- und Ausströme durch die Zellmembran nach Zugabe von 50 ng/ml EGF. Dieser EGF-induzierte Anstieg der Ströme wurde in Gegenwart von 20 μM SKF 96365 unterdrückt. Insgesamt deutet diese Studie eine Beteiligung von TRPC Kanäle am EGF-induzierten Ca2+-Anstieg in HCEC an. Die Ergebnisse weisen auch darauf hin, dass dieser Effekt eher auf ein EGFRassoziierte Aktivierung von TRPC Kanäle auf der Zellmembran zurückzuführen ist, als auf die Entleerung von intrazellulären Ca2+-Speichern.

Background Members of the transient receptor potential canonical (TRPC) protein family are widely distributed in the hippocampus of mammals and exert respective and cooperative influences on the functions of neurons. The relationship between specific TRPC subtypes and neuroinflammation is receiving increasing attention. Methods Using Cx3cr1CreERIL-10−/− transgenic mice and their littermates to study the relationship between TRPC channels and memory impairment. Results We demonstrated that Cx3cr1CreERIL-10−/− mice displayed spatial memory deficits in object location recognition (OLR) and Morris water maze (MWM) tasks. The decreased levels of TRPC4 and TRPC5 in the hippocampal regions were verified via reverse transcription polymerase chain reaction, western blotting, and immunofluorescence tests. The expression of postsynaptic density protein 95 (PSD95) and synaptophysin in the hippocampus decreased with an imbalance in the local inflammatory environment in the hippocampus. The number of cells positive for ionized calcium-binding adaptor molecule 1 (Iba1), a glial fibrillary acidic protein (GFAP), increased with the high expression of interleukin 6 (IL-6) in Cx3cr1CreERIL-10−/− mice. The nod-like receptor protein 3 (NLRP3) inflammasome was also involved in this process, and the cytokines IL-1β and IL-18 activated by NLRP3 were also elevated by western blotting. The co-localization of TRPC5 and calmodulin-dependent protein kinase IIα (CaMKIIα) significantly decreased TRPC5 expression in excitatory neurons. AAV9-CaMKIIα-TRPC5 was used to upregulate TRPC5 in excitatory neurons in the hippocampus. Conclusions The results showed that the upregulation of TRPC5 improved the memory performance of Cx3cr1CreERIL-10−/− mice related to inhibiting NLRP3 inflammasome-associated neuroinflammation. Graphical Abstract

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). Cx3cr1CreERIL-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 Cx3cr1CreERIL-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 Cx3cr1CreER 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 Cx3cr1CreERIL-10-/- mice.

The transient receptor potential canonical (TRPC) channels have been implicated in various types of malignancies including gastric cancer (GC). However, the detailed mechanisms of TRPC channels underlying cell proliferation and apoptosis of GC cells remain largely unknown. Here, we report that TRPC3 was highly expressed in clinical GC specimens and correlated with GC malignant progression and poor prognosis. Forced expression of TRPC3 in GC cells enhanced both receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) and promoted the nuclear factor of activated T cell 2 (NFATc2) nuclear translocation by AKT/GSK-3β and CNB2 signaling. Pharmacological inhibition of TRPC3 or CRISPR/Cas9-mediated TRPC3 knockout effectively inhibited the growth of GC cells both in vitro and in vivo. These effects were reversible by the rescue of TRPC3 expression. Furthermore, we confirmed the role of TRPC3 and the ROCE-AKT/GSK3β-CNB2/NFATc2 signaling cascade in regulating cell cycle checkpoint, apoptosis cascade, and intracellular ROS production in GC. Overall, our findings suggest an oncogenic role of TRPC3 in GC and may highlight a potential target of TRPC3 for therapeutic intervention of GC and its malignant progression.

Transient receptor potential canonical (TRPC) channels are the most prominent nonselective cation channels involved in various diseases. However, the function, clinical significance, and molecular mechanism of TRPCs in colorectal cancer (CRC) progression remain unclear. In this study, we identified that TRPC1 was the major variant gene of the TRPC family in CRC patients. TRPC1 was upregulated in CRC tissues compared with adjacent normal tissues and high expression of TRPC1 was associated with more aggressive tumor progression and poor overall survival. TRPC1 knockdown inhibited cell proliferation, cell-cycle progression, invasion, and migration in vitro, as well as tumor growth in vivo; whereas TRPC1 overexpression promoted colorectal tumor growth and metastasis in vitro and in vivo. In addition, colorectal tumorigenesis was significantly attenuated in Trpc1-/- mice. Mechanistically, TRPC1 could enhance the interaction between calmodulin (CaM) and the PI3K p85 subunit by directly binding to CaM, which further activated the PI3K/AKT and its downstream signaling molecules implicated in cell cycle progression and epithelial-mesenchymal transition. Silencing of CaM attenuated the oncogenic effects of TRPC1. Taken together, these results provide evidence that TRPC1 plays a pivotal oncogenic role in colorectal tumorigenesis and tumor progression by activating CaM-mediated PI3K/AKT signaling axis. Targeting TRPC1 represents a novel and specific approach for CRC treatment.

Orexins A and B are neuropeptides synthesized by a population of lateral hypothalamic neurons. Orexin’s physiological function consists mainly in regulating the sleep-wake cycle, eating behavior, and energy homeostasis. Axons of orexin-containing neurons are projected onto many structures of brain and spinal cord, thus providing a variety of their physiological effects. Moreover, the components of the orexinergic system are identified in various peripheral organs and tissues. The effects of orexins are mediated via two receptors (OX1R and OX2R) coupled with G-proteins (GPCRs). The classical signal transmission pathway through orexin receptors in neuronal cells includes an increase of the intracellular calcium as a result of the opening of TRPC membrane channels and IP3 endoplasmic reticulum (ER) channels. In addition to the classic orexin receptors signaling, there is an alternative pathway. Signal transmission through the alternative pathway leads to apoptosis of tumor cells. This pathway is probably due to the structural feature of orexin receptors compared to other GPCRs — the presence of a tyrosine-based immunoreceptor inhibition motif (ITIM). Such motifs are not limited to GPCRs, but are a hallmark of immuno-inhibiting receptors on lymphoid and myeloid cells. ITIM recruits either SHP1 and SHP2 protein tyrosine phosphatases or SHIP1 and SHIP2 inositol phosphatases, to mediate negative signal transduction. A further mechanism of the so-called orexin-induced apoptosis seems to include the p38/MAPK phosphorylation and the cytochrome c releasing from mitochondria, followed by activation of caspases 3 and 7 and cell death. It should be emphasized that this alternative pathway is present only in certain types of tumor cells. This review summarizes the available data on orexin-induced apoptosis of tumor cells from intestines, pancreas, stomach, prostate, endometrium, adrenal glands and glia, and also considers possible mechanisms for its implementation.

Background Chronic itch is a debilitating symptom of inflammatory skin diseases, but the underlying mechanism is poorly understood. We have recently demonstrated that astrocytes in the spinal dorsal horn become reactive in models of atopic and contact dermatitis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) and critically contribute to chronic itch. In general, STAT3 is transiently activated; however, STAT3 activation in reactive astrocytes of chronic itch model mice persistently occurs via an unknown mechanism. Objective We aimed to determine the mechanisms of persistent activation of astrocytic STAT3 in chronic itch conditions. Methods To determine the factors that are required for persistent activation of astrocytic STAT3, Western blotting and calcium imaging with cultured astrocytes or spinal cord slices were performed. Thereafter, chronic itch model mice were used for genetic and behavioral experiments to confirm the role of the factors determined to mediate persistent STAT3 activation from in vitro and ex vivo experiments in chronic itch. Results IP3 receptor type 1 (IP3R1) knockdown in astrocytes suppressed IL-6–induced persistent STAT3 activation and expression of lipocalin-2 (LCN2), an astrocytic STAT3-dependent inflammatory factor that is required for chronic itch. IP3R1-dependent astrocytic Ca2+ responses involved Ca2+ influx through the cation channel transient receptor potential canonical (TRPC), which was required for persistent STAT3 activation evoked by IL-6. IL-6 expression was upregulated in dorsal root ganglion neurons in a mouse model of chronic itch. Dorsal root ganglion neuron–specific IL-6 knockdown, spinal astrocyte–specific IP3R1 knockdown, and pharmacologic spinal TRPC inhibition attenuated LCN2 expression and chronic itch. Conclusion Our findings suggest that IP3R1/TRPC channel–mediated Ca2+ signals elicited by IL-6 in astrocytes are necessary for persistent STAT3 activation, LCN2 expression, and chronic itch, and they may also provide new targets for therapeutic intervention.

It has been shown by fluorescence microscopy that membranes of mouse skeletal muscle fibers contain canonical TRP channels (TRPCs) of four subfamilies, including seven known classified subtypes. In the area of the neuromuscular junction the most represented are TRPC5 channels and the least are TRPC2 channels. Some subtypes of TRPC channels (1, 3, 4, and 5) are closely associated with the membranes of the sarcoplasmic reticulum of muscle fibers, with TRPC5 channels being the most represented in these structures. There is no local concentration of TRPC channels of all subtypes (1–7) exclusively in the area of neuromuscular junction, which does not support the hypothesis of a possible neurotrophic control over the distribution of channels of this family in the muscle fiber.

Simple Summary The detection of sub-fertile boars has been a difficult task, and despite their prevalence being low, its impact is very significant because it implies economic drawbacks for artificial insemination (AI) centers and farms. Unfortunately, some crucial reproductive processes fall beyond the routine analysis performed in the porcine model, such as sperm capacitation, which is a necessary event for fertilization. A synergistic action of bicarbonate (HCO3-) with calcium (Ca2+) is needed to achieve capacitation. The transport of Ca2+ is mediated by CatSper channels and Canonical Transient Potential Channels (TRPC). We quantified mRNA transcripts of different subunits of CatSper (beta, gamma and delta) and TRPC (1, 3, 4, 6 and 7) before and after in vitro capacitation by HCO3- ions. Our results showed that in vitro capacitation using HCO3- increases the relative abundance of mRNA transcripts of almost all subunits of Ca2+ channels, except CatSper-delta and TRPC1, which were significantly reduced. More studies are needed to elucidate the specific roles of the TRPC channels at a physiological and functional level. Sperm capacitation is a stepwise complex biochemical process towards fertilization. It includes a crucial early calcium (Ca2+) transport mediated by CatSper channels and Canonical Transient Potential Channels (TRPC). We studied the relative abundance of mRNA transcripts changes of the CatSper beta, gamma and delta subunits and TRPC-channels 1, 3, 4, 6 and 7 in pig spermatozoa, after triggering in vitro capacitation by bicarbonate ions at levels present in vivo at the fertilization site. For this purpose, we analyzedfive5 ejaculate pools (from three fertile adult boars) before (control-fresh samples) and after in vitro exposure to capacitation conditions (37 mM NaHCO3, 2.25 mM CaCl2, 2 mM caffeine, 0.5% bovine serum albumin and 310 mM lactose) at 38 degrees C, 5% CO2 for 30 min. In vitro capacitation using bicarbonate elicits an increase in the relative abundance of mRNA transcripts of almost all studied Ca2+ channels, except CatSper-delta and TRPC1 (significantly reduced). These findings open new avenues of research to identify the specific role of each channel in boar sperm capacitation and elucidate the physiological meaning of the changes on sperm mRNA cargo. Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; MCIN/AEI (Spain) [PID2019-108320RJ-I00, IJCI-2015-24380]; FEDER funds (EU)European Commission [PID2019-108320RJ-I00, IJCI-2015-24380]; MECD ("Ministerio de Educacion, Cultura y Deporte"), Madrid, Spain [16/05745]; Spanish Ministry of Science, Innovation and Universities (MICINN), Madrid, Spain [RTI2018-095183-B-I00]

Betulinic acid (BA) is a natural pentacyclic triterpenoid with protective effects against inflammation, metabolic diseases, and cardiovascular diseases. We have previously shown that BA prevents endothelial dysfunction by increasing nitric oxide (NO) synthesis through activating endothelial nitric oxide synthase (eNOS) in human endothelial cells. However, the effect of BA on eNOS expression remains unclear. Thus, the aim of our study was to investigate the intracellular pathways associated with the effect of BA to regulate eNOS expression in human endothelial cells. BA significantly increased eNOS expression in a time- and concentration-dependent manner. Additionally, BA upregulated the expression of the transcription factor KLF2, which is known to regulate eNOS expression. KLF2 silencing in human endothelial cells attenuated the ability of BA to upregulate eNOS. BA also increased levels of intracellular Ca2+, activating CaMKKβ, CaMKIIα, and AMPK. Inhibition of the TRPC calcium channel abolished BA-mediated effects on intracellular Ca2+ levels. Moreover, BA increased the phosphorylation levels of ERK5, HDAC5, and MEF2C. Pretreatment of cells with compound C (AMPK inhibitor), LMK235 (HDAC5 inhibitor), and XMD8-92 (ERK5 inhibitor) attenuated the BA-induced eNOS expression. Collectively, these findings suggest that BA induces eNOS expression by activating the HDAC5/ERK5/KLF2 pathway in endothelial cells. The data presented here provide strong evidence supporting the use of BA to prevent endothelial dysfunction and treat vascular diseases, such as atherosclerosis.

Obstructive sleep apnea (OSA), a breathing disorder featured by chronic intermittent hypoxia (CIH) is associated with pulmonary hypertension (PH). Rodents exposed to CIH develop pulmonary vascular remodeling and PH, but the pathogenic mechanisms are not well known. Overexpression of Stim-activated Transient Receptor Potential Channels (TRPC) and Calcium Release-Activated Calcium Channel Protein (ORAI) TRPC-ORAI Ca 2+ channels (STOC) has been involved in pulmonary vascular remodeling and PH in sustained hypoxia. However, it is not known if CIH may change STOC levels. Accordingly, we studied the effects of CIH on the expression of STOC subunits in the lung and if these changes paralleled the progression of the vascular pulmonary remodeling and PH in a preclinical model of OSA. Male Sprague-Dawley rats (∼200 g) were exposed to CIH (5%O 2 , 12 times/h for 8 h) for 14, 21, and 28 days. We measured right ventricular systolic pressure (RVSP), cardiac morphometry with MRI, pulmonary vascular remodeling, and wire-myographic arterial responses to KCl and endothelin-1 (ET-1). Pulmonary RNA and protein STOC levels of TRPC1, TRPC4, TRPC6, ORAI 1, ORAI 2, and STIM1 subunits were measured by qPCR and western blot, and results were compared with age-matched controls. CIH elicited a progressive increase of RVSP and vascular contractile responses to KCl and ET-1, leading to vascular remodeling and augmented right ventricular ejection fraction, which was significant at 28 days of CIH. The levels of TRPC1, TRPC4, TRPC 6, ORAI 1, and STIM 1 channels increased following CIH, and some of them paralleled morphologic and functional changes. Our findings show that CIH increased pulmonary STOC expression, paralleling vascular remodeling and PH.

Ca2+ function as second messenger and changes in cytosolic Ca2+ levels dictate neuronal physiology. Although several Ca2+ entry channels are present in neuronal cells, release of Ca2+ from intracellular endoplasmic reticulum (ER) stores modulates store-operated Ca2+ entry (SOCE) that restore ER Ca2+ levels and maintains Ca2+ homeostasis in organelles such as: mitochondria, Golgi apparatus, lysosomes and associated vesicles, and nucleus. Members of the Orai and canonical TRPC channels that are gated by ER Ca2+ sensor STIM1 induces SOCE in neuronal and associated cells. Similarly, mitochondrial Ca2+ uniporter is essential for mitochondrial Ca2+ uptake; whereas two-pore Ca2+ channels and the mucolipin are essential for lysosomal functions. Interestingly, spatiotemporal compartmentalization of Ca2+ in various organelle modulate diverse and opposing functions. Thus, interplay between organelle Ca2+ and Ca2+ influx provide the spatial resolution that is imperative for executing the precise neuronal responses and alterations in Ca2+ signaling leads to neuronal loss.

TRPC channels are Ca2+-permeable nonselective cation channels activated downstream from phospholipase C (PLC). Although most TRPC channels can be activated by stimulating Gq/11-coupled receptors, TRPC4 requires simultaneous stimulation of Gi/o-coupled receptors, making it a perfect detector of coincident Gi/o and Gq/11 signaling. Evidence shows that activated Gαi/o proteins work together with PLCδ1 to induce robust TRPC4 activation and the process is accelerated by stimulation of other PLC isozymes, such as PLCβ through Gq/11 proteins. Mechanistically, Gq/11-PLCβ activation produces triggering proton and calcium signals to initiate self-propagating PLCδ1 activity, crucial for Gi/o-mediated TRPC4 function. Thus, TRPC4-containing channels are activated under conditions not only when coincident Gi/o and Gq/11 stimulation occurs, but also when Gi/o stimulation coincides with proton and Ca2+ signals. The resulting cytosolic Ca2+ rise and membrane depolarization switch the inhibitory Gi/o response to excitation. The conditions and implications of Gi/o-mediated TRPC4 activation in physiology and pathophysiology warrant further investigation.

The transient receptor potential (TRP) superfamily of plasma membrane cation channels has been recognized as a signaling hub in highly diverse settings of human physiopathology. In the past three decades of TRP research, attention was focused mainly on the channels Ca2+ signaling function, albeit additional cellular functions, aside of providing a Ca2+ entry pathway, have been identified. Our understanding of Ca2+ signaling by TRP proteins has recently been advanced by a gain in high-resolution structure information on these pore complexes, and by the development of novel tools to investigate their role in spatiotemporal Ca2+ handling. This review summarizes recent discoveries as well as remaining, unresolved aspects of the canonical subfamily of transient receptor potential channels (TRPC) research. We aim at a concise overview on current mechanistic concepts of Ca2+ entry through- and Ca2+ signaling by TRPC channels.

To investigate the effect of carboxyl terminus of Hsc70-interacting protein (CHIP) on pulmonary arterial smooth muscle cell (PASMC) proliferation and the underlying mechanism. Materials and Methods...

Cell-cell communication plays a central role in the guidance of migrating neurons during the development of the cerebral cortex. Neuregulins (NRGs) are essential mediators for migration and maintenance of the radial glial scaffold. We show, in this study that soluble NRG reduces neuronal motility, causes transition of bipolar cells to multipolar ones, and induces neuronal mitosis. Blocking the NRG receptor, ErbB4, results in reduction of neuron-neuron and neuron-radial glial contacts and causes an increase in neuronal motility. Blocking the radial glial metabotropic glutamate receptor 5 (mGluR5), the nonselective cation channel transient receptor potential 3 (TRPC3), or matrix metalloproteinases (MMPs) results in similar effects as ErbB4 blockade. Soluble NRG counteract the changes in motility pattern. Stimulation of other radial glial G-protein-coupled receptors (GPCRs), such as muscarinic acetylcholine receptors or endothelin receptors counteract all the effect of mGluR5 blockade, but not that of ErbB4, TRPC3, and MMP blockade. The results indicate that neurotransmitters and endothelins acting on radial glial GPCRs are, through proteolytic NRG/ErbB4 activation, able to modify the migratory behavior of neurons.

Background An accumulating amount of evidence has suggested that there is a contributive role of sympathetic nervous hyperactivity in the pathogenesis of chronic kidney disease (CKD). α1-AR promotes an increase in calcium levels in podocytes and adjusts podocyte contraction. Changes in TRPC6 expression and function can directly affect the podocyte cytoskeleton, which is a key component in podocyte injury. This study proposed to clarify the correlation between α1-AR activation-induced signal cascade reaction and TRPC6 in human podocytes. Methods Human podocytes were incubated with the calcium probe Fluo-3/AM. Next, the effects of the α1-AR agonists or antagonists and nonselective TRPC6 blockers on intracellular calcium were observed under laser confocal microscopy. FITC-phalloidin was employed to stain podocytes, and the change of F-actin under the α1-AR activation condition was observed. Results The α1-AR agonist PE (phenylephrine hydrochloride) induced an increase in intracellular Ca2+ ([Ca2+]i) in human podocytes. Moreover, the downregulation of TRPC6 by siRNA or TRPC blocker could attenuate the PE-induced [Ca2+]i elevation in a phospholipase C (PLC)-dependent pattern. When podocytes were stimulated to the PE, their F-actin fiber cytoskeletal structure was lost. PE subsequently increased the expression of RhoA, and the TRPC6-dependent Ca2+ influx was involved in this process. The abnormal activation of RhoA could result in disturbance of the podocyte skeleton structure, thus leading to podocyte injury. Conclusions We concluded that TRPC6 is involved in α1-AR activation-induced calcium signal changes in podocytes. Meanwhile, the α1-AR agonists can destroy the cell's cytoskeletal structure, which is mediated by TRPC6 via the RhoA/ROCK pathway.

Dysfunctions of glomerular podocytes and triggering of apoptotic processes in them are the main molecular causes of diabetic nephropathy and other kidney diseases. Pathogenetic factors causing these dysfunctions of podocytes are hyperglycemia, increased levels of advanced glycation end-products, oxidative stress, increased activity of inflammatory factors, and endoplasmic reticulum stress. These factors and their combinations result in the triggering of a number of intracellular signaling pathways that cause activation of apoptosis and reduce the survival of podocytes. Among these pathways are: (1) the Wnt/β-catenin pathway, which is activated by the Wnt-proteins when they bind to the complex of Frizzled receptors and LRP co-receptors; (2) the mTOR-dependent signaling pathway, including the mTORC1 and mTORC2 complexes, which are involved in the regulation of autophagy and endoplasmic reticulum stress and are regulated by various stimuli and effectors, in particular, the AMP-activated protein kinase; (3) the Rho/ROCK signaling pathway, including GTPases of the Rho family and Rho-associated protein kinase ROCK1; (4) calcium-dependent signaling pathways triggered by an increase in the concentration of intracellular Ca2+, primarily through the activation of calcium channels of the TRPC family. The review provides a detailed analysis of the current state of knowledge about the molecular mechanisms responsible for the regulation of apoptotic processes in glomerular podocytes and also considers hormonal and other factors that regulate them both under normal conditions and under the conditions of lesions induced by hyperglycemia and diabetic nephropathy.

BACKGROUND AND PURPOSE: NALCN is a Na(+) leak, GPCR‐activated channel that regulates the resting membrane potential and neuronal excitability. Despite numerous possible roles for NALCN in both normal physiology and disease processes, lack of specific blockers hampers further investigation. EXPERIMENTAL APPROACH: The effect of N‐benzhydryl quinuclidine compounds on NALCN channels was demonstrated using whole‐cell patch‐clamp recordings in HEK293T cells overexpressing NALCN and acutely isolated nigral dopaminergic neurons that express NALCN endogenously. Src kinase activity was measured using a Src kinase assay kit, and voltage and current‐clamp recordings from nigral dopaminergic neurons were used to measure NALCN currents and membrane potentials. KEY RESULTS: N‐benzhydryl quinuclidine compounds inhibited NALCN channels without affecting TRPC channels, another important route for Na(+) leak. In HEK293T cells overexpressing NALCN, N‐benzhydryl quinuclidine compounds potently suppressed muscarinic M(3) receptor‐activated NALCN currents. Structure–function relationship studies suggest that the quinuclidine ring with a benzhydryl group imparts the ability to inhibit NALCN currents regardless of Src family kinases. Moreover, N‐benzhydryl quinuclidine compounds inhibited not only GPCR‐activated NALCN currents but also background Na(+) leak currents and hyperpolarized the membrane potential in native midbrain dopaminergic neurons that express NALCN endogenously. CONCLUSION AND IMPLICATIONS: These findings suggest that N‐benzhydryl quinuclidine compounds have a pharmacological potential to directly inhibit NALCN channels and could be a useful tool to investigate functions of NALCN channels.

Background Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca2+ release from the sarcoplasmic reticulum on exposure to triggering agents. This study tested the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca2+ dyshomeostasis in a mouse model relevant to malignant hyperthermia. Methods This study examined the mechanisms responsible for Ca2+ dyshomeostasis in RYR1-p.G2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and Western blots. Results RYR1-p.G2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous greater than heterozygous) compared with wild-type muscles. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-p.G2435R muscles (from 153 ± 11 nM/10 ± 0.5 mM to 304 ± 45 nM/14.2 ± 0.7 mM in heterozygotes P < 0.001] and from 251 ± 25 nM/13.9 ± 0.5 mM to 534 ± 64 nM/20.9 ± 1.5 mM in homozygotes [P < 0.001] compared with 123 ± 3 nM/8 ± 0.1 mM to 196 ± 27 nM/9.4 ± 0.7 mM in wild type). These increases were inhibited both by simply removing extracellular Ca2+ and by exposure to either a nonspecific (gadolinium) or a newly available, more specific pharmacologic agent (SAR7334) to block TRPC6- and TRPC3-mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene–dose–dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx. Conclusions Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces the working hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia–susceptible muscle, both at rest and during the malignant hyperthermia crisis. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Recently, possible applications of zinc oxide nanoparticles (nano-ZnO) have been extensively studied owing to their ease of synthesis. However, the effect of nano-ZnO on the nervous system remains unclear. This study investigates the action of nano-ZnO on SH-SY5Y neuroblastoma cells. We found that nano-ZnO (0-50 µg/mL) induced a significant decrease in cell survival rate in a dose-dependent manner, and increased LC3 puncta formation. However, the apoptosis was not affected by nano-ZnO, because the protein levels of cytochrome c, caspase-3, Bcl-xL, and BAX were not varied by the nano-ZnO treatment. Nano-ZnO increased Ca2+ entry and the expression of TRPC6.The results suggested that nano-ZnO increased [Ca2+] through the TRPC-dependent Ca2+ influx, since Ca2+ influx can be prevented by the TRPC inhibitor. Furthermore, cells on nano-ZnO-treatment groups displayed loss of F-actin in a dose dependent manner, which also could be prevented by TRPC inhibitor. Herein, we demonstrated that the nano-ZnO activated TRPC6 channel, thereby increasing the Ca2+ flux and resulting in increased autophagy. Nano-ZnO could have possible anticancer effects in neuroblastoma by inhibiting the proliferation of tumor cells. However, we should also pay attention toward the biosecurity of nano materials.

Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca(2+) signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVM) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca(2+) ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca(2+) signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na(+) and Ca(2+) free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca(2+) content, respectively. In isolated rabbit cardiomyocytes bathed in Na(+) and Ca(2+) free solution, we found an increased decay of the cytosolic Ca(2+) concentration [Ca(2+)](i) and increased SR Ca(2+) content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca(2+) leak through SR TRPC channels increased the systolic and diastolic [Ca(2+)](i) with only minor effects on the action potential and SR Ca(2+) content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca(2+) leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings guide us to an understanding of TRPC1 channels as modulators of [Ca(2+)](i) and contractility in cardiomyocytes.

Abstract Here we report a novel role for TRPC6, a member of the transient receptor potential (TRPC) channel family, in the CXCL1-dependent recruitment of murine neutrophil granulocytes. Representing a central element of the innate immune system, neutrophils are recruited from the blood stream to a site of inflammation. The recruitment process follows a well-defined sequence of events including adhesion to the blood vessel walls, migration, and chemotaxis to reach the inflammatory focus. A common feature of the underlying signaling pathways is the utilization of Ca2+ ions as intracellular second messengers. However, the required Ca2+ influx channels are not yet fully characterized. We used WT and TRPC6−/− neutrophils for in vitro and TRPC6−/− chimeric mice (WT mice with WT or TRPC6−/− bone marrow cells) for in vivo studies. After renal ischemia and reperfusion injury, TRPC6−/− chimeric mice had an attenuated TRPC6−/− neutrophil recruitment and a better outcome as judged from the reduced increase in the plasma creatinine concentration. In the cremaster model CXCL1-induced neutrophil adhesion, arrest and transmigration were also decreased in chimeric mice with TRPC6−/− neutrophils. Using atomic force microscopy and microfluidics, we could attribute the recruitment defect of TRPC6−/− neutrophils to the impact of the channel on adhesion to endothelial cells. Mechanistically, TRPC6−/− neutrophils exhibited lower Ca2+ transients during the initial adhesion leading to diminished Rap1 and β2 integrin activation and thereby reduced ICAM-1 binding. In summary, our study reveals that TRPC6 channels in neutrophils are crucial signaling modules in their recruitment from the blood stream in response to CXCL1. Key point Neutrophil TRPC6 channels are crucial for CXCL1-triggered activation of integrins during the initial steps of neutrophil recruitment.

Objective: Transient receptor potential canonical (TRPC) channels are involved in neovascularization repairing after vascular injury in many tissues. However, whether TRPCs play a regulatory role in the development of diabetic retinopathy (DR) has rarely been reported. In the present study, we selected TRPC1, 3, and 6 to determine their roles and mechanism in human retina vascular endothelial cells (HREC) under high glucose (HG) conditions. Methods: HRECs were cultured in vitro under HG, hyper osmosis, and normal conditions. The expression of TRPC1, 3, and 6 in the cells at 24 and 48 h were detected by RT-polymerase chain reaction (PCR), Western blot and cell immunohistochemistry (IHC); In various concentrations, SKF96365 acted on HG cultured HRECs, the expression of vascular endothelial growth factor (VEGF) were detected by the same methods above; and the CCK-8, Transwell, cell scratch assay, and Matrigel assay were used to assess cell proliferation, migration, and lumen formation. Results: The RT-PCR, Western blot, and IHC results showed that TRPC1 expression was increased, and TRPC6 mRNA expression was increased under high-glucose conditions. SKF96365 acted on HG cultured HRECs that VEGF expression was significantly decreased. The CCK-8 assay, Transwell assay, cell scratch assay, and Matrigel assay showed that cell proliferation, migration, and lumen formation were downregulated by SKF96365. Conclusion: HG can induce increased expression of TRPC1 and 6 in HRECs. Inhibition of the TRPC pathway not only can decrease VEGF expression but also can prevent proliferation, migration, and lumen formation of HRECs induced by HG. Inhibition of TRPC channels is expected to become a drug target for DR.

Vascular smooth muscle cells (VSMCs) play a pivotal role in the stability and tonic regulation of vascular homeostasis. VSMCs can switch back and forth between highly proliferative (synthetic) and fully differentiated (contractile) phenotypes in response to changes in the vessel environment. Abnormal phenotypic switching of VSMCs is a distinctive characteristic of vascular disorders, including atherosclerosis, pulmonary hypertension, stroke, and peripheral artery disease; however, how the control of VSMC phenotypic switching is dysregulated under pathological conditions remains obscure. Canonical transient receptor potential (TRPC) channels have attracted attention as a key regulator of pathological phenotype switching in VSMCs. Several TRPC subfamily member proteins-especially TRPC1 and TRPC6-are upregulated in pathological VSMCs, and pharmacological inhibition of TRPC channel activity has been reported to improve hypertensive vascular remodeling in rodents. This review summarizes the current understanding of the role of TRPC channels in cardiovascular plasticity, including our recent finding that TRPC6 participates in aberrant VSMC phenotype switching under ischemic conditions, and discusses the therapeutic potential of TRPC channels.

Transient receptor potential canonical (TRPC) proteins assemble to form homo- or heterotetrameric, nonselective cation channels permeable to K+, Na+, and Ca2+. TRPC channels are thought to act as complex integrators of physical and chemical environmental stimuli. Although the understanding of essential physiological roles of TRPC channels is incomplete, their implication in various pathological mechanisms and conditions of the nervous system, kidneys, and cardiovascular system in combination with the lack of major adverse effects of TRPC knockout or TRPC channel inhibition is driving the search of TRPC channel modulators as potential therapeutics. Here, we review the most promising small-molecule TRPC channel modulators, the understanding of their mode of action, and their potential in the study and treatment of cardiovascular and metabolic disease. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 62 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Various physical stimulation methods are developed to minimize the invasiveness of deep brain stimulation (DBS)1–3. Among them, only magnetic field can penetrate into the biological tissues without scattering or absorption4, which makes it ideal for untethered DBS. Recently developed magnetogenetics have shown the potential of developing treatments for neurological disorders5. However, magnetogenetic approaches have potential side effects from overexpression of exogenous ion channels and gene delivery with viral vectors6,7. Here, we demonstrated that the iron oxide magnetic nanodiscs (~270 nm) can be used as transducers to trigger calcium responses in the wild-type cultured neurons during the application of slow varying weak magnetic fields (50 mT at 10 Hz). Moreover, we identified that the intrinsic mechanosensitive ion channel transient receptor potential canonical (TRPC), which were widely expressed in the brain8, plays the main roles in this magnetomechanical stimulation. Finally, when we applied magnetic fields to the awake mice with magnetic nanodiscs injecting into subthalamic nucleus, the magnetomechanical stimulation triggered neuronal activities in the targeted region and the downstream region. Overall, this research demonstrated a magnetomechanical approach that can be used for wireless neuronal stimulation in vitro and untethered DBS in awake mice in vivo without implants or genetic manipulation.

It is well established that abnormalities in [Ca2+] regulation occur in heart diseases. Actually, independent studies demonstrated that Orai1/2/3 and TRPC protein related with store-operated calcium channels (SOCC) have a role in cardiac pathologies. Ischemia/reperfusion (I/R) stimulates transcription factor activation that modifies the expression of genes implicated in the pathogenesis of this process. Previous results described an increase in the expression of Orai1 and TRPC5 in cardiomyocytes after I/R, although the molecular mechanisms that mediate this regulation are still unknown. The aim of this study is to examine the molecular mechanisms implicated in the regulation of SOCC in cardiomyocytes after I/R focusing on the handling of intracellular [Ca2+]. Experiments were performed in a rat model of myocardial I/R, in adult (ARVM) and neonatal rat ventricular myocytes (NRVM), and in ventricular samples of heart-failure patients. Immunofluorescence was used to investigate CREB activation, and the protein expression was analyzed by Western blot. Calcium diastolic studies were realized using microfluorimetric technic with FURA-2AM. To evoke intracellular Ca2+ transients, ARVMs were field stimulated at 0.5 Hz and NRVMs at 1 Hz. An activation of CREB after I/R was observed in adult and neonatal rat cardiomyocytes. Furthermore, it was demonstrated that this activation was mediated by PKA, but not for EPAC2 or ERK. I/R induced an CREB-dependent ORAI protein expression increase and also an increase in the diastolic calcium in NRVM and ARVM from I/R animal models. Additionally, it was observed that ORAI1 inhibition with SYNTA-66 or GSK reduced the calcium diastolic increase induced by I/R. We demonstrated, for the first time, the activation of the transcription factor CREB in cardiomyocytes after I/R. This activation induces an up-regulation of ORAI1, suggesting that this channel plays a role in the I/R induced calcium diastolic increase.

Pulmonary arterial hypertension (PAH) is a severe and multifactorial disease. PAH pathogenesis mostly involves pulmonary arterial endothelial and pulmonary arterial smooth muscle cell (PASMC) dysfunction, leading to alterations in pulmonary arterial tone and distal pulmonary vessel obstruction and remodeling. Unfortunately, current PAH therapies are not curative, and therapeutic approaches mostly target endothelial dysfunction, while PASMC dysfunction is under investigation. In PAH, modifications in intracellular Ca2+ homoeostasis could partly explain PASMC dysfunction. One of the most crucial actors regulating Ca2+ homeostasis is store-operated Ca2+ channels, which mediate store-operated Ca2+ entry (SOCE). This review focuses on the main actors of SOCE in human and experimental PASMC, their contribution to PAH pathogenesis, and their therapeutic potential in PAH.

We investigated the contribution of excitatory transient receptor potential canonical (TRPC) cation channels to posttraumatic hyperexcitability in the brain 7 days following controlled cortical impact model of traumatic brain injury (TBI) to the parietal cortex in male adult mice. We investigated if TRPC1/TRPC4/TRPC5 channel expression is upregulated in excitatory neurons after TBI in contribution to epileptogenic hyperexcitability in key hippocampal and cortical circuits that have substantial cholinergic innervation. This was tested by measuring TRPC1/TRPC4/TRPC5 protein and messenger RNA (mRNA) expression, assays of cholinergic function, neuronal Ca2+ imaging in brain slices, and seizure susceptibility after TBI. We found region-specific increases in expression of TRPC1, TRPC4, and TRPC5 subunits in the hippocampus and cortex following TBI. The dentate gyrus, CA3 region, and cortex all exhibited robust upregulation of TRPC4 mRNA and protein. TBI increased cFos activity in dentate gyrus granule cells (DGGCs) and layer 5 pyramidal neurons both at the time of TBI and 7 days post-TBI. DGGCs displayed greater magnitude and duration of acetylcholine-induced rises in intracellular Ca2+ in brain slices from mice subjected to TBI. The TBI mice also exhibited greater seizure susceptibility in response to pentylenetetrazol-induced kindling. Blockade of TRPC4/TRPC5 channels with M084 reduced neuronal hyperexcitation and impeded epileptogenic progression of kindling. We observed that the time-dependent upregulation of TRPC4/TRPC5-containing channels alters cholinergic responses and activity of principal neurons acting to increase proexcitatory sensitivity. The underlying mechanism includes acutely decreased acetylcholinesterase function, resulting in greater Gq/11-coupled muscarinic receptor activation of TRPC channels. Overall, our evidence suggests that TBI-induced plasticity of TRPC channels strongly contributes to overt hyperexcitability and primes the hippocampus and cortex for seizures.

Background: Doxorubicin is a widely used and effective chemotherapy, but the major limiting side effect is cardiomyopathy which in some patients leads to congestive heart failure. Genetic variants in TRPC6 have been associated with the development of doxorubicin-induced cardiotoxicity, suggesting that TRPC6 may be a therapeutic target for cardioprotection in cancer patients.Methods: Assessment of Trpc6 deficiency to prevent doxorubicin-induced cardiac damage and function was conducted in male and female B6.129 and Trpc6 knock-out mice. Mice were treated with doxorubicin intraperitoneally every other day for a total of 6 injections (4 mg/kg/dose, cumulative dose 24 mg/kg). Cardiac damage was measured in heart sections by quantification of vacuolation and fibrosis, and in heart tissue by gene expression of Tnni3 and Myh7. Cardiac function was determined by echocardiography.Results: When treated with doxorubicin, male Trpc6-deficient mice showed improvement in markers of cardiac damage with significantly reduced vacuolation, fibrosis and Myh7 expression and increased Tnni3 expression in the heart compared to wild-type controls. Similarly, male Trpc6-deficient mice treated with doxorubicin had improved LVEF, fractional shortening, cardiac output and stroke volume. Female mice were less susceptible to doxorubicin-induced cardiac damage and functional changes than males, but Trpc6-deficient females had improved vacuolation with doxorubicin treatment. Sex differences were observed in wild-type and Trpc6-deficient mice in body-weight and expression of Trpc1, Trpc3 and Rcan1 in response to doxorubicin.Conclusions: Trpc6 promotes cardiac damage following treatment with doxorubicin resulting in cardiomyopathy in male mice. Female mice are less susceptible to cardiotoxicity with more robust ability to modulate other Trpc channels and Rcan1 expression.

AIM Both human and rat myometrium express stromal interaction molecule (STIM) and Orai/transient receptor potential canonical (TRPC) proteins, which are components of plasma membrane Ca2+ store-operated channels. There are reports that these proteins mediate agonist-induced Ca2+ influx in cultured myometrial cells. In this study, we aimed to determine the effects of Pyr6, an Orai channel blocker, on different agonist-induced contractions in isolated segments of rat uterus. MAIN FINDINGS In Ca2+ -free Tyrode's solution, Pyr6 (3 μM) promoted a reduction in both the magnitude and frequency of Ca2+ (1 mM)-induced uterine contractions after the addition of carbachol (CCh, 100 μM), but not after the addition of oxytocin (OT, 150 nM). In Ca2+ (0.18 mM)-Tyrode's solution, Pyr6 completely relaxed uterine contractions induced by both CCh and cloprostenol (300 nM), but not those induced by either KCI (40-80 mM) or OT. The addition of Pyr6 abolished the oscillatory uterine contractions induced by Ca2+ after the addition of cyclopiazonic acid (CPA, 10 μM). When pre-incubated (5 min), Pyr6 reduced the magnitude of both CCh-induced phasic and tonic contractions. The addition of Pyr2 (3 μM), an Orai and TRPC channel blocker, abolished uterine contractions induced by CCh or OT. CONCLUSION Considering Pyr6 as an Orai channel blocker and its inhibitory effect on uterine contractions induced by CCh, CPA, and cloprostenol, we suggest that Orai channels are required for the maintenance of contractions induced by these agonists in rat uterus.

The transient receptor potential (TRP) channel superfamily consists of a large group of non-selective cation channels that serve as cellular sensors for a wide spectrum of physical and environmental stimuli. The 28 mammalian TRPs, categorized into six subfamilies, including TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin) and TRPP (polycystin), are widely expressed in different cells and tissues. TRPs exhibit a variety of unique features that not only distinguish them from other superfamilies of ion channels, but also confer diverse physiological functions. Located at the plasma membrane or in the membranes of intracellular organelles, TRPs are the cellular safeguards that sense various cell stresses and environmental stimuli and translate this information into responses at the organismal level. Loss- or gain-of-function mutations of TRPs cause inherited diseases and pathologies in different physiological systems, whereas up- or down-regulation of TRPs is associated with acquired human disorders. In this Cell Science at a Glance article and the accompanying poster, we briefly summarize the history of the discovery of TRPs, their unique features, recent advances in the understanding of TRP activation mechanisms, the structural basis of TRP Ca2+ selectivity and ligand binding, as well as potential roles in mammalian physiology and pathology.

Simple Summary Glioblastoma is a high-grade primary brain tumor that contains a subpopulation of cells called glioblastoma stem cells, which are responsible for tumor initiation, growth and recurrence after treatment. Recent transcriptomic studies have highlighted that calcium pathways predominate in glioblastoma stem cells. Calcium channels have the ability to transduce signals from the microenvironment and are therefore ideally placed to control cellular behavior. Using multiple approaches, we demonstrate in five different primary cultures, previously derived from surgical specimens, that glioblastoma stem cells express store-operated channels (SOC) that support calcium entry into these cells. Pharmacological inhibition of SOC dramatically reduces cell proliferation and stem cell self-renewal in these cultures. By identifying SOC as a critical mechanism involved in the maintenance of the stem cell population in glioblastoma, our study will contribute to the framework for the identification of new therapies against this deadly tumor. Abstract Glioblastoma is the most frequent and deadly form of primary brain tumors. Despite multimodal treatment, more than 90% of patients experience tumor recurrence. Glioblastoma contains a small population of cells, called glioblastoma stem cells (GSC) that are highly resistant to treatment and endowed with the ability to regenerate the tumor, which accounts for tumor recurrence. Transcriptomic studies disclosed an enrichment of calcium (Ca2+) signaling transcripts in GSC. In non-excitable cells, store-operated channels (SOC) represent a major route of Ca2+ influx. As SOC regulate the self-renewal of adult neural stem cells that are possible cells of origin of GSC, we analyzed the roles of SOC in cultures of GSC previously derived from five different glioblastoma surgical specimens. Immunoblotting and immunocytochemistry experiments showed that GSC express Orai1 and TRPC1, two core SOC proteins, along with their activator STIM1. Ca2+ imaging demonstrated that SOC support Ca2+ entries in GSC. Pharmacological inhibition of SOC-dependent Ca2+ entries decreased proliferation, impaired self-renewal, and reduced expression of the stem cell marker SOX2 in GSC. Our data showing the ability of SOC inhibitors to impede GSC self-renewal paves the way for a strategy to target the cells considered responsible for conveying resistance to treatment and tumor relapse.

Canonical members of the TRP superfamily of ion channels have long been recognized as key elements of Ca2+ handling in a plethora of cell types. The emerging role of TRPC channels in human physiopathology has generated considerable interest in their pharmacological targeting, which requires detailed understanding of their molecular function. Although consent has been reached that receptor-phospholipase C (PLC) pathways and generation of lipid mediators constitute the prominent upstream signaling process that governs channel activity, multimodal sensing features of TRPC complexes have been demonstrated repeatedly. Downstream signaling by TRPC channels is similarly complex and involves the generation of local and global cellular Ca2+ rises, which are well-defined in space and time to govern specific cellular functions. These TRPC-mediated Ca2+ signals rely in part on Ca2+ permeation through the channels, but are essentially complemented by secondary mechanisms such as Ca2+ mobilization from storage sites and Na+/Ca2+ exchange, which involve coordinated interaction with signaling partners. Consequently, the control of cell functions by TRPC molecules is critically determined by dynamic assembly and subcellular targeting of the TRPC complexes. The very recent availability of high-resolution structure information on TRPC channel complexes has paved the way towards a comprehensive understanding of signal transduction by TRPC channels. Here, we summarize current concepts of cation permeation in TRPC complexes, TRPC-mediated shaping of cellular Ca2+ signals and the associated control of specific cell functions.

Stroke is a major life-threatening and disabling disease with a restricted therapeutic approach. Bone marrow stromal cells (BMSCs) possess proliferative ability and a multi-directional differentiation potential, and secrete a range of trophic/growth factors that can protect neurons after cerebral ischemia/reperfusion. Transient receptor potential canonical (TRPC) is a family of non-selective channels permeable to Ca2+, with several functions including neuronal survival. Over-expression of TRPC6, a subtype of the TRPC family, was shown to protect neurons against cerebral ischemia/reperfusion injury. However, it remains unclear whether over-expression of TRPC6 in BMSCs can further reduce brain injury after ischemia/reperfusion. In the present study, we report that over-expression of TRPC6 via a CRISPR-based synergistic activation mediator in BMSCs provided a greater reduction of brain injury in a rat model of ischemia/reperfusion. Further, the improved neurofunctional outcomes were associated with increased TRPC6 and brain derived neurotrophic factor expression levels. Overall, these data suggest that TRPC6 over-expressing BMSCs may be a promising therapeutic agent for ischemic stroke.

Ascites syndrome (AS) in chickens is associated with profound vascular remodelling and increased pulmonary artery pressure as well as right ventricular hypertrophy. Classical transient rece...

Store operated Ca2+ entry (SOCE) is the most important Ca2+ entry pathway in non-excitable cells. However, SOCE can also play a pivotal role in excitable cells such as anterior pituitary (AP) cells. The AP gland contains five different cell types that release six major AP hormones controlling most of the entire endocrine system. AP hormone release is modulated by Ca2+ signals induced by different hypothalamic releasing hormones (HRHs) acting on specific receptors in AP cells. TRH and LHRH both induce Ca2+ release and Ca2+ entry in responsive cells while GHRH and CRH only induce Ca2+ entry. SOCE has been shown to contribute to Ca2+ responses induced by TRH and LHRH but no molecular evidence has been provided. Accordingly, we used AP cells isolated from mice devoid of Orai1 channels (noted as Orai1-/- or Orai1 KO mice) and mice lacking expression of all seven canonical TRP channels (TRPC) from TRPC1 to TRPC7 (noted as heptaTRPC KO mice) to investigate contribution of these putative channel proteins to SOCE and intracellular Ca2+ responses induced by HRHs. We found that thapsigargin-evoked SOCE is lost in AP cells from Orai1-/- mice but unaffected in cells from heptaTRPC KO mice. Conversely, while spontaneous intracellular Ca2+-oscillations related to electrical activity were not affected in the Orai1-/- mice, these responses were significantly reduced in heptaTRPC KO mice. We also found that Ca2+ entry induced by TRH and LHRH is decreased in AP cells isolated from Orai1-/-. In addition, Ca2+ responses to several HRHs, particularly TRH and GHRH, are decreased in the heptaTRPC KO mice. These results indicate that expression of Orai1, and not TRPC channel proteins, is necessary for thapsigargin-evoked SOCE and is required to support Ca2+ entry induced by TRH and LHRH in mouse AP cells. In contrast, TRPC channel proteins appear to contribute to spontaneous Ca2+-oscillations and Ca2+ responses induced by TRH and GHRH. We conclude that expression of Orai1 and TRPC channels proteins may play differential and significant roles in AP physiology and endocrine control.

The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca2+ channels that are activated when luminal [Ca2+] of the endoplasmic reticulum ([Ca2+]ER) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca2+]ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca2+ store depletion. Another family of plasma membrane Ca2+ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca2+]i elevation. Although Ca2+ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca2+ signal generated by Ca2+ entry pathways in response to depletion of ER-Ca2+ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Lung adenocarcinoma (LAD) is the most common type of lung cancer with a high possibility of tumor relapse and metastasis. ENKUR (Enkurin) was originally identified as a potential regulator or effector of TRPC channels that directly binds to several TRPC proteins and the p85 subunit of PI3K. However, the role of ENKUR in cancer development has remained unclear. In this study we analyzed the expression profile of ENKUR mRNA in clinical LAD samples and examined ENKUR mRNA expression and ENKUR protein level in LAD cells. Significant down-regulated ENKUR expression was observed in clinical tumor tissues of LAD as well as in human LAD cells. To evaluate the effects of aberrant ENKUR expression on cellular biology of LAD cells in vitro and tumor growth in vivo, ENKUR-overexpressed and -silenced LAD cell lines were constructed using lentiviral vectors. Our results showed that overexpression of ENKUR in LAD cells inhibited cell proliferation, migration and invasion, while silencing of ENKUR led to the opposite effects. Silencing of ENKUR in LAD cells also promoted tumorigenesis in nude mice model and caused epithelial to mesenchymal transition (EMT). Furthermore, using western blot and co-immunoprecipitation analyses, we demonstrated that ENKUR interacts with PI3K directly and is possibly involved in the PI3K/Akt and MAPK/ERK signaling pathways.

PURPOSE: Triple-negative breast cancers (TNBCs) represent a heterogeneous group of tumors. The lack of targeted therapies combined with the inherently aggressive nature of TNBCs results in a higher relapse rate and poorer overall survival. We evaluated the heterogeneity of TNBC cell lines for TRPC channel expression and sensitivity to cation-disrupting drugs. METHODS: The TRPC1/4/5 agonist englerin A was used to identify a group of TNBC cell lines sensitive to TRPC1/4/5 activation and intracellular cation disruption. Quantitative RT-PCR, the sulforhodamine B assay, pharmacological inhibition, and siRNA-mediated knockdown approaches were employed. Epifluorescence imaging was performed to measure intracellular Ca(2+) and Na(+) levels. Mitochondrial membrane potential changes were monitored by confocal imaging. RESULTS: BT-549 and Hs578T cells express high levels of TRPC4 and TRPC1/4, respectively, and are exquisitely, 2000- and 430-fold, more sensitive to englerin A than other TNBC cell lines. While englerin A caused a slow Na(+) and nominal Ca(2+) accumulation in Hs578T cells, it elicited rapid increases in cytosolic Ca(2+) levels that triggered mitochondrial depolarization in BT-549 cells. Interestingly, BT-549 and Hs578T cells were also more sensitive to digoxin as compared to other TNBC cell lines. Collectively, these data reveal TRPC1/4 channels as potential biomarkers of TNBC cell lines with dysfunctional mechanisms of cation homeostasis and therefore sensitivity to cardiac glycosides. CONCLUSIONS: The sensitivity of BT-549 and Hs578T cells to englerin A and digoxin suggests a subset of TNBCs are highly susceptible to cation disruption and encourages investigation of TRPC1 and TRPC4 as potential new biomarkers of sensitivity to cardiac glycosides.

Transient receptor potential canonical (TRPC) channels are calcium permeable, non-selective cation channels with wide tissue-specific distribution. Among 7 TRPC channels, TRPC 1/4/5 and TRPC3/6/7 are subdivided based on amino acid sequence homology. TRPC4 and TRPC5 channels exhibit cationic current with homotetrameric form, but they also form heterotetrameric channel such as TRPC1/4 or TRPC1/5 once TRPC1 is incorporated. The expression of TRPC1 is ubiquitous whereas the expressions of TRPC4 and TRPC5 are rather focused in nervous system. With the help of conditional knock-out of TPRC1, 4 and/or 5 genes, TRPC channels made of these constituents are reported to be involved in various pathophysiological functions such as seizure, anxiety-like behaviour, fear, Huntington's disease, Parkinson's disease and many others. In heterologous expression system, many issues such as activation mechanism, stoichiometry and relative cation permeabilites of homomeric or heteromeric channels have been addressed. In this review, we discussed the role of TRPC1 channel per se in plasma membrane, role of TRPC1 in heterotetrameric conformation (TRPC1/4 or TRPC1/5) and relationship between TRPC1/4/5 channels, calcium influx and voltage-gated calcium channels.

Although the overall structure of many classical transient receptor potential proteins (TRPC), including human and murine TRPC6, were recently resolved by cryoelectron microscopy analysis, structural changes during channel activation by 1-oleoyl-1-acetyl-sn-glycerol (OAG), the membrane-permeable analog of diacylglycerol, were not defined. Moreover, data on carboxyl- and amino-terminal interactions were not provided, as the amino-terminal regions of murine and human TRPC6 were not resolved. Therefore, we employed a Förster resonance energy transfer (FRET) approach using a small fluorescein arsenical hairpin (FlAsH) targeted to a short tetracysteine sequence at the unresolved amino-terminus and cerulean, a cyan fluorescent protein, as a tag at the carboxyl-terminus of the murine TRPC6 protein. After OAG as well as GSK-1702934A activation, FRET efficiency was simultaneously and significantly reduced, indicating a decreased interaction between the amino to carboxyl termini in the functional tagged murine TRPC6 tetramer (TRPC6 WT) heterologously expressed in human embryonic kidney 293T cells. There was a significant reduction in the FRET signal obtained from analysis of murine TRPC6 FRET constructs with homologous amino-terminal mutations (M131T, G108S) that had been identified in human patients with inherited focal segmental glomerulosclerosis, a condition that can lead to end-stage renal disease. A novel, designed loss-of-function TRPC6 mutation (N109A) in the amino-terminus in close proximity to the carboxyl-terminus produced similar FRET ratios. SIGNIFICANCE STATEMENT: Our data show for the first time that FlAsH-tagging of ion channels is a promising tool to study conformational changes after channel opening and may significantly advance the analysis of ion channel activation as well as their mutants involved in channelopathies.

Briefly review the recent structural work of transient receptor potential canonical (TRPC) ion channels by using electron cryo-microscopy (cryo-EM). The high resolution structures of TRPC3, TRPC4, TRPC5 and TRPC6 are discussed.

Mutations in the gene encoding canonical transient receptor potential-6 (TRPC6) channels result in severe nephrotic syndromes that typically lead to end-stage renal disease. Many but not all of these mutations result in a gain in the function of the resulting channel protein. Since those observations were first made, substantial work has supported the hypothesis that TRPC6 channels can also contribute to progression of acquired (non-genetic) glomerular diseases, including primary and secondary FSGS, glomerulosclerosis during autoimmune glomerulonephritis, and possibly in type-1 diabetes. Their regulation has been extensively studied, especially in podocytes, but also in mesangial cells and other cell types present in the kidney. More recent evidence has implicated TRPC6 in renal fibrosis and tubulointerstitial disease caused by urinary obstruction. Consequently TRPC6 is being extensively investigated as a target for drug discovery. Other TRPC family members are present in kidney. TRPC6 can form a functional heteromultimer with TRPC3, and it has been suggested that TRPC5 may also play a role in glomerular disease progression, although the evidence on this is contradictory. Here we review literature on the expression and regulation of TRPC6, TRPC3 and TRPC5 in various cell types of the vertebrate kidney, the evidence that these channels are dysregulated in disease models, and research showing that knock-out or pharmacological inhibition of these channels can reduce the severity of kidney disease. We also summarize several areas that remain controversial, and some of the large gaps of knowledge concerning the fundamental role of these proteins in regulation of renal function.

Transient receptor potential canonical (TRPC) 6 inhibits Aβ in Alzheimer's disease (AD) mouse brain and improves the behavioral performance.To evaluate the association of TRPC6 expression in peripheral leucocytes from AD and mild cognitive impairment (MCI) patients and to explore its potential value in early diagnosis of AD.TRPC6 mRNA levels in peripheral leucocytes were detected by quantitative real-time PCR. The Spearman correlation test was used to ascertain the associations between TRPC6 and the scores of MMSE, ADL, CSDD, CDR. The Receiver Operating Characteristic (ROC) curve was drawn to evaluate the diagnostic potential of TRPC6 for AD and MCI.There were 108 CE, 136 MCI, 164 Con and 60 PD in the study. The expression of TRPC6 mRNA level in peripheral leucocytes was significantly lower: 1) in patients with AD and MCI compared to Con; 2) in AD compared to MCI; 3) in hospitalized AD compared to AD from communities. There was a significantly positive correlation between TRPC6 mRNA and MMSE score (p = .001, R = 0.327). Significantly inverse correlations were found between TRPC6 and CDR score (p 0.001, R = -0.303) as well as between TRPC6 and ADL score (p = .001, R = -0.342) for all AD. The area under curve of ROC was 0.881 for the classification of AD, and 0.706 for the classification of MCI, respectively.TRPC6 expression is inversely correlated with cognitive performance of AD. TRPC6 in peripheral leucocytes may be a potential biomarker for the diagnosis of AD.

Transient receptor potential (TRP) channels are expressed in the endometrium but it is unknown if they are modulated through the estrous cycle (EC). This study was undertaken to identify the modulation of the TRPC gene and protein isoforms in bovine uterine epithelium, as a model for human, throughout the EC. Changes in the expression of TRPC genes in bovine uterine epithelium throughout the EC were measured using Real-Time PCR, while immunohistochemistry and immunocytochemistry were used to determine the localization of these channels. Out of the 7 members of the TRPC family, TRPC1, 2, 3, 4 and 6 genes were expressed in bovine uterine epithelial tissue and TRPC 5 and 7 were not. Gene expression levels of all TRPC isoforms underwent cyclical changes throughout the EC. Moreover, cyclical changes were detected in the protein levels of TRPC1 and TRPC6 throughout the EC. These findings show that TRPC channels are modulated through the EC and therefore may have a role in reproductive events.

Transient receptor potential canonical (TRPC) channels are highly homologous, nonselective cation channels that form many homo- and heterotetrameric channels. These channels are highly abundant in the brain and kidney and have been implicated in numerous diseases, such as depression, addiction, and chronic kidney disease, among others. Historically, there have been very few selective modulators of the TRPC family in order to fully understand their role in disease despite their physiological significance. However, that has changed recently and there has been a significant increase in interest in this family of channels which has led to the emergence of selective tool compounds, and even preclinical drug candidates, over the past few years. This review will cover these new advancements in the discovery of TRPC modulators and the emergence of newly reported structural information which will undoubtedly lead to even greater advancements.