Search

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.

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 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

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.

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.

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.

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.

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.

Canonical or classical transient receptor potential 4 and 5 proteins (TRPC4 and TRPC5) assemble as homomers or heteromerize with TRPC1 protein to form functional nonselective cationic channels with high calcium permeability. These channel complexes, TRPC1/4/5, are widely expressed in nervous and cardiovascular systems, also in other human tissues and cell types. It is debatable that TRPC1 protein is able to form a functional ion channel on its own. A recent explosion of molecular information about TRPC1/4/5 has emerged including knowledge of their distribution, function, and regulation suggesting these three members of the TRPC subfamily of TRP channels play crucial roles in human physiology and pathology. Therefore, these ion channels represent potential drug targets for cancer, epilepsy, anxiety, pain, and cardiac remodelling. In recent years, a number of highly selective small-molecule modulators of TRPC1/4/5 channels have been identified as being potent with improved pharmacological properties. This review will focus on recent remarkable small-molecule agonists: (-)-englerin A and tonantzitlolone and antagonists: Pico145 and HC7090, of TPRC1/4/5 channels. In addition, this work highlights other recently identified modulators of these channels such as the benzothiadiazine derivative, riluzole, ML204, clemizole, and AC1903. Together, these treasure troves of agonists and antagonists of TRPC1/4/5 channels provide valuable hints to comprehend the functional importance of these ion channels in native cells and in vivo animal models. Importantly, human diseases and disorders mediated by these proteins can be studied using these compounds to perhaps initiate drug discovery efforts to develop novel therapeutic agents.

Nanosecond electric pulses have been shown to open nanopores in the cell plasma membrane by fluorescent imaging of calcium uptake and fluorescent dyes, including propidium (Pr) iodide and YO-PRO-1 (YP1). Recently, we demonstrated that nsEPs also induce the phosphoinositide intracellular signaling cascade by phosphatidylinositol-4,5-bisphosphate (PIP2) depletion resulting in physiological responses similar to those observed following stimulation of Gq11-coupled receptors. In this paper, we explore the role of receptor- and store-operated calcium entry (ROCE/SOCE) mechanisms in the observed response of cells to nsEP. We show that addition of the ROCE/SOCE and transient receptor potential channel (TRPC) blocker gadolinium (Gd3+, 300 μM) slows PIP2 depletion following 1 and 20 nsEP exposures. Lipid rafts, regions of the plasma membrane rich in PIP2 and TRPC, are also disrupted by nsEP exposure suggesting that ROCE/SOCE mechanisms are likely impacted. Reducing the expression of stromal interaction molecule 1 (STIM1) protein, a key protein in ROCE and SOCE, in cells exposure to nsEP resulted in a reduction in induced intracellular calcium rise. Additionally, after exposure to 1 and 20 nsEPs (16.2 kV/cm, 5 Hz), intracellular calcium rises were significantly reduced by the addition of GD3+ and SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl-1H-imidazole hydrochloride, 100 μM), a blocker of STIM1 interaction. However, using similar nsEP exposure parameters, SKF-96365 was less effective at reducing YP1 uptake compared to Gd3+. Thus, it is possible that SKF-96365 could block STIM1 interactions within the cell, while Gd3+ could acts on TRPC/nanopores from outside of the cell. Our results present evidence of nsEP induces ROCE and SOCE mechanisms and demonstrate that YP1 and Ca2+ cannot be used solely as markers of nsEP-induced nanoporation.

Background The incidence of traumatic brain injuries (TBIs) is on the rise in the USA. Concussions, or mild TBIs without skull fracture, account for about 75% of all TBIs. Mild TBIs (mTBIs) lead to memory and cognitive deficits, headaches, intraocular pressure rises, axonal degeneration, neuroinflammation, and an array of cerebrovascular dysfunctions, including increased vascular permeability and decreased cerebral blood flow. It has been recently reported that besides vascular dysfunction in the cerebral circulation, mTBI may also cause a significant impairment of endothelial function in the systemic circulation, at least within mesenteric microvessels. In this study, we investigated whether mTBI affects endothelial function in aortas and determined the contribution of transient receptor potential canonical (TRPC) channels to modulating mTBI-associated endothelial dysfunction. Methods We used a model of closed-head mTBI in C57BL/6, 129S, 129S-C57BL/6-F2 mice, and 129S-TRPC1 and 129S-C57BL/6-TRPC6 knockout mice to determine the effect of mTBI on endothelial function in mouse aortas employing ex vivo isometric tension measurements. Aortic tissue was also analyzed using immunofluorescence and qRT-PCR for TRPC6 expression following mTBI. Results We show that in various strains of mice, mTBI induces a pronounced and long-lasting endothelial dysfunction in the aorta. Ablation of TRPC6 protects mice from mTBI-associated aortic endothelial dysfunction, while TRPC1 ablation does not impact brain injury-induced endothelial impairment in the aorta. Consistent with a role of TRPC6 activation following mTBI, we observed improved endothelial function in wild type control mice subjected to mTBI following 7-day in vivo treatment with larixyl acetate, an inhibitor of TRPC6 channels. Conversely, in vitro treatment with the pro-inflammatory endotoxin lipopolysaccharide, which activates endothelial TRPC6 in a Toll-like receptor type 4 (TLR4)-dependent manner, worsened aortic endothelial dysfunction in wild type mice. Lipopolysaccharide treatment in vitro failed to elicit endothelial dysfunction in TRPC6 knockout mice. No change in endothelial TRPC6 expression was observed 7 days following TBI. Conclusions These data suggest that TRPC6 activation may be critical for inducing endothelial dysfunction following closed-head mTBI and that pharmacological inhibition of the channel may be a feasible therapeutic strategy for preventing mTBI-associated systemic endothelial dysfunction.

Background An emerging body of evidence indicates that puerarin (PUE) plays an important role in the treatment of angina pectoris, myocardial ischemia-reperfusion injury, hypertension and other cardiovascular diseases, but how PUE affects the vascular remodeling of hypertensive rats has not been reported yet. This study aimed to investigate the effect and mechanism of PUE on carotid arteries of spontaneously hypertensive rats (SHR) to provide the basis for the clinical application of PUE. Methods Thirty male SHR and six male Wistar Kyoto rats (WKY) aged 3 months were used in this study, SHR rats were randomly divided into 5 groups, PUE(40 or 80 mg/kg/d, ip) and telmisartan (TELMI) (30 mg/kg/d, ig) were administrated for 3 months. We use DMT myography pressure-diameter system to investigate biomechanical properties of carotid arteries, 10 μM pan-classical transient receptor potential channels (TRPCs) inhibitor SKF96365, 200 nM specific TRPC6 inhibitor SAR7334 and 100 μM Orai1 inhibitor ANCOA4 were used in the mechanical test. Results PUE can significantly decrease systolic and diastolic blood pressure, long-term administration of PUE resulted in a mild reduction of thickness and inner diameter of carotid artery. PUE ameliorate NE-response and vascular remodeling mainly through inhibiting TRPCs channel activities of VSMC. Conclusion PUE can ameliorate biomechanical remodeling of carotid arteries through inhibiting TRPCs channel activities of VSMC in spontaneously hypertensive rats.

Doxorubicin (DOX), a category D pregnancy drug, is a chemotherapeutic agent that has been shown in animal studies to induce fetal toxicity, including renal abnormalities. Upregulation of the transient receptor potential cation (TRPC) 6 channel is involved in DOX-induced podocyte apoptosis. We have previously reported that TRPC6-mediated Ca2+ signaling promotes neonatal glomerular mesangial cell (GMC) death. However, it is unknown whether DOX alters mesangial TRPC expression or viability in the fetus. In this study, cell growth was tracked in control and DOX-treated primary GMCs derived from fetal pigs. Live-cell imaging demonstrated that exposure to DOX inhibited the proliferation of fetal pig GMCs and induced cell death. DOX did not alter the TRPC3 expression levels. By contrast, TRPC6 protein expression in the cells was markedly reduced by DOX. DOX treatment also attenuated the TRPC6-mediated intracellular Ca2+ elevation. DOX stimulated mitochondrial reactive oxygen species (mtROS) generation and mitophagy by the GMCs. The DOX-induced mtROS generation and apoptosis were reversed by the mitochondria-targeted antioxidant mitoquinone. These data suggest that DOX-induced fetal pig GMC apoptosis is independent of TRPC6 channel upregulation but requires mtROS production. The mtROS-dependent GMC death may contribute to DOX-induced fetal nephrotoxicity when administered prenatally.

Summary Small molecular probes designed for photopharmacology and opto-chemogenetics are rapidly gaining widespread recognition for investigations of transient receptor potential canonical (TRPC) channels. This protocol describes the use of three photoswitchable diacylglycerol analogs—PhoDAG-1, PhoDAG-3, and OptoDArG—for ultrarapid activation and deactivation of native TRPC2 channels in mouse vomeronasal sensory neurons and olfactory type B cells, as well as heterologously expressed human TRPC6 channels. Photoconversion can be achieved in mammalian tissue slices and enables all-optical stimulation and shutoff of TRPC channels. For complete details on the use and execution of this protocol, please refer to Leinders-Zufall et al. (2018)., Graphical abstract, Highlights • DAG photoswitching enables ultrarapid activation and deactivation of TRPC channels • Multiple photoswitchable DAG analogs are now available • DAG photoconversion is sufficient for the gating of TRPC2, TRPC3, and TRPC6 • Photoswitching combined with Ca2+ imaging enables all-optical stimulation and recording, Small molecular probes designed for photopharmacology and opto-chemogenetics are rapidly gaining widespread recognition for investigations of transient receptor potential canonical (TRPC) channels. This protocol describes the use of three photoswitchable diacylglycerol analogs—PhoDAG-1, PhoDAG-3, and OptoDArG—for ultrarapid activation and deactivation of native TRPC2 channels in mouse vomeronasal sensory neurons and olfactory-type B cells, as well as heterologously expressed human TRPC6 channels. Photoconversion can be achieved in mammalian tissue slices and enables all-optical stimulation and shutoff of TRPC channels.

Objectives Although both calcium-sensing receptor (CaSR) and canonical transient receptor potential (TRPC) proteins contribute to chronic hypoxia (CH)-induced pulmonary arterial smooth muscle cells (PASMCs) proliferation, the relationship between CaSR and TRPC in hypoxic PASMCs proliferation remains poorly understood. The goal of this study was to identify that CH promotes PASMCs proliferation through CaSR-TRPC pathway. Methods Rat PASMCs were isolated and treated with CH. Cell proliferation was assessed by cell counting, CCK-8 assay, and EdU incorporation. CaSR and TRPC expressions were determined by qPCR and Western blotting. Store-operated Ca2+ entry (SOCE) was assessed by extracellular Ca2+ restoration. Results In PASMCs, CH enhanced the cell number, cell viability and DNA synthesis, which is accompanied by upregulated expression of CaSR, TRPC1 and TRPC6. Negative CaSR modulators (NPS2143, NPS2390) inhibited, whereas positive modulators (spermine, R568) enhanced, the CH-induced increases in cell number, cell viability and DNA synthesis in PASMCs. Knockdown of CaSR by siRNA inhibited the CH-induced upregulation of TRPC1 and TRPC6 and enhancement of SOCE and attenuated the CH-induced enhancements of cell number, cell viability and DNA synthesis in PASMCs. However, neither siTRPC1 nor siTRPC6 had an effect on the CH-induced CaSR upregulation, although both significantly attenuated the CH-induced enhancements of cell number, cell viability and DNA synthesis in PASMCs. Conclusion These results demonstrate that upregulated CaSR-TRPC1/6 pathway mediating PASMCs proliferation is an important pathogenic mechanism under hypoxic conditions.

Consistent with their wide distribution throughout the CNS, transcripts of all transient receptor potential (TRP) cation channel superfamily members have been detected in both neuronal and non-neuronal cells of the mammalian retina. Evidence shows that members of the TRPC (canonical, TRPC1/4/5/6), TRPV (vanilloid, TRPV1/2/4), TRPM (melastatin, TRPM1/2/3/5), TRPA (ankyrin, TRPA1), and TRPP (polycystin, TRPP2) subfamilies contribute to retinal function and circulation in health and disease, but the relevance of most TRPs has yet to be determined. Their principal role in light detection is far better understood than their participation in the control of intraocular pressure, retinal blood flow, oxidative stress, ion homeostasis, and transmitter signaling for retinal information processing. Moreover, if the therapeutic potential of targeting some TRPs to treat various retinal diseases remains speculative, recent studies highlight that vision restoration strategies are very likely to benefit from the thermo- and mechanosensitive properties of TRPs. This minireview focuses on the evidence of the past 5 years about the role of TRPs in the retina and retinal circulation, raises some possibilities about the function of TRPs in the retina, and discusses the potential sources of endogenous stimuli for TRPs in this tissue, as a reflection for future studies.

Emerging evidence suggests the exercise pressor reflex is exaggerated in early-stage type 1 diabetes mellitus (T1DM). Piezo channels may play a role in this exaggeration since blocking these channels attenuates the exaggerated pressor response to tendon stretch in T1DM rats. However, tendon stretch constitutes a different mechanical and physiological stimuli than that occurring during muscle contraction. Therefore, the purpose of this study was to determine the contribution of Piezo channels in evoking the pressor reflex during an intermittent muscle contraction in T1DM. In unanaesthetized, decerebrate rats we compared the pressor and cardioaccelerator responses to intermittent muscle contraction before and after locally injecting GsMTx-4 (0.25 µM) into the hindlimb vasculature. Although GsMTx-4 has a high potency for Piezo channels, it has also been suggested to block TRPC channels. We, therefore, performed additional experiments to control for this possibility by also injecting SKF 96365 (10 µM), a TRPC channel blocker. We found that local injection of GsMTx-4, but not SKF 96365, attenuated the exaggerated peak pressor (ΔMAP before: 33±3 mmHg, after: 22±3 mmHg, p=0.007) and pressor index (ΔBPi before: 668±91 mmHg·s, after: 418±81 mmHg·s, p=0.021) response in STZ rats (n=8). GsMTx-4 attenuated the exaggerated early-onset pressor as well as the pressor response over time, which eliminated peak differences as well as those over time between T1DM and healthy controls. These data suggest that Piezo channels are an effective target to normalize the exercise pressor reflex in T1DM.

This 2020 Special Issue “TRPC channels” of Cells was dedicated to commemorating the 25th anniversary of discovery of the Transient Receptor Potential Canonical (TRPC) channel subfamily. TRPCs are calcium permeable cation channels that are localized to the plasma membrane. Cloning the first TRPC channel (TRPC1) in 1995 [1,2] opened a new vast horizon for research efforts. Since then, the accumulated knowledge has been important to understanding the roles of TRPCs in different tissues and organs of our body. In this Editorial, we briefly summarize and compare the views on TRPC channel roles in health and disease presented by the Special Issue authors.[...]

Endothelial dysfunction, characterised by impaired nitric oxide (NO) bioavailability, arises in response to a variety of cardiovascular risk factors and precedes atherosclerosis. NO is produced by tight regulation of endothelial nitric oxide synthase (eNOS) activity in response to vasodilatory stimuli. This regulation of eNOS is mediated in part by store-operated calcium entry (SOCE). We hypothesized that both ATP- and flow-induced eNOS activation are regulated by SOCE derived from Orai1 channels and members of the transient receptor potential canonical (TRPC) channel family. Bovine aortic endothelial cells (BAECs) were pre-treated with pharmacological inhibitors of TRPC channels and Orai1 to examine their effect on calcium signaling and eNOS activation in response to flow and ATP. The peak and sustained ATP-induced calcium signal and the resulting eNOS activation were attenuated by inhibition of TRPC3, which we found to be store operated. TRPC4 blockade reduced the transient peak in calcium concentration following ATP stimulation, but did not significantly reduce eNOS activity. Simultaneous TRPC3 & 4 inhibition reduced flow-induced NO production via alterations in phosphorylation-mediated eNOS activity. Inhibition of TRPC1/6 or Orai1 failed to lower ATP-induced calcium entry or eNOS activation. Our results suggest that TRPC3 is a store-operated channel in BAECs and is the key regulator of ATP-induced eNOS activation, whereas flow stimulation also recruits TRPC4 into the pathway for the synthesis of NO.

Background: Arrhythmias and heart failure are common cardiac complications leading to substantial morbidity and mortality in patients with hemochromatosis, yet mechanistic insights remain incomplete. We investigated the effects of iron (Fe) on electrophysiological properties and intracellular Ca 2+ (Ca 2+ i ) handling in mouse left ventricular cardiomyocytes. Methods: Cardiomyocytes were isolated from the left ventricle of mouse hearts and were superfused with Fe 3+ /8-hydroxyquinoline complex (5–100 μM). Membrane potential and ionic currents including TRPC (transient receptor potential canonical) were recorded using the patch-clamp technique. Ca 2+ i was evaluated by using Fluo-4. Cell contraction was measured with a video-based edge detection system. The role of TRPCs in the genesis of arrhythmias was also investigated by using a mathematical model of a mouse ventricular myocyte with the incorporation of the TRPC component. Results: We observed prolongation of the action potential duration and induction of early and delayed afterdepolarizations in myocytes superfused with 15 µmol/L Fe 3+ /8-hydroxyquinoline complex. Iron treatment decreased the peak amplitude of the L-type Ca 2+ current and total K + current, altered Ca 2+ i dynamics, and decreased cell contractility. During the final phase of Fe treatment, sustained Ca 2+ i waves and repolarization failure occurred and ventricular cells became unexcitable. Gadolinium abolished Ca 2+ i waves and restored the resting membrane potential to the normal range. The involvement of TRPC activation was confirmed by TRPC channel current recordings in the absence or presence of functional TRPC channel antibodies. Computer modeling captured the same action potential and Ca 2+ i dynamics and provided additional mechanistic insights. Conclusions: We conclude that iron overload induces cardiac dysfunction that is associated with TRPC channel activation and alterations in membrane potential and Ca 2+ i dynamics.

Background Pharmacologic modulation has previously shown that transient receptor potential canonical (TRPC) channels play an important role in the pathogenesis of malignant hyperthermia. This study tested the hypothesis that genetically suppressing the function of TRPC6 can partially ameliorate muscle cation dyshomeostasis and the response to halothane in a mouse model relevant to malignant hyperthermia. Methods This study examined the effect of overexpressing a muscle-specific nonconducting dominant-negative TRPC6 channel in 20 RYR1-p.R163C and 20 wild-type mice and an equal number of nonexpressing controls, using calcium- and sodium-selective microelectrodes and Western blots. Results RYR1-p.R163C mouse muscles have chronically elevated intracellular calcium and sodium levels compared to wild-type muscles. Transgenic expression of the nonconducting TRPC6 channel reduced intracellular calcium from 331 ± 34 nM (mean ± SD) to 190 ± 27 nM (P < 0.0001) and sodium from 15 ± 1 mM to 11 ± 1 mM (P < 0.0001). Its expression lowered the increase in intracellular Ca2+ of the TRPC6-specific activator hyperforin in RYR1-p.R163C muscle fibers from 52% (348 ± 37 nM to 537 ± 70 nM) to 14% (185 ± 11 nM to 210 ± 44 nM). Western blot analysis of TRPC3 and TRPC6 expression showed the expected increase in TRPC6 caused by overexpression of its dominant-negative transgene and a compensatory increase in expression of TRPC3. Although expression of the muscle-specific dominant-negative TRPC6 was able to modulate the increase in intracellular calcium during halothane exposure and prolonged life (35 ± 5 min vs. 15 ± 3 min; P < 0.0001), a slow, steady increase in calcium began after 20 min of halothane exposure, which eventually led to death. Conclusions These data support previous findings that TRPC channels play an important role in causing the intracellular calcium and sodium dyshomeostasis associated with RYR1 variants that are pathogenic for malignant hyperthermia. However, they also show that modulating TRPC channels alone is not sufficient to prevent the lethal effect of exposure to volatile anesthetic malignant hyperthermia–triggering agents. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Obesity is a serious global health problem because of its increasing prevalence and comorbidities, but its treatments are limited. The serotonin 2C receptor (5-HT(2C)R), a G-protein-coupled receptor, activates proopiomelanocortin (POMC) neurons in the arcuate nucleus of hypothalamus (ARH) to reduce appetite and weight gain. However, several 5-HT analogs targeting this receptor, e.g., lorcaserin (Lor), suffer from diminished efficacy to reduce weight after prolonged administration. Here, we show that barbadin (Bar), a novel β-arrestin/β2-adaptin inhibitor, can prevent 5-HT(2C)R internalization in cells and potentiate long-term effects of Lor to reduce appetite and body weight in male mice. Mechanistically, we demonstrate that Bar co-treatment can effectively maintain the sensitivity of the 5-HT(2C)R in POMC(ARH) neurons, despite prolonged Lor exposure, thereby allowing these neurons to be activated through opening the transient receptor potential cation (TRPC) channels. Thus, our results prove the concept that inhibition of 5-HT(2C)R desensitization can be a valid strategy to improve the long-term weight loss effects of Lor or other 5-HT(2C)R agonists, and also provide an intellectual framework to develop effective long-term management of weight by targeting 5-HT(2C)R desensitization. SIGNIFICANCE STATEMENT By demonstrating that the combination of barbadin (Bar) with a G-protein-coupled receptor (GPCR) agonist can provide prolonged weight-lowering benefits in a preclinical setting, our work should call for additional efforts to validate Bar as a safe and effective medicine or to use Bar as a lead compound to develop more suitable compounds for obesity treatment. These results prove the concept that inhibition of serotonin 2C receptor (5-HT(2C)R) desensitization can be a valid strategy to improve the long-term weight loss effects of lorcaserin (Lor) or other 5-HT(2C)R agonists. Since GPCRs represent a major category as therapeutic targets for various human diseases and desensitization of GPCRs is a common issue, our work may provide a conceptual framework to enhance effects of a broad range of GPCR medicines.

Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand-binding site can be transmitted to the central ion-conducting pore of TRPC4. CaM binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel CaM-induced regulatory mechanism of canonical TRP channels.

Aim The pathogenesis of obesity has been associated with high intake of dietary fat, and some recent studies have explored the cellular mechanisms of oro-sensory detection of dietary fatty acids. We further assessed the role of transient receptor potential canonical (TRPC) channels in oro-sensory perception of dietary lipids. Methods We determined by RT-qPCR and western blotting the expression of TRPC3/6/7 channels in mouse fungiform taste bud cells (mTBC). Immunocytochemistry was used to explore whether TRPC3 channels were co-expressed with fatty acid receptors. We employed wild-type (WT) mTBC, and those transfected with small interfering RNAs (siRNAs) against TRPC3 or STIM1. Ca2+ signalling was studied in TBC from TRPC3-/- mice and their WT littermates. Results We demonstrate that mouse fungiform taste bud cells (mTBC) express TRPC3, but not TRPC6 or TRPC7 channels, and their inactivation by siRNA or experiments on TBC from TRPC3-/- mice brought about a decrease in fatty acid-induced gustatory Ca2+ signalling, coupled with taste bud CD36 lipid sensor. TRPC3 channel activation was found to be under the control of STIM1 in lingual mTBC. Behavioural studies showed that spontaneous preference for a dietary long-chain fatty acid was abolished in TRPC3-/- mice, and in mice wherein lingual TRPC3 expression was silenced by employing siRNA. Conclusion We report that lingual TRPC3 channels are critically involved in fat taste perception.

Background Local anesthetics, particularly potent long acting ones such as bupivacaine, can cause cardiotoxicity by inhibiting sodium ion channels; however, the impact of left ventricular hypertrophy on the cardiotoxicity and the underlying mechanisms remain undetermined. Transient receptor potential canonical (TRPC) channels are upregulated in left ventricular hypertrophy. Some transient receptor potential channel subtypes have been reported to pass relatively large cations, including protonated local anesthetics; this is known as the “pore phenomenon.” The authors hypothesized that bupivacaine-induced cardiotoxicity is more severe in left ventricular hypertrophy due to upregulated TRPC channels. Methods The authors used a modified transverse aortic constriction model as a left ventricular hypertrophy. Cardiotoxicity caused by bupivacaine was compared between sham and aortic constriction male rats, and the underlying mechanisms were investigated by recording sodium ion channel currents and immunocytochemistry of TRPC protein in cardiomyocytes. Results The time to cardiac arrest by bupivacaine was shorter in aortic constriction rats (n =11) than in sham rats (n = 12) (mean ± SD, 1,302 ± 324 s vs. 1,034 ± 211 s; P = 0.030), regardless of its lower plasma concentration. The half-maximal inhibitory concentrations of bupivacaine toward sodium ion currents were 4.5 and 4.3 μM, which decreased to 3.9 and 2.6 μM in sham and aortic constriction rats, respectively, upon coapplication of 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3 channel activator. In both groups, sodium ion currents were unaffected by QX-314, a positively charged lidocaine derivative, that hardly permeates the cell membrane, but was significantly decreased with QX-314 and 1-oleoyl-2-acetyl-sn-glycerol coapplication (sham: 79 ± 10% of control; P = 0.004; aortic constriction: 47± 27% of control; P = 0.020; n = 5 cells per group). Effects of 1-oleoyl-2-acetyl-sn-glycerol were antagonized by a specific TRPC3 channel inhibitor. Conclusions Left ventricular hypertrophy exacerbated bupivacaine-induced cardiotoxicity, which could be a consequence of the “pore phenomenon” of TRPC3 channels upregulated in left ventricular hypertrophy. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That New

Transient receptor potentials (TRPs) are non-selective cation channels that are widely expressed in vascular beds. They contribute to the Ca2+ influx evoked by a wide spectrum of chemical and physical stimuli, both in endothelial and vascular smooth muscle cells. Within the superfamily of TRP channels, different isoforms of TRPC (canonical) and TRPV (vanilloid) have emerged as important regulators of vascular tone and blood flow pressure. Additionally, several lines of evidence derived from animal models, and even from human subjects, highlighted the role of TRPC and TRPV in vascular remodeling and disease. Dysregulation in the function and/or expression of TRPC and TRPV isoforms likely regulates vascular smooth muscle cells switching from a contractile to a synthetic phenotype. This process contributes to the development and progression of vascular disorders, such as systemic and pulmonary arterial hypertension, atherosclerosis and restenosis. In this review, we provide an overview of the current knowledge on the implication of TRPC and TRPV in the physiological and pathological processes of some frequent vascular diseases.

Photoreceptors are specialized cells devoted to the transduction of the incoming visual signals. Rods are able also to shed from their tip old disks and to synthesize at the base of the outer segment (OS) new disks. By combining electrophysiology, optical tweezers (OTs), and biochemistry, we investigate mechanosensitivity in the rods of Xenopus laevis, and we show that 1) mechanosensitive channels (MSCs), transient receptor potential canonical 1 (TRPC1), and Piezo1 are present in rod inner segments (ISs); 2) mechanical stimulation—of the order of 10 pN—applied briefly to either the OS or IS evokes calcium transients; 3) inhibition of MSCs decreases the duration of photoresponses to bright flashes; 4) bright flashes of light induce a rapid shortening of the OS; and 5) the genes encoding the TRPC family have an ancient association with the genes encoding families of protein involved in phototransduction. These results suggest that MSCs play an integral role in rods’ phototransduction., It is widely thought that sensory neurons are specialized to transduce just a single sensory modality. A combination of electrophysiology, optical tweezers, and histochemistry reveals that rod photoreceptors not only express mechanosensitive channels but display mechanosensitivity, which is crucial for phototransduction.

Key points Receptor-operated activation of TRPC4 cation channels requires Gi/o proteins and phospholipase-Cδ1 (PLCδ1) activation by intracellular Ca2+ . Concurrent stimulation of the Gq/11 pathway accelerates Gi/o activation of TRPC4, which is not mimicked by increasing cytosolic Ca2+ . The kinetic effect of Gq/11 was diminished by alkaline intracellular pH (pHi ) and increased pHi buffer capacity. Acidic pHi (6.75-6.25) together with the cytosolic Ca2+ rise accelerated Gi/o -mediated TRPC4 activation. Protons exert their facilitation effect through Ca2+ -dependent activation of PLCδ1. The data suggest that the Gq/11 -PLCβ pathway facilitates Gi/o activation of TRPC4 through hydrolysing phosphatidylinositol 4,5-bisphosphate (PIP2 ) to produce the initial proton signal that triggers a self-propagating PLCδ1 activity supported by regenerative H+ and Ca2+ . The findings provide novel mechanistic insights into receptor-operated TRPC4 activation by coincident Gq/11 and Gi/o pathways and shed light on how aberrant activation of TRPC4 may occur under pathological conditions to cause cell damage. Abstract Transient Receptor Potential Canonical 4 (TRPC4) forms non-selective cation channels activated downstream from receptors that signal through G proteins. Our recent work suggests that TRPC4 channels are particularly coupled to pertussis toxin-sensitive Gi/o proteins, with a co-dependence on phospholipase-Cδ1 (PLCδ1). The Gi/o -mediated TRPC4 activation is dually dependent on and bimodally regulated by phosphatidylinositol 4,5-bisphosphate (PIP2 ), the substrate hydrolysed by PLC, and intracellular Ca2+ . As a byproduct of PLC-mediated PIP2 hydrolysis, protons have been shown to play an important role in the activation of Drosophila TRP channels. However, how intracellular pH affects mammalian TRPC channels remains obscure. Here, using patch-clamp recordings of HEK293 cells heterologously co-expressing mouse TRPC4β and the Gi/o -coupled μ opioid receptor, we investigated the role of intracellular protons on Gi/o -mediated TRPC4 activation. We found that acidic cytosolic pH greatly accelerated the rate of TRPC4 activation without altering the maximal current density and this effect was dependent on intracellular Ca2+ elevation. However, protons did not accelerate channel activation by directly acting upon TRPC4. We additionally demonstrated that protons exert their effect through sensitization of PLCδ1 to Ca2+ , which in turn promotes PLCδ1 activity and further potentiates TRPC4 via a positive feedback mechanism. The mechanism elucidated here helps explain how Gi/o and Gq/11 co-stimulation induces a faster activation of TRPC4 than Gi/o activation alone and highlights again the critical role of PLCδ1 in TRPC4 gating.

The nonselective Ca2+ -permeable transient receptor potential channel subfamily member 5 (TRPC5) belongs to the transient receptor potential canonical (TRPC) superfamily and is widely expressed in the brain. Compelling evidence reveals that TRPC5 plays crucial roles in depression and other psychiatric disorders. To develop a TRPC5 radioligand, following up on our previous effort, we synthesized the iodine compound TZ66127 and its iodine-125-labeled counterpart [125 I]TZ66127. The synthesis of TZ66127 was achieved by replacing chloride with iodide in the structure of HC608, and the [125 I]TZ66127 was radiosynthesized using its corresponding tributylstannylated precursor. We established a stable human TRPC5-overexpressed HEK293-hTRPC5 cell line and performed Ca2+ imaging and a cell-binding assay study of TZ66127; these indicated that TZ66127 had good inhibition activity for TRPC5, and the inhibitory efficiency of TZ66127 toward TRPC5 presented in a dose-dependent manner. An in vitro autoradiography and immunohistochemistry study of rat brain sections suggested that [125 I]TZ66127 had binding specificity toward TRPC5. Altogether, [125 I]TZ66127 has high potential to serve as a radioligand for screening the binding activity of other new compounds toward TRPC5. The availability of [125 I]TZ66127 might facilitate the development of therapeutic drugs and PET imaging agents that target TRPC5.

Transient receptor potential canonical (TRPC) proteins constitute a group of receptor-operated calcium-permeable nonselective cationic membrane channels of the TRP superfamily. They are largely expressed in the hippocampus and are able to modulate neuronal functions. Accordingly, they have been involved in different hippocampal functions such as learning processes and different types of memories, as well as hippocampal dysfunctions such as seizures. This review covers the mechanisms of activation of these channels, how these channels can modulate neuronal excitability, in particular the after-burst hyperpolarization, and in the persistent activity, how they control synaptic plasticity including pre- and postsynaptic processes and how they can interfere with cell survival and neurogenesis.

Stromal interaction molecules (STIM1, 2) are acting as sensors for Ca2+ in intracellular stores and activate Orai channels at the plasma membrane for store-operated Ca2+ entry (SOCE), while classical transient receptor potential (TRPC) channel mediate receptor-operated Ca2+ entry (ROCE). Several reports, however, indicate a role for TRPC in SOCE in certain cell types. Here, we analyzed Ca2+ influx and cell function in TRPC1/6-deficient (TRPC1/6−/−) and STIM1/2- deficient (STIM1/2ΔpmLF) primary murine lung fibroblasts (pmLF). As expected, SOCE was decreased in STIM1/2- deficient pmLF and ROCE was decreased in TRPC1/6−/− pmLF compared to control cells. By contrast, SOCE was not significantly different in TRPC1/6−/− pmLF and ROCE was similar in STIM1/2-deficient pmLF compared to Wt cells. Most interestingly, cell proliferation, migration and nuclear localization of nuclear factor of activated T-cells (NFATc1 and c3) were decreased after ablation of STIM1/2 proteins in pmLF. In conclusion, TRPC1/6 channels are not involved in SOCE and STIM1/2 deficiency resulted in decreased cell proliferation and migration in pmLF.

The cardiac plexus, which contains parasympathetic ganglia, plays an important role in regulating cardiac function. Histamine is known to excite intracardiac ganglion neurons, but the underlying mechanism is obscure. In the present study, therefore, the effect of histamine on rat intracardiac ganglion neurons was investigated using perforated patch-clamp recordings. Histamine depolarized acutely isolated neurons with a half-maximal effective concentration of 4.5 μM. This depolarization was markedly inhibited by the H1 receptor antagonist triprolidine and mimicked by the H1 receptor agonist 2-pyridylethylamine, thus implicating histamine H1 receptors. Consistently, reverse transcription-PCR (RT-PCR) and Western blot analyses confirmed H1 receptor expression in the intracardiac ganglia. Under voltage-clamp conditions, histamine evoked an inward current that was potentiated by extracellular Ca2+ removal and attenuated by extracellular Na+ replacement with N-methyl-D-glucamine. This implicated the involvement of non-selective cation channels, which given the link between H1 receptors and Gq/11-protein-phospholipase C signalling, were suspected to be transient receptor potential canonical (TRPC) channels. This was confirmed by the marked inhibition of the inward current through the pharmacological disruption of either Gq/11 signalling or intracellular Ca2+ release and by the application of the TRPC blockers Pyr3, Gd3+ and ML204. Consistently, RT-PCR analysis revealed the expression of several TRPC subtypes in the intracardiac ganglia. Whilst histamine was also separately found to inhibit the M-current, the histamine-induced depolarization was only significantly inhibited by the TRPC blockers Gd3+ and ML204, and not by the M-current blocker XE991. These results suggest that TRPC channels serve as the predominant mediator of neuronal excitation by histamine.

Patients with sepsis and sepsis-related complications have a high mortality. Endothelial cell dysfunction plays a central role in sepsis pathophysiological process. In sepsis patients, endothelial cell apoptosis is associated with intracellular calcium overload. Multiple functions in the apoptotic process have been found to be regulated by calcium signaling. Our previous work had proved that LPS-induced cell injury was associated with store-operated calcium (SOC) entry mediated by stromal interaction molecule-1 (STIM 1) in Human umbilical vein endothelial cells (HUVEC), but the underlying molecular mechanism has not been adequately defined. Here we report that the LPS-induced cell injury is related to the calcium overload in HUVEC. SOC entry mediated by calcium release-activated calcium modulator (Orai) 1 and transient receptor potential canonical (TRPC) 1 was associated with LPS-induced calcium overload and cell apoptosis. Bruton's tyrosine kinase (Btk)/Phospholipase C(PLC) γ/inositol 1,4,5-triphosphate receptor (IP3R) played a major role in regulating calcium overload in LPS-induced HUVEC. Knockdown of Btk markedly inhibited the expressions of Orai 1 and its downstream molecule IP3R but not that of TRPC1 in LPS-induced HUVEC. In mice, knockdown of Btk and Orai 1 inhibited LPS-induced calcium overload, pulmonary vascular endothelial cell (VEC) injury and acute lung injury. These findings demonstrated that Btk acts as a regulator of calcium-dependent signaling, especially in the Orai 1-mediated SOC entry of the LPS-induced VEC.

Calcium (Ca2+) is perhaps the most versatile signaling molecule in cells. Ca2+ regulates a large number of key events in cells, ranging from gene transcription, motility, and contraction, to energy production and channel gating. To accomplish all these different functions, a multitude of channels, pumps, and transporters are necessary. A group of channels participating in these processes is the transient receptor potential (TRP) family of cation channels. These channels are divided into 29 subfamilies, and are differentially expressed in man, rodents, worms, and flies. One of these subfamilies is the transient receptor potential canonical (TRPC) family of channels. This ion channel family comprises of seven isoforms, labeled TRPC1−7. In man, six functional forms are expressed (TRPC1, TRPC3−7), whereas TRPC2 is a pseudogene; thus, not functionally expressed. In this review, we will describe the importance of the TRPC channels and their interacting molecular partners in the etiology of cancer, particularly in regard to regulating migration and invasion.

International audience; Pulmonary arterial hypertension is a progressive and lethal cardiopulmonary disease. The rise in right ventricular afterload leads to right ventricular hypertrophy and failure. Right ventricular failure is the most important prognostic factor for morbidity and mortality in pulmonary arterial hypertension or pulmonary hypertension caused by left heart diseases. Surprisingly, the right ventricle is not targeted by pulmonary arterial hypertension-specific therapies. The current profound lack of basic understanding of pulmonary arterial hypertension-related right ventricular remodelling can explain, at least in part, this paradox. The physiology and haemodynamic function of the right ventricle in the normal state differ considerably from those of the left ventricle, and the known mechanisms of left ventricular dysfunction cannot be generalized to right ventricular dysfunction. Ion channel activities and calcium homeostasis tightly regulate cardiac function, and their dysfunction contributes to the pathogenesis of cardiac diseases. This review focuses on the ion channels (potassium, calcium) and intracellular calcium handling remodelling involved in right ventricular hypertrophy and dysfunction caused by pulmonary arterial hypertension.

Group I metabotropic glutamate receptors (mGluR) are involved in various forms of synaptic plasticity that are believed to underlie declarative memory. We previously showed that mGluR5 specifically activates channels containing TRPC1, an isoform of the canonical family of Transient Receptor Potential channels highly expressed in the CA1-3 regions of the hippocampus. Using a tamoxifen-inducible conditional knockout model, we show here that the acute deletion of the Trpc1 gene alters the extinction of spatial reference memory. mGluR-induced long-term depression, which is partially responsible for memory extinction, was impaired in these mice. Similar results were obtained in vitro and in vivo by inhibiting the channel by its most specific inhibitor, Pico145. Among the numerous known postsynaptic pathways activated by type I mGluR, we observed that the deletion of Trpc1 impaired the activation of ERK1/2 and the subsequent expression of Arc, an immediate early gene that plays a key role in AMPA receptors endocytosis and subsequent long-term depression.

VGF gene encodes for a neuropeptide precursor of 68 kDa composed by 615 (human) and 617 (rat, mice) residues, expressed prevalently in the central nervous system (CNS), but also in the peripheral nervous system (PNS) and in various endocrine cells. This precursor undergoes proteolytic cleavage, generating a family of peptides different in length and biological activity. Among them, TLQP-21, a peptide of 21 amino acids, has been widely investigated for its relevant endocrine and extraendocrine activities. The complement complement C3a receptor-1 (C3aR1) has been suggested as the TLQP-21 receptor and, in different cell lines, its activation by TLQP-21 induces an increase of intracellular Ca2+. This effect relies both on Ca2+ release from the endoplasmic reticulum (ER) and extracellular Ca2+ entry. The latter depends on stromal interaction molecules (STIM)-Orai1 interaction or transient receptor potential channel (TRPC) involvement. After Ca2+ entry, the activation of outward K+-Ca2+-dependent currents, mainly the KCa3.1 currents, provides a membrane polarizing influence which offset the depolarizing action of Ca2+ elevation and indirectly maintains the driving force for optimal Ca2+ increase in the cytosol. In this review, we address the main endocrine and extraendocrine actions displayed by TLQP-21, highlighting recent findings on its mechanism of action and its potential in different pathological conditions.

Hippocampal GABAergic interneurons play key roles in regulating principal cell activity and plasticity. Interneurons located in stratum oriens/alveus (O/A INs) receive excitatory inputs from CA1 pyramidal cells and express a Hebbian form of long-term potentiation (LTP) at their excitatory input synapses. This LTP requires the activation of metabotropic glutamate receptors 1a (mGluR1a) and Ca2+ entry via transient receptor potential (TRP) channels. However, the type of TRP channels involved in synaptic transmission at these synapses remains largely unknown. Using patch-clamp recordings, we show that slow excitatory postsynaptic currents (EPSCs) evoked in O/A INs are dependent on TRP channels but may be independent of phospholipase C. Using reverse transcription polymerase chain reaction (RT-PCR) we found that mRNA for TRPC 1, 3–7 was present in CA1 hippocampus. Using single-cell RT-PCR, we found expression of mRNA for TRPC 1, 4–7, but not TRPC3, in O/A INs. Using co-immunoprecipitation assays in HEK-293 cell expression system, we found that TRPC1 and TRPC4 interacted with mGluR1a. Co-immunoprecipitation in hippocampus showed that TRPC1 interacted with mGluR1a. Using immunofluorescence, we found that TRPC1 co-localized with mGluR1a in O/A IN dendrites, whereas TRPC4 localization appeared limited to O/A IN cell body. Down-regulation of TRPC1, but not TRPC4, expression in O/A INs using small interfering RNAs prevented slow EPSCs, suggesting that TRPC1 is an obligatory TRPC subunit for these EPSCs. Our findings uncover a functional role of TRPC1 in mGluR1a-mediated slow excitatory synaptic transmission onto O/A INs that could be involved in Hebbian LTP at these synapses.

The seven-member transient receptor potential canonical genes (TRPC1-7) encode cation channels linked to several human diseases. There is little understanding of the participation of each TRPC in each pathology, considering functional redundancy. Also, most of the inhibitors available are not specifc. Thus, we developed mice that lack all of the TRPCs and performed a transcriptome analysis in eight tissues. The aim of this research was to address the impact of the absence of all TRPC channels on gene expression. We obtained a total of 4305 diferentially expressed genes (DEGs) in at least one tissue where spleen showed the highest number of DEGs (1371). Just 21 genes were modifed in all the tissues. Performing a pathway enrichment analysis, we found that many important signaling pathways were modifed in more than one tissue, including PI3K (phosphatidylinositol 3-kinase/protein kinase-B) signaling pathway, cytokine-cytokine receptor interaction, extracellular matrix (ECM)-receptor interaction and circadian rhythms. We describe for the frst time the changes at the transcriptome level due to the lack of all TRPC proteins in a mouse model and provide a starting point to understand the function of TRPC channels and their possible roles in pathologies. Fil: Formoso, Karina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina Fil: Susperreguy, Sebastian. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina Fil: Freichel, Marc. Universität Heidelberg; Alemania Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina

Transient Receptor Potential Canonical (TRPC) channels are homologues of Drosophila TRP channel first cloned in mammalian cells. TRPC family consists of seven members which are nonselective cation channels with a high Ca2+ permeability and are activated by a wide spectrum of stimuli. These channels are ubiquitously expressed in different tissues and organs in mammals and exert a variety of physiological functions. Post-translational modifications (PTMs) including phosphorylation, N-glycosylation, disulfide bond formation, ubiquitination, S-nitrosylation, S-glutathionylation, and acetylation play important roles in the modulation of channel gating, subcellular trafficking, protein-protein interaction, recycling, and protein architecture. PTMs also contribute to the polymodal activation of TRPCs and their subtle regulation in diverse physiological contexts and in pathological situations. Owing to their roles in the motor coordination and regulation of kidney podocyte structure, mutations of TRPCs have been implicated in diseases like cerebellar ataxia (moonwalker mice) and focal and segmental glomerulosclerosis (FSGS). The aim of this review is to comprehensively integrate all reported PTMs of TRPCs, to discuss their physiological/pathophysiological roles if available, and to summarize diseases linked to the natural mutations of TRPCs.

Background This study aimed to investigate the effects of topotecan (TPT) on the hypoxia-induced pulmonary arterial hypertension (PAH) in a rat model, and to explore the underlying mechanism. Methods The experiments were carried out in vitro using rat PASMCs and in vivo using a rat model of hypoxia-induced PAH. Results TPT significantly suppressed the hypoxia-induced upregulation of HIF-1α and TRPC1/4/6 expression both in pulmonary arterial smooth muscle cells (PASMCs) from normal rats and in pulmonary arteries from PAH model rats. Furthermore, TPT effectively inhibited intracellular Ca2+ concentration ([Ca2+]i) change (Ca2+ influx) in PASMCs from both normal rats and PAH model rats. Importantly, TPT treatment significantly inhibited the hypoxia-induced proliferation, migration and a contractile-to-synthetic phenotypic switching of normal rat PASMCs in vitro, where the effect was abrogated by overexpression of TRPC1/4/6. Furthermore, TPT administration potently attenuated the hypoxia-induced PAH-associated pulmonary arteriolar remodeling in PAH model rats, as evidenced by amelioration of elevated hemodynamic parameters, and enhanced right ventricle hypertrophy and wall thickening. Conclusion TPT ameliorates the hypoxia-induced pulmonary vascular remodeling in PAH, and the mechanism is associated with TPT-mediated inhibition of hypoxia-induced upregulation of HIF-1α and TRPC1/4/6 expression, Ca2+ influx, and PASMCs proliferation.

All of the canonical transient receptor potential channels (TRPC) with the exception of TRPC 2 are expressed in hypothalamic neurons and are involved in multiple homeostatic functions. Although the metabotropic glutamate receptors have been shown to be coupled to TRPC channel activation in cortical and sub-cortical brain regions, in the hypothalamus multiple amine and peptidergic G protein-coupled receptors (GPCRs) and growth factor/cytokine receptors are linked to activation of TRPC channels that are vital for reproduction, temperature regulation, arousal and energy homeostasis. In addition to the neurotransmitters, circulating hormones like insulin and leptin through their cognate receptors activate TRPC channels in POMC neurons. Many of the post-synaptic effects of the neurotransmitters and hormones are regulated in different physiological states by expression of TRPC channels in the post-synaptic neurons. Therefore, TRPC channels are key targets not only for neurotransmitters but circulating hormones in their vital role to control multiple hypothalamic functions, which is the focus of this review.