Upregulation of TRPC1 protects against high glucose-induced HUVECs dysfunction by inhibiting oxidative stress.

-To explore the effect of TRPC1 on endothelial cell function damage under a high glucose environment and its downstream molecular mechanism, and provide new theory and strategy for improving diabetic endothelial cell function and promoting vascular injury repair. In vitro, we use high glucose to treat human umbilical vein endothelial cells (HUVECs) and upregulated TRPC1 with adenovirus infection. HUVECs were split into 4 groups: (i) NG Group: Treated with normal glucose; (ii) HG Group: Treated with high glucose; (iii) HG + adGFP Group: High glucose + the control adenovirus (adGFP); (iv) HG + adTRPC1 Group: High glucose + recombinant adenovirus encoding TRPC1. We found that high glucose significantly decreased the expression level of TRPC1 protein, and impaired the proliferation and migration of HUVECs, which could be reversed by overexpression of TRPC1. In addition, high glucose induced an increase in ROS and MDA and a decrease in SOD activity, whereas TRPC1 overexpression could inhibit the growth of oxidative stress level. These findings suggest that overexpression of TRPC1 prevents HUVECs proliferation and migration dysfunction induced by high glucose via inhibiting oxidative stress injuries.

Biomass fuels related-PM2.5 promotes lung fibroblast-myofibroblast transition through PI3K/AKT/TRPC1 pathway.

Emerging evidence has suggested that exposure to PM2.5 is a significant contributing factor to the development of chronic obstructive pulmonary disease (COPD). However, the underlying biological effects and mechanisms of PM2.5 in COPD pathology remain elusive. In this study, we aimed to investigate the implication and regulatory effect of biomass fuels related-PM2.5 (BRPM2.5) concerning the pathological process of fibroblast-to-myofibroblast transition (FMT) in the context of COPD. In vivo experimentation revealed that exposure to biofuel smoke was associated with airway inflammation in rats. After 4 weeks of exposure, there was inflammation in the small airways, but no significant structural changes in the airway walls. However, after 24 weeks, airway remodeling occurred due to increased collagen deposition, myofibroblast proliferation, and tracheal wall thickness. In vitro, cellular immunofluorescence results showed that with stimulation of BRPM2.5 for 72 h, the cell morphology of fibroblasts changed significantly, most of the cells changed from spindle-shaped to star-shaped irregular, α-SMA stress fibers appeared in the cytoplasm and the synthesis of type I collagen increased. The collagen gel contraction experiment showed that the contractility of fibroblasts was enhanced. The expression level of TRPC1 in fibroblasts was increased. Specific siRNA-TRPC1 blocked BRPM2.5-induced FMT and reduced cell contractility. Additionally, specific siRNA-TRPC1 resulted in a decrease in the augment of intracellular Ca2+ concentration ([Ca2+]i) induced by BRPM2.5. Notably, it was found that the PI3K inhibitor, LY294002, inhibited enhancement of AKT phosphorylation level, FMT occurrence, and elevation of TRPC1 protein expression induced by BRPM2.5. The findings indicated that BRPM2.5 is capable of inducing the FMT, with the possibility of mediation by PI3K/AKT/TRPC1. These results hold potential implications for the understanding of the molecular mechanisms involved in BRPM2.5-induced COPD and may aid in the development of novel therapeutic strategies for pathological conditions characterized by fibrosis.

Selective Assembly of TRPC Channels in the Rat Retina during Photoreceptor Degeneration.

Transient receptor potential canonical (TRPC) channels are calcium channels with diverse expression profiles and physiological implications in the retina. Neurons and glial cells of rat retinas with photoreceptor degeneration caused by retinitis pigmentosa (RP) exhibit basal calcium levels that are above those detected in healthy retinas. Inner retinal cells are the last to degenerate and are responsible for maintaining the activity of the visual cortex, even after complete loss of photoreceptors. We considered the possibility that TRPC1 and TRPC5 channels might be associated with both the high calcium levels and the delay in inner retinal degeneration. TRPC1 is known to mediate protective effects in neurodegenerative processes while TRPC5 promotes cell death. In order to comprehend the implications of these channels in RP, the co-localization and subsequent physical interaction between TRPC1 and TRPC5 in healthy retina (Sprague-Dawley rats) and degenerating (P23H-1, a model of RP) retina were detected by immunofluorescence and proximity ligation assays. There was an overlapping signal in the innermost retina of all animals where TRPC1 and TRPC5 physically interacted. This interaction increased significantly as photoreceptor loss progressed. Both channels function as TRPC1/5 heteromers in the healthy and damaged retina, with a marked function of TRPC1 in response to retinal degenerative mechanisms. Furthermore, our findings support that TRPC5 channels also function in partnership with STIM1 in Müller and retinal ganglion cells. These results suggest that an increase in TRPC1/5 heteromers may contribute to the slowing of the degeneration of the inner retina during the outer retinal degeneration.

Pore residues of transient receptor potential channels canonical 1 and 4 heteromer determine channel properties.

Transient receptor potential channels canonical 1 and 4 (TRPC1 and TRPC4) are proteins belonging to the same TRPC channel family, and the two are known to form a heterotetrameric channel. TRPC4 can form a homotetrameric, nonselective cation channel by itself, but the involvement of the TRPC1 subunit changes several major characteristics of the channel. In this study, we focused on the pore region (selectivity filter, pore helix, and S6 helix) of TRPC1 and TRPC4 as a determinant of the identity and characteristics of a heteromeric TRPC1/4 channel: decreased calcium permeability of the channel and outward-rectifying current-voltage (I-V) curve. Mutants and chimeras of the pore residues were created, and their currents were recorded using whole cell patch clamp. The lower gate mutants of TRPC4 exhibited diminished calcium permeability as measured by GCaMP6 fluorescence. Also, chimeric channels substituting the pore region of TRPC1 to TRPC4 were made to locate the pore region that is critical in the production of an outward-rectifying I-V curve characteristic of TRPC1/4 heteromeric channels.NEW & NOTEWORTHY Heteromer research has been a challenging field due to lack of structural studies. Using chimeras and single mutants, we present evidence that the pore region of TRPC1/4 heteromer contributes to determining the channel's characteristics such as calcium permeability, I-V curve, and conductance.

Orai1α, but not Orai1ß, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells.

The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1ß, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1ß differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1ß are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α-TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

The Molecular Heterogeneity of Store-Operated Ca2+ Entry in Vascular Endothelial Cells: The Different roles of Orai1 and TRPC1/TRPC4 Channels in the Transition from Ca2+-Selective to Non-Selective Cation Currents.

Store-operated Ca2+ entry (SOCE) is activated in response to the inositol-1,4,5-trisphosphate (InsP3)-dependent depletion of the endoplasmic reticulum (ER) Ca2+ store and represents a ubiquitous mode of Ca2+ influx. In vascular endothelial cells, SOCE regulates a plethora of functions that maintain cardiovascular homeostasis, such as angiogenesis, vascular tone, vascular permeability, platelet aggregation, and monocyte adhesion. The molecular mechanisms responsible for SOCE activation in vascular endothelial cells have engendered a long-lasting controversy. Traditionally, it has been assumed that the endothelial SOCE is mediated by two distinct ion channel signalplexes, i.e., STIM1/Orai1 and STIM1/Transient Receptor Potential Canonical 1(TRPC1)/TRPC4. However, recent evidence has shown that Orai1 can assemble with TRPC1 and TRPC4 to form a non-selective cation channel with intermediate electrophysiological features. Herein, we aim at bringing order to the distinct mechanisms that mediate endothelial SOCE in the vascular tree from multiple species (e.g., human, mouse, rat, and bovine). We propose that three distinct currents can mediate SOCE in vascular endothelial cells: (1) the Ca2+-selective Ca2+-release activated Ca2+ current (ICRAC), which is mediated by STIM1 and Orai1; (2) the store-operated non-selective current (ISOC), which is mediated by STIM1, TRPC1, and TRPC4; and (3) the moderately Ca2+-selective, ICRAC-like current, which is mediated by STIM1, TRPC1, TRPC4, and Orai1.

Differential immunostaining patterns of transient receptor potential (TRP) ion channels in the rat nodose ganglion.

Vagal afferents regulate numerous physiological functions including arterial blood pressure, heart rate, breathing, and nociception. Cell bodies of vagal afferents reside in the inferior vagal (nodose) ganglia and their stimulation by various means is being considered as a way to regulate cardiorespiratory responses and control pain sensations. Stimulation of the nodose by exposure to infrared light is recently being considered as a precise way to elicit responses. These responses would likely involve the activity of temperature-sensitive membrane-bound channels. While papers have been published to track the expression of these transient receptor potential ion channels (TRPs), further studies are warranted to determine the in situ expression of the endogenous TRP proteins in the nodose ganglia to fully understand their pattern of expression, subcellular locations, and functions in this animal model. TRP ion channels are a superfamily of Na+ /Ca2+ -channels whose members are temperature- and/or mechano-sensitive and therefore represent a potential set of proteins that will be activated directly or indirectly by infrared light. Here, we report the spatial localization of six TRP channels, TRPV1, TRPV4, TRPM3, TRPM8, TRPA1, and TRPC1, from nodose ganglia taken from juvenile male Sprague-Dawley rats. The channels were detected using immunohistology with fluorescent tags on cryosections and imaged using confocal microscopy. All six TRP channels were detected with different levels of intensity in neuronal cell bodies and some were also detected in axonal fibers and blood vessels. The TRP receptors differed in their prevalence, in their patterns of expression, and in subcellular expression/localization. More specifically, TRPV1, TRPV4, TRPA1, TRPM8, TRPC1, and TRPM3 were found in vagal afferent cell bodies with a wide range of immunostaining intensity from neuron to neuron. Immunostaining for TRPV1, TRPV4, and TRPA1 appeared as fine particles scattered throughout the cytoplasm of the cell body. Intense TRPV1 immunostaining was also evident in a subset of axonal fibers. TRPM8 and TRPC1 were expressed in courser particles suggesting different subcellular compartments than for TRPV1. The localization of TRPM3 differed markedly from the other TRP channels with an immunostaining pattern that was localized to the periphery of a subset of cell bodies, whereas a scattering or no immunostaining was detected within the bulk of the cytoplasm. TRPV4 and TRPC1 were also expressed on the walls of blood vessels. The finding that all six TRP channels (representing four subfamilies) were present in the nodose ganglia provides the basis for studies designed to understand the roles of these channels in sensory transmission within vagal afferent fibers and in the responses elicited by exposure of nodose ganglia to infrared light and other stimuli. Depending on the location and functionality of the TRP channels, they may regulate the flux of Na+ /Ca2+ -across the membranes of cell bodies and axons of sensory afferents, efferent (motor) fibers coursing through the ganglia, and in vascular smooth muscle.

Structural basis of Klotho binding to VEGFR2 and TRPC1 and repurposing calcium channel blockers as TRPC1 antagonists for the treatment of age-related cardiac hypertrophy.

Cardiac hypertrophy results in the higher rate of heart failures among aged groups. Klotho is an anti-aging protein that is involved in the regulation of VEGF-mediated Ca2+ entry by direct interaction with Vascular endothelial growth factor receptor 2 (VEGFR2) and transient receptor potential canonical Ca2+ channel 1 (TRPC1). Here, in this study, through in silico analysis, we modeled TRPC1 3-dimensional structure and followed by its optimization, characterized the interaction pattern of TRPC1, Klotho and VEGFR2. Subsequent molecular dynamics (MD) simulation analysis revealed that Klotho-specific (P520-N630) region exhibited interaction with VEGFR2, while its C-terminal region (I822-A931) demonstrated binding to the 3rd extracellular loop of TRPC1 that is adjacent to pore region. Through TRPC1 homotetramer formation, the residues in the periphery of pore region were carefully evaluated. In order to scrutinize known Ca2+ channel blockers for their ability to bind at the pore region of TRPC1, 31 known compounds were tested through docking runs and three hits, named as diltiazem impurity B (b3), diltiazem (b5) and felodipine (b6) were selected for detailed binding analysis through MD runs. Evidently, inhibitor-bound TRPC1 pore area was more constricted (8.6 Å2, 25.1 Å2 and 18.8 Å2, respectively) than apo-TRPC1 (60 Å2). These findings suggest that Ca+2 channel blockers may serve as promising agents to impair the TRPC1 functional store-operated calcium channel (SOCC) activity in the old patients lacking Klotho expression. Thus, pore region of homotetrameric TRPC1 may be blocked via repurposing of known Ca+2 blockers to antagonize TRPC signaling for the treatment of cardiac hypertrophy.

TRPC1 contributes to endotoxemia-induced myocardial dysfunction via mediating myocardial apoptosis and autophagy.

Cardiac dysfunction is a vital complication of endotoxemia (ETM) with limited therapeutic options. Transient receptor potential canonical channel (TRPC)1 was involved in various heart diseases. While, the role of TRPC1 in ETM-induced cardiac dysfunction remains to be defined. In this study, we found that TRPC1 protein expression was significantly upregulated in hearts of lipopolysaccharide (LPS)-challenged mice. What's more, TRPC1 knockdown significantly alleviated LPS-induced cardiac dysfunction and injury. Further myocardial mRNA-sequencing analysis revealed that TRPC1 might participate in pathogenesis of ETM-induced cardiac dysfunction via mediating myocardial apoptosis and autophagy. Data showed that knockdown of TRPC1 significantly ameliorated LPS-induced myocardial apoptotic injury, cardiomyocytes autophagosome accumulation, and myocardial autophagic flux. Simultaneously, deletion of TRPC1 reversed LPS-induced molecular changes of apoptosis/autophagy signaling pathway in cardiomyocytes. Moreover, TRPC1 could promote LPS-triggered intracellular Ca2+ release, subsequent calpain activation and caveolin-1 degradation. Either blocking calpain by PD150606 or enhancing the amount of caveolin-1 scaffolding domain that interacts with TRPC1 by cell-permeable peptide cavtratin significantly alleviated the LPS-induced cardiac dysfunction and cardiomyocytes apoptosis/autophagy. Furthermore, cavtratin could inhibit LPS-induced calpain activation in cardiomyocytes. caveolin-1 could directly interact with calpain 2 both in vivo and in vitro. Importantly, cecal ligation and puncture-stimulated cardiac dysfunction and mortality were significantly alleviated in Trpc1-/- and cavtratin-treated mice, which further validated the contribution of TRPC1-caveolin-1 signaling axis in sepsis-induced pathological process. Overall, this study indicated that TRPC1 could promote LPS-triggered intracellular Ca2+ release, mediate caveolin-1 reduction, and in turn activates calpain to regulate myocardial apoptosis and autophagy, contributing to ETM-induced cardiac dysfunction of mice.

STIM1 promotes IPEC-J2 porcine epithelial cell restitution by TRPC1 signaling.

Intestinal epithelial restitution is partly dependent on cell migration, which reseals superficial wounding after injury. Here, we tested the hypothesis that stromal interaction molecule 1(STIM1) regulates porcine intestinal epithelial cell migration by activating transient receptor potential canonical 1 (TRPC1) signaling. Results showed that the knockdown of STIM1 repressed cell migration after wounding, reduced the protein concentration of STIM1 and TRPC1, and decreased the inositol trisphosphate (IP3) content in IPEC-J2 cells (p < 0.05). However, overexpression of STIM1 obtained opposite results (p < 0.05). The inhibition of TRPC1 activity by treatment with SKF96365 in cells overexpressing wild-type and mutant STIM1 attenuated the STIM1 overexpression-induced increase of cell migration, STIM1, TRPC1 and IP3 (p < 0.05). In addition, polyamine depletion caused by α-difluoromethylornithine (DFMO) resulted in the decrease of above-mentioned parameters, and exogenous polyamine could attenuate the negative effects of DFMO on IPEC-J2 cells (p < 0.05). Moreover, the overexpression of STIM1 could rescue cell migration, the protein level of STIM1 and TRPC1, and IP3 content in polyamine-deficient IPEC-J2 cells (p < 0.05). These results indicated that STIM1 could enhance porcine intestinal epithelial cell migration via the TRPC1 signaling pathway. Inhibition of cell migration by polyamine depletion resulted from the reduction of STIM1 activity.

The TRPC1 Channel Forms a PI3K/CaM Complex and Regulates Pancreatic Ductal Adenocarcinoma Cell Proliferation in a Ca2+-Independent Manner.

Dysregulation of the transient receptor canonical ion channel (TRPC1) has been found in several cancer types, yet the underlying molecular mechanisms through which TRPC1 impacts pancreatic ductal adenocarcinoma (PDAC) cell proliferation are incompletely understood. Here, we found that TRPC1 is upregulated in human PDAC tissue compared to adjacent pancreatic tissue and this higher expression correlates with low overall survival. TRPC1 is, as well, upregulated in the aggressive PDAC cell line PANC-1, compared to a duct-like cell line, and its knockdown (KD) reduced cell proliferation along with PANC-1 3D spheroid growth by arresting cells in the G1/S phase whilst decreasing cyclin A, CDK2, CDK6, and increasing p21CIP1 expression. In addition, the KD of TRPC1 neither affected Ca2+ influx nor store-operated Ca2+ entry (SOCE) and reduced cell proliferation independently of extracellular calcium. Interestingly, TRPC1 interacted with the PI3K-p85α subunit and calmodulin (CaM); both the CaM protein level and AKT phosphorylation were reduced upon TRPC1 KD. In conclusion, our results show that TRPC1 regulates PDAC cell proliferation and cell cycle progression by interacting with PI3K-p85α and CaM through a Ca2+-independent pathway.

Similarities and Differences between the Orai1 Variants: Orai1α and Orai1ß.

Orai1, the first identified member of the Orai protein family, is ubiquitously expressed in the animal kingdom. Orai1 was initially characterized as the channel responsible for the store-operated calcium entry (SOCE), a major mechanism that allows cytosolic calcium concentration increments upon receptor-mediated IP3 generation, which results in intracellular Ca2+ store depletion. Furthermore, current evidence supports that abnormal Orai1 expression or function underlies several disorders. Orai1 is, together with STIM1, the key element of SOCE, conducting the Ca2+ release-activated Ca2+ (CRAC) current and, in association with TRPC1, the store-operated Ca2+ (SOC) current. Additionally, Orai1 is involved in non-capacitative pathways, as the arachidonate-regulated or LTC4-regulated Ca2+ channel (ARC/LRC), store-independent Ca2+ influx activated by the secretory pathway Ca2+-ATPase (SPCA2) and the small conductance Ca2+-activated K+ channel 3 (SK3). Furthermore, Orai1 possesses two variants, Orai1α and Orai1ß, the latter lacking 63 amino acids in the N-terminus as compared to the full-length Orai1α form, which confers distinct features to each variant. Here, we review the current knowledge about the differences between Orai1α and Orai1ß, the implications of the Ca2+ signals triggered by each variant, and their downstream modulatory effect within the cell.

Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca2+ into Neurons.

The neural cell adhesion molecule (NCAM) plays important functional roles in the developing and mature nervous systems. Here, we show that the transient receptor potential canonical (TRPC) ion channels TRPC1, -4, and -5 not only interact with the intracellular domains of the transmembrane isoforms NCAM140 and NCAM180, but also with the glycan polysialic acid (PSA) covalently attached to the NCAM protein backbone. NCAM antibody treatment leads to the opening of TRPC1, -4, and -5 hetero- or homomers at the plasma membrane and to the influx of Ca2+ into cultured cortical neurons and CHO cells expressing NCAM, PSA, and TRPC1 and -4 or TRPC1 and -5. NCAM-stimulated Ca2+ entry was blocked by the TRPC inhibitor Pico145 or the bacterial PSA homolog colominic acid. NCAM-stimulated Ca2+ influx was detectable neither in NCAM-deficient cortical neurons nor in TRPC1/4- or TRPC1/5-expressing CHO cells that express NCAM, but not PSA. NCAM-induced neurite outgrowth was reduced by TRPC inhibitors and a function-blocking TRPC1 antibody. A characteristic signaling feature was that extracellular signal-regulated kinase 1/2 phosphorylation was also reduced by TRPC inhibitors. Our findings indicate that the interaction of NCAM with TRPC1, -4, and -5 contributes to the NCAM-stimulated and PSA-dependent Ca2+ entry into neurons thereby influencing essential neural functions.

Heteromeric channels formed by TRPC1, TRPC4 and TRPC5 define hippocampal synaptic transmission and working memory.

Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high-resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from Trpc1/Trpc4/Trpc5-triple-knockout (Trpc1/4/5-/-) mice, lacking any TRPC1-, TRPC4-, or TRPC5-containing channels, action potential-triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. In vivo, Trpc1/4/5-/- mice displayed impaired cross-frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. Trpc1/4/5-/- animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.

Ca2+ entry via TRPC1 is essential for cellular differentiation and modulates secretion via the SNARE complex.

Properties of adipocytes, including differentiation and adipokine secretion, are crucial factors in obesity-associated metabolic syndrome. Here, we provide evidence that Ca2+ influx in primary adipocytes, especially upon Ca2+ store depletion, plays an important role in adipocyte differentiation, functionality and subsequently metabolic regulation. The endogenous Ca2+ entry channel in both subcutaneous and visceral adipocytes was found to be dependent on TRPC1-STIM1, and blocking Ca2+ entry with SKF96365 or using TRPC1-/- knockdown adipocytes inhibited adipocyte differentiation. Additionally, TRPC1-/- mice have decreased organ weight, but increased adipose deposition and reduced serum adiponectin and leptin concentrations, without affecting total adipokine expression. Mechanistically, TRPC1-mediated Ca2+ entry regulated SNARE complex formation, and agonist-mediated secretion of adipokine-loaded vesicles was inhibited in TRPC1-/- adipose. These results suggest an unequivocal role of TRPC1 in adipocyte differentiation and adiponectin secretion, and that loss of TRPC1 disturbs metabolic homeostasis.This article has an associated First Person interview with the first author of the paper.

Loss of Ca2+ entry via Orai-TRPC1 induces ER stress, initiating immune activation in macrophages.

Activation of cellular stresses is associated with inflammation; however, the mechanisms are not well identified. Here, we provide evidence that loss of Ca2+ influx induces endoplasmic reticulum (ER) stress in primary macrophages and in murine macrophage cell line Raw 264.7, in which the unfolded protein response is initiated to modulate cytokine production, thereby activating the immune response. Stressors that initiate the ER stress response block store-dependent Ca2+ entry in macrophages prior to the activation of the unfolded protein response. The endogenous Ca2+ entry channel is dependent on the Orai1-TRPC1-STIM1 complex, and the presence of ER stressors decreased expression of TRPC1, Orai1 and STIM1. Additionally, blocking Ca2+ entry with SKF96365 also induced ER stress, promoted cytokine production, activation of autophagy, increased caspase activation and induced apoptosis. Furthermore, ER stress inducers inhibited cell cycle progression, promoted the inflammatory M1 phenotype, and increased phagocytosis. Mechanistically, restoration of Orai1-STIM1 expression inhibited the ER stress-mediated loss of Ca2+ entry that prevents ER stress and inhibits cytokine production, and thus induced cell survival. These results suggest an unequivocal role of Ca2+ entry in modulating ER stress and in the induction of inflammation.

STIM1-dependent membrane insertion of heteromeric TRPC1-TRPC4 channels in response to muscarinic receptor stimulation.

Muscarinic receptor stimulation results in activation of nonselective cation (NSC) channels in guinea pig adrenal medullary (AM) cells. The biophysical and pharmacological properties of the NSC channel suggest the involvement of heteromeric channels of TRPC1 with TRPC4 or TRPC5. This possibility was explored in PC12 cells and guinea pig AM cells. Proximity ligation assay (PLA) revealed that when exogenously expressed in PC12 cells, TRPC1 forms a heteromeric channel with TRPC4, but not with TRPC5, in a STIM1-dependent manner. The heteromeric TRPC1-TRPC4 channel was also observed in AM cells and trafficked to the cell periphery in response to muscarine stimulation. To explore whether heteromeric channels are inserted into the cell membrane, tags were attached to the extracellular domains of TRPC1 and TRPC4. PLA products developed between the tags in cells stimulated by muscarine, but not in resting cells, indicating that muscarinic stimulation results in the membrane insertion of channels. This membrane insertion required expression of full-length STIM1. We conclude that muscarinic receptor stimulation results in the insertion of heteromeric TRPC1-TRPC4 channels into the cell membrane in PC12 cells and guinea pig AM cells.

TRPC3, but not TRPC1, as a good therapeutic target for standalone or complementary treatment of DMD.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked inherited disease caused by mutations in the gene encoding dystrophin that leads to a severe and ultimately life limiting muscle-wasting condition. Recombinant adeno-associated vector (rAAV)-based gene therapy is promising, but the size of the full-length dystrophin cDNA exceeds the packaging capacity of a rAAV. Alternative or complementary strategies that could treat DMD patients are thus needed. Intracellular calcium overload due to a sarcolemma permeability to calcium (SPCa) increase is an early and critical step of the DMD pathogenesis. We assessed herein whether TRPC1 and TRPC3 calcium channels may be involved in skeletal muscle SPCa alterations and could represent therapeutic targets to treat DMD. METHODS: All experiments were conducted in the DMDmdx rat, an animal model that closely reproduces the human DMD disease. We measured the cytosolic calcium concentration ([Ca2+]c) and SPCa in EDL (Extensor Digitorum Longus) muscle fibers from age-matched WT and DMDmdx rats of 1.5 to 7 months old. TRPC1 and TRPC3 expressions were measured in the EDL muscles at both the mRNA and protein levels, by RT-qPCR, western blot and immunocytofluorescence analysis. RESULTS: As expected from the malignant hyperthermia like episodes observed in several DMDmdx rats, calcium homeostasis alterations were confirmed by measurements of early increases in [Ca2+]c and SPCa in muscle fibers. TRPC3 and TRPC1 protein levels were increased in DMDmdx rats. This was observed as soon as 1.5 months of age for TRPC3 but only at 7 months of age for TRPC1. A slight but reliable shift of the TRPC3 apparent molecular weight was observed in DMDmdx rat muscles. Intracellular localization of both channels was not altered. We thus focused our attention on TRPC3. Application of Pyr10, a specific inhibitor of TRPC3, abolished the differences between SPCa values measured in WT and DMDmdx. Finally, we showed that a rAAV-microdystrophin based treatment induced a high microdystrophin expression but only partial prevention of calcium homeostasis alterations, skeletal muscle force and TRPC3 protein increase. CONCLUSIONS: All together our results show that correcting TRPC3 channel expression and/or activity appear to be a promising approach as a single or as a rAAV-based complementary therapy to treat DMD.

Sigma1 Receptor Inhibits TRPC1-Mediated Ca2+ Entry That Promotes Dopaminergic Cell Death.

Regulation of Ca2+ homeostasis is essential for neuronal function and its survival. Recent data suggest that TRPC1 function as the endogenous store-mediated Ca2+ entry channel in dopaminergic cells, and loss of TRPC1 function leads to neurodegeneration; however, its regulation is not fully identified. Here we provide evidence that the sigma 1 receptor contributes to the loss of dopaminergic cells by blocking TRPC1-mediated Ca2+ entry. Importantly, downregulation of sigma 1 receptor expression significantly decreased neurotoxin-induced loss of dopaminergic cells as measured by MTT assays and caspase activity was also inhibited. Importantly, sigma 1 receptor inhibited TRPC1-mediated Ca2+ entry and silencing of sigma 1 receptor significantly restored store-dependent Ca2+ influx. Although co-immunoprecipitation failed to show an interaction between the TRPC1 and sigma 1 receptor, store depletion promoted a decrease in the sigma 1 receptor-STIM1 association. Neurotoxin-induced loss of Ca2+ entry was significantly restored in cells that had decreased sigma 1 receptor expression. Furthermore, TRPC1 or STIM1 silencing inhibited store-mediated Ca2+ entry, which was further increased upon the downregulation of the sigma 1 receptor expression. TRPC1 silencing prevented the increased neuroprotection and caspase activity observed upon the downregulation of sigma 1 receptor. Finally, sigma 1 receptor activation also significantly decreased TRPC1-mediated Ca2+ entry and lead to an increase in neurodegeneration. In contrast, addition of sigma 1 receptor antagonist prevented neurotoxin-induced neurodegeneration and facilitated TRPC1-mediated Ca2+ influx. Together these results suggest that the sigma 1 receptor is involved in the inhibition of TRPC1- mediated Ca2+ entry, which leads to the degeneration in the dopaminergic cells, and prevention of sigma 1 receptor function could protect neuronal cell death as observed in Parkinson's disease.

TRPC1/4/5 channels contribute to morphine-induced analgesic tolerance and hyperalgesia by enhancing spinal synaptic potentiation and structural plasticity.

Opioid analgesics remain the mainstay for managing intractable chronic pain, but their use is limited by detrimental side effects such as analgesic tolerance and hyperalgesia. Calcium-dependent synaptic plasticity is a key determinant in opiates tolerance and hyperalgesia. However, the exact substrates for this calcium-dependent synaptic plasticity in mediating these maladaptive processes are largely unknown. Canonical transient receptor potential 1, 4, and 5 (TRPC1, 4, 5) proteins assemble into heteromultimeric nonselective cation channels with high Ca2+ permeability and influence various neuronal functions. However, whether and how TRPC1/4/5 channels contribute to the development of opiates tolerance and hyperalgesia remains elusive. Here, we show that TRPC1/4/5 channels contribute to the generation of morphine tolerance and hyperalgesia. Chronic morphine exposure leads to upregulation of TRPC1/4/5 channels in the spinal cord. Spinally expressed TRPC1, TPRC4, and TRPC5 are required for chronic morphine-induced synaptic long-term potentiation (LTP) as well as remodeling of synaptic spines in the dorsal horn, thereby orchestrating functional and structural plasticity during the course of morphine-induced hyperalgesia and tolerance. These effects are attributed to TRPC1/4/5-mediated Ca2+ elevation in the spinal dorsal horn induced by chronic morphine treatment. This study identifies TRPC1/4/5 channels as a promising novel target to prevent the unwanted morphine tolerance and hyperalgesia.