Inhibition of TRPC4/5 Channel Is Effective in Protecting against Histamine-Induced Hyperpermeability by Blocking Ca2+ Influx in Lung Microvascular Endothelial Cells.

Dysfunction of lung microvascular endothelium is a major feature in the pathobiology of pulmonary edema and hypoxic respiratory failure. Histamine induces lung microvascular endothelial barrier disruption and hyperpermeability upon evoking intracellular Ca2+ ([Ca2+]i) dynamics via binding to its receptors. Transient receptor potential canonical (TRPC) channels are Ca2+-permeable channel and stimulated by the agonists of G-protein-coupled receptors (GPCR). Here, we assessed histamine induced [Ca2+]i increases in human lung microvascular endothelial cells (HLMVEC) by using live cell Ca2+ imaging. We found that histamine increased [Ca2+]i was maintained at a static elevated level after a transient peak. The elevated Ca2+ plateau was vanished when extracellular Ca2+ was removed, indicating Ca2+ influx from extracellular mediated the histamine-induced Ca2+ plateau. TRPC4/5 channels antagonists, ML204 (10 µM) and HC070 (1 µM), significantly inhibited the Ca2+ plateaus, which was not influenced by Pyr3 or larixyl, the antagonists of TRPC3/6. Furthermore, ML204 or HC070 effectively suppressed the permeability response to histamine in HLMVEC. Our results indicated that histamine-induced Ca2+ influx may be mediated by TRPC4/5 channels and the antagonist of the channel significantly improved histamine-induced HLMVEC dysfunction.

Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation.

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca2+ homeostasis, and whose functions are modulated by G protein-coupled receptors (GPCR). In this study, we developed bioluminescent resonance energy transfer (BRET) biosensors to better study channel conformational changes following receptor activation. For this study, two intramolecular biosensors, GFP10-TRPC7-RLucII and RLucII-TRPC7-GFP10, were constructed and were assessed following the activation of various GPCRs. We first transiently expressed receptors and the biosensors in HEK293 cells, and BRET levels were measured following agonist stimulation of GPCRs. The activation of GPCRs that engage Gαq led to a Gαq-dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (AT1R), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous AT1R and TRPC7. We detected similar BRET responses in these cells, thus validating the use of the biosensor in physiological conditions. Taken together, our results suggest that activation of Gαq-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.

TRPC1 as a negative regulator for TRPC4 and TRPC5 channels.

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.

The effects of transient receptor potential channel (TRPC) on airway smooth muscle cell isolated from asthma model mice.

This study aimed to validate whether transient receptor potential channel1 (TRPC1) and TRPC3 participate in the regulation the proliferation of airway smooth muscle cells (ASMCs) through modulating calcium ion (Ca2+ ) influx in vitro. Chronic model of murine asthma was induced and ASMCs isolated from asthmatic mice were used in this whole study. TRPC1 and TRPC3 were upregulated in asthmatic mouse ASMCs and selected for further investigation. Ca2+ concentration and the cell viability of asthmatic mouse ASMCs were significantly higher than that from non- asthma mice, however, TRPC channels blocker SKF96365 alleviated these effects. Furthermore, TRPC1 or TRPC3 overexpression markedly increased Ca2+ concentration and significantly induced the viability of ASMCs; whereas TRPC1 or TRPC3 knockdown exerted the completely conversed effects. Moreover, knockdown of TRPC1 and TRPC3 also exerted different effects on the protein expression of growth-related proteins p-p38, p-JNK, cleaved caspase-3 and Bcl-2, as well as on cell cycle. Finally, we found Ca2+ chelator EGTA or BAPTA-AM significantly diminished the effects of si-TRPC1 and si-TRPC3 on the cell viability, cell cycle, and the protein expression of p-p38, p-JNK, cleaved caspase-3, and Bcl-2 in asthmatic mouse ASMCs. Our findings demonstrated that the effects of TRPC1 and TRPC3 on the cell viability and cell cycle of ASMCs were, at least partially, through regulating Ca2+ influx.

The effect of the electrophilic fatty acid nitro-oleic acid on TRP channel function in sensory neurons.

Nitro-oleic acid (NO2-OA) and related nitroalkenes are electrophilic fatty acid derivatives that are present in normal tissues at nanomolar concentrations and can increase significantly during inflammation. These substances can suppress multiple intracellular signaling pathways contributing to inflammation by reversible Michael addition reactions with nucleophilic residues such as cysteine and histidine leading to post-translational modification of proteins. NO2-OA also can influence inflammation and pain by acting on transient receptor potential (TRP) channels in primary sensory neurons. TRPV1, TRPA1 and TRPC can respond to electrophilic fatty acids because they have ankyrin-like repeats in their N terminus that are rich in cysteine residues that react with electrophiles and other thiol modifying species. NO2-OA acts on TRP channels to initially depolarize and induce firing in sensory neurons followed by desensitization and suppression of firing. In vivo experiments revealed that pretreatment with NO2-OA reduces nociceptive behavior evoked by local administration of a TRPA1 agonist (AITC) to the rat hind paw. These results raise the possibility that NO2-OA might be useful clinically to reduce neurogenic inflammation and certain types of painful sensations by desensitizing TRPA1 expressing nociceptive afferents.

Proarrhythmic effect of sustained EPAC activation on TRPC3/4 in rat ventricular cardiomyocytes.

The Exchange Protein directly Activated by cAMP (EPAC) participates to the pathological signaling of cardiac hypertrophy and heart failure, in which the role of Ca(2+) entry through the Transient Receptor Potential Canonical (TRPC) channels begin to be appreciated. Here we studied whether EPAC activation could influence the activity and/or expression of TRPC channels in cardiac myocytes. In adult rat ventricular myocytes treated for 4 to 6h with the selective EPAC activator, 8-pCPT (10µM), we observed by Fluo-3 confocal fluorescence a Store-Operated Ca(2+) Entry (SOCE) like-activity, which was blunted by co-incubation with EPAC inhibitors (ESI-05 and CE3F4 at 10 µM). This SOCE-like activity, which was very small in control incubated cells, was sensitive to 30-µM SKF-96365. Molecular screening showed a specific upregulation of TRPC3 and C4 protein isoforms after 8-pCPT treatment. Moreover, sustained EPAC activation favored proarrhythmic Ca(2+) waves, which were reduced either by co-incubation with EPAC inhibitors or bath perfusion with TRPC inhibitors. Our study provides the first evidence that sustained selective EPAC activation leads to an increase in TRPC3 and C4 protein expression and induces a proarrhythmic SOCE-like activity in adult rat ventricular cardiomyocytes, which might be of importance during the development of cardiac diseases.

Structural and accessibility studies highlight the differential binding of clemizole to TRPC5 and TRPC6.

Transient Receptor Potential Canonical 5 (T RP C5) and T RP C6 channels play critical physiological roles in various cell types. Their involvement in numerous disease progression mechanisms has led to extensive searches for their inhibitors. Although several potent T RP C inhibitors have been developed and the structure of their binding sites were mapped using cryo electron microscopy, a comprehensive understanding of the molecular interactions within the inhibitor binding site of T RP Cs remains elusive. This study aimed to decipher the structural determinants and molecular mechanisms contributing to the differential binding of clemizole to T RP C5 and T RP C6, with a particular focus on the accessibility of binding site residues. This information can help better understand what molecular features allow for selective binding, which is a key characteristic of clinically effective pharmacological agents. Using computational methodologies, we conducted an in-depth molecular docking analysis of clemizole with T RP C5 and T RP C6 channels. The protein structures were retrieved from publicly accessible protein databases. Discovery Studio 2020 Client Visualizer and Chimera software facilitated our in-silico mutation experiments and enabled us to identify the critical structural elements influencing clemizole binding. Our study reveals key molecular determinants at the clemizole binding site, specifically outlining the role of residues' Accessible Surface Area (ASA) and Relative Accessible Surface Area (RASA) in differential binding. We found that lower accessibility of T RP C6 binding site residues, compared to those in T RP C5, could account for the lower affinity binding of clemizole to T RP C6. This work illuminates the pivotal role of binding site residue accessibility in determining the affinity of clemizole to T RP C5 and T RP C6. A nuanced understanding of the distinct binding properties between these homologous proteins may pave the way for the development of more selective inhibitors, promising improved therapeutic efficacy and fewer off-target effects. By demystifying the structural and molecular subtleties of T RP C inhibitors, this research could significantly accelerate the drug discovery process, offering hope to patients afflicted with T RP C-related diseases.

Store-Operated Calcium Entry Increases Nuclear Calcium in Adult Rat Atrial and Ventricular Cardiomyocytes.

Store-operated calcium entry (SOCE) in cardiomyocytes may be involved in cardiac remodeling, but the underlying mechanisms remain elusive. We hypothesized that SOCE may increase nuclear calcium, which alters gene expression via calcium/calmodulin-dependent enzyme signaling, and elucidated the underlying cellular mechanisms. An experimental protocol was established in isolated adult rat cardiomyocytes to elicit SOCE by re-addition of calcium following complete depletion of sarcoplasmic reticulum (SR) calcium and to quantify SOCE in relation to the electrically stimulated calcium transient (CaT) measured in the same cell before SR depletion. Using confocal imaging, calcium changes were recorded simultaneously in the cytosol and in the nucleus of the cell. In ventricular myocytes, SOCE was observed in the cytosol and nucleus amounting to ≈15% and ≈25% of the respective CaT. There was a linear correlation between the SOCE-mediated calcium increase in the cytosol and nucleus. Inhibitors of TRPC or Orai channels reduced SOCE by ≈33-67%, whereas detubulation did not. In atrial myocytes, SOCE with similar characteristics was observed in the cytosol and nucleus. However, the SOCE amplitudes in atrial myocytes were ≈two-fold larger than in ventricular myocytes, and this was associated with ≈1.4- to 3.6-fold larger expression of putative SOCE proteins (TRPC1, 3, 6, and STIM1) in atrial tissue. The results indicated that SOCE in atrial and ventricular myocytes is able to cause robust calcium increases in the nucleus and that both TRPC and Orai channels may contribute to SOCE in adult cardiomyocytes.

Roles of transient receptor potential canonical (TRPC) channels and reverse-mode Na+/Ca2+ exchanger on cell proliferation in human cardiac fibroblasts: effects of transforming growth factor ß1.

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-ß1 (TGF-ß1) on Ca2+ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca2+ concentration [Ca2+]i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca(2+)-mediated [Ca2+]i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca2+ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca2+ entry. KB-R7943, a reverse-mode Na+/Ca2+ exchanger (NCX) inhibitor, and/or replacement of Na+ with NMDG+ inhibited thapsigargin-, OAG- and Ang II-induced Ca2+ entry. Treatment with TGF-ß1 increased thapsigargin-, OAG- and Ang II-induced Ca2+ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-ß1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca2+ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca2+ influx pathways, which activate the reverse-mode NCX. TGF-ß1 enhances the Ca2+ influx pathways requiring Ca2+ signals for its effect on fibroblast proliferation.