Canonical Transient Receptor Potential Research Articles

#4317 CLINICAL DIVERSITY OF STEROID-RESISTANT NEPHROTIC SYNDROME CAUSED BY TRPC6 MUTATIONS

Abstract Background and Aims Steroid-resistant nephrotic syndrome (SRNS) is a clinically and genetically heterogeneous disorder caused by either genetic or immunological factors or their combination. Approximately 30–40% of patients with SRNS make a fast progression to ESRD. SRNS represents the second leading cause of end-stage renal disease in individuals under the age of 25 years worldwide. More than 60 podocyte-related gene mutations have thus far been reported in monogenic SRNS. Recent studies indicate that almost 30% of patients with childhood-onset SRNS have monogenic causative variants in one of the 27 SRNS genes. Among these, a mutation in Transient Receptor Potential Canonical 6 (TRPC6) gene, encoding non-selective cation channel, accounts for 6% of familial SRNS and approximately 2% of sporadic cases. We recently detected eight patients with TRPC6 mutations in our SRNS cohort (n = 39, 21%) and found inter- and intra-familial heterogeneity of clinical phenotypes in terms of disease onset and progression. Method A total of 39 Japanese SRNS patients were studied according to the protocol approved by the institutional review board of each affiliation. Subjects who manifested SRNS at age one to 14-year-old with biopsy-proven FSGS were enrolled. A total of 39 SRNS patients, whose average onset of proteinuria at median age 4 (range 0.9 to 17 yr) and ESRD at median age 7 (49% of total n = 19, range 3.0 to 16.0 yr) were studied. Family history was found in 44% (n = 17), including 13 autosomal recessive, 3 dominant, and one X-linked transmission. Sixteen percent (n = 10) of the patients who underwent renal transplantation win no recurrence. Sequence analysis revealed the pathogenic variants in 62% (n = 24) of all cases. Library for whole-exome sequencing or targeted panel sequencing were prepared from genomic DNA by use of SureSelect Human All Exon V5 (Agilent) or a HaloPlex target enrichment system kit (Agilent Technologies), or Ampli-Seq (Thermo), Prepared samples were run on a HiSeq 2000, or MiSeq (Illumina), or Ion PGM (Thermo). The sequence reads with 101-bp paired-end reads and 7-bp index reads were mapped to the human reference sequence (hg19, GRCh38.p7). After filtering the superfluous begin variants, only variants that fulfill the “pathogenic” criteria of ACMG and/or ClinVar, Varsome were selected. Results Sequence analysis revealed the pathogenic variants in 62% (n = 24) of all cases. TRPC6 mutations were the most frequent cause of SRNS (n = 8), which was followed by NUP107 (n = 5), PLCE1 (n = 3), COL4A3 (n = 2), COL4A5 (n = 1), and others were identified in only single case. The eight TRPC6 mutations clustered into the two distinctive cytoplasmic domains; N-terminal ANK (Y173D, R175W, R175G) and C-terminal coiled-coil domain (p.E875V, p.867_868Del, p.S893N, p.R895C). The p.R895C is deposited as the pathogenic in ClinVar and may be a gain-of-function. The remaining 7 variants are of unknown functions: six have not yet been deposited in ClinVar, whereas one is VUS. Four patients were de novo occurrence, three followed the dominant transmission, and one arose from gonadal mosaicism. Two index patients exhibited SRNS in early childhood (< age 3), four manifested it in childhood (age 3–12), while the remaining two developed it in adolescence (age >12). In familial cases, for example, affected ones harboring R175W or p.E875V showed a remarkable discordant age onset and disease severity. Conclusion TRPC6 channelopathy was found to be the most frequent cause in our SRNS cohort, of which age onset ranges from age 0.9 to 17 year. Heterogeneity of disease severity may reflect that channel activity is regulated by the mode of action (gain- or loss-of-function), the combination of channel subunit assembly, and interaction with other modifying factors. Mutations in these TRPCs are associated with relatively common kidney diseases as well as other pathologies. Further expression studies will improve our mechanistic understanding of TRP channel pathology and may help the development of novel therapeutic targets.

A multi-stimuli-responsive nanochannel inspired by biological disulfide bond

Disulfide bonds exist widely in channel protein and play an essential role in matter exchange and signal transduction (e.g., rhodopsin, canonical transient receptor potential 5 (TRPC5)). The research on disulfide bond in nanochannel is significant for the cognition of their biological functions. However, the fragility of biological channel limits the in-situ study and practical application. Herein, an innovative biologically-inspired artificial nanochannel based on disulfide bond (NCDS) with excellent durability, adjustable surface property is proposed. The constructed NCDS has a multi-response to UV-light, thiol (e.g., cysteine (Cys)) or pH stimulation, and can obtain reversibility after regulation by hydrogen peroxide (H2O2) or H+. The biomimetic NCDS shows great potential in biosensor and intelligent response design. This study also shines new light to channel protein based on disulfide bond that despite the nanochannel has specificity, it will be modulated by the change of nature environment, such as UV-light and chemical microenvironment (e.g., redox state and pH), which might be the reason of some disease.

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.

Diacerein modulates TLR4/ NF-κB/IL-1β and TRPC1/CHOP signalling pathways in gentamicin-induced parotid toxicity in rats.

The present study aimed to identify the possible protective effect of diacerein (DIA) on gentamicin (GNT)-induced parotid toxicity in rats. DIA was administered in the presence and absence of GNT. Thirty-two Wistar adult male rats were randomly arranged into four groups: control, DIA (50 mg/kg/day), GNT (100 mg/kg) and GNT+DIA groups for 8 days. Parotid oxidative stress parameters, besides inflammatory and apoptotic biomarkers, were evaluated. Salivary flow rate, transient receptor potential canonical 1 (TRCP1), and C/EBP homologous protein (CHOP) in parotid tissue were measured. A parotid histopathological examination and an interleukin-1 beta (IL-1β) immunohistochemical study were also performed. GNT significantly increased parotid oxidative stress, inflammatory, apoptotic and CHOP biomarkers with decreased salivary flow rate and TRCP1 level. A histopathological picture of parotid damage and high IL-1β immunoexpression were detected. DIA significantly normalized the distributed oxidative, inflammatory and apoptotic indicators, CHOP and TRCP1, with a prompt improvement in the histopathological picture and a decrease in IL-1β immunoexpression. These results reported that DIA protects against GNT-induced parotid toxicity via modulation of TLR4/NF-κB/IL-1β and TRPC1/CHOP signalling pathways.

Molecular architecture of the Gαi-bound TRPC5 ion channel

G-protein coupled receptors (GPCRs) and ion channels serve as key molecular switches through which extracellular stimuli are transformed into intracellular effects, and it has long been postulated that ion channels are direct effector molecules of the alpha subunit of G-proteins (Gα). However, no complete structural evidence supporting the direct interaction between Gα and ion channels is available. Here, we present the cryo-electron microscopy structures of the human transient receptor potential canonical 5 (TRPC5)-Gαi3 complexes with a 4:4 stoichiometry in lipid nanodiscs. Remarkably, Gαi3 binds to the ankyrin repeat edge of TRPC5 ~ 50 Å away from the cell membrane. Electrophysiological analysis shows that Gαi3 increases the sensitivity of TRPC5 to phosphatidylinositol 4,5-bisphosphate (PIP2), thereby rendering TRPC5 more easily opened in the cell membrane, where the concentration of PIP2 is physiologically regulated. Our results demonstrate that ion channels are one of the direct effector molecules of Gα proteins triggered by GPCR activation–providing a structural framework for unraveling the crosstalk between two major classes of transmembrane proteins: GPCRs and ion channels.

Myoglobin-driven ET-1 production and downstream activation of TRPC3- but not TRPC6-dependent vasoconstriction contributes to rhabdomyolysis-induced AKI

Acute kidney injury (AKI) is the most severe and life-threatening complication of rhabdomyolysis, with 10-40% of patients developing some form of kidney insufficiency within days. Experimental evidence indicates that renal vasoconstriction plays a significant role in the pathophysiology of rhabdomyolysis-induced AKI. Rodents subjected to glycerol-induced rhabdomyolysis exhibited increased plasma concentrations of endothelin-1 (ET-1). Treating glomerular mesangial cells with myoglobin has also been shown to increase the cellular production of ET-1. The downstream effectors of ET-1-induced vasoconstriction are smooth muscle Ca2+ permeable transient receptor potential canonical (TRPC) channels. There are seven members of the TRPC gene family (TRPC1-7). Whereas TRPC2 and 7 are absent, TRPC1, 3, 4, 5, and 6 are expressed in renal vessels, with TRPC3 predominant. TRPC3, TRPC6, and TRPC7 are highly homologous. Although the ET system is known to be associated with rhabdomyolysis-induced AKI, the function of renal vascular ion channels in the disease is unclear. Here, we investigated the involvement of TRPC3 and TRPC6 in glycerol-induced rhabdomyolysis in rats. ET-1 production is increased in myoglobin-treated epithelial cells, the primary source of renal ET. Rhabdomyolysis also increased renal ET-1 production, decreased renal blood flow (RBF), and increased renal vascular resistance, effects attenuated by ET receptor and TRPC channel blocker Pyr3. Furthermore, we used TRPC3 and TRPC6 knockout (KO) rats to support the pharmacological approaches. Basal day and night arterial pressure, heart rate, GFR, plasma creatinine, and BUN were unchanged in WT vs. KO rats. Twenty-four h rhabdomyolysis led to comparable increases in urinary ET-1 production in WT and KO rats. However, rhabdomyolysis-induced decreases in GFR, elevations in BUN, and plasma creatinine were all attenuated in the TRPC3 but not TRPC6 KO rats. Similarly, morphological kidney damage (tubular necrosis, casts, and dilatation) was mitigated in the TRPC3 KO with no protection offered by the KO of TRPC6 channels. Together, our data suggest that myoglobin-driven ET-1 production and downstream activation of TRPC3- but not TRPC6-dependent vasoconstriction contributes to rhabdomyolysis-induced AKI. American Heart Association Predoctoral Fellowship This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

P38 MAPK activation and STIM1-Orai3 association mediate TRPC6 externalization.

Endothelial cell (EC) migration is critical for the repair of monolayer disruption following angioplasties, but migration is inhibited by lipid oxidation products, including lysophosphatidylcholine (lysoPC), which open canonical transient receptor potential 6 (TRPC6) channels. TRPC6 activation requires an increase in intracellular Ca2+ concentration ([Ca2+]i), the source of which is unknown. LysoPC can activate phospholipase A2 to release arachidonic acid (ArA). ArA can activate arachidonic acid-regulated calcium (ARC) channels that are formed by stromal interaction molecule 1 (STIM1) and Orai1 and Orai3 proteins. Both lysoPC and ArA can activate p38 mitogen-activated protein kinase (MAPK) that induces the phosphorylation required for STIM1-Orai3 association. This is accompanied by an increase in [Ca2+]i and TRPC6 externalization. The effect of lysoPC and ArA is not additive, suggesting activation of the same pathway. The increase in [Ca2+]i activates an Src kinase that leads to TRPC6 activation. Downregulation of Orai3 using siRNA blocks the lysoPC- or ArA-induced increase in [Ca2+]i and TRPC6 externalization and preserves EC migration. These data show that lysoPC induces activation of p38 MAPK, which leads to STIM1-Orai3 association and increased [Ca2+]i. This increase in [Ca2+]i activates an Src kinase leading to TRPC6 externalization, which initiates a cascade of events ending in cytoskeletal changes that disrupt EC migration. Blocking this pathway preserves EC migration in the presence of lipid oxidation products.NEW & NOTEWORTHY The major lysophospholipid component in oxidized LDL, lysophosphatidylcholine (lysoPC), can activate p38 MAP kinase, which in turn promotes externalization of Orai3 and STIM1-Orai3 association, suggesting involvement of arachidonic acid-regulated calcium (ARC) channels. The subsequent increase in intracellular calcium activates an Src kinase required for TRPC6 externalization. TRPC6 activation, which has been shown to inhibit endothelial cell migration, is blocked by p38 MAP kinase or Orai3 downregulation, and this partially preserves endothelial migration in lysoPC.

Transient Receptor Potential Canonical 6 (TRPC6) Channel in the Pathogenesis of Diseases: A Jack of Many Trades.

The mammalian Transient Receptor Potential Canonical (TRPC) subfamily comprises seven transmembrane proteins (TRPC1-7) forming cation channels in the plasma membrane of mammalian cells. TRPC channels mediate Ca2+ and Na+ influx into the cells. Amongst TRPCs, TRPC6 deficiency or increased activity due to gain-of-function mutations has been associated with a multitude of diseases, such as kidney disease, pulmonary disease, and neurological disease. Indeed, the TRPC6 protein is expressed in various organs and is involved in diverse signalling pathways. The last decade saw a surge in the investigative studies concerning the physiological roles of TRPC6 and describing the development of new pharmacological tools modulating TRPC6 activity. The current review summarizes the progress achieved in those investigations.

Reduction of TRPC1/TRPC3 mediated Ca2+-signaling protects oxidative stress-induced COPD

Oxidative stress is a predisposing factor in Chronic Obstructive Pulmonary Disease (COPD). Specifically, pulmonary epithelial (PE) cells reduce antioxidant capacity during COPD because of the continuous production of reactive oxygen species (ROS). However, the molecular pathogenesis that governs such ROS activity is unclear. Here we show that the dysregulation of intracellular calcium concentration ([Ca2+]i) in PE cells from COPD patients, compared to the healthy PE cells, is associated with the robust functional expressions of Transient Receptor Potential Canonical (TRPC)1 and TRPC3 channels, and Ca2+ entry (SOCE) components, Stromal Interaction Molecule 1 (STIM1) and ORAI1 channels. Additionally, the elevated expression levels of fibrotic, inflammatory, oxidative, and apoptotic markers in cells from COPD patients suggest detrimental pathway activation, thereby reducing the ability of lung remodeling. To further delineate the mechanism, we used human lung epithelial cell line, A549, since the behavior of SOCE and the expression patterns of TRPC1/C3, STIM1, and ORAI1 were much like PE cells. Notably, the knockdown of TRPC1/C3 in A549 cells substantially reduced the SOCE-induced [Ca2+]i rise, and reversed the ROS-mediated oxidative, fibrotic, inflammatory, and apoptotic responses, thus confirming the role of TRPC1/C3 in SOCE driven COPD-like condition. Higher TRPC1/C3, STIM1, and ORAI1 expressions, along with a greater Ca2+ entry, via SOCE in ROS-induced A549 cells, led to the rise in oxidative, fibrotic, inflammatory, and apoptotic gene expression, specifically through the extracellular signal-regulated kinase (ERK) pathway. Abatement of TRPC1 and/or TRPC3 reduced the mobilization of [Ca2+]i and reversed apoptotic gene expression and ERK activation, signifying the involvement of TRPC1/C3. Together these data suggest that TRPC1/C3 and SOCE facilitate the COPD condition through ROS-mediated cell death, thus implicating their likely roles as potential therapeutic targets for COPD. SummaryAlterations in Ca2+ signaling modalities in normal pulmonary epithelial cells exhibit COPD through oxidative stress and cellular injury, compromising repair, which was alleviated through inhibition of store-operated calcium entry. Subject areaCalcium, ROS, Cellular signaling, lung disease.

IPLA2 inhibition blocks LysoPC-induced TRPC6 externalization and promotes Re-endothelialization of carotid injuries in hypercholesterolemic mice

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), accumulate at the site of arterial injury after vascular interventions and hinder re-endothelization. LysoPC activates calcium-permeable channels, specifically canonical transient receptor potential 6 (TRPC6) channels that induce a sustained increase in intracellular calcium ion concentration [Ca2+]i and contribute to dysregulation of the endothelial cell (EC) cytoskeleton. Activation of TRPC6 leads to inhibition of EC migration in vitro and delayed re-endothelization of arterial injuries in vivo. Previously, we demonstrated the role of phospholipase A2 (PLA2), specifically calcium-independent PLA2 (iPLA2), in lysoPC-induced TRPC6 externalization and inhibition of EC migration in vitro. The ability of FKGK11, an iPLA2-specific pharmacological inhibitor, to block TRPC6 externalization and preserve EC migration was assessed in vitro and in a mouse model of carotid injury. Our data suggest that FKGK11 prevents lysoPC-induced PLA2 activity, blocks TRPC6 externalization, attenuates calcium influx, and partially preserves EC migration in vitro. Furthermore, FKGK11 promotes re-endothelization of an electrocautery carotid injury in hypercholesterolemic mice. FKGK11 has similar arterial healing effects in male and female mice on a high-fat diet. This study suggests that iPLA2 is a potential therapeutic target to attenuate calcium influx through TRPC6 channels and promote EC healing in cardiovascular patients undergoing angioplasty.

The synthesis and effects of a novel TRPC6 inhibitor, BP3112, on hepatocellular carcinoma.

Aims: Transient receptor potential canonical-6 (TRPC6) is a therapeutic target for hepatocellular carcinoma. The authors aimed to synthesize and determine whether indole-2-carboxamide derivatives have anti-hepatocellular carcinoma activities targeting TRPC6. Materials & methods: Molecular docking was carried out to design these derivatives. The top five compounds were synthesized for activity validation using microscale thermophoresis. Cell cytotoxicity, flow cytometry, western blottingand cell transfection were used to investigate the anti-hepatocellular carcinoma activities and mechanisms in vitro. Xenografts of nude mice were used for in vivo evaluation. Results: The indole-2-carboxamide derivative, BP3112, promoted apoptosis and G1-phase arrest in HCCs via inhibiting TRPC6, and dose-dependently inhibit tumor growth in vivo. Conclusion: BP3112 as a specific inhibitor of TRPC6 is a potential therapeutic agent for hepatocellular carcinoma.

Targeting alarmin release reverses Sjogren's syndrome phenotype by revitalizing Ca2+ signalling.

Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease that is embodied by the loss of salivary gland function and immune cell infiltration, but the mechanism(s) are still unknown. The aim of this study was to understand the mechanisms and identify key factors that leads to the development and progression of pSS. Immunohistochemistry staining, FACS analysis and cytokine levels were used to detect immune cells infiltration and activation in salivary glands. RNA sequencing was performed toidentify the molecular mechanisms involved in the development of pSS. The function assays include in vivo saliva collection along with calcium imaging and electrophysiology on isolated salivary gland cells in mice models of pSS. Western blotting, real-time PCR, alarmin release, and immunohistochemistry was performed to identify the channels involved in salivary function in pSS. We provide evidence that loss of Ca2+ signaling precedes a decrease in saliva secretion and/or immune cell infiltration in IL14α, a mouse model for pSS. We also showed that Ca2+ homeostasis was mediated by transient receptor potential canonical-1 (TRPC1) channels and inhibition of TRPC1, resulting in the loss of salivary acinar cells, which promoted alarmin release essential for immune cell infiltration/release of pro-inflammatory cytokines. In addition, both IL14α and samples from human pSS patients showed a decrease in TRPC1 expression and increased acinar cell death. Finally, paquinimod treatment in IL14α restored Ca2+ homeostasis that inhibited alarmin release thereby reverting the pSS phenotype. These results indicate that loss of Ca2+ signaling is one of the initial factors, which induces loss of salivary gland function along with immune infiltration that exaggerates pSS. Importantly, restoration of Ca2+ signaling upon paquinimod treatment reversed the pSS phenotype thereby inhibiting the progressive development of pSS.

Anti-inflammatory role of extracellular l-arginine through calcium sensing receptor in human renal proximal tubular epithelial (HK-2) cells.

Renal tubular epithelial cells are capable of synthesizing interleukins (IL) in response to a variety of proinflammatory cytokines. Moreover, elevated urinary levels of IL have been shown in patients with various forms of nephritic diseases. However, the underlying intracellular signaling mechanism is unclear. Here we show the immunological signaling role of l-Arginine (l-Arg) through Ca2+-sensing receptor (CaSR) in human kidney 2 (HK-2) renal proximal tubular epithelial cells, using Ca2+ imaging and patch clamp techniques and its mechanistic link to the downstream cellular function. Both pharmacological and siRNA inhibitors support the activation CaSR by extracellular l-Arg to induced Ca2+ entry via a Transient receptor potential canonical (TRPC) channel in HK-2 cells mainly through the receptor operated Ca2+ entry (ROCE). Activation of CaSR by l-Arg led to the rise in p-p38/p38 expression suggesting [Ca2+]i as a regulator for p38-signaling pathways. Notably, l-Arg activated CaSR-induced Ca2+ signaling reduced the expressions of key fibrotic, inflammatory, and apoptotic genes, suggesting its nephroprotective role via Ca2+ signaling through CaSR in HK-2 cells. Since we found that the IL-6 expressions were inversely proportional to the increasing concentrations of l-Arg in HK-2 cells, we measured the release of IL-6, which steadily decreased as the concentrations of l-Arg were elevated. Taken together, extracellular l-Arg is a negative regulator for IL-6-induced inflammatory process, through the activation of CaSR and TRPC channel by ROCE pathway and can have a potential to alleviate inflammatory renal diseases.

P85α regulatory subunit isoform controls PI3-kinase and TRPC6 membrane translocation

Activation of phosphatidylinositol 3-kinase (PI3K) by lipid oxidation products, including lysophosphatidylcholine (lysoPC), increases the externalization of canonical transient receptor potential 6 (TRPC6) channels leading to a subsequent increase in intracellular calcium that contributes to cytoskeletal changes which inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo. The PI3K p110α and p110δ catalytic subunit isoforms regulate lysoPC-induced TRPC6 externalization in vitro, but have many other functions. The goal of the current study is to identify the PI3K regulatory subunit isoform involved in TRPC6 externalization to potentially identify a more specific treatment regimen to improve EC migration and arterial healing, while minimizing off-target effects. Decreasing the p85α regulatory subunit isoform protein levels, but not the p85β and p55γ regulatory subunit isoforms, with small interfering RNA inhibits lysoPC-induced translocation of the PI3K catalytic subunit to the plasma membrane, dramatically decreased phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production and TRPC6 externalization, and significantly improves EC migration in the presence of lysoPC. These results identify the important and specific role of p85α in controlling translocation of PI3K from the cytosol to the plasma membrane and PI3K-mediated TRPC externalization by oxidized lipids. Current PI3K inhibitors block the catalytic subunit, but our data suggest that the regulatory subunit is a novel therapeutic target to promote EC migration and healing after arterial injuries that occur with angioplasty.

Interruption of TRPC6-NFATC1 signaling inhibits NADPH oxidase 4 and VSMCs phenotypic switch in intracranial aneurysm

Intracranial aneurysm (IA) is a frequent cerebrovascular disorder with unclear pathogenesis. The vascular smooth muscle cells (VSMCs) phenotypic switch is essential for IA formation. It has been reported that Ca2+ overload and excessive reactive oxygen species (ROS) are involved in VSMCs phenotypic switch. The transient receptor potential canonical 6 (TRPC6) and NADPH oxidase 4 (NOX4) are the main pathway to participate in Ca2+ overload and ROS production in VSMCs. Ca2+ overload can activate calcineurin (CN), leading to nuclear factor of activated T cell (NFAT) dephosphorylation to regulate the target gene's transcription. We hypothesized that activation of TRPC6-NFATC1 signaling may upregulate NOX4 and involve in VSMCs phenotypic switch contributing to the progression of IA. Our results showed that the expressions of NOX4, p22phox, p47phox, TRPC6, CN and NFATC1 were significantly increased, and VSMCs underwent a significant phenotypic switch in IA tissue and cellular specimens. The VIVIT (NFATC1 inhibitor) and BI-749327 (TRPC6 inhibitor) treatment reduced the expressions of NOX4, p22phox and p47phox and the production of ROS, and significantly improved VSMCs phenotypic switch in IA rats and cells. Consistent results were obtained from IA Trpc6 knockout (Trpc6-/-) mice. Furthermore, the results also revealed that NFATC1 could regulate NOX4 transcription by binding to its promoter. Our findings reveal that interrupting the TRPC6-NFATC1 signaling inhibits NOX4 and improves VSMCs phenotypic switch in IA, and regulating Ca2+ homeostasis may be an important therapeutic strategy for IA.

Negative self-regulation of transient receptor potential canonical 4 by the specific interaction with phospholipase C-δ1

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. It is suggested that TRPC4β is regulated by phospholipase C (PLC) signaling and is especially maintained by phosphatidylinositol 4,5-bisphosphate (PIP2). In this study, we present the regulation mechanism of the TRPC4 channel with PIP2 hydrolysis which is mediated by a channel-bound PLCδ1 but not by the GqPCR signaling pathway. Our electrophysiological recordings demonstrate that the Ca2+ via an open TRPC4 channel activates PLCδ1 in the physiological range, and it causes the decrease of current amplitude. The existence of PLCδ1 accelerated PIP2 depletion when the channel was activated by an agonist. Interestingly, PLCδ1 mutants which have lost the ability to regulate PIP2 level failed to reduce the TRPC4 current amplitude. Our results demonstrate that TRPC4 self-regulates its activity by allowing Ca2+ ions into the cell and promoting the PIP2 hydrolyzing activity of PLCδ1.

Transient Receptor Potential Canonical 5 (TRPC5) Channels Activator, BTD [N-{3-(adamantan-2-yloxy)-propyl}-3-(6-methyl-1,1-dioxo-2H-1λ6,2,4- benzothiadiazin-3-yl)-propanamide)] Ameliorates Diabetic Cardiac Autonomic Neuropathy in Rats.

Diabetic cardiac autonomic neuropathy (DCAN) is a serious diabetic complication with no approved pharmacological agents for its treatment. Parasympathetic system dysfunction characterized by vagal nerve damage is one of the major drivers of DCAN. The TRPC5 or transient receptor potential canonical 5 channel is a promising target in autonomic dysfunction; however, its role in vagal nerve damage and subsequent DCAN has not yet been elucidated. The present study investigated the role of the TRPC5 channel in DCAN using [N-{3-(adamantan-2-yloxy)-propyl}-3-(6-methyl-1,1-dioxo-2H-1λ6,2,4-benzothiadiazin-3-yl) propanamide)] or BTD, which is a potent TRPC5 activator. The role of the TRPC5 channel and its activator, BTD, was investigated in the treatment of parasympathetic dysfunction associated with DCAN. Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin. The alterations in cardiac autonomic parameters in diabetic animals were assessed by heart rate variability, hemodynamic parameters, and baroreflex sensitivity. TRPC5's role in DCAN was investigated by treating diseased rats with BTD (1 and 3 mg/kg, i.p. for 14 days). BTD's beneficial effects in parasympathetic dysfunction were assessed by western blotting, estimating oxidative stress and inflammatory markers in the vagus nerve. BTD treatment (3 mg/kg, i.p.) once daily for 14 days ameliorated heart rate variability, hemodynamic dysfunction, and baroreflex sensitivity in diseased rats. BTD treatment down regulated TRPC5 expression by increasing the activity of protein kinase C in the vagus nerve. It also down-regulated the apoptotic marker CASPASE-3 and also exerted potent anti-inflammatory action on pro-inflammatory cytokines levels in the vagus. BTD ameliorated parasympathetic dysfunction associated with DCAN by virtue of its TRPC5 modulatory, anti-inflammatory, and anti-apoptotic properties.

TRPC absence induces pro-inflammatory macrophages and gut microbe disorder, sensitizing mice to colitis.

The transient receptor potential canonical (TRPC) channels, encoded in seven non-allelic genes, are important contributors to calcium fluxes, are strongly associated with various diseases. Here we explored the consequences of ablating all seven TRPCs in mice focusing on colitis. We discovered that absence of all seven TRPC proteins in mice (TRPC HeptaKO mice) promotes the development of dextran sulfate sodium (DSS)-induced colitis. RNA-sequence analysis highlighted an extremely pro-inflammatory profile in colons of DSS-treated TRPC HeptaKO mice, with an amount of increased pro-inflammatory cytokines and chemokines. Flow cytometry analysis showed that the infiltration of Ly6Chi monocytes and neutrophils in colonic lamina propria was significantly increased in DSS-treated TRPC HeptaKO mice. Results also revealed that macrophages from TRPC HeptaKO mice exhibited M1 polarization and enhanced secretion of pro-inflammatory factors. In addition, the composition of gut microbiota was markedly disturbed in DSS-treated TRPC HeptaKO mice. However, upon antibiotic cocktail (Abx)-treatment, TRPC HeptaKO mice showed no significant differences with WT mice in disease severity. Collectively, these data suggest that ablation of all TRPCs promotes the development of DSS-induced colitis by inducing pro-inflammatory macrophages and gut microbiota disorder.

Electrophysiological properties of TRPC1/4 heteromer determined by its pore residues.

Transient receptor potential canonical 1 (TRPC1) is widely expressed in various mammalian tissues and is involved in many physiological functions. It is known to heteromerize with TRPC4 to form a cationic channel, and it induces significant changes in channel characteristics. The huge differences in channel characteristics in the physiological environment indicates the importance of channel regulation in many tissues expressing both TRPC1 and TRPC4. In this study, we focused on the pore region (selectivity filter, pore helix, and S6 helix) of TRPC1 and TRPC4, compared their homology, and investigated how they contribute to the electrophysiological characteristics of the ion channel. We observed changes in G-V curve and I-V curve with mutations of the pore region and demonstrated that TRPC4 selectivity filter mutants lost sensitivity to G protein mediated activation. Moreover, lower gate mutants of TRPC4 exhibited diminished calcium permeability compared to wild type channels. Through chimeric channels where we substituted the pore region of TRPC1 with that of TRPC4, we were able to determine regions that are critical in producing the double-rectifying I-V curve of the homomeric TRPC4 channel.

TRP434 and TRP435 residues are crucial for sensing calcium ions and PI(4,5)P2 molecules in TRPC5 channel.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. Among 7 subunits, TRPC4, 5 channels are well known to be potentiated by direct bindings of calcium ion and phosphatidylinositol 4,5-biphosphate. Both calcium and PIP2 were therefore also implicated in activation and desensitization of the channel. The binding site for calcium ion was revealed but PIP2 binding site remains controversial. Previously, we found two tryptophan residues in TRPC4 channel as a candidate by which calcium sensing in S2-S3 linker is delivered to channel gating. Here, we suggest the WW site in TRPC5 channel conducts delivering of calcium sensing like in TRPC4, and also acts as a potential PIP2 binding site. We made two single mutants (W434A, W435A) and a double mutant (WW/AA) from human TRPC5 channel. Wild-type and mutant TRPC5 channels were expressed in HEK293 cells for macroscopic current recordings in whole-cell or inside-out mode. Generally, our data demonstrate W434 and W435 residues participate in PIP2 binding and calcium sensing. Interestingly, when co-expressed with human muscarinic acetylcholine receptor type 3 (mAChR3) and stimulated by carbachol, W434A channel didn’t show desensitization characteristically observed in wild-type TRPC5 channel. Loss of desensitization in W434A channel implies that it has lost the ability of sensing PIP2 hydrolysis by PLC or the ability of phosphorylation by PKC. The latter possibility is ruled out because the C-terminus of the channels we used were truncated so they do not contain the phosphorylation site. Recently, PIP2-bound ion channel structures were revealed including TRPs. Many channels had PIP2 molecules near S3, S4, and S5 helices. Our electrophysiological data also indicate that PIP2 binds near S3 helix in TRPC5 channel, and the WW site is a specific candidate for sensing PIP2 and calcium.