TRPC6 Research Articles

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.

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.

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.

Role of IL-17A in different stages of ischemic stroke

Interleukin-17A (IL-17A) plays an important role in the progression of ischemic stroke. IL-17A mediates the endothelial inflammatory response, promotes water and sodium retention, and changes the electrophysiological structure of the atrium, accelerating the progression of ischemic stroke risk factors such as atherosclerotic plaques, hypertension, and atrial fibrillation. In the acute phase of ischemic stroke, IL-17A mediates neuronal injury through neutrophil chemotaxis to the site of injury, the induction of neuronal apoptosis, and activation of the calpain-TRPC-6 (transient receptor potential channel-6) pathway. During ischemic stroke recovery, IL-17A, which is mainly derived from reactive astrocytes, promotes and maintains the survival of neural precursor cells (NPCs) in the subventricular zone (SVZ), neuronal differentiation, and synapse formation and participates in the repair of neurological function. Therapies targeting IL-17A-associated inflammatory signaling pathways can reduce the risk of ischemic stroke and neuronal damage and are a new therapeutic strategy for ischemic stroke and its risk factors. In this paper, we will briefly discuss the pathophysiological role of IL-17A in ischemic stroke risk factors, acute and chronic inflammatory responses, and the potential therapeutic value of targeting IL-17A.

Correction to: Endothelial Transient Receptor Potential Canonical Channel Regulates Angiogenesis and Promotes Recovery after Myocardial Infarction.

HomeJournal of the American Heart AssociationVol. 12, No. 7Correction to: Endothelial Transient Receptor Potential Canonical Channel Regulates Angiogenesis and Promotes Recovery after Myocardial Infarction Open AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citations ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toOpen AccessCorrectionPDF/EPUBCorrection to: Endothelial Transient Receptor Potential Canonical Channel Regulates Angiogenesis and Promotes Recovery after Myocardial Infarction Originally published28 Mar 2023https://doi.org/10.1161/JAHA.121.027660Journal of the American Heart Association. 2023;12:e027660This article corrects the followingEndothelial Transient Receptor Potential Canonical Channel Regulates Angiogenesis and Promotes Recovery After Myocardial InfarctionOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: March 28, 2023: Ahead of Print In the article by Xin Wen et al, “Endothelial Transient Receptor Potential Canonical Channel Regulates Angiogenesis and Promotes Recovery After Myocardial Infarction,” which published online March 5, 2022 (J Am Heart Assoc. 2022;11:e023678. DOI: 10.1161/JAHA.121.023678) and was included in the March 15, 2022 issue of the journal, corrections were needed.In Figure 1E, the image for tube formation in primary coronary artery endothelial cells from floxed TRPC1 (TRPC1fl/fl) mice was incorrect. This figure has been corrected.In the Supplemental Material, in Table S2, the reverse PCR primer sequence for HIF‐1α was incorrect. This primer sequence has been corrected.In the Supplemental Material, in Figure S3C, the immunofluorescence image for CD31 in mesenteric artery smooth muscle cells was incorrect. This figure has been corrected.The authors regret the errors.The online version of the article has been updated and is available here: https://www.ahajournals.org/doi/full/10.1161/JAHA.121.023678. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesEndothelial Transient Receptor Potential Canonical Channel Regulates Angiogenesis and Promotes Recovery After Myocardial InfarctionXin Wen, et al. Journal of the American Heart Association. 2022;11 April 4, 2023Vol 12, Issue 7Article InformationMetrics Copyright © 2023 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley BlackwellThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.https://doi.org/10.1161/JAHA.121.027660PMID: 36974768 Originally publishedMarch 28, 2023 PDF download

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.

Inhibition of Canonical Transient Receptor Potential Channels 4/5 with Highly Selective and Potent Small-Molecule HC-070 Alleviates Mechanical Hypersensitivity in Rat Models of Visceral and Neuropathic Pain.

Transient receptor potential channels C4/C5 are widely expressed in the pain pathway. Here, we studied the putative analgesic efficacy of the highly selective and potent TRPC4/C5 antagonist HC-070 in rats. Inhibitory potency on human TRPC4 was assessed by using the whole-cell manual patch-clamp technique. Visceral pain sensitivity was assessed by the colonic distension test after intra-colonic trinitrobenzene sulfonic acid injection and partial restraint stress. Mechanical pain sensitivity was assessed by the paw pressure test in the chronic constriction injury (CCI) neuropathic pain model. We confirm that HC-070 is a low nanomolar antagonist. Following single oral doses (3-30 mg/kg in male or female rats), colonic hypersensitivity was significantly and dose-dependently attenuated, even fully reversed to baseline. HC-070 also had a significant anti-hypersensitivity effect in the established phase of the CCI model. HC-070 did not have an effect on the mechanical withdrawal threshold of the non-injured paw, whereas the reference compound morphine significantly increased it. Analgesic effects are observed at unbound brain concentrations near the 50% inhibitory concentration (IC50) recorded in vitro. This suggests that analgesic effects reported here are brought about by TRPC4/C5 blocking in vivo. The results strengthen the idea that TRPC4/C5 antagonism is a novel, safe non-opioid treatment for chronic pain.

Ghrelin signaling in the cerebellar cortex enhances GABAergic transmission onto Purkinje cells

Ghrelin, an orexigenic peptide ligand for growth hormone secretagogue receptor 1a (GHS-R1a), occurs not only in the stomach but also in the brain, and modulates neuronal activity and synaptic efficacy. Previous studies showed that GHS-R1a exists in the cerebellum, and ghrelin facilitates spontaneous firing of Purkinje cells (PCs). However, the effects of ghrelin on cerebellar GABAergic transmission have yet to be elucidated. We found that ghrelin enhanced GABAergic transmission between molecular layer interneurons (MLIs) and PCs using electrophysiological recordings in mouse cerebellar slices. This finding was consistent with the possibility that blocking synaptic transmission enhanced the ghrelin-induced facilitation of PC firing. Ghrelin profoundly increased the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) in PCs without affecting miniature or stimulation-evoked IPSCs, whereas it significantly facilitated spontaneous firing of MLIs. This facilitation of MLI spiking disappeared during treatments with blockers of GHS-R1a, type 1 transient receptor potential canonical (TRPC1) channels and KCNQ channels. These results suggest that both activating TRPC1 channels and inhibiting KCNQ channels occur downstream the ghrelin-GHS-R1a signaling pathway probably in somatodendritic sites of MLIs. Thus, ghrelin can control PC firing directly and indirectly via its modulation of GABAergic transmission, thereby impacting activity in cerebellar circuitry.

The small GTPase regulatory protein Rac1 drives podocyte injury independent of cationic channel protein TRPC5

Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies.

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.

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.

Soluble epoxide hydrolase and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy: Interrelation and regulation by C/EBPβ

ObjectivesStudies in certain cardiac hypertrophy models suggested the individual role of soluble epoxide hydrolase (sEH) and canonical transient receptor potential 3 (TRPC3) channels, however, whether they jointly mediate hypertrophic process remains unexplored. Hyperhomocysteinemia promotes cardiac hypertrophy while the involvement of sEH and TRPC3 channels remains unknown. This study aimed to explore the role of, and interrelation between sEH and TRPC3 channels in homocysteine-induced cardiac hypertrophy. MethodsRats were fed methionine-enriched diet to induce hyperhomocysteinemia. H9c2 cells and neonatal rat cardiomyocytes were incubated with homocysteine. Cardiac hypertrophy was evaluated by echocardiography, histological examination, immunofluorescence imaging, and expressions of hypertrophic markers. Epoxyeicosatrienoic acids (EETs) were determined by ELISA. TRPC3 current was recorded by patch-clamp. Gene promotor activity was measured using dual-luciferase reporter assay. ResultsInhibition of sEH by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) reduced ventricular mass, lowered the expression of hypertrophic markers, decreased interstitial collagen deposition, and improved cardiac function in hyperhomocysteinemic rats, associated with restoration of EETs levels in myocardium. TPPU or knockdown of sEH suppressed TRPC3 transcription and translation as well as TRPC3 current that were enhanced by homocysteine. Exogenous 11,12-EET inhibited homocysteine-induced TRPC3 expression and cellular hypertrophy. Silencing C/EBPβ attenuated, while overexpressing C/EBPβ promoted homocysteine-induced hypertrophy and expressions of sEH and TRPC3, resulting respectively from inhibition or activation of sEH and TRPC3 gene promoters. ConclusionssEH and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy. Homocysteine transcriptionally activates sEH and TRPC3 genes through a common regulatory element C/EBPβ. sEH activation leads to an upregulation of TRPC3 channels via a 11,12-EET-dependent manner.

Capsazepine (CPZ) Inhibits TRPC6 Conductance and Is Protective in Adriamycin-Induced Nephropathy and Diabetic Glomerulopathy.

Reactive oxygen species (ROS), which excessively arise in diabetes and systemic inflammatory diseases, modify cellular lipids and cellular lipid composition leading to altered biophysical properties of cellular membranes. The impact of lipid peroxidation on transmembrane signaling routes is not yet well studied. The canonical transient receptor potential channel 6 (TRPC6) is implicated in the pathogenesis of several forms of glomerular diseases. TRPC6 is sensitive to membrane stretch and relies on a distinct lipid environment. This study investigates the effect of oxidative alterations to plasma membrane lipids on TRPC6 activity and the function of the glomerular filter. Knockout of the anti-oxidative, lipid modifying enzyme paraoxonase 2 (PON2) leads to altered biophysical properties of glomerular epithelial cells, which are called podocytes. Cortical stiffness, quantified by atomic force microscopy, was largely increased in PON2-deficient cultured podocytes. PON2 deficiency markedly enhanced TRPC6 channel currents and channel recovery. Treatment with the amphiphilic substance capsazepine in micromolar doses reduced cortical stiffness and abrogated TRPC6 conductance. In in vivo studies, capsazepine reduced the glomerular phenotype in the model of adriamycin-induced nephropathy in PON2 knockout mice and wildtype littermates. In diabetic AKITA mice, the progression of albuminuria and diabetic kidney disease was delayed. In summary, we provide evidence that the modification of membrane characteristics affects TRPC6 signaling. These results could spur future research to investigate modification of the direct lipid environment of TRPC6 as a future therapeutic strategy in glomerular disease.

Screening of rosmarinic acid from Salvia miltiorrhizae acting on the novel target TRPC1 based on the ‘homology modelling–virtual screening–molecular docking–affinity assay–activity evaluation’ method

Context Salvia miltiorrhizae Bunge (Lamiaceae) is a traditional Chinese medicine (TCM) for the treatment of ‘thoracic obstruction’. Transient receptor potential canonical channel 1 (TRPC1) is a important target for myocardial injury treatment. Objective This work screens the active component acting on TRPC1 from Salvia miltiorrhizae. Materials and methods TCM Systems Pharmacology Database and Analysis Platform (TCMSP) was used to retrieve Salvia miltiorrhiza compounds for preliminary screening by referring to Lipinski’s rule of five. Then, the compound group was comprehensively scored by AutoDock Vina based on TRPC1 protein. Surface plasmon resonance (SPR) was used to determine the affinity of the optimal compound to TRPC1 protein. Western blot assay was carried out to observe the effect of the optimal compound on TRPC1 protein expression in HL-1 cells, and Fura-2/AM detection was carried out to observe the effect of the optimal compound on calcium influx in HEK293 cells. Results Twenty compounds with relatively good characteristic parameters were determined from 202 compounds of Salvia miltiorrhiza. Rosmarinic acid (RosA) was obtained based on the molecular docking scoring function. RosA had a high binding affinity to TRPC1 protein (KD value = 1.27 µM). RosA (50 μM) could reduce the protein levels (417.1%) of TRPC1 after oxygen-glucose deprivation/reperfusion (OGD/R) in HL-1 cells and it could inhibit TRPC1-mediated Ca2+ influx injury (0.07 ΔRatio340/380) in HEK293 cells. Discussion and conclusions We obtained the potential active component RosA acting on TRPC1 from Salvia miltiorrhizae, and we speculate that RosA may be a promising clinical candidate for myocardial injury therapy.

Optical Activation of Photoswitchable TRPC Ligands in the Mammalian Olfactory System Using Laser Scanning Confocal Microscopy.

The transient receptor potential canonical (TRPC) ion channels play important biological roles, but their activation mechanisms are incompletely understood. Here, we describe recent methodological advances using small molecular probes designed for photopharmacology of TRPC channels by focusing on results obtained from the mouse olfactory system. These studies developed and used photoswitchable diacylglycerol (DAG) analogs for ultrarapid activation of native TRPC2 channels in vomeronasal sensory neurons and type B cells of the main olfactory epithelium. Further studies investigated the role of TRPC5 channels in prolactin regulation of dopamine neurons in the arcuate nucleus of the hypothalamus. Here, the first photoswitchable TRPC5 modulator, BTDAzo, was developed and shown to control endogenous TRPC5-based neuronal Ca2+ responses in mouse brain slices. Thus, photoswitchable reagents are rapidly gaining widespread recognition for investigating various types of TRPC channels including TRPC2, TRPC3, TRPC5, and TRPC6, enabling to gain new insights into the gating mechanisms and functions of these channels.

Syndrome similar to Familial Hypocalciuric Hypercalcemia (FHH) produced in mice deleted of the gene encoding transient receptor potential canonical channel 1 (TRPC1)

We recently found that global deletion of TRPC1 produces phenotypes similar to FHH [1]. These TRPC1 null mice have mildly elevated serum Ca in both fasted and unfasted conditions from 3.5 through 21.5 months of age, and inappropriately elevated parathyroid hormone (PTH) levels.

TRPC1 channels underlie stretch-modulated sarcoplasmic reticulum calcium leak in cardiomyocytes.

Transient receptor potential canonical 1 (TRPC1) channels are Ca2+-permeable ion channels expressed in cardiomyocytes. An involvement of TRPC1 channels in cardiac diseases is widely established. However, the physiological role of TRPC1 channels and the mechanisms through which they contribute to disease development are still under investigation. Our prior work suggested that TRPC1 forms Ca2+ leak channels located in the sarcoplasmic reticulum (SR) membrane. Prior studies suggested that TRPC1 channels in the cell membrane are mechanosensitive, but this was not yet investigated in cardiomyocytes or for SR localized TRPC1 channels. We applied adenoviral transfection to overexpress or suppress TRPC1 expression in neonatal rat ventricular myocytes (NRVMs). Transfections were evaluated with RT-qPCR, western blot, and fluorescent imaging. Single-molecule localization microscopy revealed high colocalization of exogenously expressed TRPC1 and the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2). To test our hypothesis that TRPC1 channels contribute to mechanosensitive Ca2+ SR leak, we directly measured SR Ca2+ concentration ([Ca2+]SR) using adenoviral transfection with a novel ratiometric genetically encoded SR-targeting Ca2+ sensor. We performed fluorescence imaging to quantitatively assess [Ca2+]SR and leak through TRPC1 channels of NRVMs cultured on stretchable silicone membranes. [Ca2+]SR was increased in cells with suppressed TRPC1 expression vs. control and Transient receptor potential canonical 1-overexpressing cells. We also detected a significant reduction in [Ca2+]SR in cells with Transient receptor potential canonical 1 overexpression when 10% uniaxial stretch was applied. These findings indicate that TRPC1 channels underlie the mechanosensitive modulation of [Ca2+]SR. Our findings are critical for understanding the physiological role of TRPC1 channels and support the development of pharmacological therapies for cardiac diseases.

Functional cooperation between IKCa and TRPC1 channels regulates serum-induced vascular smooth muscle cell proliferation via mediating Ca2+ influx and ERK1/2 activation.

The increased proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of vascular diseases. The intermediate conductance calcium-activated potassium (IKCa ) channel plays a critical role in VSMC proliferation by raising the intracellular calcium concentration ([Ca2+ ]i ), but the underlying mechanism is still not unclear. Here we investigated the cooperation between IKCa and transient receptor potential canonical 1 (TRPC1) channels in mediating extracellular Ca2+ entry, which in turn activates downstream Ca2+ signalling in the regulation of VSMC proliferation using serum-induced cell proliferation model. Serum-induced cell proliferation was accompanied with up-regulation of IKCa expression and an increase in [Ca2+ ]i . Serum-induced cell proliferation and increase in [Ca2+ ]i were suppressed by IKCa inhibition with TRAM-34 or IKCa knockdown. Serum-induced cell proliferation was strongly reduced by the removal of extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA-AM and, additionally, by TRPC1 knockdown. Moreover, the increase in [Ca2+ ]i induced by serum or by IKCa activation with 1-EBIO was attenuated by TRPC1 knockdown. Finally, serum induced ERK1/2 activation, which was attenuated by treatment with TRAM-34 or BAPTA-AM, as well as TRPC1 knockdown. Consistently, serum-induced cell proliferation was suppressed by ERK1/2 inhibition with PD98059. Taken together, these results suggest that the IKCa and TRPC1 channels cooperate in mediating Ca2+ influx that activates the ERK1/2 pathway to promote cell proliferation, thus providing new mechanistic insights into VSMC proliferation.

TRPC channels blockade abolishes endotoxemic cardiac dysfunction by hampering intracellular inflammation and Ca2+ leakage

Intracellular Ca2+ dysregulation is a key marker in septic cardiac dysfunction; however, regulation of the classic Ca2+ regulatory modules cannot successfully abolish this symptom. Here we show that the knockout of transient receptor potential canonical (TRPC) channel isoforms TRPC1 and TRPC6 can ameliorate LPS-challenged heart failure and prolong survival in mice. The LPS-triggered Ca2+ release from the endoplasmic reticulum both in cardiomyocytes and macrophages is significantly inhibited by Trpc1 or Trpc6 knockout. Meanwhile, TRPC’s molecular partner — calmodulin — is uncoupled during Trpc1 or Trpc6 deficiency and binds to TLR4’s Pococurante site and atypical isoleucine-glutamine-like motif to block the inflammation cascade. Blocking the C-terminal CaM/IP3R binding domain in TRPC with chemical inhibitor could obstruct the Ca2+ leak and TLR4-mediated inflammation burst, demonstrating a cardioprotective effect in endotoxemia and polymicrobial sepsis. Our findings provide insight into the pathogenesis of endotoxemic cardiac dysfunction and suggest a novel approach for its treatment.

Mechanism of Action of Flavin-Dependent Halogenases.

To rationally engineer the substrate scope and selectivity of flavin-dependent halogenases (FDHs), it is essential to first understand the reaction mechanism and substrate interactions in the active site. FDHs have long been known to achieve regioselectivity through an electrophilic aromatic substitution at C7 of the natural substrate Trp, but the precise role of a key active-site Lys residue remains ambiguous. Formation of hypochlorous acid (HOCl) at the cofactor-binding site is achieved by the direct reaction of molecular oxygen and a single chloride ion with reduced FAD and flavin hydroxide, respectively. HOCl is then guided 10 Å into the halogenation active site. Lys79, located in this site, has been proposed to direct HOCl toward Trp C7 through hydrogen bonding or a direct reaction with HOCl to form an -NH2Cl+ intermediate. Here, we present the most likely mechanism for halogenation based on molecular dynamics (MD) simulations and active-site density functional theory "cluster" models of FDH PrnA in complex with its native substrate l-tryptophan, hypochlorous acid, and the FAD cofactor. MD simulations with different protonation states for key active-site residues suggest that Lys79 directs HOCl through hydrogen bonding, which is confirmed by calculations of the reaction profiles for both proposed mechanisms.