TRPC6 Activation Research Articles

All-Optical Analysis of TRPC3/6 Signalling in Mast Cells

Transient receptor potential canonical channels TRPC3/6 are expressed in various cell types of the immune system. These Ca2+ permeable channels are typically activated downstream of the phospholipase C (PLC) pathway, but their role in immune cell Ca2+ handling is still elusive. We set out to explore the impact of TRPC3 and TRPC6 function in RBL mast cells at the level of local, sub-plasmalemmal and global cellular Ca2+ signalling, independently of PLC activity by using an all-optical approach. C-terminal fusions of TRPC3 and TRPC6 with R-GECO1.0 were generated to record Ca2+ changes in the vicinity of the TRPC channels. A new photochromic benzimidazole (OptoBI-1) was used to generate temporally precise pattern of channel activity in the absence of PLC signalling. When expressed in HEK293 cells, TRPC3 or TRPC6 activation generated both local and global Ca2+ rises, while TRPC channels expressed in RBL mast cells produced large local Ca2+ elevation that was associated with barely detectable global Ca2+ rises. Interestingly, disruption of lipid accommodation within the channels' pore domain (mutation in the lipid gating window) only moderately modified kinetics of local Ca2+ rises but completely restored the associated global cytosolic Ca2+ response. Our all-optical analysis of TRPC signal transduction in RBL mast cells suggest that minor changes in TRPC lipid coordination and current kinetics are of crucial impact on overall TRPC signalling features in immune cells.

Direct Activation of TRPC3 Channels by the Antimalarial Agent Artemisinin.

(1) Background: Members of the TRPC3/TRPC6/TRPC7 subfamily of canonical transient receptor potential (TRP) channels share an amino acid similarity of more than 80% and can form heteromeric channel complexes. They are directly gated by diacylglycerols in a protein kinase C-independent manner. To assess TRPC3 channel functions without concomitant protein kinase C activation, direct activators are highly desirable. (2) Methods: By screening 2000 bioactive compounds in a Ca2+ influx assay, we identified artemisinin as a TRPC3 activator. Validation and characterization of the hit was performed by applying fluorometric Ca2+ influx assays and electrophysiological patch-clamp experiments in heterologously or endogenously TRPC3-expressing cells. (3) Results: Artemisinin elicited Ca2+ entry through TRPC3 or heteromeric TRPC3:TRPC6 channels, but did not or only weakly activated TRPC6 and TRPC7. Electrophysiological recordings confirmed the reversible and repeatable TRPC3 activation by artemisinin that was inhibited by established TRPC3 channel blockers. Rectification properties and reversal potentials were similar to those observed after stimulation with a diacylglycerol mimic, indicating that artemisinin induces a similar active state as the physiological activator. In rat pheochromocytoma PC12 cells that endogenously express TRPC3, artemisinin induced a Ca2+ influx and TRPC3-like currents. (4) Conclusions: Our findings identify artemisinin as a new biologically active entity to activate recombinant or native TRPC3-bearing channel complexes in a membrane-confined fashion.

Abstract A019: Role of MTI-101 in disrupting calcium homeostasis leading to cell death in multiple myeloma

Abstract Introduction: Multiple Myeloma (MM) is a hematologic cancer characterized by neoplastic proliferation of the plasma cells in the bone marrow. Newly diagnosed MM patients typically respond to currently available treatments. However, the disease is not curable and unfortunately all patients eventually will relapse with drug resistant disease. Clinical data continues to indicate that novel treatment strategies are required to improve patient outcomes. Our group has developed a novel agent cyclic peptide called MTI-101 for the treatment of MM. MTI-101 was optimized form the D-amino acid linear peptide HYD-1 (1). HYD-1 and the more potent MTI-101 were discovered to induce necroptotic cell death by disrupting Ca2+ homeostasis in MM cell lines. Our laboratory previously reported that acquisition towards resistance to MTI-101 correlated with repression of genes predicted to activate SOCE including decreased PLCβ, SERCA, ITPR3, and TRPC1 expression (2). Moreover, our data showed that pharmacological inhibition of SOCE or reducing the expression of TRPC1 blocks MTI-101 induced cell death (2). Activation of SOCE is dependent on STIM1-ORAI1 recruitment to endoplasmic reticulum- plasma membrane junction (ER-PM junction), allowing an oscillatory Ca2+ flux via activation of ICRAC (Calcium Release Activated Calcium channel) (3). Kaczmarek et al showed that TRPC5 (TRPC1 heterodimer) activity can be positively regulated by calpain truncation (4). In this study, we sought to determine the role of TRPC1 in MTI-101 Ca2+ induced necrosis, as we hypothesize that MTI-101 induced prolonged SOCE activation is due to TRPC1 truncation by calpains activation, leading to disruption of Ca2+ homeostasis and cell death Methods: To evaluate determinants of activity of MTI-101, we developed an assay to measure intracellular Ca2+ levels and cell death by using the Ca2+ indicator Fluo-4 AM and DAPI as a marker for cell death. Further, to evaluate the role of MTI-101 on TRPC1 activation, MTI-101 treated cells were lysed using RIPA detergent, and the detergent resistant component was lysed using RIPA and 2% SDS, followed by sonication with an ultrasonic homogenizer. Both compartments were analyzed via western blot. Results: Our data showed that MTI-101-induced robust, delayed calcium flux is causative for cell death due to irreversible activation of SOCE, while thapsigargin causes rapid, transient, reversible activation. Further, the data indicated that MTI-101 treatment induces TRPC1 truncation and translocation to a detergent-resistant compartment of the cell. Moreover, inhibiting the Ca2+ activated proteases calpain1 and calpain2 abrogated MTI-101 induced Ca2+ flux and cell death, and interferes with TRPC1 truncation following MTI-101 treatment in a time dependent manner. Conclusions: MTI-101 induces a robust, and sustained activation of SOCE causative for cell death. Increased intracellular Ca2+ levels following MTI-101 treatment induces calpains activation, furthering TRPC1 truncation and activation leading to necroptotic cell death. Our future studies will focus on evaluating the role of mutants TRPC1 on MTI-101 activity, identify calpains consensus sites on TRPC1, and determine the role of ORAI1 in MTI-101 Ca2+ induced cell death. Citation Format: Osama M Elzamzamy, Karen Hayes, Werner Geldenhuys, Lori A Hazlehurst. Role of MTI-101 in disrupting calcium homeostasis leading to cell death in multiple myeloma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A019. doi:10.1158/1535-7163.TARG-19-A019

Effect of Exercise Training and Aging on Klotho Signaling in the Heart

: Aging, if accompanied by a sedentary lifestyle, can result in different diseases including cardiovascular diseases. Klotho, a protein found in the kidneys and other tissues of the body, is influenced by age and level of physical activity. In this paper, using 66 papers published in PubMed and SID between 1997 and 2018, we examined the mechanism of action of Klotho on cardiac hypertrophy under senescence conditions. Studies have shown that aging results in the development of oxidative stress as well as increased MAPK and TRPC6. These factors push the heart towards pathological hypertrophy, where deficiency of Klotho accelerates this trend. Nevertheless, exercise training causes enhanced Klotho levels. This protein is able to inhibit oxidative stress as an upstream factor in the activity of MAPK and TRPC6.

Inhibition of Canonical Transient Receptor Potential 5 Channels Polarizes Macrophages to an M1 Phenotype

Background: Transient receptor potential (TRP) channels are widely expressed in the mammals. However, the functions of canonical TRP (TRPC) in inflammatory responses are largely unknown. The present study was focused on the effect of canonical TRP5 (TRPC5) channel on the polarization of macrophage to an M1 phenotype. Methods: Polarization of macrophages was studied in TRPC5 knockout (TRPC5^–/–) mice and in Raw264.7, a mouse macrophage cell line. Indicators of M1 type polarized macrophage were measured in the aorta of mice. Inhibition of TRPC5 in macrophages was achieved by the administration of ML204 (a non-selective TRPC5 antagonist) or the silencing of the TRPC5 gene with short hairpin RNA. Lipopolysaccharide (LPS) was used to stimulate Raw264.7 cells to an M1 type polarization. Proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were measured in mice or cells, and protein expressions of Akt, phosphorylated (p)-Akt, IκBα, p-IκBα, and NF-κB were analyzed in Raw264.7 cells. Results: In TRPC5^–/– mice the number of M1 type polarization of macrophages infiltrating into the aortic walls were significantly increased. The serum levels of inflammatory cytokines, TNF-α and IL-6 were also increased. Furthermore, after treated with ML204 or silenced the gene of TRPC5, the releases of TNF-α, IL-1β and IL-6 from lipopolysaccharide-stimulated RAW264.7 cells were significantly increased. Meanwhile, phosphorylations of Akt and IκBα were upregulated, and the shift of NF-κB from the cytoplasm to nucleus was markedly enhanced. Conclusion: The activation of TRPC5 may inhibit the polarization of macrophage to an M1 phenotype by regulating Akt/IκB/NF-κB signaling pathways.

FKBP52 regulates TRPC3-dependent Ca2+ signals and the hypertrophic growth of cardiomyocyte cultures.

The transient receptor potential (TRP; C-classical, TRPC) channel TRPC3 allows a cation (Na+/Ca2+) influx that is favored by the stimulation of Gq protein-coupled receptors (GPCRs). An enhanced TRPC3 activity is related to adverse effects, including pathological hypertrophy in chronic cardiac disease states. In the present study, we identified FK506-binding protein 52 (FKBP52, also known as FKBP4) as a novel interaction partner of TRPC3 in the heart. FKBP52 was recovered from a cardiac cDNA library by a C-terminal TRPC3 fragment (amino acids 742-848) in a yeast two-hybrid screen. Downregulation of FKBP52 promoted a TRPC3-dependent hypertrophic response in neonatal rat cardiomyocytes (NRCs). A similar effect was achieved by overexpressing peptidyl-prolyl isomerase (PPIase)-deficient FKBP52 mutants. Mechanistically, expression of the FKBP52 truncation mutants elevated TRPC3-mediated currents and Ca2+ fluxes, and the activation of calcineurin and the nuclear factor of activated T-cells in NRCs. Our data demonstrate that FKBP52 associates with TRPC3 via an as-yet-undescribed binding site in the C-terminus of TRPC3 and modulates TRPC3-dependent Ca2+ signals in a PPIase-dependent manner. This functional interaction might be crucial for limiting TRPC3-dependent signaling during chronic hypertrophic stimulation.

Transmembrane insertases and N-glycosylation critically determine synthesis, trafficking, and activity of the nonselective cation channel TRPC6

Transient receptor potential cation channel subfamily C member 6 (TRPC6) is a widely expressed ion channel. Gain-of-function mutations in the human TRPC6 channel cause autosomal-dominant focal segmental glomerulosclerosis, but the molecular components involved in disease development remain unclear. Here, we found that overexpression of gain-of-function TRPC6 channel variants is cytotoxic in cultured cells. Exploiting this phenotype in a genome-wide CRISPR/Cas screen for genes whose inactivation rescues cells from TRPC6-associated cytotoxicity, we identified several proteins essential for TRPC6 protein expression, including the endoplasmic reticulum (ER) membrane protein complex transmembrane insertase. We also identified transmembrane protein 208 (TMEM208), a putative component of a signal recognition particle-independent (SND) ER protein-targeting pathway, as being necessary for expression of TRPC6 and several other ion channels and transporters. TRPC6 expression was also diminished by loss of the previously uncharacterized WD repeat domain 83 opposite strand (WDR83OS), which interacted with both TRPC6 and TMEM208. Additionally enriched among the screen hits were genes involved in N-linked protein glycosylation. Deletion of the mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT1), necessary for the generation of complex N-linked glycans, abrogated TRPC6 gain-of-function variant-mediated Ca2+ influx and extracellular signal-regulated kinase activation in HEK cells, but failed to diminish cytotoxicity in cultured podocytes. However, mutating the two TRPC6 N-glycosylation sites abrogated the cytotoxicity of mutant TRPC6 and reduced its surface expression. These results expand the targets of TMEM208-mediated ER translocation to include multipass transmembrane proteins and suggest that TRPC6 N-glycosylation plays multiple roles in modulating channel trafficking and activity.

Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes.

TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved. We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes. Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton. The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.

Angiotensin II-mediated MYH9 downregulation causes structural and functional podocyte injury in diabetic kidney disease

MYH9, a widely expressed gene encoding nonmuscle myosin heavy chain, is also expressed in podocytes and is associated with glomerular pathophysiology. However, the mechanisms underlying MYH9-related glomerular diseases associated with proteinuria are poorly understood. Therefore, we investigated the role and mechanism of MYH9 in diabetic kidney injury. MYH9 expression was decreased in glomeruli from diabetic patients and animals and in podocytes treated with Ang II in vitro. Ang II treatment and siRNA-mediated MYH9 knockdown in podocytes resulted in actin cytoskeleton reorganization, reduced cell adhesion, actin-associated protein downregulation, and increased albumin permeability. Ang II treatment increased NOX4 expression and ROS generation. The Ang II receptor blocker losartan and the ROS scavenger NAC restored MYH9 expression in Ang II-treated podocytes, attenuated disrupted actin cytoskeleton and decreased albumin permeability. Furthermore, MYH9 overexpression in podocytes restored the effects of Ang II on the actin cytoskeleton and actin-associated proteins. Ang II-mediated TRPC6 activation reduced MYH9 expression. These results suggest that Ang II-mediated MYH9 depletion in diabetic nephropathy may increase filtration barrier permeability by inducing structural and functional podocyte injury through TRPC6-mediated Ca2+ influx by NOX4-mediated ROS generation. These findings reveal a novel MYH9 function in maintaining urinary filtration barrier integrity. MYH9 may be a potential target for treating diabetic nephropathy.

WNK1 promotes renal tumor progression by activating TRPC6-NFAT pathway.

Deregulation of Ca2+ signaling has been regarded as one of the key features of cancer progression. Lysine-deficient protein kinase 1 (WNK1), a major regulator of renal ion transport, regulates Ca2+ signaling through stimulating the phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to activate Gαq-coupled receptor/PLC-β signaling. However, the contribution of WNK1-mediated Ca2+ signaling in the development of clear-cell renal-cell carcinoma (ccRCC) is yet unknown. We found that the canonical transient receptor potential channel (TRPC)6 was widely expressed in ccRCC tissues and functioned as a primary Ca2+ influx mechanism. We further identified that the expressions of WNK1, PI4KIIIα, TRPC6, and the nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were elevated in the tumor tissues compared with the adjacent normal tissues. WNK1 expression was directly associated with the nuclear grade of ccRCC tissues. Functional experiments showed that WNK1 activated TRPC6-mediated Ca2+ influx and current by stimulating PI4KIIIα. Notably, the inhibition of WNK1-mediated TRPC6 activation and its downstream substrate calcineurin attenuated NFATc1 activation and the subsequent migration and proliferation of ccRCC. These findings revealed a novel perspective of WNK1 signaling in targeting the TRPC6-NFATc1 pathway as a therapeutic potential for renal-cell carcinoma.-Kim, J.-H., Hwang, K.-H., Eom, M., Kim, M., Park, E. Y., Jeong, Y., Park, K.-S., Cha, S.-K. WNK1 promotes renal tumor progression by activating TRPC6-NFAT pathway.

How the calcium influx and homeostasis might be disrupted in the Purkinje cell degeneration and cerebellar dysfunction?

In cerebellar circuitry the Purkinje cells (PCs) are the most prominent neuronal element and are characterized by thick and highly branched dendritic tree. The signaling molecule PKCγ is critically linked to Purkinje cell function and has a strong negative impact on the dendritic growth and development of PCs. Recent evidence links enhanced PKCγ to long‐term alteration in calcium equilibrium in PCs. Observations also show that blocking the voltage gated calcium channels could partially or completely protect Purkinje cell dendrites from stunted growth thought to be associated with enhanced PKCγ activity. Additional evidence from moon walker mutant mice also link the increased calcium influx through TRPC3 channels to dampened dendritic morphology. In this study we investigate that how the calcium influx and homoeostasis is regulated in PCs? Here, we propose an integrated model of calcium equilibrium in PCs, by converging the mGluR1 induced membrane generation of DAG and calcium influx through TRPC3 channels into an integrated signaling cascade. The model proposed here link the DAG generation after membrane depolarization to PKCγ activation as well as the activation of TRPC3 through direct binding of DAG. This proposed signaling cascade suggests that DAG signaling in Purkinje cells is tightly regulated through a double‐negative feed‐back loop. The results show that DAG‐ PKCγ‐DGKγ axis forms the first negative feedback loop whereas, the DAG‐TRPC3‐PKCγ‐DGKγ axis forms the second negative feedback loop where, both PKCγ and DGKγ molecules in second negative feedback loop are contributing due to Ca+2 influx through TRPC3 channel, which in turn, stimulates the cytosol‐to‐membrane translocation of these molecules in PCs. Our results suggest that for wild‐type case the double negative feedback on DAG and subsequently on TRPC3 channel is exquisite and specific enough to maintain the calcium homeostasis in PCs, thus ensuring their viability. However, for PKCγ with enhanced activity the second double negative feedback loop is not able to tightly regulate the gating characteristics of TRPC3 channels thus disrupting the calcium homeostasis and leading to degeneration and dysfunction of PCs.Support or Funding InformationPrivate FundingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The activity of transient receptor potential channel C-6 modulates the differentiation of fat cells.

Previously, the V1-3 isoforms of the transient receptor potential channel (TRP) have been shown to promote or prevent adipocyte differentiation. In the current study, the C isoforms were screened for blocking adipogenesis. The hypothesis that the TRP classic or canonical (TRPC) deters adipocyte differentiation was investigated in 3T3-L1 cells employing the channel-specific activator and antagonist, silencing, and overexpression techniques. Fat accumulation in cells was visualized by Oil Red O staining. Intracellular calcium inflow was estimated by confocal microscopy. A high-fat (HF) feeding study was also performed on C57BL/6J mice to verify the findings in the cell model. Among the 6 C isoforms tested, only TRPC-6 inhibited the differentiation of fat cells. The phytochemical quercetin induced the channel protein expression. Calcium-imaging results also revealed that the flavonoid could trigger calcium inflow. Coadministration of quercetin (1 or 20 mg/kg body weight) in an HF diet prevented TRPC-6 from declining and attenuated phosphorylated (p)-PKB and PI3k, as well as the proliferation of visceral fat cells. The present study illustrated that TRPC-6 activation could perturb adipocyte differentiation. The food flavonoid quercetin was a TRPC-6 inducer and activator and it could prevent adipogenesis in mice.-Tan, Y. Q., Kwan, H. Y., Yao, X., Leung, L. K. The activity of transient receptor potential channel C-6 modulates the differentiation of fat cells.

Structural and Functional Analyses of TRPC3 Reveal Allosteric Gating Modulation by the Cytoplasmic Domain

TRPC3 is associated with neurodegenerative diseases, memory loss, and hypertension. In blood vessels, TRPC3 is an essential component of the cellular mechanisms by which vasoconstrictors regulate blood pressure. Vasoconstrictors, such as angiotensin II, bind to phospholipase C (PLC)-coupled receptors, increasing the intracellular levels of DAG, which in turn activates TRPC3. Upon activation, TRPC3 channels induce depolarization increases intracellular Ca2+and promotes vasoconstriction. Here, we show the full-length and cytoplasmic domain (CPD) cryoEM structures for human TRPC3 in the apo state at 5.8 and 4.0 Å resolution. A TRPC3 subunit consists of an N-terminus formed by four ankyrin repeats, the linker helical domain (LHD1-9), and the pre-S1 elbow. The N-terminus is followed by six membrane-spanning helices (S1-S6) and a pore helix connecting to a re-entrant loop that forms the selectivity filter. The TRP helix is immediately adjacent to S6, and the C-terminus consists of two alpha-helices connected by a small linker. TRPC3 transmembrane domain resembles those of other TRP channels, whereas the CPD features major differences. A C-terminal domain swap occurs at the center of the CPD, where horizontal helices (HH) transition into a coiled-coil bundle, highlighting a unique fold for the TRPC subfamily. Comparison of TRPC3 structures reveals that the HH can reside in two distinct positions. Electrophysiological analyses demonstrate that TRPC3 activity increases by shortening the length of the C-terminal loop connecting the HH with the TRP helices; likewise elongating the length of the loop has the opposite effect. Our findings demonstrate that the C-terminal loop impacts channel gating by altering the allosteric coupling between the cytoplasmic and transmembrane domains. Future functional and structural experiments are underway to validate whether the upward movement of the HHs corresponds to a positive modulation of the TRPC3 gating cycle.

The TRPC6 inhibitor, larixyl acetate, is effective in protecting against traumatic brain injury-induced systemic endothelial dysfunction

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

Lipid-independent control of endothelial and neuronal TRPC3 channels by light.

Lipid-gated TRPC channels are highly expressed in cardiovascular and neuronal tissues. Exerting precise pharmacological control over their activity in native cells is expected to serve as a basis for the development of novel therapies. Here we report on a new photopharmacological tool that enables manipulation of TRPC3 channels by light, in a manner independent of lipid metabolism and with higher temporal precision than lipid photopharmacology. Using the azobenzene photoswitch moiety, we modified GSK1702934A to generate light-controlled TRPC agonists. We obtained one light-sensitive molecule (OptoBI-1) that allows us to exert efficient, light-mediated control over TRPC3 activity and the associated cellular Ca2+ signaling. OptoBI-1 enabled high-precision, temporal control of TRPC3-linked cell functions such as neuronal firing and endothelial Ca2+ transients. With these findings, we introduce a novel photopharmacological strategy to control native TRPC conductances.

Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation.

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

HERV-W env regulates calcium influx via activating TRPC3 channel together with depressing DISC1 in human neuroblastoma cells.

The activation and involvement of human endogenous retroviruses W family envelope gene (HERV-W env, also called ERVWE1) have been reported in several neuropsychiatric disorders, including schizophrenia, as well as in multiple sclerosis (MS). Dysregulation of intracellular calcium content is also involved in the pathogenesis of these diseases. Our previous studies showed that HERV-W env overexpression results in activation of small conductance Ca2+-activated K+ channel protein 3 (SK3), a potential risk factor for schizophrenia. In the present study, we aimed to elucidate the relationship between HERV-W env and calcium signaling in schizophrenia. Our results showed that HERV-W env could induce Ca2+ influx in two human neuroblastoma cell lines and upregulate the expression and activation of TRPC3 in cells. The abnormal increase in intracellular Ca2+ concentration was inhibited by addition of the TRPC3 channel blocker pyr3, demonstrating that the Ca2+ influx induced by HERV-W env was TRPC3-dependent. Further experiments showed that HERV-W env overexpression downregulated DISC1, while knockdown of DISC1 promoted activation of TRPC3 without affecting TRPC3 expression. In conclusion, HERV-W env induced Ca2+ influx in human neuroblastoma cells by activating the TRPC3 channel through directly regulating its expression or downregulating DISC1, which could also increase TRPC3 activation without affecting TRPC3 expression. These findings provide new insights into how HERV-W env affects neuronal activity and contributes to the pathogenesis of schizophrenia.

Structure–function analyses of the ion channel TRPC3 reveal that its cytoplasmic domain allosterically modulates channel gating

The transient receptor potential ion channels support Ca2+ permeation in many organs, including the heart, brain, and kidney. Genetic mutations in transient receptor potential cation channel subfamily C member 3 (TRPC3) are associated with neurodegenerative diseases, memory loss, and hypertension. To better understand the conformational changes that regulate TRPC3 function, we solved the cryo-EM structures for the full-length human TRPC3 and its cytoplasmic domain (CPD) in the apo state at 5.8- and 4.0-Å resolution, respectively. These structures revealed that the TRPC3 transmembrane domain resembles those of other TRP channels and that the CPD is a stable module involved in channel assembly and gating. We observed the presence of a C-terminal domain swap at the center of the CPD where horizontal helices (HHs) transition into a coiled-coil bundle. Comparison of TRPC3 structures revealed that the HHs can reside in two distinct positions. Electrophysiological analyses disclosed that shortening the length of the C-terminal loop connecting the HH with the TRP helices increases TRPC3 activity and that elongating the length of the loop has the opposite effect. Our findings indicate that the C-terminal loop affects channel gating by altering the allosteric coupling between the cytoplasmic and transmembrane domains. We propose that molecules that target the HH may represent a promising strategy for controlling TRPC3-associated neurological disorders and hypertension.

Mechanisms underlying modulation of podocyte TRPC6 channels by suPAR: Role of NADPH oxidases and Src family tyrosine kinases

The soluble urokinase receptor (suPAR) has been implicated in the pathogenesis of chronic kidney diseases (CKD) and may function as a circulating “permeability factor” driving primary focal and segmental glomerulosclerosis (FSGS). Here we examined the mechanisms whereby suPAR causes mobilization and increased activation of Ca2+-permeable TRPC6 channels, which are also implicated in FSGS. Treatment of immortalized mouse podocytes with recombinant suPAR for 24 h caused a marked increase in cytosolic reactive oxygen species (ROS) that required signaling through integrins. This effect was associated with increased assembly of active cell surface NADPH oxidase 2 (Nox2) complexes and was blocked by the Nox2 inhibitor apoycynin. Treatment with suPAR also evoked a functionally measurable increase in TRPC6 channels that was blocked by concurrent treatment with the ROS-quencher TEMPOL as well as by inhibition of Rac1, an essential component of active Nox2 complexes. Elevated ROS evoked by exposing cells to suPAR or H2O2 caused a marked increase in the abundance of tyrosine-phosphorylated proteins including Src, and suPAR-evoked Src activation was blocked by TEMPOL. Moreover, mobilization and increased activation of TRPC6 by suPAR or H2O2 was blocked by concurrent exposure to PP2, an inhibitor of Src family tyrosine kinases. These data suggest that suPAR induces oxidative stress in podocytes that in turn drives signaling through Src family kinases to upregulate TRPC6 channels. The combination of oxidative stress and altered Ca2+ signaling may contribute to loss of podocytes and progression of various forms of CKD.

Permeation and Rectification in Canonical Transient Receptor Potential-6 (TRPC6) Channels.

Transient receptor potential-6 channels are widely expressed cation channels that play a role in regulating Ca2+ dynamics, especially during G protein-coupled receptor signaling. The permeation of cations through TRPC6 is complex and the relative permeability to Ca2+ relative to monovalent cations appears to be highly voltage-dependent and is reduced upon membrane depolarization. Many investigators have observed complex current-voltage (I-V) relationships in recordings of TRPC6 channels, which often manifest as flattening of I-V curves between 0 and +40 mV and negative to -60 mV. These features are especially common in recordings from TRPC6 channels expressed in heterologous expression systems. Indeed, it is sometimes argued that marked rectification at both negative and positive membrane potentials is a defining feature of TRPC6, and that recordings in which these features are reduced or absent cannot reflect activity of TRPC6. Here we present a review of the literature to show that complex rectification is not seen in every cell type expressing TRPC6, even when comparing recordings made from the same groups of investigators, or in recordings from what is nominally the same heterologous expression system. Therefore other criteria, such as gene knockout or knockdown, or the use of newly emerging selective blockers, must be used to ascertain that a given current reflects activity of endogenously expressed TRPC6 channels. We also discuss the possibility that complex rectification may not be an intrinsic property of TRPC6 in cells where it is observed, and may instead reflect presence of endogenous substances that cause voltage-dependent inhibition of the channels.