TRPC6 Research Articles

Control of PTH secretion by the TRPC1 ion channel.

Familial hypocalciuric hypercalcemia (FHH) is a genetic condition associated with hypocalciuria, hypercalcemia, and, in some cases, inappropriately high levels of circulating parathyroid hormone (PTH). FHH is associated with inactivating mutations in the gene encoding the Ca2+-sensing receptor (CaSR), a GPCR, and GNA11 encoding G protein subunit α 11 (Gα11), implicating defective GPCR signaling as the root pathophysiology for FHH. However, the downstream mechanism by which CaSR activation inhibits PTH production/secretion is incompletely understood. Here, we show that mice lacking the transient receptor potential canonical channel 1 (TRPC1) develop chronic hypercalcemia, hypocalciuria, and elevated PTH levels, mimicking human FHH. Ex vivo and in vitro studies revealed that TRPC1 serves a necessary and sufficient mediator to suppress PTH secretion from parathyroid glands (PTGs) downstream of CaSR in response to high extracellular Ca2+ concentration. Gα11 physically interacted with both the N- and C-termini of TRPC1 and enhanced CaSR-induced TRPC1 activity in transfected cells. These data identify TRPC1-mediated Ca2+ signaling as an essential component of the cellular apparatus controlling PTH secretion in the PTG downstream of CaSR.

TRPC3-Based Protein Signaling Complex as a Therapeutic Target of Myocardial Atrophy.

Transient receptor potential (TRP) channels, especially canonical TRP channel subfamily members 3 (TRPC3) and 6 (TRPC6), have gained attention as a putative therapeutic target of heart failure. Moreover, TRPC3 and TRPC6 channels are physiologically important for maintaining cellular homeostasis. How TRPC3/C6 channels alter intracellular signaling from adaptation to maladaptation, has been discussed for many years. We have recently shown that the formation of a protein signal complex between TRPC3 and NADPH oxidase (Nox) 2 caused by environmental stresses (e.g., hypoxia, nutritional deficiency, and anti-cancer drug treatment) promotes Nox2-dependent reactive oxygen species production and cardiac stiffness, including myocardial atrophy and interstitial fibrosis, in rodents. In fact, pharmacological prevention of the TRPC3 -Nox2 protein complex can maintain cardiac flexibility in mice after anti-cancer drug treatment. In this mini-review, we discuss the relationship between TRPC3/C6 channels and cardiovascular disease, and propose a new therapeutic strategy by focusing on pathology-specific protein-protein interactions.

Structure‐Function Relationship for Spermine‐mediated Inward Rectification of Transient Receptor Potential Canonical (TRPC) 4 Channels.

Transient Receptor Potential Canonical (TRPC) channels constitute calcium‐permeable, non‐selective cation channels in mammalian cells. The expression of TRPC4 channels can be found in gastrointestinal (GI) tract, endometrium, ventricular myocytes, vascular smooth muscle cells, endothelium and many others. In such tissues, it is well‐known that TRPC4 channels exert membrane excitatory effect due to inwardly‐rectifying nature of current‐voltage (I‐V) relationship of the channels. Based on the electrophysiological similarity between inwardly‐rectifying potassium channel and TRPC4 channel, we investigated the molecular mechanism of inward‐rectification of TRPC4 channels, especially by intracellular magnesium and polyamine, which is two major cytoplasmic blocking agent of IRKs.Based on the pre‐established theory for inward‐rectification of IRKs, we have constructed a number of mutant TRPC4 channels whose acidic amino acid (glutamate or aspartate) at cytoplasmic side (C‐terminus) was changed to alanine. Such mutant channels were overexpressed in HEK293 cells and the activity and efficacy of cytoplasmic blocking action of spermine was measured using whole‐cell patch‐clamp technique. Structural analysis was based on published Cryo‐EM structure of mouse TRPC4 channels (PDB Accession Number: 5z96). The modeled 3D structures of TRPC4 mutants were visualized and analyzed using the PyMOL software. For the calculation of electrical distance, two‐barrier model with one reaction‐intermediate potential was conveyed.As a result, the electrical distance of spermine demonstrated that spermine barely penetrates into the pore but rather exerts its action on the entrance of putative inner vestibule of the channel. The voltage‐dependency of Kd value was not significant compared to other well‐known blocking agents such as proton in Na channels and polyamine in IRKs. Among various mutant channels, D629A, E648A, E728A and E729A channels showed significant recovery from spermine block, demonstrating that such residues are crucial for spermine‐mediated inward rectification. Identification of such residues in Cryo‐EM structure of TRPC4 channel showed rather consistent structural implication for residues D629, E728 and E729; they all lined inner vestibule of the channel. E648 is located at the connecting‐helix of the channel and faces the into cytoplasmic face. Hence we postulated that the residue may contribute to the inward‐rectification by reducing access resistance of spermine from channel periphery into inner vestibule. The location of residues fitted well with electrical distance.Support or Funding Information*This work was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology), and Future Planning (MSIP) of the Korean Government (2018R1A41023822 to I. So) and by the Education and Research Encouragement Fund of Seoul National University Hospital (I. So). Christine Haewon Park was supported by the BK plus program from the MSIP.

Regulation of transient receptor potential canonical 4 activity by phospholipase C δ1

Transient receptor potential canonical (TRPC)4 and TRPC5 channels are non‐selective calciumpermeable cation channels that maintained by phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) and inactivated due to its depletion. Phospholipase C (PLC) is an enzyme that cleaves phospholipids and the PLCδ is the most calcium‐sensitive form among the isozymes that stimulated by physiological concentration of Ca2+. Here, we investigated the regulation mechanism of TRPC4 by the Ca2+‐PLCδ‐PI(4,5)P2 signaling cascade. In order to identify the interaction between ion channel and PLCδ protein, we implied co‐immunoprecipitation and fluorescence imaging including Föster Resonance Energy Transfer. We evaluated the activity of channels with electrophysiological recording in HEK293 cells expressing TRPC channels. Here, we demonstrate that TRPC4 directly interacts with PLCδ1. We also found that PLCδ isozymes are activated by the calcium through opened channels but not by the cytosolic calcium increase. Our study established the PLCδ1 inhibits overall TRPC4’s currents activity, but mainly regulates the basal TRPC4 currents to have a characteristic low basal activity. These regulation of PLCδ1 on TRPC4 activity occurs depend on PI(4,5)P2 hydrolysis. Resultantly, PLCδ1 is considered to be a negative regulator of TRPC4.Support or Funding InformationThis research was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology), and Future Planning (MSIP) of the Korean government (2018R1A4A1023822 to I.S.S.). H.Kang, J.Ko., J.Myeong., and M.Kwak. were supported by the BK plus program from the MSIP.

Cellular mechanisms underlying the rapid depolarization caused by oxygen and glucose deprivation in layer III pyramidal cells of the somatosensory cortex

Cortical pyramidal neurons show rapid and irreversible membrane depolarization in response to oxygen-glucose depolarization (OGD). In this study, we investigated cellular mechanisms responsible for rapid depolarization caused by OGD in layer III pyramidal neurons of the mouse somatosensory cortex. When OGD solution was perfused in the presence of Ca2+ chelator and inhibitors of ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in the pipette solution or in the presence of inhibitors of NMDA receptors (NMDARs), voltage-gated Ca2+ channels (VGCCs), and canonical transient receptor potential (TRPC) channels in the perfusion solution, the latency of the rapid depolarization was significantly prolonged compared to the control. In addition, when OGD solution was perfused in the presence of scavengers of nitric oxide and reactive oxygen species in the perfusion solution or in the presence of calcineurin inhibitors in the pipette solution, the latency of the rapid depolarization was significantly prolonged compared to the control. These data indicate that OGD-induced intracellular Ca2+ increases mediated by Ca2+ influx through NMDARs, VGCCs and TRPC channels as well as by Ca2+ release from RyRs and IP3Rs lead to mitochondrial impairment, which may facilitate the generation of the rapid depolarization via dysfunction of Na+-K+-ATPase due to decreased ATP production.

Canonical Transient Receptor Potential (TRPC) Channels in Nociception and Pathological Pain.

Chronic pathological pain is one of the most intractable clinical problems faced by clinicians and can be devastating for patients. Despite much progress we have made in understanding chronic pain in the last decades, its underlying mechanisms remain elusive. It is assumed that abnormal increase of calcium levels in the cells is a key determinant in the transition from acute to chronic pain. Exploring molecular players mediating Ca2+ entry into cells and molecular mechanisms underlying activity-dependent changes in Ca2+ signaling in the somatosensory pain pathway is therefore helpful towards understanding the development of chronic, pathological pain. Canonical transient receptor potential (TRPC) channels form a subfamily of nonselective cation channels, which permit the permeability of Ca2+ and Na+ into the cells. Initiation of Ca2+ entry pathways by these channels triggers the development of many physiological and pathological functions. In this review, we will focus on the functional implication of TRPC channels in nociception with the elucidation of their role in the detection of external stimuli and nociceptive hypersensitivity.

Novel Targets for Stroke Therapy: Special Focus on TRPC Channels and TRPC6.

Stroke remains a leading cause of death, disability, and medical care burden worldwide. However, transformation from laboratory findings toward effective pharmacological interventions for clinical stroke has been unsatisfactory. Novel evidence has been gained on the underlying mechanisms and therapeutic potential related to the transient receptor potential (TRP) channels in several disorders. The TRP superfamily consists of a diverse group of Ca2+ permeable non-selective cation channels. In particular, the members of TRP subfamilies, TRP canonical (TRPC) channels and TRPC6, have been found in different cell types in the whole body and have high levels of expression in the central nervous system (CNS). Notably, the TRPCs and TRPC6 channel have been implicated in neurite outgrowth and neuronal survival during normal development and in a range of CNS pathological conditions. Recent studies have shown that suppression of TRPC6 channel degradation prevents ischemic neuronal cell death in experimental stroke. Accumulating evidence supports the important functions of TRPC6 in brain ischemia. We have highlighted some crucial advancement that points toward an important involvement of TRPCs and TRPC6 in ischemic stroke. This review will make an overview of the TRP and TRPC channels due to their roles as targets for clinical trials and CNS disorders. Besides, the primary goal is to discuss and update the critical role of TRPC6 channels in stroke and provide a promising target for stroke prevention and therapy.

Transient Receptor Potential Canonical (TRPC) Channels as Modulators of Migration and Invasion.

Calcium (Ca2+) is perhaps the most versatile signaling molecule in cells. Ca2+ regulates a large number of key events in cells, ranging from gene transcription, motility, and contraction, to energy production and channel gating. To accomplish all these different functions, a multitude of channels, pumps, and transporters are necessary. A group of channels participating in these processes is the transient receptor potential (TRP) family of cation channels. These channels are divided into 29 subfamilies, and are differentially expressed in man, rodents, worms, and flies. One of these subfamilies is the transient receptor potential canonical (TRPC) family of channels. This ion channel family comprises of seven isoforms, labeled TRPC1–7. In man, six functional forms are expressed (TRPC1, TRPC3–7), whereas TRPC2 is a pseudogene; thus, not functionally expressed. In this review, we will describe the importance of the TRPC channels and their interacting molecular partners in the etiology of cancer, particularly in regard to regulating migration and invasion.

Role of the TRPC1 Channel in Hippocampal Long-Term Depression and in Spatial Memory Extinction.

Group I metabotropic glutamate receptors (mGluR) are involved in various forms of synaptic plasticity that are believed to underlie declarative memory. We previously showed that mGluR5 specifically activates channels containing TRPC1, an isoform of the canonical family of Transient Receptor Potential channels highly expressed in the CA1-3 regions of the hippocampus. Using a tamoxifen-inducible conditional knockout model, we show here that the acute deletion of the Trpc1 gene alters the extinction of spatial reference memory. mGluR-induced long-term depression, which is partially responsible for memory extinction, was impaired in these mice. Similar results were obtained in vitro and in vivo by inhibiting the channel by its most specific inhibitor, Pico145. Among the numerous known postsynaptic pathways activated by type I mGluR, we observed that the deletion of Trpc1 impaired the activation of ERK1/2 and the subsequent expression of Arc, an immediate early gene that plays a key role in AMPA receptors endocytosis and subsequent long-term depression.

Expression of TRPC3 in cortical lesions from patients with focal cortical dysplasia

Focal cortical dysplasia (FCD) is one of the main causes of medically intractable epilepsy. Some studies have reported that transient receptor potential canonical channel 3 (TRPC3) may play an important role in the occurrence of seizures. In this study, we investigated the expression patterns of TRPC3 in different types of FCD. Forty-five FCD specimens and 12 control samples from autopsies were used in our study. Western blotting, immunohistochemistry, and immunofluorescence staining were employed to detect protein expression and distribution. The amount of TRPC3 protein was markedly elevated in the FCD group. The immunohistochemistry results revealed that TRPC3 staining was strong in the malformed cells and microcolumns. Most of the TRPC3-positive cells were colabeled with glutamatergic and GABAergic markers. The overexpression and altered cellular distribution of TRPC3 in the FCD samples suggest that TRPC3 may be related to epileptogenesis in FCD.

Upregulation of TRPC6 Mediated by PAX6 Hypomethylation Is Involved in the Mechanical Allodynia Induced by Chemotherapeutics in Dorsal Root Ganglion.

BackgroundAlthough the action mechanism of antineoplastic agents is different, oxaliplatin, paclitaxel, or bortezomib as first-line antineoplastic drugs can induce painful neuropathy. In rodents, mechanical allodynia is a common phenotype of painful neuropathy for 3 chemotherapeutics. However, whether there is a common molecular involved in the different chemotherapeutics-induced painful peripheral neuropathy remains unclear.MethodsMechanical allodynia was tested by von Frey hairs following i.p. injection of vehicle, oxaliplatin, paclitaxel, or bortezomib in Sprague-Dawley rats. Reduced representation bisulfite sequencing and methylated DNA immunoprecipitation were used to detect the change of DNA methylation. Western blot, quantitative polymerase chain reaction, chromatin immunoprecipitation, and immunohistochemistry were employed to explore the molecular mechanisms.ResultsIn 3 chemotherapeutic models, oxaliplatin, paclitaxel, or bortezomib accordantly upregulated the expression of transient receptor potential cation channel, subfamily C6 (TRPC6) mRNA and protein without affecting the DNA methylation level of TRPC6 gene in DRG. Inhibition of TRPC6 by using TRPC6 siRNA (i.t., 10 consecutive days) relieved mechanical allodynia significantly following application of chemotherapeutics. Furthermore, the downregulated recruitment of DNA methyltransferase 3 beta (DNMT3b) at paired box protein 6 (PAX6) gene led to the hypomethylation of PAX6 gene and increased PAX6 expression. Finally, the increased PAX6 via binding to the TPRC6 promoter contributes to the TRPC6 increase and mechanical allodynia following chemotherapeutics treatment.ConclusionsThe TRPC6 upregulation through DNMT3b-mediated PAX6 gene hypomethylation participated in mechanical allodynia following application of different chemotherapeutic drugs.

TRPC6-dependent Ca2+ signaling mediates airway inflammation in response to oxidative stress via ERK pathway

Ozone (O3) plays an extremely important role in airway inflammation by generating reactive oxygen species (ROS) including hydrogen peroxide, then promoting redox actions and causing oxidative stress. Evidences indicate that TRPC6 (canonical transient receptor potential channel 6) is a redox-regulated Ca2+ permeable nonselective cation channel, but its role in the setting of oxidative stress-related airway inflammation remains unknown. Here, we found that both TRPC6−/− mice and mice pretreated with SAR7334, a potent TRPC6 inhibitor, were protected from O3-induced airway inflammatory responses. In vitro, both knockdown of TRPC6 expression with shRNA and TRPC6 blockage markedly attenuated the release of cytokines IL-6 and IL-8 induced by O3 or H2O2 in 16HBE cells (human bronchial epithelial cell line). Treatment with O3 or H2O2 enhanced TRPC6 protein expression in vivo and vitro. We also observed that TRPC6-dependent increase of intracellular Ca2+ concentration ([Ca2+]i) was triggered by H2O2, which consisted of the release from intracellular calcium store and the influx of extracellular Ca2+ and could be further strengthened by 6-h O3 exposure in both 16HBE cells and HBEpiCs (primary human bronchial epithelial cells). Moreover, we confirmed that the activation of MAPK signals (ERK1/2, p38, JNK) was required for the inflammatory response induced by O3 or H2O2 while only the phosphorylation of ERK pathway was diminished in the TRPC6-knockdown situation. These results demonstrate that oxidative stress regulates TRPC6-mediated Ca2+ cascade, which leads to the activation of ERK pathway and inflammation and could become a potential target to treat oxidative stress-associated airway inflammatory diseases.

Corrigendum] Cortex Mori Radicis Extract promotes neurite outgrowth of diabetic rats by activating PI3K/AKT signaling and inhibiting Ca2+influx associated with upregulation of transient receptor potential canonical channel1.

Subsequently to the publication of the above paper, the authors have realized that Fig.6E and F contained errors. Essentially, some of the data shown in Fig.6E were incorrect, and consequently, the quantification of the data illustrated in Fig.6F were likewise incorrect. The corrected version of Fig.6, showing the correct data for these figure parts, is shown opposite. Note that these errors in the data selection for this figure did not seriously affect the overall conclusions reported in the study. The authors are grateful to the Editor for allowing them the opportunity to publish this Corrigendum, and wish to apologize to the readership of the Journal for any inconvenience caused. [the original article was published in Molecular Medicine Reports21: 320‑328, 2020; DOI:10.3892/mmr.2019.10839].

Light-Mediated Control over TRPC3-Mediated NFAT Signaling.

Canonical transient receptor potential (TRPC) channels were identified as key players in maladaptive remodeling, with nuclear factor of activated T-cells (NFAT) transcription factors serving as downstream targets of TRPC-triggered Ca2+ entry in these pathological processes. Strikingly, the reconstitution of TRPC-NFAT signaling by heterologous expression yielded controversial results. Specifically, nuclear translocation of NFAT1 was found barely responsive to recombinant TRPC3, presumably based on the requirement of certain spatiotemporal signaling features. Here, we report efficient control of NFAT1 nuclear translocation in human embryonic kidney 293 (HEK293) cells by light, using a new photochromic TRPC benzimidazole activator (OptoBI-1) and a TRPC3 mutant with modified activator sensitivity. NFAT1 nuclear translocation was measured along with an all-optical protocol to record local and global Ca2+ pattern generated during light-mediated activation/deactivation cycling of TRPC3. Our results unveil the ability of wild-type TRPC3 to produce constitutive NFAT nuclear translocation. Moreover, we demonstrate that TRPC3 mutant that lacks basal activity enables spatiotemporally precise control over NFAT1 activity by photopharmacology. Our results suggest tight linkage between TRPC3 activity and NFAT1 nuclear translocation based on global cellular Ca2+ signals.

Role of Transient Receptor Potential Canonical Channels in Heart Physiology and Pathophysiology.

Transient receptor potential canonical (TRPC) channels are involved in the regulation of cardiac function under (patho)physiological conditions and are closely associated with the pathogenesis of cardiac hypertrophy, arrhythmias, and myocardial infarction. Understanding the molecular mechanisms and the regulatory pathway/locus of TRPC channels in related heart diseases will provide potential new concepts for designing novel drugs targeting TRPC channels. We will present the properties and regulation of TRPC channels and their roles in the development of various forms of heart disease. This article provides a brief review on the role of TRPC channels in the regulation of myocardial function as well as how TRPC channels may serve as a therapeutic target in heart failure and cardiac arrhythmias including atrial fibrillation.

Is the Loss of Trpc2 Gene Function a Real Working Hypothesis for the Emergence of Same-Sex Sexual Behavior in Old World Primates?

Is the Loss of Trpc2 Gene Function a Real Working Hypothesis for the Emergence of Same-Sex Sexual Behavior in Old World Primates?

Transient receptor potential channels TRPC1/TRPC6 regulate lamina cribrosa cell extracellular matrix gene transcription and proliferation

The lamina cribrosa (LC) in glaucoma is with augmented production of extracellular matrix proteins (ECM) and connective tissue fibrosis. Fundamental pathological mechanisms for this fibrosis comprise fibrotic growth factors and oxidative stress. Transient receptor potential canonical channels (TRPC) channels play a key role in ECM fibrosis. Here, we study TRPC expression in glaucomatous LC cells, and investigate the role of TRPC in oxidative stress induced-profibrotic ECM gene transcription and cell proliferation in normal LC cells. Age-matched human LC cells (normal, n = 3 donors; glaucoma, n = 3 donors) were used. Hydrogen peroxide (H2O2, 100 μM), was used to induce oxidative stress in LC cells in the presence or absence of the pan TRPC inhibitor SKF96365 (10 μM) or knockdown of TRPC1/6 with siRNA. After treatments, ECM gene transcription, LC cell viability and proliferation and the phosphorylation of the transcription factor NFATc3, were measured using real time RT-PCR, colorimetric cell counting with the methyl-thiazolyl tetrazolium salt (MTS) assay, and Western immunoblotting, respectively. Results showed that TRPC1/C6 transcript and protein expression levels were significantly (p < 0.05) enhanced in glaucoma LC cells. Both SKF96365 and siRNA-TRPC1/C6 treatments significantly reduced the oxidative stress induced-ECM gene expression (transforming growth factor-β1 (TGFβ1), alpha smooth muscle actin (α-SMA), and collagen type 1A1 (Col1A1)), and cell proliferation in normal and glaucoma LC cells. Also, SKF96365 treatment inhibited the H2O2-induced NFATc3 protein dephosphorylation in LC cells. In conclusion, TRPC1/C6 expression is enhanced in glaucoma LC cells. These channels may contribute to oxidative stress-induced ECM gene transcription and cell proliferation in normal and glaucoma LC cells through Ca2+-NFATc3 signaling pathway mechanism. TRPC1 and TRPC6 channels could be important therapeutic targets to prevent ECM remodeling and fibrosis development in glaucoma optic neuropathy.

Modulation of Calcium Transients in Cardiomyocytes by Transient Receptor Potential Canonical 6 Channels.

Transient receptor potential canonical 6 (TRPC6) channels are non-selective cation channels that are thought to underlie mechano-modulation of calcium signaling in cardiomyocytes. TRPC6 channels are involved in development of cardiac hypertrophy and related calcineurin-nuclear factor of activated T cells (NFAT) signaling. However, the exact location and roles of TRPC6 channels remain ill-defined in cardiomyocytes. We used an expression system based on neonatal rat ventricular myocytes (NRVMs) to investigate the location of TRPC6 channels and their role in calcium signaling. NRVMs isolated from 1- to 2-day-old animals were cultured and infected with an adenoviral vector to express enhanced-green fluorescent protein (eGFP) or TRPC6-eGFP. After 3 days, NRVMs were fixed, immunolabeled, and imaged with confocal and super-resolution microscopy to determine TRPC6 localization. Cytosolic calcium transients at 0.5 and 1 Hz pacing rates were recorded in NRVMs using indo-1, a ratio-metric calcium dye. Confocal and super-resolution microscopy suggested that TRPC6-eGFP localized to the sarcolemma. NRVMs infected with TRPC6-eGFP exhibited higher diastolic and systolic cytosolic calcium concentration as well as increased sarcoplasmic reticulum (SR) calcium load compared to eGFP infected cells. We applied a computer model comprising sarcolemmal TRPC6 current to explain our experimental findings. Altogether, our studies indicate that TRPC6 channels play a role in sarcolemmal and intracellular calcium signaling in cardiomyocytes. Our findings support the hypothesis that upregulation or activation of TRPC6 channels, e.g., in disease, leads to sustained elevation of the cytosolic calcium concentration, which is thought to activate calcineurin-NFAT signaling and cardiac hypertrophic remodeling. Also, our findings support the hypothesis that mechanosensitivity of TRPC6 channels modulates cytosolic calcium transients and SR calcium load.

Involvement of TRPC4 and 5 Channels in Persistent Firing in Hippocampal CA1 Pyramidal Cells

Persistent neural activity has been observed in vivo during working memory tasks, and supports short-term (up to tens of seconds) retention of information. While synaptic and intrinsic cellular mechanisms of persistent firing have been proposed, underlying cellular mechanisms are not yet fully understood. In vitro experiments have shown that individual neurons in the hippocampus and other working memory related areas support persistent firing through intrinsic cellular mechanisms that involve the transient receptor potential canonical (TRPC) channels. Recent behavioral studies demonstrating the involvement of TRPC channels on working memory make the hypothesis that TRPC driven persistent firing supports working memory a very attractive one. However, this view has been challenged by recent findings that persistent firing in vitro is unchanged in TRPC knock out (KO) mice. To assess the involvement of TRPC channels further, we tested novel and highly specific TRPC channel blockers in cholinergically induced persistent firing in mice CA1 pyramidal cells for the first time. The application of the TRPC4 blocker ML204, TRPC5 blocker clemizole hydrochloride, and TRPC4 and 5 blocker Pico145, all significantly inhibited persistent firing. In addition, intracellular application of TRPC4 and TRPC5 antibodies significantly reduced persistent firing. Taken together these results indicate that TRPC4 and 5 channels support persistent firing in CA1 pyramidal neurons. Finally, we discuss possible scenarios causing these controversial observations on the role of TRPC channels in persistent firing.

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.