TRPC6 Research Articles

Placental canonical transient receptor potential channel 6 expression and gestational trimester-specific fine particle air pollution exposure in the ENVIRONAGE birth cohort

INTRODUCTION: Ion channels are indispensable for embryonic development. Transient receptor potential (TRP) channels encompass a family of Ca2+ transporter channels of which canonical channel TRPC6 ...

Resistance to pathologic cardiac hypertrophy and reduced expression of CaV1.2 in Trpc3-depleted mice.

Sustained elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) reprograms cardiovascular cell fate, leading to cellular hypertrophy via Ca(2+)-calmodulin/calcineurin (Cn)/NFAT activation. Accumulating evidence suggests that transient receptor potential canonical (Trpc) channels play important roles in the development of pathologic cardiac hypertrophy. Here, we demonstrated that Trpc3 mediates pathologic cardiac hypertrophy in neurohumoral elevation via direct regulation of CaV1.2 expressions. Elevated PE (phenylephrine) was maintained in mice by continuous infusion using an osmotic pump. Wild-type (WT) mice, but not Trpc3 (-/-) showed a sudden decrease in blood pressure (BP) or death following elevation of BP under conditions of elevated PE. Trpc3 (-/-) mesenteric artery showed decreased PE-stimulated vasoconstriction. Analysis of morphology, function, and pathologic marker expression revealed that PE elevation caused pathologic cardiac hypertrophy in WT mice, which was prevented by deletion of Trpc3. Interestingly, protection by Trpc3 deletion seemed to be a result of reduced cardiac CaV1.2 expressions. Basal and PE induced increased expression of protein and mRNA of CaV1.2 was decreased in Trpc3 (-/-) heart. Accordingly, altered expression of CaV1.2 was observed by knockdown or stimulation of Trpc3 in cardiomyocytes. These findings suggest that Trpc3 is a mediator of pathologic cardiac hypertrophy not only through mediating part of the Ca(2+) influx, but also through control of CaV1.2 expressions.

Upregulation of Transient Receptor Potential Canonical Channels Contributes to Endotoxin-Induced Pulmonary Arterial Stenosis.

BackgroundSeptic shock is a pathologic condition caused by endotoxin-producing bacteria, and often associated with severe pulmonary hypertension. Inflammation is a major systemic response to endotoxin; however, it is unknown whether endotoxin has a direct impact on pulmonary arteries that contributes to pathogenesis of pulmonary hypertension.Material/MethodsRat pulmonary arteries and primary pulmonary arterial smooth muscle cells (PASMCs) were cultured in vitro and treated with lipopolysaccharide (LPS) and blockers of transient receptor potential canonical (TRPC) channels. Neointimal growth and arterial stenosis were observed on cryosections of cultured pulmonary arteries. Proliferation of PASMCs was examined by a WST-1 (water-soluble tetrazolium salt) assay. Expression of TRPC genes in pulmonary arteries and PASMCs were detected and quantified by real-time polymerase chain reaction and Western blotting.ResultsLPS significantly induced neointimal growth and stenosis of pulmonary arteries and promoted proliferation of PASMCs. TRPC channel blockers 2-aminoethoxydiphenyl borate and SKF-96365 inhibited LPS-induced remodeling of pulmonary arteries and PASMC proliferation. Expression of TRPC1/3/4/6 was detected in pulmonary arteries and PASMCs. LPS treatment dramatically increased the expression of TRPC3 and TRPC4 at both messenger RNA and protein levels.ConclusionsLPS stimulates stenosis of pulmonary arteries through enhancement of TRPC-mediated Ca2+ entry into PASMCs, which is caused by upregulation of TRPC3 and TRPC4 channels.

Abstract 296: Loss of Trpc6 Function in Cardiomyocytes Improves Survival and Attenuates Progression of Cardiac Dysfunction After Mi

Ischemic heart disease (IHD) commonly culminates in myocardial infarction (MI), which causes cardiac myocyte death and depressed cardiac pump function. Surviving myocytes can usually maintain pump function by increasing Ca 2+ influx and contractility. However, elevated intracellular Ca 2+ also can activate pathological hypertrophic signaling that promotes cardiac dysfunction and progression into heart failure. Ca 2+ influx through Canonical Transient Receptor Potential (TRPC) channels has been classified as a potential source of hypertrophic signaling. Specifically, TRPC6 gene expression and biological activity is significantly upregulated in cardiac myocytes after MI. Our aim was to determine if TRPC6 loss of function is cardioprotective in MI mouse model. We performed an MI study on cardiac specific dominant negative (dn) TRPC6 transgenic mice that express 3 mutated amino acids (L678A-W680A) in the pore region, which disables TRPC6 channel function. In the course of 6 weeks post MI, dnTRPC6 mice had significantly greater survival (69.2%) than wild-type (WT) mice (47.5%). Cardiac function at 2 weeks post MI was decreased to the same extent in WT and dnTRPC6 mice. Ejection fraction (EF) in WT group was 30.6% vs. 57.1% and in dnTRPC6 EF was 31% vs. 61% (MI vs. sham, respectively). The EF in dnTRPC6 mice 6 weeks post MI suggested attenuation of heart failure progression compared to WT mice (32.5% vs. 26.5%, *P=0.05). Mice in the WT group demonstrated significant elevation in end diastolic and end systolic volumes (EDV and ESV) 6 weeks post MI, while dnTRPC6 had reduced end diastolic and end systolic volumes (116μL vs. 88μL and 106μL vs. 76μL, WT vs. dnTRPC6, EDV and ESV, respectively. ***P<0.0001). Hypertrophy measures of heart weight to body weight ratio and heart weight to tibia length ratio were significantly reduced in dnTRPC6 mice 6 weeks post MI as opposed to WT mice (8.4 vs. 7.3 and 13.4 vs. 11.7, respectively *P<0.05). Hypertrophic markers of ANP, BNP and βMHC followed similar downregulation trend in the dnTRPC6 mice. In conclusion, loss of TRPC6 function slows the progression of cardiac dysfunction and cardiac remodeling in the post MI heart.

Expression of TRPC6 and BDNF in Cortical Lesions From Patients With Focal Cortical Dysplasia.

Focal cortical dysplasia (FCD) likely results from abnormal migration of neural progenitor cells originating from the subventricular zone. To elucidate the roles in molecules that are involved in neural migration pathway abnormalities in FCDs, we investigated the expression patterns of transient receptor potential canonical channel 6 (TRPC6) and brain-derived neurotrophic factor (BDNF) in cortical lesions from FCD patients and in samples of normal control cortex. TRPC6 and BDNF mRNA and protein levels were increased in FCD lesions. By immunohistochemistry, they were strongly expressed in microcolumns, heterotopic neurons, dysmorphic neurons, and balloon cells (BCs). Colocalization assays revealed that most of the misshapen TRPC6-positive or heterotopic cells had a neuronal lineage with the exception of TRPC6-positive FCDiib patient BCs, which had both neuronal and glial features. Most TRPC6-positive cells were glutamatergic neurons. There was also greater expression of calmodulin-dependent kinase IV (CaMKIV), the downstream factor of TRPC6, in FCD lesions, suggesting that TRPC6 expression promoted dendritic growth and the development of dendritic spines and excitatory synapses via the CaMKIV-CREB pathway in FCD. Thus, overexpression of BDNF and TRPC6 and activation of the TRPC6 signal transduction pathway in cortical lesions of FCD patients may contribute to FC pathogenesis and epileptogenesis.

Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels: NEW TARGETS OF ALDOSTERONE IN CARDIOMYOCYTES

Store-operated Ca(2+) entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca(2+) influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca(2+)]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway.

Pyr3, a TRPC3 channel blocker, potentiates dexamethasone sensitivity and apoptosis in acute lymphoblastic leukemia cells by disturbing Ca2+ signaling, mitochondrial membrane potential changes and reactive oxygen species production

Dexamethasone (Dex) is used as a chemotherapeutic drug in the treatment of acute lymphoblastic leukemia (ALL) because of its capacity to induce apoptosis. However, some ALL patients acquire resistance to glucocorticoids (GC). Thus, it is important to explore new agents to overcome GC resistance. The aim of the present work was to assess the ability of Pyr3, a selective inhibitor of transient receptor potential canonical 3 (TRPC3), to sensitize human ALL cells to Dex. We show here, for the first time, that Pyr3 enhances Dex sensitivity through the distraction of Dex-mediated Ca2+ signaling in ALL cells (in vitro) and primary blasts (ex vivo) associated with mitochondrial-mediated reactive oxygen species production in ALL cells. Pyr3 alone induced Ca2+ signaling via only endoplasmic reticulum-released Ca2+ and exerted inhibitory effect on store-operated Ca2+ entry in dose-dependent manner in ALL cell lines. Pre-incubation of cells with Pyr3 significantly curtailed the thapsigargin- and Dex-evoked Ca2+ signaling in ALL cell lines. Pyr3 synergistically potentiated Dex lethality, as shown by the induction of cell mortality, G2/M cell cycle arrest and apoptosis in ALL cell lines. Moreover, Pyr3 disrupted Dex-mediated Ca2+ signaling and increased the sensitivity of Dex-induced cell death in primary blasts from ALL patients. Additional analysis showed that co-treatment with Dex and Pyr3 results in mitochondrial membrane potential depolarization and reactive oxygen species production in ALL cells. Together, Pyr3 exhibited potential therapeutic benefit in combination with Dex to inverse glucocorticoid resistance in human ALL and probably in other lymphoid malignancies.

Klotho May Ameliorate Proteinuria by Targeting TRPC6 Channels in Podocytes.

Klotho is a type-1 membrane protein predominantly produced in the kidney, the extracellular domain of which is secreted into the systemic circulation. Membranous and secreted Klotho protect organs, including the kidney, but whether and how Klotho directly protects the glomerular filter is unknown. Here, we report that secreted Klotho suppressed transient receptor potential channel 6 (TRPC6)-mediated Ca2+ influx in cultured mouse podocytes by inhibiting phosphoinositide 3-kinase-dependent exocytosis of the channel. Furthermore, soluble Klotho reduced ATP-stimulated actin cytoskeletal remodeling and transepithelial albumin leakage in these cells. Overexpression of TRPC6 by gene delivery in mice induced albuminuria, and exogenous administration of Klotho ameliorated the albuminuria. Notably, immunofluorescence and in situ hybridization revealed Klotho expression in podocytes of mouse and human kidney. Heterozygous Klotho-deficient CKD mice had aggravated albuminuria compared with that in wild-type CKD mice with a similar degree of hypertension and reduced clearance function. Finally, disrupting the integrity of glomerular filter by saline infusion-mediated extracellular fluid volume expansion increased urinary Klotho excretion. These results reveal a potential novel function of Klotho in protecting the glomerular filter, and may offer a new therapeutic strategy for treatment of proteinuria.

Lipopolysaccharide potentiates endothelin-1-induced proliferation of pulmonary arterial smooth muscle cells by upregulating TRPC channels.

Lipopolysaccharide (LPS) and endothelin-1 (ET-1) are critical pathogenic factors in sepsis-induced pulmonary hypertension; however it is unknown whether they have a coordinated action in the pathogenesis of this disease. Here we found that although LPS did not change the contractility of rat pulmonary arterial smooth muscle cells (PASMCs) in response to ET-1, it significantly promoted ET-1-induced PASMC proliferation. Measurement of ET-1-evoked Ca(2+) transients in PASMCs showed that LPS dramatically enhanced Ca(2+) influx mediated by transient receptor potential canonical (TRPC) channels. LPS did not directly activate TRPC channels, instead it selectively upregulated the expression of TRPC3 and TRPC4 in pulmonary arteries. Small interfering RNA (siRNA) and chemical blockers against TRPC channels abolished LPS-induced PASMC proliferation. LPS-induced cell proliferation and TRPC expression was mediated by the Ca(2+)-dependent calcineurin/NFAT signaling pathway. We suggest that blocking TRPC channels could be an effective strategy in controlling pulmonary arterial remodeling after endotoxin exposure.

The interaction domains of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heteromultimeric channels

Transient receptor potential canonical (TRPC) family contains a non-selective cation channel, and four TRPC subunits form a functional tetrameric channel. TRPC4/5 channels form not only the homotetrameric channel but also a heterotetrameric channel with TRPC1. We investigated the interaction domain required for TRPC1/4 or TRPC1/5 heteromultimeric channels using FRET and the patch-clamp technique. TRPC1 only localized at the plasma membrane (PM) when it was coexpressed with TRPC4 or TRPC5. The TRPC1/4 or TRPC1/5 heteromultimeric showed the typical outward rectifying I/V curve. When TRPC1 and TRPC4 form a heteromeric channel, the N-terminal coiled-coil domain (CCD) and C-terminal 725–745 region of TRPC1 interact with the N-terminal CCD and C-terminal 700–728 region of TRPC4. However, when TRPC1 and TRPC5 form a heteromeric channel, the N-terminal CCD and C-terminal 673–725 region of TRPC1 interact with the N-terminal CCD and C-terminal 707–735 region of TRPC5. In conclusion, the N-terminal CCD of TRPC channels is essential for the heteromultimeric structure of TRPC channels, whereas specific C-terminal regions are required for unique heteromerization between subgroups of TRPC channels.

Astragaloside IV prevents high glucose-induced podocyte apoptosis via downregulation of TRPC6.

Diabetic nephropathy (DN) is one of the most important causes of end‑stage renal disease. AstragalosideIV (AS-IV) is a saponin isolated from Astragalusmembranaceus, which possesses various pharmacological activities. AS‑IV prevents podocyte apoptosis and ameliorates renal injury in DN; however, few studies have focused on its effects on ion channels. The transient receptor potential channel6(TRPC6) is an important Ca2+‑permeable ion channel in podocytes, which is involved in high glucose (HG)-induced podocyte apoptosis. The aim of the present study was to investigate whether AS‑IV prevented HG‑induced podocyte apoptosis via TRPC6. Cultured podocytes were pre‑treated with 10,20or40µM AS‑IV for 1h prior to HG exposure for 24h. Apoptosis, cell viability, expression of TRPC6, nuclear factor of activated Tcells (NFAT2) and B‑cell lymphoma2‑associated X protein (Bax), as well as the intracellular Ca2+ concentration were subsequently analyzed. The results indicated that HG induced podocyte apoptosis and upregulation of TRPC6, and increased intracellular Ca2+. Furthermore, enhanced NFAT2 and Bax expression was detected. Conversely, AS‑IV protected HG‑induced podocyte apoptosis, downregulated TRPC6 expression and suppressed intracellular Ca2+ in HG-stimulated podocytes. AS‑IV also suppressed NFAT2 and Bax expression. These results suggest that AS‑IV may prevent HG-induced podocyte apoptosis via downregulation of TRPC6, which is possibly mediated via the calcineurin/NFAT signaling pathway.

Transient Receptor Potential Channel 4 Encodes a Vascular Permeability Defect and High-Frequency Ca2+ Transients in Severe Pulmonary Arterial Hypertension

The canonical transient receptor potential channel 4 (TRPC4) comprises an endothelial store-operated Ca(2+) entry channel, and TRPC4 inactivation confers a survival benefit in pulmonary arterial hypertension (PAH). Endothelial Ca(2+) signals mediated by TRPC4 enhance vascular permeability invitro, but the contribution of TRPC4-dependent Ca(2+) signals to the regulation of endothelial permeability in PAH is poorly understood. We tested the hypothesis that TRPC4 increases vascular permeability and alters the frequency of endothelial Ca(2+) transients in PAH. We measured permeability in isolated lungs, and found that TRPC4 exaggerated permeability responses to thapsigargin in Sugen/hypoxia-treated PAH rats. We compared endothelial Ca(2+) activity of wild-type with TRPC4-knockout rats using confocal microscopy, and evaluated how Ca(2+) signals were influenced in response to thapsigargin and sequential treatment with acetylcholine. We found that thapsigargin-stimulated Ca(2+) signals were increased in PAH, and recovered by TRPC4 inactivation. Store depletion revealed bimodal Ca(2+) responses to acetylcholine, with both short- and long-duration populations. Our results show that TRPC4 underlies an exaggerated endothelial permeability response in PAH. Furthermore, TRPC4 increased the frequency of endothelial Ca(2+) transients in severe PAH, suggesting that TRPC4 provides a Ca(2+) source associated with endothelial dysfunction in the pathophysiology of PAH. This phenomenon represents a new facet of the etiology of PAH, and may contribute to PAH vasculopathy by enabling inflammatory mediator fluxacross the endothelial barrier.

Calcium‐sensing Receptor, Coupled with Transient Receptor Potential Canonical Channels 6, Regulates Cytosolic [Ca2+] in the Development and Progression of Pulmonary Hypertension

RationaleElevated pulmonary arterial pressure (PAP) in patients with pulmonary arterial hypertension (PAH) is mainly caused by increased pulmonary vascular resistance (PVR), due primarily to sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMCs) is not only a major trigger for pulmonary vasoconstriction but also an important stimulus for pulmonary vascular remodeling. Our previous data showed that the extracellular calcium sensing receptor (CaSR) expression and function were both enhanced in PASMC from idiopathic PAH patients. However, the implication of CaSR in the development of PAH remains elusive.MethodsCasr double knockout mice (Casr−/−Pth−/−) and transient receptor potential canonical Channels 6 (Trpc6−/−) have been exposed to hypoxia for 4 weeks to develop pulmonary hypertension. Following hypoxic exposure, right ventricle systolic pressures (RVSP), right ventricular hypertrophy by RV/(LV+S) ratios, and small pulmonary artery smooth muscle layer thickness were measured. The PAP was measured using the isolated perfused/ventilated mouse lung system. PASMCs were isolated from patients with IPAH, followed with [Ca2+]cyt measurement, proliferation and migration assays.ResultsIn the current study, we found blockade of TRPC6 channels significantly inhibits Ca2+‐induced increase in [Ca2+]cyt and OAG caused a larger increase in [Ca2+]cyt in IPAH PASMCs. Overexpression of CaSR and TRPC6 channels increase the extracellular Ca2+‐induced [Ca2+]cyt in normal PASMCs. Compared to wild mice, both Casr−/−Pth−/−and Trpc6−/− mice had significantly lower RVSP, less right ventricular hypertrophy, and displayed less pulmonary vascular remodeling after exposure to hypoxia. Deletion of Casr or Trpc6 gene significantly attenuates acute hypoxic vasoconstriction.ConclusionsThese data indicate that CaSR functionally interacts with TRPC6 and regulate [Ca2+]cyt in IPAH‐PASMC. Deletions of CaSR or TRPC6 prevent the development of hypoxia‐induced PAH in mice via its modulation of extracellular Ca2+ entry in PASMC, suggesting that CaSR may be a novel therapeutic candidate for the treatment of PAH.

Synaptopodin Limits TRPC6 Podocyte Surface Expression and Attenuates Proteinuria.

Gain-of-function mutations of classic transient receptor potential channel 6 (TRPC6) were identified in familial FSGS, and increased expression of wild-type TRPC6 in glomeruli is observed in several human acquired proteinuric diseases. Synaptopodin, an actin binding protein that is important in maintaining podocyte function, is downregulated in various glomerular diseases. Here, we investigated whether synaptopodin maintains podocyte function by regulating podocyte surface expression and activity of TRPC6. We show indirect interaction and nonrandom association of synaptopodin and TRPC6 in podocytes. Knockdown of synaptopodin in cultured mouse podocytes increased the expression of TRPC6 at the plasma membrane, whereas overexpression of synaptopodin decreased it. Mechanistically, synaptopodin-dependent TRPC6 surface expression required functional actin and microtubule cytoskeletons. Overexpression of wild-type or FSGS-inducing mutant TRPC6 in synaptopodin-depleted podocytes enhanced TRPC6-mediated calcium influx and induced apoptosis. In vivo, knockdown of synaptopodin also caused increased podocyte surface expression of TRPC6. Administration of cyclosporin A, which stabilizes synaptopodin, reduced LPS-induced proteinuria significantly in wild-type mice but to a lesser extent in TRPC6 knockout mice. Furthermore, administration of cyclosporin A reversed the LPS-induced increase in podocyte surface expression of TRPC6 in wild-type mice. Our findings suggest that alteration in synaptopodin levels under disease conditions may modify intracellular TRPC6 channel localization and activity, which further contribute to podocyte dysfunction. Reducing TRPC6 surface levels may be a new approach to restoring podocyte function.

Spatial memory impairment by TRPC1 depletion is ameliorated by environmental enrichment.

Canonical transient receptor potential (TRPC) channels are widely expressed throughout the nervous system whereas their functions remain largely unclear. Here we investigated the effects of TRPC1 deletion on spatial memory ability of mice and the potential intervention by environmental enrichment (EE). Significant spatial memory impairment assessed by conditional fearing test, Y maze test and step-down test in TRPC1 knockout mice was revealed. The behavioral abnormality were attenuated by the treatment of EE. Proteomic analysis by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with a matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) and tandem mass spectrometry (MS) revealed that TRPC1 deletion caused differential expression of a total of 10 proteins (8 up-regulated and 2 down-regulated) in hippocampus. EE treatment resulted in differential expression of a total of 22 proteins (2 up-regulated and 20 down-regulated) in hippocampus of TRPC1 knockout mice. Among these differentially expressed proteins, the expression of α-internexin and glia maturation factor β (GMF-β), two proteins shown to impair memory, were significantly down-regulated in hippocampus of TRPC1 knockout mice by EE treatment. Taken together, these data suggested that TRPC1 regulated directly or indirectly the expression of multiple proteins, which may be crucial for the maintenance of memory ability, and that EE treatment modulated spatial memory impairment caused by TRPC1 depletion and the mechanisms may involve the modulation of EE on the expression of those dys-regulated proteins such as α-internexin and GMF-β in hippocampus.

Melittin, the Major Pain-Producing Substance of Bee Venom.

Melittin is a basic 26-amino-acid polypeptide that constitutes 40-60% of dry honeybee (Apis mellifera) venom. Although much is known about its strong surface activity on lipid membranes, less is known about its pain-producing effects in the nervous system. In this review, we provide lines of accumulating evidence to support the hypothesis that melittin is the major pain-producing substance of bee venom. At the psychophysical and behavioral levels, subcutaneous injection of melittin causes tonic pain sensation and pain-related behaviors in both humans and animals. At the cellular level, melittin activates primary nociceptor cells through direct and indirect effects. On one hand, melittin can selectively open thermal nociceptor transient receptor potential vanilloid receptor channels via phospholipase A2-lipoxygenase/cyclooxygenase metabolites, leading to depolarization of primary nociceptor cells. On the other hand, algogens and inflammatory/pro-inflammatory mediators released from the tissue matrix by melittin's pore-forming effects can activate primary nociceptor cells through both ligand-gated receptor channels and the G-protein-coupled receptor-mediated opening of transient receptor potential canonical channels. Moreover, subcutaneous melittin up-regulates Nav1.8 and Nav1.9 subunits, resulting in the enhancement of tetrodotoxin-resistant Na(+) currents and the generation of long-term action potential firing. These nociceptive responses in the periphery finally activate and sensitize the spinal dorsal horn pain-signaling neurons, resulting in spontaneous nociceptive paw flinches and pain hypersensitivity to thermal and mechanical stimuli. Taken together, it is concluded that melittin is the major pain-producing substance of bee venom, by which peripheral persistent pain and hyperalgesia (or allodynia), primary nociceptive neuronal sensitization, and CNS synaptic plasticity (or metaplasticity) can be readily induced and the molecular and cellular mechanisms underlying naturally-occurring venomous biotoxins can be experimentally unraveled.

Loss of the trpc4 gene is associated with a reduction in cocaine self-administration and reduced spontaneous ventral tegmental area dopamine neuronal activity, without deficits in learning for natural rewards

Among the canonical transient receptor potential (TRPC) channels, the TRPC4 non-selective cation channel is one of the most abundantly expressed subtypes within mammalian corticolimbic brain regions, but its functional and behavioral role is unknown. To identify a function for TRPC4 channels we compared the performance of rats with a genetic knockout of the trpc4 gene (trpc4 KO) to wild-type (WT) controls on the acquisition of simple and complex learning for natural rewards, and on cocaine self-administration (SA). Despite the abundant distribution of TRPC4 channels through the corticolimbic brain regions, we found trpc4 KO rats exhibited normal learning in Y-maze and complex reversal shift paradigms. However, a deficit was observed in cocaine SA in the trpc4 KO group, which infused significantly less cocaine than WT controls despite displaying normal sucrose SA. Given the important role of ventral tegmental area (VTA) dopamine neurons in cocaine SA, we hypothesized that TRPC4 channels may regulate basal dopamine neuron excitability. Double-immunolabeling showed a selective expression of TRPC4 channels in a subpopulation of putative dopamine neurons in the VTA. Ex vivo recordings of spontaneous VTA dopamine neuronal activity from acute brain slices revealed fewer cells with high-frequency firing rates in trpc4 KO rats compared to WT controls. Since deletion of the trpc4 gene does not impair learning involving natural rewards, but reduces cocaine SA, these data demonstrate a potentially novel role for TRPC4 channels in dopamine systems and may offer a new pharmacological target for more effective treatment of a variety of dopamine disorders.

Inhibition of store-operated calcium entry by sub-lethal levels of proteasome inhibition is associated with STIM1/STIM2 degradation

Dysfunction of the ubiquitin-proteasome system (UPS) and calcium homeostasis has been implicated in the neurodegeneration of Alzheimer’s and Parkinson’s diseases. The cytosolic calcium concentration is maintained by store-operated calcium entry (SOCE), which is repressed by Alzheimer’s disease-associated mutants, such as mutant presenilins. We hypothesized that inhibition of UPS impacts SOCE. This study showed that pretreatment with sub-lethal levels of proteasome inhibitors, including MG-132 and clasto-lactacystin-β-lactone (LA), reduced SOCE after depletion of endoplasmic reticulum calcium in rat neurons. With the same treatment, MG-132 and LA reduced the protein levels of stromal interaction molecule 1and 2 (STIM1/2), but not the levels of Orai1 and canonical transient receptor potential channel 1 (TRPC1). STIM1 or STIM2 protein was mobilized to lysosome by MG-132/LA treatment as observed under an immunofluorescence confocal laser microscope. In the neurons, MG-132 and LA degraded p62/SQSTM1, promoted autophagy, converted LC3I to LC3II, and promoted co-localization of LC3 and lysosomes. Rapamycin, which enhances autophagy, reduced STIM1/2 protein levels, whereas bafilomycin, which inhibits autophagy, increased their protein levels. The protein levels of STIM1/2 and the amplitude of SOCE were decreased in SH-SY5Y with decreased protein level of proteasome subunit beta type-5 induced by shRNA. We conclude that sub-lethal levels of proteasome inhibition reduce SOCE and promote autophagy-mediated degradation of STIM1/2. UPS inhibition, a common finding in neurodegenerative diseases, interferes with calcium homeostasis via repression of SOCE.

Modulation of Neuronal Activity by Synthetic Activators of Lipid-Gated TRPC Channels

Transient receptor potential canonical (TRPC) cation channels are highly expressed in the central nervous system. Neuner et al. showed an inverse correlation between TRPC3 expression/activity and firing of hippocampal neurons. These results lead to the concept of TRPC3 being crucially involved in regulation of hippocampal excitability and constitution of fear memory.A small molecule TRPC3/6 agonist GSK1702934A has recently been introduced as a tool to activate lipid-sensitive TRPC channels directly, bypassing phospholipase C signaling. In an attempt to test the potential and pharmacological value of TRPC3 as a direct pharmacological target for interference with hippocampal functions, we characterized the effects of a series of novel synthetic TRPC3 activators, including molecules that are suitable for optical control of agonist activity. All compounds that proved effective in eliciting TRPC3 conductances in TRPC3 overexpressing, but not in wild type HEK293 cells, significantly reduced firing frequency in hippocampal neurons. Experiments with selective TRPC channel inhibitors as well as TRPC knock down strategies confirmed direct modulation of lipid-sensitive TRPC channels by small molecule activators as a suitable basis for pharmacological and/or optopharmacological interventions to govern neuronal functions.Supported by FWF, DK-MCD.

IL-17 induces MIP-1α expression in primary mouse astrocytes via TRPC channel.

Our previous study demonstrated IL-17-mediated induction of MIP-1α through its binding to the cognate IL-17RA and MIP-1α was involved in astrocyte activation. Transient receptor potential canonical (TRPC) channel was involved in astrocyte activation, however, whether TRPC channel regulates MIP-1α expression in the context of multiple sclerosis (MS) remains largely unknown. In this study we identify the essential role of TRPC channel in IL-17-mediated MIP-1α expression and astrocyte activation. Moreover, treatment of astrocytes with IL-17 activated MAPKs and PI3K/Akt signaling pathways with downstream NF-κB pathways. Interestingly, the TRPC blocker-SKF96365 (10μM) and Norgestimate (10μM) significantly inhibited the increased expression of MIP-1α via suppression of IL-17-mediated ERK, p38 and JNK MAPKs and PI3K/Akt pathway activation, thereby underscoring the role of TRPC channel in this process. Together these data underpin the role of TRPC channel as a novel target that regulates MIP-1α expression and cell activation-mediated by IL-17 with implications for therapeutic intervention for reversal of neuroinflammation inflicted by IL-17. Understanding the regulation of MIP-1α expression may provide insights into the development of potential therapeutic targets for neuroinflammation associated with MS.