Transient Receptor Potential Isoforms Research Articles

TRPC3-mediated NFATc1 calcium signaling promotes triple negative breast cancer migration through regulating glypican-6 and focal adhesion

Canonical transient receptor potential isoform 3 (TRPC3), a calcium-permeable non-selective cation channel, has been reported to be upregulated in breast cancers and a modulator of cell migration. Calcium-sensitive transcription factor NFATc1, which is important for cell migration, was shown to be frequently activated in triple negative breast cancer (TNBC) biopsy tissues. However, whether TRPC3-mediated calcium influx would activate NFATc1 and affect the migration of TNBC cells, and, if yes, the underlying mechanisms involved, remain to be investigated. By immunostaining followed by confocal microscopy, TNBC lines MDA-MB-231 and BT-549 were both found to express TRPC3 on their plasma membrane while ER+ line MCF-7 and HER2+ line SK-BR3 do not. Blockade of TRPC3 by pharmacological inhibitor Pyr3 or stable knockdown of TRPC3 by lentiviral vector both inhibited cell migration as measured by wound healing assay. Importantly, blocking TRPC3 by Pyr3 or knockdown of TRPC3 both caused the translocation of NFATc1 from the nucleus to the cytosol as revealed by confocal microscopy. Interestingly, NFATc1 was found to bind to the promoter of glypican 6 (GPC6) as determined by chromatin immunoprecipitation assay. Consistently, knockdown of TRPC3 decreased the expression of GPC6 as revealed by western blotting. Moreover, long-term knockdown of GPC6 by lentiviral vector also consistently decreased the migration of TNBC cells. Intriguingly, GPC6 proteins physically interact with vinculin in MDA-MB-231 as determined by co-immunoprecipitation. Blockade of TRPC3, knockdown of TRPC3 or knockdown of GPC6 all induced larger, stabilized actin-bound peripheral focal adhesion (FA) formations in TNBC cells as determined by co-staining of actin and vinculin followed by confocal microscopy. These large, stabilized actin-bound peripheral FAs indicated a defective FA turnover, and were reported to be responsible for impairing directed cell migration. Our results suggest that, in TNBC cells, calcium influx through TRPC3 channel positively regulates NFATc1 nuclear translocation and GPC6 expression, which maintains the dynamics of FA turnover and optimal cell migration. Our study reveals a novel TRPC3-NFATc1-GPC6-vinculin signaling cascade in maintaining the migration of TNBC cells.

Soluble Epoxide Hydrolase Inhibitor Prevents Homocysteine‐induced Cardiac Hypertrophy via Epoxyeicosatrienoic Acid–mediated Inhibition of TRPC3 Channels

ObjectivesActivation of soluble epoxide hydrolase (sEH), a key enzyme catalyzing the degradation of epoxyeicosatrienoic acids (EETs), has been implicated in cardiac remodeling. We previously demonstrated the involvement of sEH and transient receptor potential isoform 3 (TRPC3) channels in homocysteine‐induced hypertrophy of cardiac cells. This study investigated the anti‐hypertrophic effect of sEH inhibitor with the aim to reveal the link between sEH/EETs and TRPC3 channels in cardiac hypertrophy associated with hyperhomocysteinemia.Materials and methodsMale Wistar rats on 10‐week standard or 2% (WT/WT) high methionine diet were given drinking water supplemented with or without the specific sEH inhibitor 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl) urea (TPPU, 5mg/L). In vitro, H9c2 cells were treated with homocysteine (100 μmol/L) in the presence or absence of TPPU (10 μmol/L) or 11,12‐EET (1 μmol/L). Cardiac remodeling was evaluated by echocardiography and histological analysis. Plasma homocysteine level and EETs content were determined by ELISA. Hypertrophic markers were detected by western blot and qPCR. TRPC3 channel current was recorded by patch clamp in whole‐cell mode.ResultsTPPU lowered the heart to body weight ratio, inhibited expressions of the hypertrophic markers atrial natriuretic factor (ANP) and β‐myosin heavy chain (β‐MHC), and reduced the size of cardiomyocytes in hyperhomocysteinemic rats. TPPU treatment ameliorated ejection fraction (85.50±0.87% vs. 74.18±2.19%, p<0.05) and fraction shortening (50.30±1.32% vs. 42.27±0.68%, p<0.01) of the hyperhomocysteinemic rats. Rats with hyperhomocysteinemia showed reduced levels of EETs and increased expression of TRPC3 channels, which were reversed by TPPU treatment. In vitro, TPPU and 11,12‐EET inhibited H9c2 cell enlargement induced by homocysteine, along with a downregulation of hypertrophic markers and TRPC3 channels. Homocysteine significantly increased TRPC3 channel current (4.74±0.49 vs. 1.76±0.21 pA/pF in control, p<0.001) and the enhancement was inhibited by TPPU (2.44±0.29 pA/pF, p<0.01).ConclusionsInactivation of sEH alleviates homocysteine‐induced cardiac hypertrophy and improves cardiac function. EET‐mediated inhibition of TRPC3 channels contributes to the protective effect of the sEH inhibitor TPPU.

TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes

BackgroundBiological pacemakers consisting of pluripotent stem cell-derived cardiomyocytes are potentially useful for treating bradycardia. However, tachyarrhythmia caused by derived cardiomyocytes themselves is one of main barriers hampering their clinical translation. An in-depth understanding of the mechanisms underlying the spontaneous action potential (a.k.a. automaticity) might provide potential approaches to solve this problem. The aim of this project is to study the role of canonical transient receptor potential isoform 7 (TRPC7) channels in regulating the automaticity of embryonic stem cell-derived cardiomyocytes (ESC-CMs).Methods and resultsBy Western blotting, the expression of TRPC7 was found to be increased during the differentiation of mouse ESC-CMs (mESC-CMs). Adenovirus-mediated TRPC7 knockdown decreased while overexpression increased the frequency of Ca2+ transients (CaTs), local Ca2+ releases (LCRs), and action potentials (APs) as detected by confocal microscopy and whole-cell patch-clamping. TRPC7 was found to be positively associated with the activity of ryanodine receptor 2 (RyR2), sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), and sodium-calcium exchanger (NCX) but not hyperpolarization-activated, cyclic nucleotide-gated channel (HCN), and inositol trisphosphate receptor (IP3R). Knockdown or overexpression of TRPC7 did not alter the expression of HCN4, Cav1.3, Cav3.1, Cav3.2, IP3R1, RyR2, and SERCA but positively regulated the phosphorylation of RyR2 at S2814 and phospholamban (PLN) at T17. Moreover, the positive regulation of APs by TRPC7 was Ca2+-dependent, as overexpression of N-terminus of TRPC7 (dominant negative of TRPC7) which diminished the Ca2+ permeability of TRPC7 decreased the AP frequency.ConclusionsTRPC7 regulates the automaticity of mESC-CMs through two mechanisms. On the one hand, TRPC7 positively regulates the intracellular Ca2+ clock through the regulation of activities of both RyR2 and SERCA; on the other hand, TRPC7 also positively regulates the membrane clock via its influence on NCX activity. Altogether, our study reveals that TRPC7 is a potential drug target to manipulate the action potential firing rate of pluripotent stem cell-derived cardiomyocyte-based biological pacemakers to prevent tachyarrhythmia, a condition that might be encountered after transplantation.

Association Between Soluble Epoxide Hydrolase and TRPC3 Channels in Pathological Cardiac Hypertrophy

Objectives Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Although previous studies have shown a positive correlation between homocysteine levels and the incidence of cardiac hypertrophy and remodeling, the molecular mechanisms by which homocysteine promotes the development of cardiac hypertrophy remain inadequately understood. In this study, we investigated the role of soluble epoxide hydrolase (sEH) and transient receptor potential isoform 3 (TRPC3) channels in homocysteine-induced cardiac hypertrophy with further elucidating the association between sEH and TRPC3. Methods H9c2 cardiomyocytes were exposed to different concentrations of homocysteine ranging from 25 to 200 μmol/L for different duration. The cell surface area was measured by phase contrast microscope and image analysis software. The mRNA and protein expressions of sEH, TRPC3 channels, and hypertrophic markers beta-smooth muscle myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) were detected by quantitative polymerase chain reaction and western blot. Results Homocysteine treatment dose-dependently and time-dependently increased the expression of β-MHC and ANP in H9c2 cardiomyocytes. Both mRNA and protein levels of sEH and TRPC3 channels were unregulated by homocysteine. Inhibition of sEH with 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) significantly suppressed homocysteine-induced upregulation of β-MHC and ANP and attenuated the increase of cell size. Pyr3, a selective TRPC3 channel blocker, also exerted a strong anti-hypertrophic effect against homocysteine. Treatment with TPPU significantly suppressed the expression of TRPC3 channels in homocysteine-exposed H9c2 cells. Conclusions: Both sEH and TRPC3 channels are involved in homocysteine-induced hypertrophy of cardiomyocytes. TRPC3 channels may act as a downstream mediator for sEH activation induced by homocysteine to promote cardiac hypertrophy.

Evaluation of AQP4/TRPV4 Channel Co-expression, Microvessel Density, and its Association with Peritumoral Brain Edema in Intracranial Meningiomas

Apart from VEGF-A pathway activation, the existence of peritumoral edema (PTBE) in meningiomas has been correlated with the expression levels of water transporter aquaporin 4 (AQP4). A novel cooperation of AQP4 with the transient receptor potential isoform 4 (TRPV4), a polymodal swelling-sensitive cation channel, has been proposed for regulating cell volume in glial cells. We investigated AQP4/TRPV4 channel co-expression in meningiomas along with the neovascularization of tumors and associate with PTBE. Immunohistochemical staining for AQP4 and TRPV4 expression was quantitatively analyzed in semi-serial sections of archival tissue from 174 patients. Microvessel density was expressed as microvessel count (MVC). PTBE was measured and edema index (EI) was assessed in 23 patients, based on magnetic resonance images (MRI) whereas mRNA levels of AQP4 and TRPV4 were evaluated in these patients using quantitative real-time PCR. High AQP4 was associated with lower–tumor grade (p < 0.05). AQP4 and TRPV4 were correlated in benign (WHO, grade I) (p < 0.0001) but not in high-grade (WHO, grades II and III) meningiomas (p > 0.05). AQP4/TRPV4 levels were independent of EI and MVC (p > 0.05). In contrast, EI was correlated to MVC (p = 0.02). AQP4/TRPV4 co-expression was detected in both edematous and non-edematous meningiomas. However, most of tumors with larger edema (EI ≥ 2) demonstrated increased levels of AQP4 and TRPV4. Importantly, peri-meningioma tissue of edematous meningiomas demonstrated significantly increased expression for AQP4 (p = 0.007) but not for TRPV4 (p > 0.05) compared with the main tumor. AQP4 and TRPV4 expression is rather associated with a response to vasogenic edema of meningiomas than with edema formation.

Delineating the Role of TRP Channels in Metabolic Disease (P10-083-19)

ObjectivesThe objective of the present study is to elucidate the role of Transient Receptor Potential (TRP) channels in the process of adipogenesis and diabetes mellitus, in hopes of getting more understanding of the role of TRP channels in the process as well as in hopes of discovering a novel therapeutic target against metabolic diseases. MethodsThe role of TRP channels in adipogenesis and diabetes mellitus was investigated by using in vivo (C57/BL6J mice) and in vitro (3T3-L1 cells). The expressions of TRP isoforms were studied by using RT-PCR and western blotting assay. TRP channels agonist and antagonist were used to study the role of TRP channels while fat accumulation in cells was visualized by Oil Red O staining. Intracellular calcium inflow was estimated by confocal microscopy. ResultsAmong the TRP channels screened, the authors identified the differential expressions of TRPC isoforms by using in vivo model. The results were further confirmed by using western blotting analysis. The changes in expression suggested the importance of the specific isoforms in the adipogenesis process. The agonist-antagonist study illustrated that the treatment of TRPC antagonists induced the maturation process while TRPC agonist attenuated adipocyte differentiation of 3T3-L1 cells. ConclusionsThe present study serves to illustrate the role of TRP channels in adipocyte biology. In conclusion, the current study demonstrated that the TRPC isoforms have differential expression during the maturation process of fat. Further, the modulation of TRPC could affect the adipocyte differentiation of 3T3-L1 cells. The understanding of TRPC channels in adipocyte biology serves as a novel therapeutic target against metabolic diseases such as obesity and diabetes mellitus. Funding SourcesThe research is funded by The Chinese University of Hong Kong Direct Grant. Supporting Tables, Images and/or Graphs▪▪

TRPC3 Regulates the Proliferation and Apoptosis Resistance of Triple Negative Breast Cancer Cells through the TRPC3/RASA4/MAPK Pathway.

Currently, there is no effective molecular-based therapy for triple-negative breast cancer (TNBC). Canonical transient receptor potential isoform 3 (TRPC3) was previously shown to be upregulated in breast cancer biopsy tissues when compared to normal breast tissues. However, the biological role of TRPC3 in breast cancer still remains to be elucidated. In this study, subcellular fractionation followed by Western blot and immunocytochemistry showed that TRPC3 was over-expressed on the plasma membrane of TNBC line MDA-MB-231 when compared to an estrogen receptor-positive cell line MCF-7. TRPC3 blocker Pyr3 and dominant negative of TRPC3 attenuated proliferation, induced apoptosis and sensitized cell death to chemotherapeutic agents in MDA-MB-231 as measured by proliferation assays. Interestingly, Ras GTPase-activating protein 4 (RASA4), a Ca2+-promoted Ras-MAPK pathway suppressor, was found to be located on the plasma membrane of MDA-MB-231. Blocking TRPC3 decreased the amount of RASA4 located on the plasma membrane, with concomitant activation of MAPK pathways. Our results suggest that, in TNBC MDA-MB-231 cells, Ca2+ influx through TRPC3 channel sustains the presence of RASA4 on the plasma membrane where it inhibits the Ras-MAPK pathway, leading to proliferation and apoptosis resistance. Our study reveals the novel TRPC3-RASA4-MAPK signaling cascade in TNBC cells and suggests that TRPC3 may be exploited as a potential therapeutic target for TNBC.

Suppression of neovascularization in corneal stroma in a TRPA1-null mouse

Corneal neovascularization and inflammatory fibrosis induced by severe injury or infection leads to tissue opacification and even blindness. Transient receptor potential (TRP) channel subtypes contribute to mediating these maladaptive responses through their interactions with other receptors. TRPV1 is one of the contributing channel isoforms inducing neovascularization in an alkali burn mouse wound healing model. VEGF-A upregulation contributes to neovascularization through interaction with its cognate receptors (VEGFR). Since the TRP isoform in this tissue, TRPA1, is also involved, we determined here if one of the pathways mediating neovascularization and immune cell infiltration involve an interaction between VEGFR and TRPA1 in a cauterization corneal mouse wound healing model. Localization of TRPA1 and endothelial cell (EC) CD31 immunostaining pattern intensity determined if TRPA1 expression was EC delimited during cauterization induced angiogenesis. Quantitative RT-PCR evaluated the effects of the absence of TRPA1 function on VEGF-A and TGF-β1 mRNA expression during this process. Macrophage infiltration increased based on rises in F4/80 antigen immunoreactivity. TRPA1 immunostaining was absent on CD31-immunostained EC cells undergoing neovascularization, but it was present on other cell type(s) adhering to EC in vivo. Absence of TRPA1 expression suppressed both stromal neovascularization and inhibited macrophage infiltration. Similarly, the increases occurring in both VEGF-A and TGF-β1 mRNA expression levels in WT tissue were blunted in the TRPA1−/− counterpart. On the other hand, in the macrophages their levels were invariant and their infiltration was inhibited. To determine if promotion by TRPA1 of angiogenesis was dependent on its expression on other unidentified cell types, the effects were compared of pharmacological manipulation of TRPA1 activity on EC proliferation tube formation and migration. In the presence and absence of a fibroblast containing feeder layer. Neither VEGF-induced increases in human vascular endothelial cell (HUVEC) proliferation nor migration were changed by a TRPA1 antagonist HC-030031 in the absence of a feeder layer. However, on a fibroblast feeder layer this antagonist suppressed HUVEC tube formation. In conclusion, during corneal wound healing transactivation by VEGFR of TRPA1 contributes to mediating neovascularization and macrophage infiltration. Such crosstalk is possible because of close proximity between VEGFR delimited expression on EC and TRPA1 expression restricted to cell types adhering to EC.

Calcium influx through TRPV4 channels modulates the adherens contacts between retinal microvascular endothelial cells.

Endothelial cells employ transient receptor potential isoform 4 (TRPV4) channels to sense ambient mechanical and chemical stimuli. In retinal microvascular endothelial cells, TRPV4 channels regulate calcium homeostasis, cytoskeletal signalling and the organization of adherens junctional contacts. Intracellular calcium increases induced by TRPV4 agonists include a significant contribution from calcium release from internal stores. Activation of TRPV4 channels regulates retinal endothelial barriers in vitro and in vivo. TRPV4 sensing may provide a feedback mechanism between sensing shear flow and eicosanoid modulators, vascular permeability and contractility at the inner retinal endothelial barrier. The identity of microvascular endothelial (MVE) mechanosensors that sense blood flow in response to mechanical and chemical stimuli and regulate vascular permeability in the retina is unknown. Using immunohistochemistry, calcium imaging, electrophysiology, impedance measurements and vascular permeability assays, we show that the transient receptor potential isoform 4 (TRPV4) plays a major role in Ca2+ /cation signalling, cytoskeletal remodelling and barrier function in retinal microvasculature in vitro and in vivo. Human retinal MVE cells (HrMVECs) predominantly expressed Trpv1 and Trpv4 transcripts, and TRPV4 was broadly localized to the plasma membrane of cultured cells and intact blood vessels in the inner retina. Treatment with the selective TRPV4 agonist GSK1016790A (GSK101) activated a nonselective cation current, robustly elevated [Ca2+ ]i and reversibly increased the permeability of MVEC monolayers. This was associated with disrupted organization of endothelial F-actin, downregulated expression of occludin and remodelling of adherens contacts consisting of vascular endothelial cadherin (VE-cadherin) and β-catenin. In vivo, GSK101 increased the permeability of retinal blood vessels in wild type but not in TRPV4 knockout mice. Agonist-evoked effects on barrier permeability and cytoskeletal reorganization were antagonized by the selective TRPV4 blocker HC 067047. Human choroidal endothelial cells expressed lower TRPV4 mRNA/protein levels and showed less pronounced agonist-evoked calcium signals compared to MVECs. These findings indicate a major role for TRPV4 in Ca2+ homeostasis and barrier function in human retinal capillaries and suggest that TRPV4 may differentially contribute to the inner vs. outer blood-retinal barrier function.

Adenosine A1 receptor‐operated calcium entry in renal afferent arteriolar smooth muscle cells is dependent on postnatal maturation of canonical transient receptor potential isoform 3 channels

Adenosine, a regulator of cardiovascular development and renal function constricts renal afferent arterioles by activating its cognate A1 receptors (AA1Rs). Activation of AA1Rs elevates intracellular Ca2+ ([Ca2+]i) concentration in renal vascular smooth muscle cells, mechanisms of which are not fully resolved. Whether AA1R expression and function in preglomerular microvessels are dependent on postnatal kidney maturation are also unclear. Here, we show that selective activation of AA1R by 2‐chloro‐N6‐cyclopentyladenosine (CCPA) does not stimulate store‐operated Ca2+ entry in afferent arterioles isolated from infant pigs. However, CCPA‐induced [Ca2+]i elevation is dependent on phospholipase C, protein kinase C, and canonical transient receptor potential isoform 3 (TRPC3) channels. The basal [Ca2+]i concentration was unchanged in afferent arterioles isolated from newly farrowed (0 day) pigs when compared with their 20‐day old counterparts. Whereas, CCPA‐induced [Ca2+]i elevation was ~ 43% larger in afferent arterioles from 20‐day old pigs. AA1R protein expression levels in the kidneys and afferent arterioles were unaltered in 0‐day versus 20‐day old pigs. By contrast, TRPC3 channel protein expression was ~ 92 and 78% higher in 20‐day old pig kidneys and afferent arterioles, respectively. These data suggest that activation of AA1Rs elicits receptor‐operated Ca2+ entry in porcine afferent arterioles, an effect dependent on postnatal maturation of TRPC3 channels.Support or Funding InformationNIH R01 DK101668

Bortezomib alleviates experimental pulmonary hypertension by regulating intracellular calcium homeostasis in PASMCs.

The ubiquitin-proteasome system is considered to be the key regulator of protein degradation. Bortezomib (BTZ) is the first proteasome inhibitor approved by the US Food and Drug Administration for treatment of relapsed multiple myeloma and mantle cell lymphoma. Recently, BTZ treatment was reported to inhibit right ventricular hypertrophy and vascular remodeling in hypoxia-exposed and monocrotaline-injected rats. However, the underlying mechanisms remain poorly understood. We previously confirmed that hypoxia-elevated basal intracellular Ca(2+) concentration ([Ca(2+)]i) and store-operated Ca(2+) entry (SOCE) in pulmonary artery smooth muscle cells (PASMCs) are involved in pulmonary vascular remodeling. In this study we aim to determine whether BTZ attenuates the hypoxia-induced elevation of [Ca(2+)] in PASMCs and the signaling pathway involved in this mechanism. Our results showed that 1) in hypoxia- and monocrotaline-induced rat pulmonary hypertension (PH) models, BTZ markedly attenuated the development and progression of PH, 2) BTZ inhibited the hypoxia-induced increase in cell proliferation, basal [Ca(2+)]i, and SOCE in PASMCs, and 3) BTZ significantly normalized the hypoxia-upregulated expression of hypoxia-inducible factor-1α, bone morphogenetic protein 4, canonical transient receptor potential isoforms 1 and 6, and the hypoxia-downregulated expression of peroxisome proliferator-activated receptor-γ in rat distal pulmonary arteries and PASMCs. These results indicate that BTZ exerts its protective role in the development of PH potentially by inhibiting the canonical transient receptor potential-SOCE-[Ca(2+)]i signaling axis in PASMCs.

Subcellular propagation of calcium waves in Müller glia does not require autocrine/paracrine purinergic signaling

ABSTRACTThe polarized morphology of radial glia allows them to functionally interconnect different layers of CNS tissues including the retina, cerebellum, and cortex. A likely mechanism involves propagation of transcellular Ca2+ waves which were proposed to involve purinergic signaling. Because it is not known whether ATP release is required for astroglial Ca2+ wave propagation we investigated this in mouse Müller cells, radial astroglia-like retinal cells in which in which waves can be induced and supported by Orai/TRPC1 (transient receptor potential isoform 1) channels. We found that depletion of endoplasmic reticulum (ER) stores triggers regenerative propagation of transcellular Ca2+ waves that is independent of ATP release and activation of P2X and P2Y receptors. Both the amplitude and kinetics of transcellular, depletion-induced waves were resistant to non-selective purinergic P2 antagonists such as pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). Thus, store-operated calcium entry (SOCE) is itself sufficient for the initiation and subcellular propagation of calcium waves in radial glia.

TRPC3 regulates the automaticity of embryonic stem cell-derived cardiomyocytes

BackgroundCardiac pacemaking is a complex phenomenon that is not completely understood. Canonical transient receptor potential isoform 3 (TRPC3) channel is a cation channel that permeates both Ca2+ and Na+. TRPC3 was previously found to express in adult cardiomyocytes. However, its role in cardiac pacemaking is unexplored. Here we used mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) to investigate whether TRPC3 regulates the spontaneous automaticity and the underlying mechanism involved. Methods and resultsImmunocytochemistry results showed that TRPC3 is expressed at the T-tubules of mESC-CMs. Whole-cell patch clamping showed that single mESC-CMs contain TRPC3 current. Confocal Ca2+ imaging showed that the TRPC3-specific blocker Pyr3 decreased Ca2+ transients and local Ca2+ release (LCR) of mESC-CMs. Combined current and voltage clamp recordings from the same cell showed that reducing the TRPC3 current, either by Pyr3 or a dominant negative (loss-of-function) construct of TRPC3, decreased the pacemaker activity of mESC-CMs as reflected by a decrease in action potential rate, a depolarized maximum diastolic potential and a decrease in slope of phase 4 diastolic depolarization. Furthermore, decreasing the TRPC3 current diminished, while increasing the TRPC3 current augmented the sodium–calcium exchanger (NCX) current in mESC-CMs. Lastly, decrease in TRPC3 current decreased the phosphorylation of ryanodine receptor isoform 2 at Ser2809 and phospholamban at Thr17. ConclusionsTRPC3 positively regulates diastolic depolarization of spontaneous action potential by increasing LCR and NCX current and therefore is an important determinant in pacemaking of mESC-CMs.

TRPV4 and AQP4 Channels Synergistically Regulate Cell Volume and Calcium Homeostasis in Retinal Müller Glia.

Brain edema formation occurs after dysfunctional control of extracellular volume partly through impaired astrocytic ion and water transport. Here, we show that such processes might involve synergistic cooperation between the glial water channel aquaporin 4 (AQP4) and the transient receptor potential isoform 4 (TRPV4), a polymodal swelling-sensitive cation channel. In mouse retinas, TRPV4 colocalized with AQP4 in the end feet and radial processes of Müller astroglia. Genetic ablation of TRPV4 did not affect the distribution of AQP4 and vice versa. However, retinas from Trpv4(-/-) and Aqp4(-/-) mice exhibited suppressed transcription of genes encoding Trpv4, Aqp4, and the Kir4.1 subunit of inwardly rectifying potassium channels. Swelling and [Ca(2+)]i elevations evoked in Müller cells by hypotonic stimulation were antagonized by the selective TRPV4 antagonist HC-067047 (2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide) or Trpv4 ablation. Elimination of Aqp4 suppressed swelling-induced [Ca(2+)]i elevations but only modestly attenuated the amplitude of Ca(2+) signals evoked by the TRPV4 agonist GSK1016790A [(N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide]. Glial cells lacking TRPV4 but not AQP4 showed deficits in hypotonic swelling and regulatory volume decrease. Functional synergy between TRPV4 and AQP4 during cell swelling was confirmed in the heterologously expressing Xenopus oocyte model. Importantly, when the swelling rate was osmotically matched for AQP4-positive and AQP4-negative oocytes, TRPV4 activation became independent of AQP4. We conclude that AQP4-mediated water fluxes promote the activation of the swelling sensor, whereas Ca(2+) entry through TRPV4 channels reciprocally modulates volume regulation, swelling, and Aqp4 gene expression. Therefore, TRPV4-AQP4 interactions constitute a molecular system that fine-tunes astroglial volume regulation by integrating osmosensing, calcium signaling, and water transport and, when overactivated, triggers pathological swelling. Significance statement: We characterize the physiological features of interactions between the astroglial swelling sensor transient receptor potential isoform 4 (TRPV4) and the aquaporin 4 (AQP4) water channel in retinal Müller cells. Our data reveal an elegant and complex set of mechanisms involving reciprocal interactions at the level of glial gene expression, calcium homeostasis, swelling, and volume regulation. Specifically, water influx through AQP4 drives calcium influx via TRPV4 in the glial end foot, which regulates expression of Aqp4 and Kir4.1 genes and facilitates the time course and amplitude of hypotonicity-induced swelling and regulatory volume decrease. We confirm the crucial facets of the signaling mechanism in heterologously expressing oocytes. These results identify the molecular mechanism that contributes to dynamic regulation of glial volume but also provide new insights into the pathophysiology of glial reactivity and edema formation.

Molecular and clinical analysis of TRPC6 and AGTR1 genes in patients with pulmonary arterial hypertension.

BackgroundPulmonary arterial hypertension (PAH) is a rare and progressive vascular disorder characterized by increased pulmonary vascular resistance and right heart failure. The aim of this study was to analyze 5′UTR region in canonical transient receptor potential isoform 6 (TRPC6) and 3′UTR region in Angiotensin II type I receptor (AGTR1) genes in patients with idiopathic and associated PAH. Correlation among mutations and clinical and functional parameters was further analyzed.MethodsAnalysis of TRPC6 and AGTR1 genes was performed by polymerase chain reaction (PCR) and direct sequencing. We used a non-parametric test to determine if significant differences were found between the groups studied and chi-square test to compare clinical and hemodynamic variables among genotypes.ResultsFifty five patients and fifty two controls were included in this study. We found statistically significant differences for c.1-361A > T (p = 0.0077), c.1-254C > G (p < 0.0001) and c.1-218C > T (p = 0.0021) in TRPC6 gene and c.1166A > C (p < 0.001) in AGTR1 gene, between patients and controls. Idiopathic PAH patients (IPAH) and controls presented significant differences for all 3 TRPC6 polymorphisms (p = 0.020), (p = 0.002) and (p = 0.008) respectively, and also showed differences for AGTR1 gene (p < 0.001). In associated PAH (APAH) patients we found statistical differences for c.1-254C > G (p < 0.001) and c.1-218C > T (p = 0.001) in TRPC6 gene and c.1166A > C (p = 0.001) in AGTR1 gene. Several clinical and hemodynamic parameters showed significant differences between carriers and non-carriers of these single nucleotide polymorphisms (SNPs). Nineteen patients were carriers of all 3 SNPs in TRPC6 gene and presented a more severe phenotype with differences in mean pulmonary arterial pressure (p = 0.016), systolic pulmonary arterial pressure (p = 0.040), cardiac index (p < 0.001) and 6 minute walking test (p = 0.049). 16 of these patients harbored the SNP in AGTR1 gene. These patients showed differences in age at diagnosis (p = 0.049), mean pulmonary arterial pressure (p = 0.033), cardiac index (p = 0.002) and 6 minute walking test (p = 0.039).ConclusionsPAH is a rare disease with pulmonary vascular remodeling caused in part by a heterogeneous constellation of genetic arrangements. This study seems to suggest that c.1-361A > T, c.1-254C > G and c.1-218C > T polymorphisms in TRPC6 gene and c.1166A > C polymorphism in AGTR1 could have a role in the development of this disease.

TRPV4, TRPC1, and TRPP2 assemble to form a flow-sensitive heteromeric channel.

Transient receptor potential (TRP) channels, a superfamily of ion channels, can be divided into 7 subfamilies, including TRPV, TRPC, TRPP, and 4 others. Functional TRP channels are tetrameric complexes consisting of 4 pore-forming subunits. The purpose of this study was to explore the heteromerization of TRP subunits crossing different TRP subfamilies. Two-step coimmunoprecipitation (co-IP) and fluorescence resonance energy transfer (FRET) were used to determine the interaction of the different TRP subunits. Patch-clamp and cytosolic Ca(2+) measurements were used to determine the functional role of the ion channels in flow conditions. The analysis demonstrated the formation of a heteromeric TRPV4-C1-P2 complex in primary cultured rat mesenteric artery endothelial cells (MAECs) and HEK293 cells that were cotransfected with TRPV4, TRPC1, and TRPP2. In functional experiments, pore-dead mutants for each of these 3 TRP isoforms nearly abolished the flow-induced cation currents and Ca(2+) increase, suggesting that all 3 TRPs contribute to the ion permeation pore of the channels. We identified the first heteromeric TRP channels composed of subunits from 3 different TRP subfamilies. Functionally, this heteromeric TRPV4-C1-P2 channel mediates the flow-induced Ca(2+) increase in native vascular endothelial cells.

Multiple Roles of Transient Receptor Potential (TRP) Channels in Inflammatory Conditions and Current Status of Drug Development

During inflammation, several Transient Receptor Potential (TRP) channels are directly or indirectly activated by inflammatory signaling molecules and microenvironmental changes including heat, oxidative conditions or low pH. In either case, specific TRP isoforms participate in chains of pro- or anti-inflammatory signaling cascades often including activation of transcription factors, protein kinases and phospholipases, which result in signal integration or amplification. In a few cases, their potentials as therapeutic targets for inflammatory conditions like pruritis, cystitis, dermatitis, asthma among other conditions are investigated pre-clinically or clinically by pioneering academic groups and industries. Significant efforts are still devoted to the understanding of the detailed physiological roles played by TRP channels during inflammation. This review intends to summarize key biological findings and reports of drug discovery activities when available, in an overview of the current status and recent developments in the field.

Role of Reactive Oxygen Species and Redox in Regulating the Function of Transient Receptor Potential Channels

Cellular redox status, regulated by production of reactive oxygen species (ROS), greatly contributes to the regulation of vascular smooth muscle cell contraction, migration, proliferation, and apoptosis by modulating the function of transient receptor potential (TRP) channels in the plasma membrane. ROS functionally interact with the channel protein via oxidizing the redox-sensitive residues, whereas nitric oxide (NO) regulates TRP channel function by cyclic GMP/protein kinase G-dependent and -independent pathways. Based on the structural differences among different TRP isoforms, the effects of ROS and NO are also different. In addition to regulating TRP channels in the plasma membrane, ROS and NO also modulate Ca(2+) release channels (e.g., IP(3) and ryanodine receptors) on the sarcoplasmic/endoplasmic reticulum membrane. This review aims at briefly describing (a) the role of TRP channels in receptor-operated and store-operated Ca(2+) entry, and (b) the role of ROS and redox status in regulating the function and structure of TRP channels.

A brief history of trp: commentary and personal perspective

The history of the discovery of the transient receptor potential (TRP) cation channel superfamily began in 1969 with Cosens and Manning's isolation of the Drosophila transient receptor potential mutant, in which the photoreceptor response decays during continuous illumination. Early studies from Minke found that the elementary light response was unaffected in trp mutants, and he attributed the defect to an intermediate stage of phototransduction. Montell and Rubin cloned the trp gene in 1989: they recognised it as a transmembrane protein, but also concluded that it did not encode the light-sensitive channels. In 1991, Minke and Selinger proposed that TRP represented a Ca2+ transporter required for refilling intracellular InsP3-sensitive Ca2+ stores, in turn required for activation of the light-sensitive channels. Also in 1991, after developing a photoreceptor patch clamp preparation, I showed that the light-sensitive channels themselves were highly permeable to Ca2+, questioning the need for such a dedicated Ca2+ transporter. In 1992, in collaboration with Minke, I resolved this paradox by showing there were two classes of light-sensitive channels, one highly Ca2+ permeable and eliminated in trp mutants. This represented the first and compelling evidence that TRP represented a light-sensitive channel and was supported by the cloning of the second light-sensitive channel, TRPL, by Kelly's lab. Three years later, in 1995, the labs of Montell and Birnbaumer independently cloned TRPC1, the first of 29 vertebrate TRP isoforms distributed amongst seven subfamilies.

Reply to “Letter to the editor: ‘Is menthol- or icilin-induced vasodilation mediated by the activation of TRPM8?'”

LETTERS TO THE EDITORReply to “Letter to the editor: ‘Is menthol- or icilin-induced vasodilation mediated by the activation of TRPM8?'”Christopher Johnson and Alexander ZholosChristopher Johnson and Alexander ZholosPublished Online:01 Aug 2009https://doi.org/10.1152/ajpheart.00506.2009MoreSectionsPDF (30 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat reply: The letter from Dr. Ma (3) highlights the problems that are inevitable in filling in the gaps of our knowledge of the properties of heterologously expressed transient receptor potential (TRP) channels, their expression profiles, and the functional roles in tissues and whole organisms. Not only are multiple TRP isoforms commonly expressed in native cells, but also they often do not fulfill our expectations from studies of exogenously expressed channels in cultured cells (7).Although TRPA1 has only a very limited sequence homology with TRP melastatin 8 (TRPM8), both channels can be activated by icilin and menthol. It is thus difficult for pharmacological approaches to be conclusive. However, we feel strongly that our combination of methods and the absence of evidence that TRPA1 channels are expressed on vascular myocytes (2) justify our conclusions that TRPM8 channels may, indeed, contribute to vascular tone in the vessels examined in our study with the use of menthol and icilin. The experimental approaches suggested by Ma (3) are all extremely valid. However, nonpharmacological approaches also have their own inherent problems. There are several notable examples within the field of TRP channel study where molecular interventions, such as the deletion of the gene for specific channels, have not had the effects predicted (6, 7). These studies should be done but may not be conclusive in isolation. Thus future and imminent work in the field of vascular TRPM8 and TRPA1 channels should use a combination of all the approaches available, including molecular approaches outlined by Ma (3), but should also have a strong element of functional physiology/pharmacology, using the best ligands available at the time, which are constantly being reviewed (1, 4). Indeed, controversy surrounding the roles of TRPM8 and TRPA1 proteins in cold sensation, and especially conflicting reports on the role of TRPA1 in sensory neurons (5), raises awareness that the answer to the above questions may not be simple.REFERENCES1 Bödding M, Wissenbach U, Flockerzi V. Characterisation of TRPM8 as a pharmacophore receptor. Cell Calcium 42: 618–628, 2007.Crossref | PubMed | ISI | Google Scholar2 Earley S, Gonzales AL, Crnich R. Endothelium-dependent cerebral artery dilation mediated by TRPA1 and Ca2+-activated K+ Channels. Circ Res 104: 987–994, 2009.Crossref | PubMed | ISI | Google Scholar3 Ma S. Letter to the editor: “Is menthol- or icilin-induced vasodilation mediated by the activation of TRPM8?” Am J Physiol Heart Circ Physiol. doi:10.1152/ajpheart.00460.2009.Link | ISI | Google Scholar4 Ma S, G G, Ak VE, Jf D, H H. Menthol derivative WS-12 selectively activates transient receptor potential melastatin-8 (TRPM8) ion channels. Pak J Pharm Sci 21: 370–378, 2008.PubMed | ISI | Google Scholar5 McKemy DD. How cold is it? TRPM8 and TRPA1 in the molecular logic of cold sensation. Mol Pain 1: 16, 2005.Crossref | PubMed | ISI | Google Scholar6 Nilius B, Owsianik G, Voets T, Peters JA. Transient receptor potential cation channels in disease. Physiol Rev 87: 165–217, 2007.Link | ISI | Google Scholar7 Venkatachalam K, Montell C. TRP Channels. Annu Rev Biochem 76: 387–417, 2007.Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: C. Johnson, Centre for Vision and Vascular Science, School of Medicine, Medical Biology Ctr., Queen's Univ. Belfast, 97 Lisburn Rd., Belfast BT9 7BL, UK (e-mail: [email protected]) Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 297Issue 2August 2009Pages H888-H888 Copyright & PermissionsCopyright © 2009 the American Physiological Societyhttps://doi.org/10.1152/ajpheart.00506.2009History Published online 1 August 2009 Published in print 1 August 2009 Metrics