ABSTRACT: Acetaminophen (paracetamol) is the most frequently used analgesic and antipyretic drug available over the counter. At the same time, acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. Acetaminophen overdose causes a multitude of interrelated biochemical reactions in hepatocytes including the formation of reactive oxygen species, deregulation of Ca(2+) homeostasis, covalent modification and oxidation of proteins, lipid peroxidation, and DNA fragmentation. Although an increase in intracellular Ca(2+) concentration in hepatocytes is a known consequence of acetaminophen overdose, its importance in acetaminophen-induced liver toxicity is not well understood, primarily due to lack of knowledge about the source of the Ca(2+) rise. Here we report that the channel responsible for Ca(2+) entry in hepatocytes in acetaminophen overdose is the Transient Receptor Potential Melanostatine 2 (TRPM2) cation channel. We show by whole-cell patch clamping that treatment of hepatocytes with acetaminophen results in activation of a cation current similar to that activated by H2O2 or the intracellular application of ADP ribose. siRNA-mediated knockdown of TRPM2 in hepatocytes inhibits activation of the current by either acetaminophen or H2O2. In TRPM2 knockout mice, acetaminophen-induced liver damage, assessed by the blood concentration of liver enzymes and liver histology, is significantly diminished compared with wild-type mice. The presented data strongly suggest that TRPM2 channels are essential in the mechanism of acetaminophen-induced hepatocellular death.
Project description:Reactive oxygen species (ROS) have been commonly accepted as inducers of autophagy, and autophagy in turn is activated to relieve oxidative stress. Yet, whether and how oxidative stress, generated in various human pathologies, regulates autophagy remains unknown. Here, we mechanistically studied the role of TRPM2 (transient receptor potential cation channel subfamily M member 2)-mediated Ca(2+) influx in oxidative stress-mediated autophagy regulation. On the one hand, we demonstrated that oxidative stress triggered TRPM2-dependent Ca(2+) influx to inhibit the induction of early autophagy, which renders cells more susceptible to death. On the other hand, oxidative stress induced autophagy (and not cell death) in the absence of the TRPM2-mediated Ca(2+) influx. Moreover, in response to oxidative stress, TRPM2-mediated Ca(2+) influx activated CAMK2 (calcium/calmodulin dependent protein kinase II) at levels of both phosphorylation and oxidation, and the activated CAMK2 subsequently phosphorylated BECN1/Beclin 1 on Ser295. Ser295 phosphorylation of BECN1 in turn decreased the association between BECN1 and PIK3C3/VPS34, but induced binding between BECN1 and BCL2. Clinically, acetaminophen (APAP) overdose is the most common cause of acute liver failure worldwide. We demonstrated that APAP overdose also activated ROS-TRPM2-CAMK2-BECN1 signaling to suppress autophagy, thereby causing primary hepatocytes to be more vulnerable to death. Inhibiting the TRPM2-Ca(2+)-CAMK2 cascade significantly mitigated APAP-induced liver injury. In summary, our data clearly demonstrate that oxidative stress activates the TRPM2-Ca(2+)-CAMK2 cascade to phosphorylate BECN1 resulting in autophagy inhibition.
Project description:The human non-selective cation channel TRPM2 represents a mediator of apoptosis triggered by oxidative stress. The principal agonist ADP-ribose binds to the cytosolic domain of TRPM2, which is homologous to the human ADP-ribose pyrophosphatase NUDT9. To further elucidate the structure-function relationship of this channel, we characterised a TRPM2 orthologue from the cnidarian Nematostella vectensis, after its expression in a human cell line. This far distant relative shows only 31% total sequence similarity to hTRPM2, while its C-terminal domain has a greater resemblance to the NUDT9 enzyme. Current through nvTRPM2 was induced by ADPR, with a more pronounced sensitivity and faster kinetics than in hTRPM2. In contrast to hTRPM2, there was no response to H2O2 and hardly any modulatory effect by intracellular Ca(2+). The deletion of a stretch of 15 residues from the NUDT9 domain of nvTRPM2, which is absent in hTRPM2, did not change the response to ADPR but enabled activation of the channel by H2O2 and increased the effects of intracellular Ca(2+). These findings shed new light on the evolution of TRPM2 and establish nvTRPM2 as a promising tool to decipher its complex gating mechanisms.
Project description:Transient potential receptor melastatin-2 (TRPM2) is a non-selective Ca(2+)-permeable cation channel of the TRPM channel subfamily and is mainly activated by intracellular adenosine diphosphate ribose (ADPR). Here we synthesized a 1-(2-nitrophenyl)ethyl caged ADPR (NPE-ADPR) and found that uncaging of NPE-ADPR efficiently stimulated Ca(2+), Mg(2+), and Zn(2+) influx in a concentration-dependent manner in intact human Jurkat T-lymphocytes. The cation influx was inhibited by inhibitors or knockdown of TRPM2. Likewise, uncaging of NPE-ADPR markedly induced cation entry in HEK 293 cells that overexpress TRPM2. As expected, high temperature increased the ability of the photolyzed NPE-ADPR to induce cation entry, whereas acidic pH inhibited. Moreover, the absence of extracellular Ca(2+) significantly inhibited Mg(2+) and Zn(2+) influx after uncaging NPE-ADPR. On the other hand, the absence of extracellular Na(+) or Mg(2+) had no effect on photolyzed NPE-ADPR induced Ca(2+) entry. Taken together, our results indicated that NPE-ADPR is a cell permeable ADPR analogue that is useful for studying TRPM2-mediated cation entry in intact cells.
Project description:Melastatin-related transient receptor potential channel 2 (TRPM2) is a Ca(2+)-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular Ca(2+) ([Ca(2+)](i)) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [Ca(2+)](i), indicating that [Ca(2+)](i) gating of TRPM2 is independent of ADPR. The mechanism by which [Ca(2+)](i) activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas Ca(2+)-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [Ca(2+)](i) and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular Ca(2+) release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced Ca(2+) release is capable of activating TRPM2. These results indicate that [Ca(2+)](i) is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [Ca(2+)](i)-mediated activation of TRPM2 and its alternative spliced isoforms may represent a major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [Ca(2+)](i).
Project description:Recent studies showed that KGN cells, derived from a human granulosa cell tumor (GCT), express NADPH oxidase 4 (NOX4), an important source of H2O2. Transient receptor potential melastatin 2 (TRPM2) channel is a Ca2+ permeable cation channel that can be activated by H2O2 and plays an important role in cellular functions. It is also able to promote susceptibility to cell death. We studied expression and functionality of TRPM2 in KGN cells and examined GCT tissue microarrays (TMAs) to explore in vivo relevance. We employed live cell, calcium and mitochondrial imaging, viability assays, fluorescence activated cell sorting (FACS) analysis, Western blotting and immunohistochemistry. We confirmed that KGN cells produce H2O2 and found that they express functional TRPM2. H2O2 increased intracellular Ca2+ levels and N-(p-Amylcinnamoyl)anthranilic acid (ACA), a TRPM2 inhibitor, blocked this action. H2O2 caused mitochondrial fragmentation and apoptotic cell death, which could be attenuated by a scavenger (Trolox). Immunohistochemistry showed parallel expression of NOX4 and TRPM2 in all 73 tumor samples examined. The results suggest that GCTs can be endowed with a system that may convey susceptibility to cell death. If so, induction of oxidative stress may be beneficial in GCT therapy. Our results also imply a therapeutic potential for TRPM2 as a drug target in GCTs.
Project description:The Ca(2+)-permeable cation channel transient receptor potential melastatin 2 (TRPM2) plays a key role in pathogen-evoked phagocyte activation, postischemic neuronal apoptosis, and glucose-evoked insulin secretion, by linking these cellular responses to oxidative stress. TRPM2 channels are coactivated by binding of intracellular ADP ribose and Ca(2+) to distinct cytosolically accessible sites on the channels. These ligands likely regulate the activation gate, conserved in the voltage-gated cation channel superfamily, that comprises a helix bundle formed by the intracellular ends of transmembrane helix six of each subunit. For several K(+) and TRPM family channels, activation gate opening requires the presence of phosphatidylinositol-bisphosphate (PIP(2)) in the inner membrane leaflet. Most TRPM family channels inactivate upon prolonged stimulation in inside-out patches; this "rundown" is due to PIP(2) depletion. TRPM2 currents also run down within minutes, but the molecular mechanism of this process is unknown. Here we report that high-affinity PIP(2) binding regulates Ca(2+) sensitivity of TRPM2 activation. Nevertheless, TRPM2 inactivation is not due to PIP(2) depletion; rather, it is state dependent, sensitive to permeating ions, and can be completely prevented by mutations in the extracellular selectivity filter. Introduction of two negative charges plus a single-residue insertion, to mimic the filter sequence of TRPM5, results in TRPM2 channels that maintain unabated maximal activity for over 1 h, and display altered permeation properties but intact ADP ribose/Ca(2+)-dependent gating. Thus, upon prolonged stimulation, the TRPM2 selectivity filter undergoes a conformational change reminiscent of that accompanying C-type inactivation of voltage-gated K(+) channels. The noninactivating TRPM2 variant will be invaluable for gating studies.
Project description:Acetaminophen (APAP) is a safe analgesic antipyretic drug at prescribed doses. Its overdose, however, can cause life-threatening liver damage. Though, involvement of oxidative stress is widely acknowledged in APAP-induced hepatocellular death, the mechanism of this increased oxidative stress and the associated alterations in Ca(2+) homeostasis are still unclear. Among members of transient receptor potential (TRP) channels activated in response to oxidative stress, we here identify that redox-sensitive TRPV1, TRPC1, TRPM2, and TRPM7 channels underlie Ca(2+) entry and downstream cellular damages induced by APAP in human hepatoma (HepG2) cells. Our data indicate that APAP treatment of HepG2 cells resulted in increased reactive oxygen species (ROS) production, glutathione (GSH) depletion, and Ca(2+) entry leading to increased apoptotic cell death. These responses were significantly suppressed by pretreatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt monohydrate (Tiron), and also by preincubation of cells with the glutathione inducer Dimethylfumarate (DMF). TRP subtype-targeted pharmacological blockers and siRNAs strategy revealed that suppression of either TRPV1, TRPC1, TRPM2, or TRPM7 reduced APAP-induced ROS formation, Ca(2+) influx, and cell death; the effects of suppression of TRPV1 or TRPC1, known to be activated by oxidative cysteine modifications, were stronger than those of TRPM2 or TRPM7. Interestingly, TRPV1 and TRPC1 were labeled by the cysteine-selective modification reagent, 5,5'-dithiobis (2-nitrobenzoic acid)-2biotin (DTNB-2Bio), and this was attenuated by pretreatment with APAP, suggesting that APAP and/or its oxidized metabolites act directly on the modification target cysteine residues of TRPV1 and TRPC1 proteins. In human liver tissue, TRPV1, TRPC1, TRPM2, and TRPM7 channels transcripts were localized mainly to hepatocytes and Kupffer cells. Our findings strongly suggest that APAP-induced Ca(2+) entry and subsequent hepatocellular death are regulated by multiple redox-activated cation channels, among which TRPV1 and TRPC1 play a prominent role.
Project description:1. TRPM2 is a Ca2+ -permeable nonselective cation channel activated by intracellular ADP-ribose (ADPR) and by hydrogen peroxide (H2O2). We investigated the modulation of TRPM2 activity by N-(p-amylcinnamoyl)anthranilic acid (ACA). ACA has previously been reported to inhibit phospholipase A2 (PLA2). 2. Using patch-clamp and calcium-imaging techniques, we show that extracellular application of 20 microM ACA completely blocked ADPR-induced whole-cell currents and H2O2-induced Ca2+ signals (IC50 = 1.7 microM) in HEK293 cells transfected with human TRPM2. Two other PLA2 inhibitors, p-bromophenacyl bromide (BPB; 100 microM) and arachidonyl trifluoromethyl ketone (20 microM), had no significant effect on ADPR-stimulated TRPM2 activity. 3. Inhibition of TRPM2 whole-cell currents by ACA was voltage independent and accelerated at decreased pH. ACA was ineffective when applied intracellularly. The single-channel conductance was not changed during ACA treatment, suggesting a reduction of TRPM2 open probability by modulating channel gating. 4. ACA (20 microM) also blocked currents through human TRPM8 and TRPC6 expressed in HEK293 cells, while BPB (100 microM) was ineffective. TRPC6-mediated currents (IC50 = 2.3 microM) and TRPM8-induced Ca2+ signals (IC50 = 3.9 microM) were blocked in a concentration-dependent manner. 5. ADPR-induced currents in human U937 cells, endogeneously expressing TRPM2 protein, were fully suppressed by 20 microM ACA. 6. Our data indicate that ACA modulates the activity of different TRP channels independent of PLA2 inhibition. Owing to its high potency and efficacy ACA can serve, in combination with other blockers, as a useful tool for studying the unknown function of TRPM2 in native cells.
Project description:OBJECTIVE: TRPM2 is a Ca²(+)-permeable nonselective cation channel activated by adenosine dinucleotides. We previously demonstrated that TRPM2 is activated by coapplication of heat and intracellular cyclic adenosine 5'-diphosphoribose, which has been suggested to be involved in intracellular Ca²(+) increase in immunocytes and pancreatic ?-cells. To clarify the involvement of TRPM2 in insulin secretion, we analyzed TRPM2 knockout (TRPM2-KO) mice. RESEARCH DESIGN AND METHODS: Oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT) were performed in TRPM2-KO and wild-type mice. We also measured cytosolic free Ca²(+) in single pancreatic cells using fura-2 microfluorometry and insulin secretion from pancreatic islets. RESULTS: Basal blood glucose levels were higher in TRPM2-KO mice than in wild-type mice without any difference in plasma insulin levels. The OGTT and IPGTT demonstrated that blood glucose levels in TRPM2-KO mice were higher than those in wild-type mice, which was associated with an impairment in insulin secretion. In isolated ?-cells, smaller intracellular Ca²(+) increase was observed in response to high concentrations of glucose and incretin hormone in TRPM2-KO cells than in wild-type cells. Moreover, insulin secretion from the islets of TRPM2-KO mice in response to glucose and incretin hormone treatment was impaired, whereas the response to tolbutamide, an ATP-sensitive potassium channel inhibitor, was not different between the two groups. CONCLUSIONS: These results indicate that TRPM2 is involved in insulin secretion stimulated by glucose and that further potentiated by incretins. Thus, TRPM2 may be a new target for diabetes therapy.
Project description:Neutrophil extracellular trap (NET) formation constitutes an important extracellular antimicrobial function of neutrophils that plays a protective role in bacterial pneumonia. Formation of reactive oxygen species (ROS) such as highly diffusible hydrogen peroxide (H2O2) is a hallmark of oxidative stress during inflammatory lung conditions including pneumonia. However, the impact of exogenous ROS on NET formation and the signaling pathway involved in the process is not completely understood. Here we demonstrate that the ROS-sensing, nonselective, calcium-permeable channel transient receptor potential melastatin 2 (TRPM2) is required for NET formation in response to exogenous H2O2. This TRPM2-dependent H2O2-mediated NET formation involved components of autophagy and activation of AMPK and p38 MAPK, but not PI3K and AKT. Primary neutrophils from Trpm2-/- mice fail to activate this pathway with a block in NET release and a concomitant decrease in their antimicrobial capacity. Consequently, Trpm2-/- mice were highly susceptible to pneumonic infection with Klebsiella pneumoniae owing to an impaired NET formation and high bacterial burden despite increased neutrophil infiltration in their lungs. These results identify a key role of TRPM2 in regulating NET formation by exogenous ROS via AMPK/p38 activation and autophagy machinery, as well as a protective antimicrobial role of TRPM2 in pneumonic bacterial infection.-Tripathi, J. K., Sharma, A., Sukumaran, P., Sun, Y., Mishra, B. B., Singh, B. B., Sharma, J. Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.