Aminoethoxydiphenyl Borate and Flufenamic Acid Inhibit Ca<Superscript>2+</Superscript> Influx Through TRPM2 Channels in Rat Dorsal Root Ganglion Neurons Activated by ADP-Ribose and Rotenone

Exposure to oxidative stress causes health problems, including sensory neuron neuropathy and pain. Rotenone is a toxin used to generate intracellular oxidative stress in neurons. However, the mechanism of toxicity in dorsal root ganglion (DRG) neurons has not been characterized. Melastatin-like transient receptor potential 2 (TRPM2) channel activation and inhibition in response to oxidative stress, ADP-ribose (ADPR), flufenamic acid (FFA) and 2-aminoethoxydiphenyl borate (2-APB) in DRG neurons are also not clear. We tested the effects of FFA and 2-APB on ADPR and rotenone-induced TRPM2 cation channel activation in DRG neurons of rats. DRG neurons were freshly isolated from rats and studied with the conventional whole-cell patch-clamp technique. Rotenone, FFA and 2-APB were extracellularly added through the patch chamber, and ADPR was applied intracellularly through the patch pipette. TRPM2 cation currents were consistently induced by ADPR and rotenone. Current densities of the neurons were higher in the ADPR and rotenone groups than in control. The time courses (gating times) in the neurons were longer in the rotenone than in the ADPR group. ADPR and rotenone-induced TRPM2 currents were totally blocked by 2-APB and partially blocked by FFA. In conclusion, TRPM2 channels were constitutively activated by ADPR and rotenone, and 2-APB and FFA induced an inhibitory effect on TRPM2 cation channel currents in rat DRG neurons. Since oxidative stress is a common feature of neuropathic pain and diseases of sensory neurons, the present findings have broad application to the etiology of neuropathic pain and diseases of DRG neurons.     

Glutathione modulates Ca(2+) influx and oxidative toxicity through TRPM2 channel in rat dorsal root ganglion neurons

Glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells and maintains thiol redox in the cells. GSH depletion has been implicated in the neurobiology of sensory neurons. Because the mechanisms that lead to melastatin-like transient receptor potential 2 (TRPM2) channel activation/inhibition in response to glutathione depletion and 2-aminoethyldiphenyl borinate (2-APB) administration are not understood, we tested the effects of 2-APB and GSH on oxidative stress and buthionine sulfoximine (BSO)-induced TRPM2 cation channel currents in dorsal root ganglion (DRG) neurons of rats. DRG neurons were freshly isolated from rats and the neurons were incubated for 24 h with BSO. In whole-cell patch clamp experiments, TRPM2 currents in the rat were consistently induced by H₂O₂ or BSO. TRPM2 channels current densities and cytosolic free Ca²⁺ content of the neurons were higher in BSO and H₂O₂ groups than in control. However, the current densities and cytosolic Ca²⁺ release were also higher in the BSO + H₂O₂ group than in the H₂O₂ alone. When intracellular GSH is introduced by pipette TRPM2 channel currents were not activated by BSO, H₂O₂ or rotenone. BSO and H₂O₂-induced Ca²⁺ gates were blocked by the 2-APB. Glutathione peroxidase activity, lipid peroxidation and GSH levels in the DRG neurons were also modulated by GSH and 2-APB inhibition. In conclusion, we observed the protective role of 2-APB and GSH on Ca²⁺ influx through a TRPM2 channel in intracellular GSH depleted DRG neurons. Since cytosolic glutathione depletion is a common feature of neuropathic pain and diseases of sensory neuron, our findings are relevant to the etiology of neuropathology in DRG neurons.     

TRPM2 Cation Channels,Oxidative Stress and Neurological Diseases: Where Are We Now?

The Na+ and Ca²⁺-permeable melastatin related transient receptor potential 2 (TRPM2) channels can be gated either by ADP-ribose (ADPR) in concert with Ca²⁺ or by hydrogen peroxide (H₂O₂), an experimental model for oxidative stress, binding to the channel’s enzymatic Nudix domain. Since the mechanisms that lead to TRPM2 gating in response to ADPR and H₂O₂ are not understood in neuronal cells, I summarized previous findings and important recent advances in the understanding of Ca²⁺ influx via TRPM2 channels in different neuronal cell types and disease processes. Considering that TRPM2 is activated by oxidative stress, mediated cell death and inflammation, and is highly expressed in brain, the channel has been investigated in the context of central nervous system. TRPM2 plays a role in H₂O₂ and amyloid β-peptide induced striatal cell death. Genetic variants of the TRPM2 gene confer a risk of developing Western Pacific amyotropic lateral sclerosis and parkinsonism-dementia complex and bipolar disorders. TRPM2 also contributes to traumatic brain injury processes such as oxidative stress, inflammation and neuronal death. There are a limited number of TRPM2 channel blockers and they seem to be cell specific. For example, ADPR-induced Ca²⁺ influx in rat hippocampal cells was not blocked by N-(p-amylcinnomoyl)anthralic acid (ACA), the IP₃ receptor inhibitor 2-aminoethoxydiphenyl borate or PLC inhibitor flufenamic acid (FFA). However, the Ca²⁺ entry in rat primary striatal cells was blocked by ACA and FFA. In conclusion TRPM2 channels in neuronal cells can be gated by either ADPR or H₂O₂. It seems to that the exact relationship between TRPM2 channels activation and neuronal cell death still remains to be determined.     

Identification of Direct and Indirect Effectors of the Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable cation channel involved in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca²⁺ and ADP-ribose (ADPR) but also modulated in intact cells by several additional factors. Superfusion of TRPM2-expressing cells with H₂O₂ or intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) activates, whereas dialysis of AMP inhibits, TRPM2 whole-cell currents. Additionally, H₂O₂, cADPR, and NAADP enhance ADPR sensitivity of TRPM2 currents in intact cells. Because in whole-cell recordings the entire cellular machinery for nucleotide and Ca²⁺ homeostasis is intact, modulators might affect TRPM2 activity either directly, by binding to TRPM2, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca²⁺. To identify direct modulators of TRPM2, we have studied the effects of H₂O₂, AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide in inside-out patches from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H₂O₂ (1 mm) and enzymatically purified cADPR (10 μm) failed to activate, whereas AMP (200 μm) failed to inhibit TRPM2 currents. NAADP was a partial agonist (maximal efficacy, ∼50%), and nicotinic acid adenine dinucleotide was a full agonist, but both had very low affinities (K_0.5 = 104 and 35 μm). H₂O₂, cADPR, and NAADP did not enhance activation by ADPR. Considering intracellular concentrations of these compounds, none of them are likely to directly affect the TRPM2 channel protein in a physiological context.     

Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H₂O₂), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1–5 mM H₂O₂ for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H₂O₂ and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H₂O₂ in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia. nonselective cation channel; transient receptor potential channel; H₂O₂; activated microglia     

Synergistic regulation of endogenous TRPM2 channels by adenine dinucleotides in primary human neutrophils

The Ca²⁺-permeable TRPM2 channel is a dual function protein that is activated by intracellular ADPR through its enzymatic pyrophosphatase domain with Ca²⁺ acting as a co-factor. Other TRPM2 regulators include cADPR, NAADP and H₂O₂, which synergize with ADPR to potentiate TRPM2 activation. Although TRPM2 has been thoroughly characterized in overexpression or cell-line systems, little is known about the features of TRPM2 in primary cells. We here characterize the regulation of TRPM2 activation in human neutrophils and report that ADPR activates TRPM2 with an effective half-maximal concentration (EC₅₀) of 1 μM. Potentiation by Ca²⁺ is dose-dependent with an EC₅₀ of 300 nM. Both cADPR and NAADP activate TRPM2, albeit with lower efficacy than in the presence of subthreshold levels of ADPR (100 nM), which significantly shifts the EC₅₀ for cADPR from 44 to 3 μM and for NAADP from 95 to 1 μM. TRPM2 activation by ADPR can be suppressed by AMP with an IC₅₀ of 10 μM and cADPR-induced activation can be blocked by 8-Bromo-cADPR. We further show that 100 μM H₂O₂ enables subthreshold concentrations of ADPR (100 nM) to activate TRPM2. We conclude that agonistic and antagonistic characteristics of TRPM2 as seen in overexpression systems are largely compatible with the functional properties of TRPM2 currents measured in human neutrophils, but the potencies of agonists in primary cells are significantly higher.     

New Molecular Mechanisms on the Activation of TRPM2 Channels by Oxidative Stress and ADP-Ribose

The Na⁺ and Ca²⁺-permeable melastatin related transient receptor potential (TRPM2) cation channels can be gated either by ADP-ribose (ADPR) in concert with Ca²⁺ or by hydrogen peroxide (H₂O₂), an experimental model for oxidative stress, and binding to the channel’s enzymatic Nudix domain. Since the mechanisms that lead to TRPM2 inhibiting in response to ADPR and H₂O₂ are not understood, I reviewed the effects of various inhibitors such as flufenamic acid and PARP inhibitors on ADPR, NAD⁺ and H₂O₂-induced TRPM2 currents. In our experimental study, TRPM2 cation channels in chinese hamster ovary transected cells were gated both by ADPR and NAD⁺. In addition, H₂O₂ seems to activate TRPM2 by changing to the hydroxyl radical in the intracellular space after passing the plasma membrane. Experimental studies with respect to patch-clamp and Ca²⁺ imaging, inhibitor roles of antioxidants are also summarized in the review.     

A critical role of the transient receptor potential melastatin 2 channel in a positive feedback mechanism for reactive oxygen species-induced delayed cell death

Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage via altering intracellular Zn²⁺ homeostasis, but a mechanistic understanding is still lacking. Here, we showed that H₂O₂ induced neuroblastoma SH-SY5Y cell death with a significant delay, dependently of the TRPM2 channel and increased [Zn²⁺]_i, and therefore used this cell model to investigate the mechanisms underlying ROS-induced TRPM2-mediated delayed cell death. H₂O₂ increased concentration-dependently the [Zn²⁺]_i and caused lysosomal dysfunction and Zn²⁺ loss and, furthermore, mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation. Such effects were suppressed by preventing poly(adenosine diphosphate ribose, ADPR) polymerase-1-dependent TRPM2 channel activation with PJ34 and 3,3′,5,5′-tetra-tert-butyldiphenoquinone, inhibiting the TRPM2 channel with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid, or chelating Zn²⁺ with N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Bafilomycin-induced lysosomal dysfunction also resulted in mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation that were inhibited by PJ34 or 2-APB, suggesting that these mitochondrial events are TRPM2 dependent and sequela of lysosomal dysfunction. Mitochondrial TRPM2 expression was detected and exposure to ADPR-induced Zn²⁺ uptake in isolated mitochondria, which was prevented by TPEN. H₂O₂-induced delayed cell death was inhibited by apocynin and diphenyleneiodonium, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) inhibitors, GKT137831, an NOX1/4-specific inhibitor, or Gö6983, a protein kinase C (PKC) inhibitor. Moreover, inhibition of PKC/NOX prevented H₂O₂-induced ROS generation, lysosomal dysfunction and Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, fragmentation and ROS generation. Collectively, these results support a critical role for the TRPM2 channel in coupling PKC/NOX-mediated ROS generation, lysosomal Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, and ROS generation to form a vicious positive feedback signaling mechanism for ROS-induced delayed cell death.     

Regulation of TRPM2 by extra- and intracellular calcium

TRPM2 is a calcium-permeable nonselective cation channel that is opened by the binding of ADP-ribose (ADPR) to a C-terminal nudix domain. Channel activity is further regulated by several cytosolic factors, including cyclic ADPR (cADPR), nicotinamide adenine dinucleotide phosphate (NAADP), Ca(2+) and calmodulin (CaM), and adenosine monophosphate (AMP). In addition, intracellular ions typically used in patch-clamp experiments such as Cs(+) or Na(+) can alter ADPR sensitivity and voltage dependence, complicating the evaluation of the roles of the various modulators in a physiological context. We investigated the roles of extra- and intracellular Ca(2+) as well as CaM as modulators of ADPR-induced TRPM2 currents under more physiological conditions, using K(+)-based internal saline in patch-clamp experiments performed on human TRPM2 expressed in HEK293 cells. Our results show that in the absence of Ca(2+), both internally and externally, ADPR alone cannot induce cation currents. In the absence of extracellular Ca(2+), a minimum of 30 nM internal Ca(2+) is required to cause partial TRPM2 activation with ADPR. However, 200 microM external Ca(2+) is as efficient as 1 mM Ca(2+) in TRPM2 activation, indicating an external Ca(2+) binding site important for proper channel function. Ca(2+) facilitates ADPR gating with a half-maximal effective concentration of 50 nM and this is independent of extracellular Ca(2+). Furthermore, TRPM2 currents inactivate if intracellular Ca(2+) levels fall below 100 nM irrespective of extracellular Ca(2+). The facilitatory effect of intracellular Ca(2+) is not mimicked by Mg(2+), Ba(2+), or Zn(2+). Only Sr(2+) facilitates TRPM2 as effectively as Ca(2+), but this is due to Sr(2+)-induced Ca(2+) release from internal stores rather than a direct effect of Sr(2+) itself. Together, these data demonstrate that cytosolic Ca(2+) regulates TRPM2 channel activation. Its facilitatory action likely occurs via CaM, since the addition of 100 microM CaM to the patch pipette significantly enhances ADPR-induced TRPM2 currents at fixed [Ca(2+)](i) and this can be counteracted by calmidazolium. We conclude that ADPR is responsible for TRPM2 gating and Ca(2+) facilitates activation via calmodulin.     

Vitamin E modulates oxidative stress and protein kinase C activator (PMA)-induced TRPM2 channel gate in dorsal root ganglion of rats

It is well known that Ca²⁺ influx through cation channels induces peripheral pain in dorsal root ganglion (DRG) neurons. Melastatin-like transient receptor potential 2 (TRPM2) channel is a oxidative redox sensitive Ca²⁺-permeable cation channel. There is scarce report on block of the channels. Since the mechanisms that lead to TRPM2 inhibition in response to oxidative stress and protein kinase C (PKC) activation are not understood, we investigated effects of the antioxidants on the inhibition of TRPM2 channel currents in the DRG neurons of rats. The DRG peripheral neurons were freshly isolated from rats and the neurons were incubated by phorbol 12-myristate 13-acetate (PMA) which leads to activation of PKC and cause oxidative stress. In whole-cell patch clamp experiments, TRPM2 currents in the DRG incubated with PMA were stimulated by H₂O₂. In addition, the PMA-induced activation of TRPM2 channels were blocked by nonspecific TRPM2 channels inhibitors [2-aminoethyl diphenylborinate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid (ACA)]. The currents in the neurons are also totally blocked by vitamin E incubation. However, administration of catalase and vitamin C with/without the vitamin E incubation did not block the currents. In conclusion, we indicated that vitamin E modulated oxidative stress-induced TRPM2 channel activation in the DRG neurons. The results may be useful modulation of oxidative stress-induced peripheral pain in sensory neurons.     

Effects of Antagonists and Heat on TRPM8 Channel Currents in Dorsal Root Ganglion Neuron Activated by Nociceptive Cold Stress and Menthol

Transient receptor potential ion channel melastatin subtype 8 (TRPM8) is activated by cold temperature and cooling agents, such as menthol and icilin. Compounds containing peppermint are reported to reduce symptoms of environmental cold stress such as cold allodynia in dorsal root ganglion (DRG) neuron; however, the underlying mechanisms of action are unclear. We tested the effects of physiological heat (37°C), anthralic acid (ACA and 0.025 mM), 2-aminoethyl diphenylborinate (2-APB and 0.05) on noxious cold (10°C) and menthol (0.1 mM)-induced TRPM8 cation channel currents in the DRG neurons of rats. DRG neurons were freshly isolated from rats. In whole-cell patch clamp experiments, TRPM8 currents were consistently induced by noxious cold or menthol. TRPM8 channels current densities of the neurons were higher in cold and menthol groups than in control. When the physiological heat is introduced by chamber TRPM8 channel currents were inhibited by the heat. Noxious cold-induced Ca²⁺ gates were blocked by the ACA although menthol-induced TRPM8 currents were not blocked by ACA and 2-APB. In conclusion, the results suggested that activation of TRPM8 either by menthol or nociceptive cold can activate TRPM8 channels although we observed the protective role of heat, ACA and 2-APB through a TRPM8 channel in nociceptive cold-activated DRG neurons. Since cold allodynia is a common feature of neuropathic pain and diseases of sensory neuron, our findings are relevant to the etiology of neuropathology in DRG neurons.     

Sites of the NUDT9-H domain critical for ADP-ribose activation of the cation channel TRPM2

TRPM2 is a cation channel unique within the transient receptor potential family because of its gating by ADP-ribose (ADPR). ADPR gating is enabled by a cytosolic C-terminal Nudix box sequence motif embedded into a region homologous to the NUDT9 ADPR pyrophosphatase. A recently discovered splice variant of TRPM2 (TRPM2-DeltaC) lacks 34 amino acid residues in the NUDT9 domain and is insensitive to ADPR. To analyze in detail which parts of the deleted sequence (DeltaC-stretch) are critical for ADPR gating, we tested mutants that lacked 19, 25, and 29 amino acid residues in the N-terminal part or had amino acid residues substituted in the remaining C-terminal part of the DeltaC-stretch. All of these mutants displayed typical ADPR-induced currents. However, the deletion or substitution of the amino acid residue Asn-1326 immediately downstream of the DeltaC-stretch abrogated ADPR gating. We furthermore analyzed the mutation I1405E/L1406F in the Nudix box of TRPM2, because a considerably decreased AD-PRase activity of the TRPM2 NUDT9-H protein in comparison to the NUDT9 pyrophosphatase has been attributed to the reverse exchange EF --> IL. The I1405E/L1406F variant of TRPM2 failed to respond to ADPR even at concentrations up to 10 mM. We concluded that the DeltaC-stretch contains no individual amino acid residues essential for ADPR gating but may act as a spacer segment stabilizing a conformation necessary for the essential residue Asn-1326 to interact with other channel regions. Enhancing the enzymatic activity of the Nudix box abolishes the ADPR gating of TRPM2, pointing to the requirement of prolonged binding rather than degradation.     

Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes

TRPM2 (transient receptor potential melastatin 2) is a Ca2+-permeable cation channel gated by ADPR (ADP-ribose) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC method to determine ADPR contents in HClO4 cell extracts. The reversed-phase ion-pair HPLC method with an Mg2+-containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were approx. 5 muM in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP (N-formylmethionyl-leucylphenylalanine). We furthermore determined intracellular concentrations of cADPR (cyclic ADPR) with a cyclase assay involving enzymatic conversion of cADPR into NAD+ and fluorimetric determination of NAD+. Intracellular cADPR concentrations were approx. 0.2 microM and not altered by fMLP. In patch-clamp experiments, ADPR (0.1-100 microM) was dialysed into granulocytes to analyse its effects on whole-cell currents characteristic for TRPM2, in the presence of a low (<10 nM) or a high (1 microM) intracellular Ca2+ concentration. TRPM2 currents were significantly larger at high than at low [Ca2+] (e.g. -225+/-27.1 versus -7+/-2.0 pA/pF at 5 muM ADPR), but no currents at all were observed in the absence of ADPR (ADPR concentration < or =0.3 microM). cADPR (0.1, 0.3 and 10 microM) was without effect even in the presence of subthreshold ADPR (0.1 microM). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca2+. Thus ADPR and Ca2+ in concert behave as a messenger system for agonist-induced influx of Ca2+ through TRPM2 in granulocytes.     

Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe2+ in human monocytic U937 cells

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca²⁺-permeable channel. In monocytes/macrophages, H₂O₂-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H₂O₂ are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H₂O₂ were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30 °C to 37 °C enhanced H₂O₂-induced TRPM2-mediated increase in intracellular free Ca²⁺ ([Ca²⁺]_i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H₂O₂-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe²⁺-accumulation following pretreatment with FeSO₄. Thus intracellular Fe²⁺-accumulation sensitizes H₂O₂-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe²⁺-accumulation increased poly(ADP-ribose) levels in nuclei by H₂O₂ treatment, and the sensitization of H₂O₂-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe²⁺-accumulation enhances H₂O₂-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO₄ stimulated H₂O₂-induced TRPM2 activation at 37 °C in U937 cells and enhanced H₂O₂-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H₂O₂ to cells under conditions of intracellular Fe²⁺-accumulation caused cell death, concentration of H₂O₂ required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe²⁺-accumulation sensitizes TRPM2 channels to H₂O₂ and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H₂O₂-induced TRPM2 channels by Fe²⁺ may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe²⁺ is released by heme degradation under intracerebral hemorrhage.     

Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2′-phosphate

Transient receptor potential melastatin 2 (TRPM2), a Ca²⁺-permeable cation channel implicated in postischemic neuronal cell death, leukocyte activation, and insulin secretion, is activated by intracellular ADP ribose (ADPR). In addition, the pyridine dinucleotides nicotinamide-adenine-dinucleotide (NAD), nicotinic acid–adenine-dinucleotide (NAAD), and NAAD-2′-phosphate (NAADP) have been shown to activate TRPM2, or to enhance its activation by ADPR, when dialyzed into cells. The precise subset of nucleotides that act directly on the TRPM2 protein, however, is unknown. Here, we use a heterologously expressed, affinity-purified–specific ADPR hydrolase to purify commercial preparations of pyridine dinucleotides from substantial contaminations by ADPR or ADPR-2′-phosphate (ADPRP). Direct application of purified NAD, NAAD, or NAADP to the cytosolic face of TRPM2 channels in inside-out patches demonstrated that none of them stimulates gating, or affects channel activation by ADPR, indicating that none of these dinucleotides directly binds to TRPM2. Instead, our experiments identify for the first time ADPRP as a true direct TRPM2 agonist of potential biological interest.     

Four Ca2+ Ions Activate TRPM2 Channels by Binding in Deep Crevices near the Pore but Intracellularly of the Gate

TRPM2 is a tetrameric Ca²⁺-permeable channel involved in immunocyte respiratory burst and in postischaemic neuronal death. In whole cells, TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca²⁺, but the mechanism and the binding sites for Ca²⁺ activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages and Ca²⁺ dependence of steady-state single-channel gating kinetics provide unprecedented insight into the molecular mechanism of Ca²⁺ activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca²⁺ activated ∼50-pS nonselective cation channels; K_1/2 for ADPR was ∼1 µM at saturating Ca²⁺. Intracellular Ca²⁺ dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca²⁺) reveals that Ca²⁺ activation is a consequence of tighter binding of Ca²⁺ in the open rather than in the closed channel conformation. Four Ca²⁺ ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant ∼33-fold, producing altogether 10⁶-fold activation. Experiments in the presence of 1 mM of free Ca²⁺ on the extracellular side clearly show that closed channels do not sense extracellular Ca²⁺, but once channels have opened Ca²⁺ entering passively through the pore slows channel closure by keeping the “activating sites” saturated, despite rapid continuous Ca²⁺-free wash of the intracellular channel surface. This effect of extracellular Ca²⁺ on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca²⁺ influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells that contain micromolar ADPR a single brief puff of Ca²⁺ likely triggers prolonged, self-sustained TRPM2 activity.     

A Cell Permeable NPE Caged ADP-Ribose for Studying TRPM2

Transient potential receptor melastatin-2 (TRPM2) is a non-selective Ca²⁺-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 Ca²⁺, Mg²⁺, and Zn²⁺ 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 Ca²⁺ significantly inhibited Mg²⁺ and Zn²⁺ influx after uncaging NPE-ADPR. On the other hand, the absence of extracellular Na⁺ or Mg²⁺ had no effect on photolyzed NPE-ADPR induced Ca²⁺ 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.     

Mg2+- and ATP-dependent inhibition of transient receptor potential melastatin 7 by oxidative stress

Transient receptor potential melastatin 7 (TRPM7) is a Ca²⁺- and Mg²⁺-permeable nonselective cation channel that contains a unique carboxyl-terminal serine/threonine protein kinase domain. It has been reported that reactive oxygen species associated with hypoxia or ischemia activate TRPM7 current and then induce Ca²⁺ overload resulting in neuronal cell death in the brain. In this study, we aimed to investigate the molecular mechanisms of TRPM7 regulation by hydrogen peroxide (H₂O₂) using murine TRPM7 expressed in HEK293 cells. Using the whole-cell patch-clamp technique, it was revealed that the TRPM7 current was inhibited, not activated, by the application of H₂O₂ to the extracellular solution. This inhibition was not reversed after washout or treatment with dithiothreitol, suggesting irreversible oxidation of TRPM7 or its regulatory factors by H₂O₂ under whole-cell recording. Application of an electrophile, N-methylmaleimide (NMM), which covalently modifies cysteine residues in proteins, also inhibited TRPM7 current irreversibly. The effects of H₂O₂ and NMM were dependent on free [Mg²⁺]_i; the inhibition was stronger when cells were perfused with higher free [Mg²⁺]_i solutions via pipette. In addition, TRPM7 current was not inhibited by H₂O₂ when millimolar ATP was included in the intracellular solution, even in the presence of substantial free [Mg²⁺]_i, which is sufficient for TRPM7 inhibition by H₂O₂ in the absence of ATP. Moreover, a kinase-deficient mutant of TRPM7 (K1645R) was similarly inhibited by H₂O₂ just like the wild-type TRPM7 in a [Mg²⁺]_i- and [ATP]_i-dependent manner, indicating no involvement of the kinase activity of TRPM7. Thus, these data suggest that oxidative stress inhibits TRPM7 current under pathological conditions that accompany intracellular ATP depletion and free [Mg²⁺]_i elevation.     

A Calcium Influx Pathway Regulated Separately by Oxidative Stress and ADP-Ribose in TRPM2 Channels: Single Channel Events

A melastatin-like transient receptor potential 2 (TRPM2) channel is activated in concert with Ca2+ by intracellular adenosine diphosphoribose (ADPR) binding to the channel's enzyme Nudix domain. Channel activity is also seen with nicotinamide dinucleotide (NAD+) and hydrogen peroxide (H2O2) although the mechanisms remain unknown. Hence, we tested the effects of ADPR, NAD+ and H2O2 on the activation of TRPM2 currents in transfected Chinese hamster ovary (CHO) cells. The CHO cells were transfected with cDNA coding for TRPM2. The intracellular solution used EDTA (10 mM) as a chelator for Ca2+ and heavy metal ions. Moreover, we balanced the intracellular Ca2+ concentration at 1 microM. H2O2 (10 mM) in the bath chamber was extracellularly added although ADPR (0.3 mM) and NAD+ (1 mM) in pipette solution were intracellularly added. Using these conditions, the channel currents were evoked by the three stimulators. The time course of ADPR, NAD+ and H2O2 effects was characterized by a delay of 0.6, 3.0 min and 2-5 min, respectively and a slow current induction reached a clear plateau with ADPR and NAD+ although H2O2 currents continued to gain in amplitude over at least 15 min and it did not reach a clear plateau in many experiments. Furthermore, H2O2-induced a single-channel conductance in the current study; the first time that this has been resolved in CHO. The conductance of ADPR and H2O2 was 48.80 pS and 39.14 pS, respectively and the cells seem to be separately activated by ADPR and H2O2. In conclusion, we observed further support for a calcium influx pathway regulated separately by oxidative stress and ADPR in TRPM2 channels in transfected cells. A second novel result of the present study was that the TRPM2 channels were constitutionally activated by H2O2.