Chemical physiology of oxidative stress-activated TRPM2 and TRPC5 channels

Progress in Biophysics and Molecular Biology 103 (2010) 18e27

Contents lists available at ScienceDirect

Progress in Biophysics and Molecular Biology journal homepage: www.elsevier.com/locate/pbiomolbio

Review

Chemical physiology of oxidative stress-activated TRPM2 and TRPC5 channels Shinichiro Yamamoto a, b, Nobuaki Takahashi a, Yasuo Mori a, * a b

Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 27 May 2010

The ability to sense and adapt to a wide variety of environmental changes is crucial for the survival of all cells. Transient receptor potential (TRP) channels play pivotal roles in these sensing and adaptation reactions. In vertebrates, there are about 30 TRP channels; these are divided into six subfamilies by homology of the protein sequences. We have previously revealed that a group of TRP channels senses oxidative stress and induces cellular signaling and gene expression. TRPM2, a member of the TRPM subfamily, is activated by reactive oxygen species (ROS) via second-messenger production. Recently, we demonstrated that Ca2þ influx through TRPM2 activated by ROS induces chemokine production in monocytes, which aggravates inflammatory neutrophil infiltration. Additionally, we also revealed that nitric oxide, chemical compounds containing reactive disulfide, and inflammatory mediators directly activate the TRPC, TRPV, and TRPA subfamilies via oxidative modification of cysteine residues. In this review, we describe how these TRP channels sense oxidative stress and induce adaptation reactions, and we discuss the biological importance of oxidative stress-activated TRP channels. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: TRP channel Ca2þ signaling Oxidative stress ROS Oxidative modification

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 TRPM2 channel sensing oxidative stress via second-messenger production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.1. Activation mechanism of TRPM2 by oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.2. Involvement of TRPM2 with chemokine production in monocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.3. TRPM2-mediated Ca2þ influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration . . . . . . . . . . . 22 TRP channels that sense oxidative stress directly via oxidative modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.1. Activation mechanisms of TRP channels via cysteine residue modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.2. Involvement of Ca2þ influx via nitrosylated TRPC5 channels in the positive feedback regulation of Ca2þ-dependent NO production . . . . . . . . 24 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

1. Introduction

Abbreviations: TRP, transient receptor potential; ROS, reactive oxygen species; NADþ, nicotinamide adenine dinucleotide; H2O2, hydrogen peroxide; PARP-1, poly (ADP-ribose) polymerase-1; PARG, poly(ADP-ribose) glycohydrolase; NF-kB, nuclear factor-kB; [Ca2þ]i, intracellular Ca2þ concentration; Erk, extracellular signal-regulated kinase; Pyk2, proline-rich tyrosine kinase 2; PTP, protein tyrosine phosphatase; HIF-1, hypoxia inducible factor-1; DSS, dextran sulfate sodium; BMDM, bone marrow-derived macrophages; NO, nitric oxide; DTT, dithiothreitol; 15d-PGJ2, 15deoxy-D12,14-prostaglandin J2; NOS, nitric oxide synthase; BAEC, bovine aortic endothelial cells. * Corresponding author. Tel.: þ81 75 383 2761; fax: þ81 75 383 2765. E-mail address: [email protected] (Y. Mori). 0079-6107/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.pbiomolbio.2010.05.005

The trp gene was identified through the genetic studies of the Drosophila visual transduction mutation (Montell and Rubin, 1989). The term “TRP” is derived from “transient receptor potential”, because the trp gene mutant photoreceptors fail to generate the Ca2þdependent “sustained” phase of receptor potential and therefore fail to induce subsequent Ca2þ-dependent adaptation to light. Approximately 30 TRP homologs in vertebrates have been discovered since the cloning of the Drosophila trp gene (Montell and Rubin, 1989). They are divided into six subfamilies (canonical (C), vanilloid (V), melastatin (M), polycystic kidney disease (P), mucolipin (ML), and ankyrin

S. Yamamoto et al. / Progress in Biophysics and Molecular Biology 103 (2010) 18e27

(A)) by homology of the protein sequences (Table 1). The TRP protein has six putative transmembrane domains and a pore region between the fifth and sixth transmembrane domains, and it assembles into homo- or hetero-tetramers to form channels (Fig. 1) (Goel et al., 2002; Hofmann et al., 2002). And in some TRP subfamilies, there are several ankyrin repeats at N-terminal domain (Fig. 1). The TRPC subfamily contains proteins with the greatest homology to the Drosophila TRP protein, and its members are activated in response to phospholipase C activation following receptor stimulation (Zhu et al., 1996; Hofmann et al., 1999; Okada et al., 1999; Inoue et al., 2001). The receptor stimulation eventually induces a biphasic Ca2þ signal, which is composed of an initial Ca2þ release from the endoplasmic reticulum followed by a sustained and/or oscillatory intracellular Ca2þ concentration ([Ca2þ]i) increase due to Ca2þ influx via TRPC channels. Different modes of receptor-operated Ca2þ entry include storeoperated Ca2þ entry or capacitative Ca2þ entry, resulting from the depletion of intracellular Ca2þ stores, as well as Ca2þ entry activated in a Ca2þ depletion-independent manner. It has been shown that TRPC1 channel is activated by intracellular Ca2þ-store depletion (Zitt et al., 1996), and TRPC3 is also likely to be stimulated, at least in part, by intracellular Ca2þ-store depletion (Zitt et al., 1997; Zhu et al., 1998), whereas TRPC5, TRPC6 and TRPC7 channels are distinguishable from store-operated Ca2þ channels (Boulay et al., 1997; Okada et al., 1998, 1999). The TRPV subfamily is composed of TRPV1, known as a vanilloid receptor, and its homologs, and is activated by physical or chemical stimuli including heat (TRPV1, TRPV2, TRPV3, and TRPV4) (Caterina et al., 1997, 1999; Güler et al., 2002; Peier et al., 2002a; Smith et al., 2002; Watanabe et al., 2002; Xu et al., 2002), protons (TRPV1) (Tominaga et al., 1998), osmotic stress (TRPV4) (Liedtke et al., 2000; Strotmann et al., 2000), and capsaicin (TRPV1) (Caterina et al., 1997). Electrophysiologic studies showed that Currentevoltage relationship of these TRPV channels exhibits outward rectification. By contrast, it has been suggested that currentevoltage relationship of

19

TRPV5 and TRPV6 exhibit inward rectification (Clapham, 2003). The TRPM subfamily, named after melastatin (TRPM1), which is a tumor suppressor protein isolated in a screen for genes whose level of expression was inversely correlated with the severity of metastatic potential of a melanoma cell line (Duncan et al., 1998), contains eight mammalian members. TRPM8 channels, in contrast to TRPV channels, are activated by cold temperature and menthol (McKemy et al., 2002; Peier et al., 2002b). The sole member of the TRPA subfamily, TRPA1, has a large N-terminal domain with 17 predicted ankyrin repeats (Takahashi et al., 2008). Pungent compounds, such as allyl isothiocyanate included in mustard oil, and noxiously cold stimuli trigger TRPA1 activation (Story et al., 2003; Jordt et al., 2004). Thus, TRP channels serve as sensors for a variety of environmental factors (Table 1). Furthermore, Ca2þ influx via the TRP channel is involved in the regulation of diverse cellular processes. We have demonstrated that TRPM2 and TRPC5 sense oxidative stress and induces cellular signaling and gene expression. TRPM2 is a Ca2þ-permeable nonselective cation channel activated by reactive oxygen species (ROS) (Hara et al., 2002). Although details of mechanism are not clarified, it is thought that ROS-induced TRPM2 activation is mediated via second-messenger production (Fonfria et al., 2004; Perraud et al., 2005). Accumulated evidence indicates that TRPM2 channel mediates several cellular responses. For example, in pancreatic b-cells Ca2þ influx via TRPM2 participate in insulin secretion (Togashi et al., 2006). And we have demonstrated that TRPM2 is responsible for the H2O2-activated Ca2þ influx that mediates cell death (Hara et al., 2002). However, with regard to in vivo physiological or pathophysiological cellular responses, the importance of ROS-activated Ca2þ influx through TRPM2 remained to be elucidated. Recently, in studies utilizing TRPM2 knockout (TRPM2 KO) mice, we have demonstrated that Ca2þ influx through TRPM2 activated by ROS induces chemokine production in monocytes, which aggravates inflammatory neutrophil infiltration.

Table 1 Phylogenetic tree and a variety of environmental factors. TRP channels function as sensor for many environmental factors. Environmental factors that activate TRP channels are indicated.

M6 M7 M3 M1 M5 M4 M2 M8 C7 C3 C6 C4 C5 C1 C2 P3 P2 P5 P1 ML1 ML3 ML2 V2 V1 V4 V3 V5 V6 A1

Environment factors

References

[Mg2þ]i Mg$ATP, ROS Osmotic stress, D-erythro-sphingosine

Voets et al., 2004 Nadler et al., 2001, Aarts et al., 2003 Grimm et al., 2003, 2005

Taste [Ca2þ]i ADP-ribose, ROS Cold temperature, menthol

Pérez et al., 2002 Launay et al., 2002 Perraud et al., 2001, Hara et al., 2002 McKemy et al., 2002, Peier et al., 2002b

M

C

Oxidative stress Stretch Pheromone

Yoshida et al., 2006 Maroto et al., 2005 Leypold et al., 2002

Mechanical stimulation

Nauli et al., 2003

Mechanical stimulation Cathepsin B Proton

Nauli et al., 2003 Kiselyov et al., 2005 Kim et al., 2008

P

Temperature Temperature Temperature Temperature

> 52  C > 43  C, capsaicin, proton 25e34  C, osmotic stress 30e39  C

Pungent compounds noxiously cold temperature

ML

Caterina et al., 1999 Caterina et al., 1997, Tominaga et al., 1998 Güler et al., 2002, Watanabe et al., 2002, Liedtke et al., 2000 Xu et al., 2002, Peier et al., 2002a, Smith et al., 2002

V

Story et al., 2003, Jordt et al., 2004, Takahashi et al., 2008

A

20

S. Yamamoto et al. / Progress in Biophysics and Molecular Biology 103 (2010) 18e27

A

pore region

exracellular

1 2 3 4 5

2

HN

6

TRP domain

AnkR

HOOC intracellular

B

pore region

2. TRPM2 channel sensing oxidative stress via second-messenger production The second member of the TRP melastatin subfamily, TRPM2 (previously known as LTRPC2 and TRPC7), is a Ca2þ-permeable nonselective cation channel that is expressed in the brain, pancreatic b-cells, and immunocytes including monocytes/macrophages and neutrophils (Wehage et al., 2002; Perraud et al., 2003). TRPM2 possesses a NudT9-H domain in its C-terminus, which has a low level of ADP-ribose pyrophosphatase activity despite significant homology with NudT9 ADP-ribose pyrophosphatase. Intracellular ADP-ribose can activate TRPM2 by binding to the C-terminal NudT9-H domain (Perraud et al., 2001). We have demonstrated that TRPM2 is activated by ROS including hydrogen peroxide (H2O2) and that Ca2þ influx via TRPM2 induces cell death (Hara et al., 2002). Our study indicated that nicotinamide adenine dinucleotide (NADþ) can also directly induce the opening of TRPM2 upon ROS stimulation (Hara et al., 2002). The Currentevoltage relationship of NADþinduced TRPM2 currents is linear, similar to that of ADP-ribose or H2O2-induced TRPM2 currents. It is now considered that this opening may be mediated by conversion from NADþ to ADP-ribose (Kolisek et al., 2005; Perraud et al., 2005). 2.1. Activation mechanism of TRPM2 by oxidative stress

C T A

N N

AT T

N C

C

Fig. 1. Transmembrane topology of TRP channels. (A) The TRP protein has six putative transmembrane domains, a pore region between the fifth and sixth transmembrane domains, and a TRP domain in the C-terminal region. Some TRP subfamilies have several ankyrin repeats (AnkR) at N-terminal domain. (B) The TRP protein assembles into homo-tetramers or hetero-tetramers to form channels. T: TRP domain, A: ankyrin repeats.

Our previous report on TRPC5 described a moderately Ca2þ permeable, receptor-operated channel with no involvement of store depletion in channel activation (Okada et al., 1998). A dominant-negative mutant of TRPC5 harbouring an exchange of three amino acids between the fifth and sixth predicted transmembrane domain induces an increased length of neurite as well as filopodia, the finger-like projections characteristic of growth cones (Greka et al., 2003). The results suggest that TRPC5-mediated Ca2þ entry is an important determinant of axonal growth and growth cone morphology. Additionally, recent studies utilizing TRPC5 KO have suggested that TRPC5, activated via G protein-coupled neuronal receptors, is involved in fear-related behavior (Riccio et al., 2009). Like this, the physiological significance of TRPC5 as receptor-operated channel has been identified. We have recently revealed that nitric oxide (NO) and chemical compounds containing reactive disulfide directly activate the TRPC5 via oxidative modification of cysteine residues (Yoshida et al., 2006), in contrast to TRPM2 which is activated by ROS via second-messenger production. It has been suggested that Ca2þ influx via nitrosylated TRPC5 channels in endothelial cells comprises the positive feedback regulation of Ca2þ-dependent NO production. In pathophysiological condition, endothelial cell dysfunction is closely link to a loss of the capacity of these cells to produce NO. Thus, disruption of the positive feedback regulated by TRPC5 may lead to initiation of endothelial cell dysfunction. In this review, we describe how these TRP channels sense the oxidative stress and induce adaptation reactions.

It has been suggested that oxidative stress-induced TRPM2 activation is triggered via the production of ADP-ribose from mitochondria (Perraud et al., 2005). Cytosolic or mitochondrial overexpression of ADP-ribose pyrophosphatase, which degrades ADP-ribose, suppresses H2O2-induced Ca2þ responses, providing a requirement for ADP-ribose binding in H2O2-induced TRPM2 activation. ADP-ribose is produced not only by mitochondria but also by the nucleus. In the nucleus, ADP-ribose generation is attributed to a pathway involving poly(ADP-ribose) polymerase-1 (PARP-1), and may be initiated by DNA damage due to various noxious factors including oxidative stress. PARP-1 binds to damaged single-stranded and double-stranded DNA breaks and catalyses the cleavage of NADþ into nicotinamide and ADP-ribose. ADP-ribose is then polymerized onto various nuclear proteins, resulting in the activation of DNA repair mechanisms and the stimulation of nuclear factor-mediated transcription (Tanuma et al., 1985; de Murcia and Menissier de Murcia, 1994; Oliver et al., 1999; Virag and Szabo, 2002). Free ADP-ribose is generated following the degradation of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosyl protein lyase activities (Virag and Szabo, 2002). Thus, it may be hypothesized that free ADP-ribose, which activates TRPM2, is produced by the activation of PARP-1 and PARG. Evidence for a functional role for PARP-1 is derived from experiments on three structurally distinct PARP inhibitors that suppress oxidative stressdependent activation of TRPM2 (Fonfria et al., 2004). Electrophysiological studies have shown that PARP inhibitors do not interfere with the activation of TRPM2 by ADP-ribose. Thus, the inhibition by PARP inhibitors must take place upstream from the TRPM2 and ADP-ribose interaction. Additionally, it has also been reported that the PARP-deficient DT40 cell line, which expresses TRPM2, exhibits no oxidative stress-induced Ca2þ responses (Buelow et al., 2008). In addition to full-length TRPM2, some physiological splice variants of TRPM2 have been identified. TRPM2-S (short) lacks the four C-terminal transmembrane domains, the putative Ca2þ-permeable pore region, and the entire C-terminus (Zhang et al., 2003). TRPM2-S directly interacts with full-length TRPM2 to suppress Ca2þ influx through full-length TRPM2 by oxidative stress. Thus, TRPM2-S has a dominant-negative function. TRPM2-DN contains a deletion of amino acids 538e557 in the N-terminus; HEK 293 cells expressing TRPM2-DN failed to respond to H2O2 or ADP-ribose, suggesting that

S. Yamamoto et al. / Progress in Biophysics and Molecular Biology 103 (2010) 18e27

the TRPM2-DN mutation dominantly disrupts channel gating, channel assembly, or surface trafficking (Wehage et al., 2002). TRPM2-DC contains a deletion of amino acids 1292e1325 in the C-terminal CAP-domain of NudT9-H, which decreases the affinity for ADP-ribose (Wehage et al., 2002; Kuhn and Luckhoff, 2004). HEK cells expressing TRPM2-ÄC are not activated by the intracellular application of ADP-ribose. Interestingly, this variant responds to H2O2, suggesting that oxidative stress may be able to activate TRPM2 through a mechanism independent of ADP-ribose. Thus, the question that remains resolved is whether the gating of TRPM2 by ROS is due to an ADP-ribose-independent mechanism or a diffusible second messenger such as ADP-ribose or NADþ. 2.2. Involvement of TRPM2 with chemokine production in monocytes Inflammation is complex stereotypical reaction of the body expressing the response to injury or infection. Although it is known that the biological significance of inflammation is likely that it brings fluids, proteins, and inflammatory cells from the blood into the damaged tissues to eliminate the injuring agents and trigger the healing and repairing processes. However, chronic inflammation is linked to serious health risks. Cytokines are released form immunocytes that are accumulated at inflammatory sites, and they function as mediators to organize an inflammatory network. Chemotactic cytokines, known as the chemokines, play a key role in mediating the recruitment of immunocytes migrating to inflamed sites (Luster, 1998). Chemokines are divided into four subfamiliesdCXC, CC, C and CX3Cdbased on structural and genetic characteristics (Luster, 1998). CXC chemokines, such as interleukin-8 (IL-8/CXCL8) and macrophage inflammatory protein 2 (MIP-2/CXCL2), exhibit potent neutrophil chemotactic activity. At inflamed sites, ROS are secreted from immunocytes and epithelial cells (Henricks and Nijkamp, 2001; Droge, 2002; Lambeth, 2004). Thus, a large amount of ROS is present at inflamed sites. The ROS function as potent antimicrobial agents by virtue of their ability to kill microbial pathogens. However, in chronic inflammation, the continued production of ROS causes extensive tissue damage. Traditionally, ROS have been considered as nonspecific toxins that cause random damage to cellular components including membrane lipids, DNA, and proteins (Henricks and Nijkamp, 2001), but recently ROS have emerged as signal-transduction molecules (Droge, 2002). In immunocytes, ROS contribute significantly to the expression of various inflammatory cytokines, adhesion molecules, and enzymes by activating redox-sensitive transcription factors such as nuclear factor-kB (NF-kB) (Droge, 2002). Along with ROS, Ca2þ is an intracellular signal-transduction molecule that plays important roles in immunocytes (Berridge et al., 2003). Increases in [Ca2þ]i are important for the production of certain cytokines, including interleukin-2 in T cells (Feske et al., 2001) and CXCL8 in human monocytic U937 cells (Wilson et al.,1993). In human monocytes, CXCL8 production is also induced by ROS including H2O2 (Josse et al., 2001) via extracellular signal-regulated kinase (Erk)activated NF-kB (Zeng et al., 2003). Despite these interesting indications of the involvement of ROS and Ca2þ as important regulatory factors in CXCL8 production in monocytes, the molecular entities and/or signaling mechanisms that link ROS, Ca2þ, and CXCL8 production are not clear. We have suggested that TRPM2 is responsible for the H2O2activated Ca2þ influx that mediates pathophysiological cell death (Hara et al., 2002). However, with regard to normal physiological cellular responses, the importance of H2O2-activated Ca2þ influx via TRPM2 remains to be elucidated. To establish the physiological function of TRPM2 channels, it is crucial that key roles played by the TRPM2 channels are studied in the context of signaling mechanisms that control specific cellular responses. Thus, we have

21

focused on determining whether TRPM2, known as the ROSactivated Ca2þ channel, is involved in ROS-induced chemokine production in monocytes (Yamamoto et al., 2008). We investigated H2O2-evoked [Ca2þ]i increases in human monocytic U937 cells. H2O2-evoked [Ca2þ]i increases were observed only in the presence of extracellular Ca2þ. Additionally, we confirmed that H2O2-induced increases in [Ca2þ]i were almost completely suppressed by TRPM2-specific siRNA, indicating that H2O2-evoked [Ca2þ]i increases in U937 cells are attributable to Ca2þ influx through endogenous TRPM2 channels. When we examined H2O2-induced CXCL8 production in U937 cells, we observed H2O2-mediated CXCL8 production, which is consistent with previous reports (Josse et al., 2001; Zeng et al., 2003). H2O2-induced CXCL8 production was significantly reduced by the omission of extracellular Ca2þ or TRPM2specific siRNA. These results clearly suggest that Ca2þ influx via native TRPM2 plays an important role in the H2O2-induced CXCL8 production in monocytes. Erk (Zeng et al., 2003; Chiu et al., 2007) and NF-kB (Mukaida et al., 1994; Josse et al., 2001; Mizukami et al., 2005) are involved in CXCL8 production in monocytes and other cell types. We confirmed the involvement of the Erk pathway and NF-kB with CXCL8 production by performing studies with pharmacological inhibitors. We then examined H2O2-induced NF-kB activation in U937 cells by observing the translocation of RelA, an NF-kB-subunit. Nuclear translocation of RelA was triggered within 30 min of H2O2 stimulation and reached a maximum level after 60 min. H2O2mediated translocation of RelA to the nucleus was significantly attenuated by the administration of the Erk pathway inhibitor PD98059, the addition of EGTA to the extracellular solution to abolish Ca2þ influx, and treatment with TRPM2-specific siRNA. It has been suggested that H2O2-induced activation of the NF-kB pathway in monocytes is positively regulated by Erk and the TRPM2-mediated Ca2þ influx. Since the Erk pathway is involved in H2O2-induced RelA translocation and CXCL8 production, we determined the Erk activity in relation to Ca2þ influx. In the presence of extracellular Ca2þ, Erk1 was activated by H2O2 within 2e5 min, and Erk2 was activated within 1 min, and maximum Erk activation occurred after 10e20 min. Erk1 was more intensely phosphorylated than Erk2. In the absence of extracellular Ca2þ, however, the activation of Erk1 was dramatically reduced, although the Erk2 response to H2O2 was relatively intact. These results suggest that Ca2þ influx plays an important role in Erk activation, reflected by the sustained phosphorylation of mainly Erk1 after 10e20 min of H2O2 stimulation. Much of this Ca2þ influx is likely mediated by Ca2þ-permeable TRPM2 channels, since Erk1 activation after 10 min of H2O2 stimulation was significantly reduced in cells that were treated with TRPM2-specific siRNA. Since Ras is an upstream signaling molecule of Erk, we determined its activity following H2O2 stimulation. Ras was gradually activated by H2O2, and its activation level reached a plateau within 5 min in the presence of extracellular Ca2þ; however, Ras was not activated by H2O2 in the absence of extracellular Ca2þ. Thus, Erk activation amplified by Ca2þ influx through TRPM2 is mediated via Ras. A further upstream signaling molecule, the Ca2þ-sensitive, proline-rich tyrosine kinase 2 (Pyk2), has been shown to activate the Ras/Erk pathway (Lev et al., 1995; Cullen and Lockyer, 2002). A Pyk2 dominant-negative mutant suppressed Ras and Erk activation, when assayed after 10 min of H2O2 stimulation. Erk activation was also suppressed by Pyk2-specific siRNAs, which suppressed the nuclear translocation of NF-kB. Importantly, Pyk2 activation was observed within 5 min of H2O2 stimulation in the presence, but not the absence, of extracellular Ca2þ. Furthermore, Pyk2 activation after 10 min of H2O2 stimulation was attenuated in TRPM2 siRNA-treated U937 cells. Thus, Ca2þ influx via H2O2activated TRPM2 triggers Pyk2/Ras signaling, which then amplifies downstream Erk activation, leading to the nuclear translocation of NF-kB and CXCL8 production in U937 monocytes (Fig. 2).

22

S. Yamamoto et al. / Progress in Biophysics and Molecular Biology 103 (2010) 18e27

mediated CXCL8 production in colon cancer cells, which is consistent with our model for CXCL8 production in monocytes.

H2O2

plasma membrane

TRPM2

GDP

Ras

P

Erk

P GTP

Ca2+

Ras

Erk

Erk

Pyk2

RelA

Mek P P

IKK RelA

Erk P

P

Erk

Erk

nucleus

CXCL8

transcription

Fig. 2. Schematic summary of signal-transduction pathways underlying H2O2-induced CXCL8 production in monocytes. H2O2 triggers TRPM2 activation together with the Ca2þ-independent initial phase of Erk activation. Ca2þ influx via H2O2-activated TRPM2 triggers Pyk2 phosphorylation, which amplifies Erk activation in a Ras-dependent manner. The amplified Erk activates transcription of the CXCL8 gene by inducing nuclear translocation of NF-kB.

In this study, H2O2-triggered NF-kB translocation and CXCL8 production were significantly but not entirely suppressed by the omission of extracellular Ca2þ or the addition of TRPM2 siRNA. Similarly, the H2O2-induced NF-kB nuclear translocation and CXCL8 production were strongly but not completely suppressed by the Erk pathway inhibitor PD98059. In contrast, H2O2-induced CXCL8 production was essentially abolished by the NF-kB inhibitor. Therefore, while NF-kB appears to be fully responsible for H2O2-induced CXCL8 production, a TRPM2/Ca2þ- and Mek-independent (PD98059insensitive) mechanism may contribute to NF-kB nuclear translocation in response to H2O2. Consistent with this scenario, the level of H2O2-induced CXCL8 production resistant to Erk pathway inhibitor PD98059 was indistinguishable from that resistant to the removal of extracellular Ca2þ. A possible mechanism that could account for this type of Erk activation might be a decrease in phosphatase activity, since protein tyrosine phosphatase (PTP) can be inhibited by H2O2 through the modification of catalytic cysteine residues (Lee and Esselman, 2001). Specifically, the inhibition of haematopoietic PTP (HePTP) expressed in U937 cells (Seimiya and Tsuruo, 1993) might be involved, since the inhibition of HePTP by H2O2 triggers Erk activation without Mek activation (Lee and Esselman, 2001). Therefore, some Erk activation through HePTP inactivation may be responsible for the residual nuclear translocation of NF-kB and CXCL8 production independent of TRPM2 and Mek. Experiments in which hypoxia inducible factor-1 (HIF-1) is knocked-down have shown that Ras augments H2O2-induced CXCL8 production (Mizukami et al., 2005). Hypoxic conditions can lead to the increased production of ROS, and scavenging of ROS is often achieved by the increased production of pyruvate, which occurs when cells shift from oxidative to glycolytic metabolism. HIF-1 shifts cellular status from oxidative to glycolytic metabolism, leading to increased pyruvate production and the scavenging of ROS in hypoxic conditions. Thus, HIF-1-deficient cells could release more H2O2. Interestingly, hypoxia-induced CXCL8 production via NF-kB activation occurs in HIF-1-deficient colon cancer DLD-1 cells, which harbor an oncogenic KRas mutation; however, this hypoxiainduced CXCL8 production does not occur in HIF-1-deficient colon cancer Caco2 cells, which carry wild-type (WT) KRas. These results suggest that sustained activation of KRas is critical for H2O2-

2.3. TRPM2-mediated Ca2þ influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration To study the physiological significance of TRPM2 channels in vivo and their impact on the cytokine signal-transduction pathways revealed above, we generated transgenic mice in which TRPM2 expression was knocked out. In monocytes from WT mice, intracellular perfusion with ADP-ribose evoked ionic currents. However, monocytes isolated from TRPM2 KO mice failed to respond, and no significant changes in current were ever observed. Similarly, WT cells typically responded to H2O2 by robust increases in cationic current and [Ca2þ]i, whereas these responses were nearly abolished in monocytes from TRPM2 KO mice. Mice do not carry the gene encoding CXCL8; however, CXCL2 is the murine CXC chemokine functional homologue of CXCL8 that shows potent chemotactic activity for neutrophils (Singer and Sansonetti, 2004). Notably, CXCL2 production is induced by H2O2 in the murine macrophage cell line B10R (Jaramillo and Olivier, 2002). We therefore compared H2O2-induced CXCL2 expression in monocytes from WT and TRPM2 KO mice. The exposure of monocytes to H2O2 induced the expression of CXCL2 in WT cells, whereas the induction of CXCL2 expression in mutant monocytes was significantly impaired. Additionally, the activation of the Pyk2/ Erk/NF-kB pathway in response to H2O2 was impaired in TRPM2 KO monocytes. This result together with the suppression of H2O2induced CXCL2 expression by the Erk and NF-kB inhibitors strongly suggests that the signaling cascade proposed for H2O2-induced CXCL8 expression in U937 cells is applicable to the regulation of H2O2induced CXCL2 expression in mouse monocytes. To determine if the reduction of H2O2-induced CXCL2 expression results in decreased chemoattractant effects of monocytes in TRPM2 KO mice, we measured in vitro neutrophil migration triggered by conditioned medium from cultured WT or mutant monocytes that were pretreated with H2O2. Neutrophil migration toward the conditioned medium from WT cultures was significantly increased by H2O2 pretreatment, whereas this effect of H2O2 was nearly abolished when medium from mutant monocytes was used or when neutralizing monoclonal-antibody for CXCL2 was added to the medium from WT monocytes. Thus, the differences in H2O2-induced CXCL2 expression in WT and TRPM2 KO monocytes can influence the chemoattractant properties of monocytes and affect neutrophil migration. As described above, ROS including H2O2 are generated and released from various cell types at inflamed sites (Henricks and Nijkamp, 2001; Droge, 2002; Lambeth, 2004). Released ROS are very likely to activate TRPM2 channels and induce chemokine production in monocytes at the inflamed sites. We tested this hypothesis in a dextran sulfate sodium (DSS)-induced ulcerative colitis model for inflammation in mice (Korenaga et al., 2002). The mice in this model exhibit many symptoms that are seen in human ulcerative colitis, i.e., diarrhea, bloody feces, body weight loss, mucosal ulceration, and shortening of the colon (Blackburn et al., 1998; Korenaga et al., 2002). Studies have suggested that the enhanced release of ROS plays an important role in the pathogenesis of DSS-induced ulcerative colitis (Blackburn et al., 1998; Araki et al., 2006) and that neutrophils infiltrated into the colonic mucosa by the chemotactic action of CXCL8 are responsible for tissue damage in human ulcerative colitis (Mahida et al., 1992; Anezaki et al., 1998; Keshavarzian et al., 1999). As expected, the expression of CXCL2 was greatly increased both in monocytes and in the colon tissue of DSS-treated WT mice, whereas the DSS-treated CXCL2 expression was strongly suppressed in TRPM2 KO mice. To determine if the reduction in CXCL2 expression induced by DSS results in decreased neutrophil infiltration in TRPM2 KO mice, we measured the

S. Yamamoto et al. / Progress in Biophysics and Molecular Biology 103 (2010) 18e27

neutrophil infiltration into the colon after DSS treatment. Interestingly, the number of recruited neutrophils was significantly reduced in DSS-treated TRPM2 KO mice, whereas the number of DSS-induced macrophages that infiltrated into tissues was indistinguishable in WT and TRPM2 KO mice. With regard to other immunocytes, DSSinduced infiltration was intact in TRPM2 KO mice; the populations of T cells, dendritic cells, and NK cells were indistinguishable between WT and TRPM2 KO mice in the untreated control group or the DSStreated colon group. We confirmed that the functions of TRPM2 KO neutrophils, such as their ability to infiltrate DSS-treated colon, were intact. We also confirmed that the bone marrow output and infiltration of neutrophils into the abdominal cavity after intraperitoneal injection of chemokines including CXCL2, CCL1, CCL3, and CCL5 were intact. Thus, we concluded that the diminished expression of CXCL2 in monocytes/macrophages contributes to the paucity of neutrophil accumulation in the colon of DSS-treated TRPM2 KO mice. We next performed macrophage transfer experiments. Bone marrow-derived macrophages (BMDM) were injected into the tail veins of DSStreated TRPM2 KO mice. Twenty-four hours later, we measured neutrophil infiltration into the colon. The defect in neutrophil infiltration into DSS-treated TRPM2 KO colon was restored by the transfer of WT BMDM and TRPM2 or CXCL2 cDNA-infected TRPM2 KO BMDM. These results suggested that CXCL2 released from macrophages in a TRPM2-dependent manner contributes to the neutrophil accumulation in the colon tissue of DSS-treated mice. As described above, neutrophils that have infiltrated the colonic mucosa by the chemotactic action of CXCL8 are responsible for tissue damage in human ulcerative colitis (Mahida et al., 1992; Anezaki et al., 1998; Keshavarzian et al., 1999). We performed clinical assessments of disease activity in the DSS mouse model. The colons of WT mice exhibited profound inflammation and tissue destruction characterized by mucosal ulceration, serosa destruction, and the infiltration of inflammatory cells. In TRPM2 KO mice, the severity of the DSS-induced colitis was dramatically reduced, although epithelial injury was identified. Moreover, TRPM2 KO mice did not exhibit a DSS-induced loss of body weight or shortening of the colon. Thus, TRPM2 KO mice were largely protected from DSS-mediated colitis, suggesting that the TRPM2-mediated chemokine production in monocytes/macrophages is an important mechanism in the progressive severity of DSS-induced ulcerative colitis (Fig. 3). In other inflammatory diseases, such as rheumatoid arthritis (Hitchon and El-Gabalawy, 2004), asthma (Henricks and Nijkamp, 2001), inflammatory liver disease (Jaeschke, 2000), and chronic obstructive pulmonary disease (Rahman, 2005), ROS also function as mediators for the aggravation of symptoms and pathophysiological responses. Kupffer cells, the resident liver macrophages, produce and release various proinflammatory cytokines, including CXCL8, leading to exacerbation of inflammatory liver disease (Videla et al., 2004), and CXCL8 produced by alveolar macrophages is involved in the development of chronic obstructive pulmonary disease (Barnes, 2004). Furthermore, genetic links of TRPM2 to defective inflammatory diseases such as amyotrophic lateral sclerosis and Parkinsonism dementia have been suggested (Hermosura and Garruto, 2007). Therefore, it is conceivable that released ROS trigger inflammatory responses via TRPM2 activation. This raises the prospect that inhibition of CXCL8 production might be an effective way to reduce pathological severity in ulcerative colitis and that the suppression of TRPM2 should be considered as a viable target for the treatment of inflammatory diseases related to ROS production. 3. TRP channels that sense oxidative stress directly via oxidative modification TRPC5, a canonical TRP expressed widely in the brain, testis, kidney, ovary, adrenal gland, uterus and endothelial cells, was first

23

bone marrow neutrophils

blood vessel

CXCL2

TRPM2-dependent CXCL2 production macrophages

chemotaxis

neutrophil accumulation

ROS ulcerative colitis

epithelium

Fig. 3. TRPM2-dependent chemokine production in monocytes/macrophages aggravates inflammatory neutrophil infiltration. ROS, including H2O2, generated and released from various cells at inflamed sites activate TRPM2 channels in monocytes/macrophages. CXCL2 production in monocytes/macrophages is induced by ROS in a TRPM2-dependent manner. CXCL2-evoked neutrophil accumulation at inflamed sites plays an important role in the pathogenesis of ulcerative colitis. ROS-evoked Ca2þ influx via TRPM2 represents a key inflammatory mediator in monocytes/macrophages.

cloned from the mouse brain (Okada et al., 1998). TRPC5 is a Ca2þpermeable, receptor-operated channel with no involvement of store depletion in channel activation. Currentevoltage relationship of ionic current regulated by ATP in TRPC5-expressing cells is nonlinear, showing a significant inward current. Interestingly, in cells coexpressing TRPC1 and TRPC5, the currentevoltage relation of currents through the TRPC1/TRPC5 heteromer, which show outwardly rectifying current, is clearly distinct from the inwardly rectifying currentevoltage relation of the TRPC5 homomers (Strübing et al., 2001). We have demonstrated that TRPC5 is activated by NO via cysteine S-nitrosylation (Yoshida et al., 2006). It is also known that pungent compounds such as allyl isothiocyanate included in mustard oil also trigger TRPA1 activation via cysteine residue modification (Hinman et al., 2006; Macpherson et al., 2007). 3.1. Activation mechanisms of TRP channels via cysteine residue modification The phosphorylations of serine, threonine, and tyrosine residues in signal-transduction proteins play a pivotal role in the activation of signal cascades. Redox modifications of cysteine residues also mediate many physiological responses including gene expression following the activation of signal cascades. It is thought that these reactions are triggered by protein conformational change via thiol modification of cysteine residues. By performing labeling and functional assays with cysteine mutants, we showed that NO activates TRPC5 via the modification of cysteine residues and that cytoplasmically accessible Cys553 and nearby Cys558 on the Nterminal side of the putative pore-forming region, which is located between the fifth and sixth transmembrane domains, are essential for mediating TRPC5 activation in response to NO. The reducing agent ascorbate can reduce S-nitrosothiols to thiols without the reduction of disulfides. Dithiothreitol (DTT) is a stronger reducing agent that can reduce S-nitrosothiols and disulfides to thiols. NO-activated TRPC5 channels were significantly but not entirely suppressed by ascorbate, while DTT fully suppressed NO-activated TRPC5 channels. Thus, nitrosylation and disulfide bond formation may be involved in NO-induced TRPC5 activation.

24

S. Yamamoto et al. / Progress in Biophysics and Molecular Biology 103 (2010) 18e27

In an S-nitrosylation assay (Jaffrey et al., 2001), the S-nitrosylation was abolished by the mutation of Cys553 of TRPC5, whereas it was unaffected by the mutation of Cys558. As reported in the acid-base catalysis of hemoglobin nitrosylation in proteins with high NO sensitivity, basic and acidic amino acids surrounding S-nitrosylated cysteines have been proposed to enhance the nucleophilicity of sulfhydryl and its S-nitrosylation (Hess et al., 2005). In TRPC5, charged residues flanking Cys553 and Cys558 may confer modification susceptibility to NO. Our data indicated that the TRPC5 channel is opened via the S-nitrosylation of Cys553 in response to NO and that the free sulfhydryl group of Cys558 nucleophilically attacks nitrosylated Cys553 to form a disulfide bond that stabilizes the open state (Fig. 4). The alignment of amino acid sequences surrounding Cys553 and Cys558 of TRPC5 with counterpart sequences shows that thermosensor channels TRPV1, TRPV3 and TRPV4 have conserved cysteine residues on the Nterminal side of the putative pore-forming region located between the fifth and sixth transmembrane domains. Thermosensors TRPV1, TRPV3 and TRPV4 respond well to NO. These findings reveal the structural motif for the NO-sensitive activation gate in TRP channels and indicate that NO sensors are a new functional category of cellular receptors extending over different TRP families. In addition to NO, reactive disulfides that selectively detect free sulfhydryl groups of cysteine residues in proteins also induce TRPC5 activation via the covalent modification of cysteine residue. Currentevoltage relationship of ionic current induced by reactive disulfides in TRPC5-expressing cells is similar to that induced by ATP. We showed that Cys553 is a modification site for reactive disulfides and a nitrosylation site for NO. On the other hand, the TRPA1 channel is highly expressed by a subset of C-fiber nociceptors (Story et al., 2003; Jordt et al., 2004; Nagata et al., 2005) and plays an important role in modulating nociceptor excitability and neurogenic inflammation in injured tissue (Bautista et al., 2006; Kwan et al., 2006). Pungent compounds such as allyl isothiocyanate included

in mustard oil trigger TRPA1 activation (Jordt et al., 2004). The electrophilic attack of pungent compounds on the thiol of a cysteine residue induces TRPA1 activation via the covalent modification of the cysteine residue (Hinman et al., 2006; Macpherson et al., 2007). We recently demonstrated that endogenous inflammatory mediators including 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2), NO, H2O2, and protons also activate TRPA1 via cysteine modification (Takahashi et al., 2008). As 15d-PGJ2 has highly reactive structures that contain a,b-unsaturated aldehyde moieties, it can covalently bind to cysteine sulfhydryl groups via a Michael addition reaction. Our site-directed mutagenesis studies revealed that Cys421 and Cys621 were involved in 15d-PGJ2-induced activation. Experiments using biotinylated 15d-PGJ2 demonstrated that Cys621 is a binding site for 15d-PGJ2, while Cys421, Cys641 and Cys665 are involved in TRPA1 activation by NO and H2O2 via nitrosylation and oxidation, respectively. Cys421 is also an important site for Hþ-induced TRPA1 activation. These results suggest that the relative importance of each candidate cysteine action site varies among inflammatory mediators. The conformation surrounding cysteines modified by each inflammatory mediator may be important, because each inflammatory mediator modifies the cysteine residue by different chemical reactions. 3.2. Involvement of Ca2þ influx via nitrosylated TRPC5 channels in the positive feedback regulation of Ca2þ-dependent NO production NO is produced by nitric oxide synthase (NOS). Two constitutive isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS), and one inducible isoform (iNOS) have been cloned. NO produced by NOS is involved in diverse physiological processes including vascular smooth muscle relaxation. [Ca2þ]i increases in endothelial cells induce NO production via eNOS activation, which triggers vascular smooth muscle relaxation. However, the molecular entities and/or mechanisms that mediate [Ca2þ]i increases required for

Fig. 4. Model for TRPC5 channel activation by NO and reactive disulfides. Cys553 is nitrosylated by NO, which triggers TRPC5 channel opening. The free sulfhydryl group of Cys558 nucleophilically attacks nitrosylated Cys553 to form a disulfide bond that stabilizes the open state.

S. Yamamoto et al. / Progress in Biophysics and Molecular Biology 103 (2010) 18e27

eNOS activation are not clear. Vasodilators, such as bradykinin, ATP, substance P, and acetylcholine, can increase endothelial cell [Ca2þ]i. These vasodilators can activate TRP channels, suggesting that TRP channels may participate in vasodilator-induced [Ca2þ]i increases in endothelial cells. TRPC5 expression is induced by retinoic acid in bovine aortic endothelial cells (BAEC). To determine if TRPC5 mediates vasodilatorinduced [Ca2þ]i increases, we first investigated NO-induced [Ca2þ]i increases in BAEC. NO-evoked [Ca2þ]i increases were observed in BAEC. Additionally, we confirmed that NO-induced increases in [Ca2þ]i were suppressed by TRPC5-specific siRNA and TRPC5 dominant-negative mutant, indicating that NO-evoked [Ca2þ]i increases in BAEC are attributable to Ca2þ influx through endogenous TRPC5 channels. ATP, a vasodilator, evoked [Ca2þ]i increases and NO production in BAEC. These ATP-evoked [Ca2þ]i increases and NO productions were suppressed by TRPC5-specific siRNA, suggesting that TRPC5 is involved in ATP-evoked [Ca2þ]i increases and NO production. Additionally, the ATP-evoked [Ca2þ]i increases and NO production were suppressed by a NOS inhibitor, u-nitro-L-arginine, and eNOS-specific siRNA. Thus, NO produced by eNOS in response to ATP plays a pivotal role in the ATP-evoked [Ca2þ]i influx via TRPC5. We confirmed the S-nitrosylation of endogenous TRPC5 after ATP stimulation. These results provide evidence for the activation of native TRPC5 channels by nitrosylation via eNOS upon ATP receptor stimulation in endothelial cells. The Ca2þ influx via nitrosylated TRPC5 channels may mediate the positive feedback regulation of Ca2þ-dependent NO production (Fig. 5). It has been suggested that endothelial cells of mice deficient in TRPC4 lack a store-operated Ca2þ entry. As a consequence, agonistinduced Ca2þ entry and vasorelaxation was reduced markedly, showing that TRPC4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2þ-entry pathway essentially contributing to the regulation of blood vessel tone (Freichel et al., 2001). Although we have investigated the sensitivity of TRPC4 to NO in HEK expression system, NO-induced Ca2þ responses in TRPC4-expressing cells were not seen. However, interestingly, cells coexpressing TRPC4 and TRPC5 showed NO responses comparable to those in cells expressing TRPC5 alone. We could immunoprecipitate TRPC5 with co-expressed TRPC4, suggesting that heteromultimeric TRPC5/TRPC4 channels have intact NO sensitivity. Additionally, we have confirmed superimposable distribution of TRPC5 with TRPC4 in BAEC. Thus, it can be speculated that Ca2þ influx via nitrosylated TRPC4/TRPC5 mediates the positive feedback regulation of Ca2þ-dependent NO production in endothelial

receptor

TRPC5 PIP2 DAG

Gα

eNOS

PLCβ

eNOS

βγ

ER

IP3R

IP3

Ca2+ eNOS

NO positive feedback amplification of Ca2+ influx and NO production

Fig. 5. Schematic model for positive feedback mechanism by which Ca2þ signals from NO-activated TRPC5 channels trigger NO production. In endothelial cells, Ca2þ influx via TRPC5 is triggered in response to vasodilator, which induces NO production via eNOS activation. NO diffuse and activate TRPC5. Ca2þ influx via nitrosylated TRPC5 channels mediates the positive feedback regulation of Ca2þ-dependent NO production. PLCb: phospholipase Cb, PIP2: phosphatidylinosito1-4,5-bisphosphate, DAG: diacyl glycerol, IP3: inositol-1,4,5-trisphosphate, IP3R: IP3 receptor.

25

cells, which participate in the regulation of blood vessel tone. Endothelial dysfunction is characterized by impaired NO signaling, decreased NO-dependent vasodilatation, increased vascular inflammation, and diminished response to angiogenic factors. In addition, endothelial dysfunction in the setting of cardiovascular risk factors such as hypercholesterolemia, hypertension, diabetes mellitus, and chronic smoking as well as in patients with heart failure has been shown to be at least in part dependent on decrease in vascular bioavailability of NO. Thus, disruption of the positive feedback regulated by TRPC4/TRPC5 may lead to initiation of endothelial cell dysfunction. 4. Conclusions Our data strongly suggest that crosstalk between Ca2þ and oxidative stress in the signal cascade mediates a wide variety of strictly controlled physiological responses. Ca2þ-permeable TRP channels are the molecular entities that link Ca2þ and oxidative stress signaling. As we demonstrated, TRPM2-mediated Ca2þ influx induces chemokine production in monocytes in response to ROS that aggravate inflammatory neutrophil infiltration and that Ca2þ influx via the NO-induced oxidative modification of TRPC5 channels mediates the positive feedback regulation of Ca2þ-dependent NO production in endothelial cells. Additionally, our recent study (Yamamoto et al., 2008) suggested that DSS-induced expression of proinflammatory cytokines interferon g and IL-12 was significantly diminished in the TRPM2-deficient colon and that the chemoattractant formylmethionyl-leucyl-phenylalanine-induced Ca2þ response and in vitro migration were significantly suppressed in TRPM2-deficient neutrophils. Thus, future studies will need to address TRPM2-dependent pathophysiological responses. In the future, we would like to propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases. References Aarts, M., Iihara, K., Wei, W.L., Xiong, Z.G., Arundine, M., Cerwinski, W., MacDonald, J.F., Tymianski, M., 2003. A key role for TRPM7 channels in anoxic neuronal death. Cell 115, 863e877. Anezaki, K., Asakura, H., Honma, T., Ishizuka, K., Funakoshi, K., Tsukada, Y., Narisawa, R., 1998. Correlations between interleukin-8, and myeloperoxidase or luminol-dependent chemiluminescence in inflamed mucosa of ulcerative colitis. Intern. Med. 37, 253e258. Araki, Y., Sugihara, H., Hattori, T., 2006. The free radical scavengers edaravone and tempol suppress experimental dextran sulfate sodium-induced colitis in mice. Int. J. Mol. Med. 17, 331e334. Barnes, P.J., 2004. COPD is there light at the end of the tunnel? Curr. Opin. Pharmacol. 4, 263e272. Bautista, D.M., Jordt, S.E., Nikai, T., Tsuruda, P.R., Read, A.J., Poblete, J., Yamoah, E.N., Basbaum, A.I., Julius, D., 2006. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 124, 1269e1282. Berridge, M.J., Bootman, M.D., Roderick, H.L., 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517e529. Blackburn, A.C., Doe, W.F., Buffinton, G.D., 1998. Salicylate hydroxylation as an indicator of hydroxyl radical generation in dextran sulfate-induced colitis. Free Radic. Biol. Med. 25, 305e313. Boulay, G., Zhu, X., Peyton, M., Jiang, M., Hurst, R., Stefani, E., Birnbaumer, L., 1997. Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein. J. Biol. Chem. 272, 29672e29680. Buelow, B., Song, Y., Scharenberg, A.M., 2008. The poly(ADP-ribose) polymerase PARP-1 is required for oxidative stress-induced TRPM2 activation in lymphocytes. J. Biol. Chem. 283, 24571e24583. Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J., Julius, D., 1999. A capsaicinreceptor homologue with a high threshold for noxious heat. Nature 398, 436e441. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., 1997. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816e824. Chiu, L.L., Perng, D.W., Yu, C.H., Su, S.N., Chow, L.P., 2007. Mold allergen, pen C 13, induces IL-8 expression in human airway epithelial cells by activating proteaseactivated receptor 1 and 2. J. Immunol. 178, 5237e5244. Cullen, P.J., Lockyer, P.J., 2002. Integration of calcium and Ras signalling. Nat. Rev. Mol. Cell Biol. 3, 339e348. Clapham, D.E., 2003. TRP channels as cellular sensors. Nature 426, 517e524.

26

S. Yamamoto et al. / Progress in Biophysics and Molecular Biology 103 (2010) 18e27

de Murcia, G., Menissier de Murcia, J., 1994. Poly(ADP-ribose) polymerase: a molecular nick-sensor. Trends Biochem. Sci. 19, 172e176. Droge, W., 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47e95. Duncan, L.M., Deeds, J., Hunter, J., Shao, J., Holmgren, L.M., Woolf, E.A., Tepper, R.I., Shyjan, A.W., 1998. Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis. Cancer Res. 58, 1515e1520. Feske, S., Giltnane, J., Dolmetsch, R., Staudt, L.M., Rao, A., 2001. Gene regulation mediated by calcium signals in T lymphocytes. Nat. Immunol. 2, 316e324. Fonfria, E., Marshall, I.C., Benham, C.D., Boyfield, I., Brown, J.D., Hill, K., Hughes, J.P., Skaper, S.D., McNulty, S., 2004. TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase. Br. J. Pharmacol. 143, 186e192. Freichel, M., Suh, S.H., Pfeifer, A., Schweig, U., Trost, C., Weißgerber, P., Biel, M., Philipp, S., Freise, D., Droogmans, G., Hofmann, F., Flockerzi, V., Nilius, B., 2001. Lack of an endothelial store-operated Ca2þ current impairs agonist-dependent vasorelaxation in TRP4/ mice. Nat. Cell Biol. 3, 121e127. Goel, M., Sinkins, W.G., Schilling, W.P., 2002. Selective association of TRPC channel subunits in rat brain synaptosomes. J. Biol. Chem. 277, 48303e48310. Greka, A., Navarro, B., Oancea, E., Duggan, A., Clapham, D.E., 2003. TRPC5 is a regulator of hippocampal neurite length and growth cone morphology. Nat. Neurosci. 6, 837e845. Grimm, C., Kraft, R., Sauerbruch, S., Schultz, G., Harteneck, C., 2003. Molecular and functional characterization of the melastatin-related cation channel TRPM3. J. Biol. Chem. 278, 21493e21501. Grimm, C., Kraft, R., Schultz, G., Harteneck, C., 2005. Activation of the melastatin-related cation channel TRPM3 by D-erythro-sphingosine. Mol. Pharmacol. 67, 798e805. Güler, A.D., Lee, H., Iida, T., Shimizu, I., Tominaga, M., Caterina, M., 2002. Heatevoked activation of the ion channel, TRPV4. J. Neurosci. 22, 6408e6414. Hara, Y., Wakamori, M., Ishii, M., Maeno, E., Nishida, M., Yoshida, T., Yamada, H., Shimizu, S., Mori, E., Kudoh, J., Shimizu, N., Kurose, H., Okada, Y., Imoto, K., Mori, Y., 2002. LTRPC2 Ca2þ-permeable channel activated by changes in redox status confers susceptibility to cell death. Mol. Cell 9, 163e173. Henricks, P.A., Nijkamp, F.P., 2001. Reactive oxygen species as mediators in asthma. Pulm. Pharmacol. Ther. 14, 409e420. Hermosura, M.C., Garruto, R.M., 2007. TRPM7 and TRPM2 e candidate susceptibility genes for Western Pacific ALS and PD? Biochim. Biophys. Acta 1772, 822e835. Hess, D.T., Matsumoto, A., Kim, S.O., Marshall, H.E., Stamler, J.S., 2005. Protein Snitrosylation: purview and parameters. Nat. Rev. Mol. Cell Biol. 6, 150e166. Hinman, A., Chuang, H.H., Bautista, D.M., Julius, D., 2006. TRP channel activation by reversible covalent modification. Proc. Natl. Acad. Sci. U. S. A. 103, 19564e19568. Hitchon, C.A., El-Gabalawy, H.S., 2004. Oxidation in rheumatoid arthritis. Arthritis Res. Ther. 6, 265e278. Hofmann, T., Obukhov, A.G., Schaefer, M., Harteneck, C., Gudermann, T., Schultz, G., 1999. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259e263. Hofmann, T., Schaefer, M., Schultz, G., Sudermann, T., 2002. Subunit composition of mammalian transient receptor potential channels in living cells. Proc. Natl. Acad. Sci. U S A 99, 7461e7466. Inoue, R., Okada, T., Onoue, H., Hara, Y., Shimizu, S., Naitoh, S., Ito, Y., Mori, Y., 2001. The transient receptor potential protein homologue TRP6 is the essential component of vascular a1-adrenoceptor-activated Ca2þ-permeable cation channel. Circ. Res. 88, 325e332. Jaeschke, H., 2000. Reactive oxygen and mechanisms of inflammatory liver injury. J. Gastroenterol. Hepatol. 15, 718e724. Jaffrey, S.R., Erdjument-Bromage, H., Ferris, C.D., Tempst, P., Snyder, S.H., 2001. Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat. Cell Biol. 3, 193e197. Jaramillo, M., Olivier, M., 2002. Hydrogen peroxide induces murine macrophage chemokine gene transcription via extracellular signal-regulated kinase- and cyclic adenosine 50 -monophosphate (cAMP)-dependent pathways: involvement of NF-kB, activator protein 1, and cAMP response element binding protein. J. Immunol. 169, 7026e7038. Jordt, S.E., Bautista, D.M., Chuang, H.H., McKemy, D.D., Zygmunt, P.M., Hogestatt, E. D., Meng, I.D., Julius, D., 2004. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 427, 260e265. Josse, C., Boelaert, J.R., Best-Belpomme, M., Piette, J., 2001. Importance of posttranscriptional regulation of chemokine genes by oxidative stress. Biochem. J. 360, 321e333. Keshavarzian, A., Fusunyan, R.D., Jacyno, M., Winship, D., MacDermott, R.P., Sanderson, I.R., 1999. Increased interleukin-8 (IL-8) in rectal dialysate from patients with ulcerative colitis: evidence for a biological role for IL-8 in inflammation of the colon. Am. J. Gastroenterol. 94, 704e712. Kim, H.J., Li, Q., Tjon-Kon-Sang, S., So, I., Kiselyov, K., Soyombo, A.A., Muallem, S., 2008. A novel mode of TRPML3 regulation by extracytosolic pH absent in the varitint-waddler phenotype. EMBO J. 27, 1197e1205. Kiselyov, K., Chen, J., Rbaibi, Y., Oberdick, D., Tjon-Kon-Sang, S., Shcheynikov, N., Muallem, S., Soyombo, A., 2005. TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage. J. Biol. Chem. 280, 43218e43223. Kolisek, M., Beck, A., Fleig, A., Penner, R., 2005. Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels. Mol. Cell 18, 61e69. Korenaga, D., Takesue, F., Kido, K., Yasuda, M., Inutsuka, S., Honda, M., Nagahama, S., 2002. Impaired antioxidant defense system of colonic tissue and cancer development in dextran sulfate sodium-induced colitis in mice. J. Surg. Res. 102, 144e149.

Kuhn, F.J., Luckhoff, A., 2004. Sites of the NUDT9-H domain critical for ADP-ribose activation of the cation channel TRPM2. J. Biol. Chem. 279, 46431e46437. Kwan, K.Y., Allchorne, A.J., Vollrath, M.A., Christensen, A.P., Zhang, D.S., Woolf, C.J., Corey, D.P., 2006. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction. Neuron 50, 277e289. Lambeth, J.D., 2004. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181e189. Launay, P., Fleig, A., Perraud, A.L., Scharenberg, A.M., Penner, R., Kinet, J.P., 2002. TRPM4 is a Ca2þ-activated nonselective cation channel mediating cell membrane depolarization. Cell 109, 397e407. Lee, K., Esselman, W.J., 2001. cAMP potentiates H2O2-induced ERK1/2 phosphorylation without the requirement for MEK1/2 phosphorylation. Cell. Signal. 13, 645e652. Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J.M., Plowman, G.D., Rudy, B., Schlessinger, J., 1995. Protein tyrosine kinase PYK2 involved in Ca2þinduced regulation of ion channel and MAP kinase functions. Nature 376, 737e745. Leypold, B.G., Yu, C.R., Leinders-Zufall, T., Kim, M.M., Zufall, F., Axel, R., 2002. Altered sexual and social behaviors in trp2 mutant mice. Proc. Natl. Acad. Sci. U. S. A. 99, 6376e6381. Liedtke, W., Choe, Y., Marti-Renom, M.A., Bell, A.M., Denis, C.S., Sali, A., Hudspeth, A.J., Friedman, J.M., Heller, S., 2000. Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell 103, 525e535. Luster, A.D., 1998. Chemokinesechemotactic cytokines that mediate inflammation. N. Engl. J. Med. 338, 436e445. Macpherson, L.J., Dubin, A.E., Evans, M.J., Marr, F., Schultz, P.G., Cravatt, B.F., Patapoutian, A., 2007. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 445, 541e545. Mahida, Y.R., Ceska, M., Effenberger, F., Kurlak, L., Lindley, I., Hawkey, C.J., 1992. Enhanced synthesis of neutrophil-activating peptide-1/interleukin-8 in active ulcerative colitis. Clin. Sci. 82, 273e275. Maroto, R., Raso, A., Wood, T.G., Kurosky, A., Martinac, B., Hamill, O.P., 2005. TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nat. Cell Biol. 7, 179e185. McKemy, D.D., Neuhausser, W.M., Julius, D., 2002. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416, 52e58. Mizukami, Y., Jo, W.S., Duerr, E.M., Gala, M., Li, J., Zhang, X., Zimmer, M.A., Iliopoulos, O., Zukerberg, L.R., Kohgo, Y., Lynch, M.P., Rueda, B.R., Chung, D.C., 2005. Induction of interleukin-8 preserves the angiogenic response in HIF-1alpha-deficient colon cancer cells. Nat. Med. 11, 992e997. Montell, C., Rubin, G.M., 1989. Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron 2, 1313e1323. Mukaida, N., Okamoto, S., Ishikawa, Y., Matsushima, K., 1994. Molecular mechanism of interleukin-8 gene expression. J. Leukoc. Biol. 56, 554e558. Nadler, M.J., Hermosura, M.C., Inabe, K., Perraud, A.L., Zhu, Q., Stokes, A.J., Kurosaki, T., Kinet, J.P., Penner, R., Scharenberg, A.M., Fleig, A., 2001. LTRPC7 is a Mg$ATPregulated divalent cation channel required for cell viability. Nature 411, 590e595. Nagata, K., Duggan, A., Kumar, G., Garcia-Anoveros, J., 2005. Nociceptor and hair cell transducer properties of TRPA1, a channel for pain and hearing. J. Neurosci. 25, 4052e4061. Nauli, S.M., Alenghat, F.J., Luo, Y., Williams, E., Vassilev, P., Li, X., Elia, A.E., Lu, W., Brown, E.M., Quinn, S.J., Ingber, D.E., Zhou, J., 2003. Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat. Genet. 33, 129e137. Okada, T., Inoue, R., Yamazaki, K., Maeda, A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K., Imoto, K., Mori, Y., 1999. Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. J. Biol. Chem. 274, 27359e27370. Okada, T., Shimizu, S., Wakamori, M., Maeda, A., Kurosaki, T., Takada, N., Imoto, K., Mori, Y., 1998. Molecular cloning and functional characterization of a novel receptor-activated TRP Ca2þ channel from mouse brain. J. Biol. Chem. 273, 10279e10287. Oliver, F.J., Menissier-de Murcia, J., Nacci, C., Decker, P., Andriantsitohaina, R., Muller, S., de la Rubia, G., Stoclet, J.C., de Murcia, G., 1999. Resistance to endotoxic shock as a consequence of defective NF-kB activation in poly (ADP-ribose) polymerase-1 deficient mice. EMBO J. 18, 4446e4454. Peier, A.M., Reeve, A.J., Andersson, D.A., Moqrich, A., Earley, T.J., Hergarden, A.C., Story, G.M., Colley, S., Hogenesch, J.B., McIntyre, P., Bevan, S., Patapoutian, A., 2002a. A heat-sensitive TRP channel expressed in keratinocytes. Science 296, 2046e2049. Peier, A.M., Moqrich, A., Hergarden, A.C., Reeve, A.J., Andersson, D.A., Story, G.M., Earley, T.J., Dragoni, I., McIntyre, P., Bevan, S., Patapoutian, A., 2002b. A TRP channel that senses cold stimuli and menthol. Cell 108, 705e715. Perraud, A.L., Fleig, A., Dunn, C.A., Bagley, L.A., Launay, P., Schmitz, C., Stokes, A.J., Zhu, Q., Bessman, M.J., Penner, R., Kinet, J.P., Scharenberg, A.M., 2001. ADPribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology. Nature 411, 595e599. Perraud, A.L., Schmitz, C., Scharenberg, A.M., 2003. TRPM2 Ca2þ permeable cation channels: from gene to biological function. Cell Calcium 33, 519e531. Perraud, A.L., Takanishi, C.L., Shen, B., Kang, S., Smith, M.K., Schmitz, C., Knowles, H. M., Ferraris, D., Li, W., Zhang, J., Stoddard, B.L., Scharenberg, A.M., 2005. Accumulation of free ADP-ribose from mitochondria mediates oxidative stressinduced gating of TRPM2 cation channels. J. Biol. Chem. 280, 6138e6148. Pérez, C.A., Huang, L., Rong, M., Kozak, J.A., Preuss, A.K., Zhang, H., Max, M., Margolskee, R.F., 2002. Transient receptor potential channel expressed in taste receptor cells. Nat. Neurosci. 11, 1169e1176.

S. Yamamoto et al. / Progress in Biophysics and Molecular Biology 103 (2010) 18e27 Rahman, I., 2005. The role of oxidative stress in the pathogenesis of COPD: implications for therapy. Treat. Respir. Med. 4, 175e200. Riccio, A., Li, Y., Moon, J., Kim, K.S., Smith, K.S., Rudolph, U., Gapon, S., Yao, G.L., Tsvetkov, E., Rodig, S.J., Veer, A.V., Meloni, E.G., Carlezon Jr., W.A., Bolshakov, V. Y., Clapham, D.E., 2009. Essential role for TRPC5 in amygdala function and fearrelated behavior. Cell 137, 761e772. Seimiya, H., Tsuruo, T., 1993. Differential expression of protein tyrosine phosphatase genes during phorbol ester-induced differentiation of human leukemia U937 cells. Cell Growth Differ. 4, 1033e1039. Singer, M., Sansonetti, P.J., 2004. IL-8 is a key chemokine regulating neutrophil recruitment in a new mouse model of Shigella-induced colitis. J. Immunol. 173, 4197e4206. Smith, G.D., Gunthorpe, M.J., Kelsell, R.E., Hayes, P.D., Reilly, P., Facer, P., Wright, J.E., Jerman, J.C., Walhin, J.P., Ooi, L., Egerton, J., Charles, K.J., Smart, D., Randall, A.D., Anand, P., Davis, J.B., 2002. TRPV3 is a temperature-sensitive vanilloid receptorlike protein. Nature 418, 186e190. Story, G.M., Peier, A.M., Reeve, A.J., Eid, S.R., Mosbacher, J., Hricik, T.R., Earley, T.J., Hergarden, A.C., Andersson, D.A., Hwang, S.W., McIntyre, P., Jegla, T., Bevan, S., Patapoutian, A., 2003. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112, 819e829. Strotmann, R., Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T.D., 2000. OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat. Cell Biol. 2, 695e702. Strübing, C., Krapivinsky, G., Krapivinsky, L., Clapham, D.E., 2001. TRPC1 and TRPC5 form a novel cation channel in mammalian brain. Neuron 29, 645e655. Takahashi, N., Mizuno, Y., Kozai, D., Yamamoto, S., Kiyonaka, S., Shibata, T., Uchida, K., Mori, Y., 2008. Molecular characterization of TRPA1 channel activation by cysteine-reactive inflammatory mediators. Channels (Austin) 2, 287e298. Tanuma, S., Yagi, T., Johnson, G.S., 1985. Endogenous ADP ribosylation of high mobility group proteins 1 and 2 and histone H1 following DNA damage in intact cells. Arch. Biochem. Biophys. 237, 38e42. Togashi, K., Hara, Y., Tominaga, T., Higashi, T., Konishi, Y., Mori, Y., Tominaga, M., 2006. TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion. EMBO J. 25, 1804e1815. Tominaga, M., Caterina, M.J., Malmberg, A.B., Rosen, T.A., Gilbert, H., Skinner, K., Raumann, B.E., Basbaum, A.I., Julius, D., 1998. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 21, 531e543. Videla, L.A., Rodrigo, R., Orellana, M., Fernandez, V., Tapia, G., Quinones, L., Varela, N., Contreras, J., Lazarte, R., Csendes, A., Rojas, J., Maluenda, F., Burdiles, P., Diaz, J.C., Smok, G., Thielemann, L., Poniachik, J., 2004. Oxidative stress-related parameters in the liver of non-alcoholic fatty liver disease patients. Clin. Sci. 106, 261e268. Voets, T., Nilius, B., Hoefs, S., van der Kemp, A.W., Droogmans, G., Bindels, R.J., Hoenderop, J.G., 2004. TRPM6 forms the Mg2þ influx channel involved in intestinal and renal Mg2þ absorption. J. Biol. Chem. 279, 19e25.

27

Virag, L., Szabo, C., 2002. The therapeutic potential of poly(ADP-ribose) polymerase inhibitors. Pharmacol. Rev. 54, 375e429. Watanabe, H., Vriens, J., Suh, S.H., Benham, C.D., Droogmans, G., Nilius, B., 2002. Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells. J. Biol. Chem. 277, 47044e47051. Wehage, E., Eisfeld, J., Heiner, I., Jungling, E., Zitt, C., Luckhoff, A., 2002. Activation of the cation channel long transient receptor potential channel 2 (LTRPC2) by hydrogen peroxide. A splice variant reveals a mode of activation independent of ADP-ribose. J. Biol. Chem. 277, 23150e23156. Wilson, L., Butcher, C.J., Kellie, S., 1993. Calcium ionophore A23187 induces interleukin-8 gene expression and protein secretion in human monocytic cells. FEBS Lett. 325, 295e298. Xu, H., Ramsey, I.S., Kotecha, S.A., Moran, M.M., Chong, J.A., Lawson, D., Ge, P., Lilly, J., Silos-Santiago, I., Xie, Y., DiStefano, P.S., Curtis, R., Clapham, D.E., 2002. TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 418, 181e186. Yamamoto, S., Shimizu, S., Kiyonaka, S., Takahashi, N., Wajima, T., Hara, Y., Negoro, T., Hiroi, T., Kiuchi, Y., Okada, T., Kaneko, S., Lange, I., Fleig, A., Penner, R., Nishi, M., Takeshima, H., Mori, Y., 2008. TRPM2-mediated Ca2þ influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration. Nat. Med. 14, 738e747. Yoshida, T., Inoue, R., Morii, T., Takahashi, N., Yamamoto, S., Hara, Y., Tominaga, M., Shimizu, S., Sato, Y., Mori, Y., 2006. Nitric oxide activates TRP channels by cysteine S-nitrosylation. Nat. Chem. Biol. 2, 596e607. Zeng, X., Dai, J., Remick, D.G., Wang, X., 2003. Homocysteine mediated expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human monocytes. Circ. Res. 93, 311e320. Zhang, W., Chu, X., Tong, Q., Cheung, J.Y., Conrad, K., Masker, K., Miller, B.A., 2003. A novel TRPM2 isoform inhibits calcium influx and susceptibility to cell death. J. Biol. Chem. 278, 16222e16229. Zhu, X., Jiang, M., Peyton, M., Boulay, G., Hurst, R., Stefani, E., Birnbaumer, L., 1996. trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2þ entry. Cell 85, 661e671. Zhu, X., Jiang, M., Birnbaumer, L., 1998. Receptor-activated Ca2þ influx via human Trp3 stably expressed in human embryonic kidney (HEK) 293 cells. J. Biol. Chem. 273, 133e142. Zitt, C., Zobel, A., Obukhov, A.G., Harteneck, C., Kalkbrenner, F., Lückhoff, A., Schultz, G., 1996. Cloning and functional expression of a human Ca2þpermeable cation channel activated by calcium store depletion. Neuron 16, 1189e1196. Zitt, C., Obukhov, A.G., Strübing, C., Zobel, A., Kalkbrenner, F., Lückhoff, A., Schultz, G., 1997. Expression of TRPC3 in Chinese hamster ovary cells results in calcium-activated cation currents not related to store depletion. J. Cell Biol. 138, 1333e1341.