Transient receptor potential (TRP) channels: Biosensors for redox environmental stimuli and cellular status

Journal Pre-proof Transient receptor potential (TRP) channels: Biosensors for redox environmental stimuli and cellular status Reiko Sakaguchi, Yasuo Mori PII:

S0891-5849(19)30701-4

DOI:

https://doi.org/10.1016/j.freeradbiomed.2019.10.415

Reference:

FRB 14469

To appear in:

Free Radical Biology and Medicine

Received Date: 26 April 2019 Revised Date:

26 October 2019

Accepted Date: 29 October 2019

Please cite this article as: R. Sakaguchi, Y. Mori, Transient receptor potential (TRP) channels: Biosensors for redox environmental stimuli and cellular status, Free Radical Biology and Medicine (2019), doi: https://doi.org/10.1016/j.freeradbiomed.2019.10.415. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

Transient receptor potential (TRP) channels: biosensors for redox environmental stimuli and cellular status

Reiko Sakaguchi1,2 and Yasuo Mori1,2,*

From the 1Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan and the 2World Premier International Research Initiative-Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 615-8510, Japan.

1

Address for correspondence: Yasuo Mori, Ph.D. Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan. Tel: 81-75-383-2761, Fax: 81-75-383-2765 E-mail: [email protected]

Key words: TRP channels; redox sensing; biosensors; drug discovery

2

Abstract Transient receptor potential (TRP) channels are a family of cation channels that depolarizes the membrane potential and regulates intracellular concentrations of cations such as Ca2+. TRP channels are also known to function as “biosensors” to detect changes of the surrounding environment and cellular status. Lines of evidence have unveiled that numerous proteins are subject to redox modification and subsequent signaling. For example, TRPM2, TRPC5, TRPV1, and TRPA1 are known as redox sensors activated by hydrogen peroxide (H2O2), nitric oxide (NO), and electrophiles. Thus, these channels facilitate the influx of cations which in turn triggers the appropriate cellular responses against environmental redox stimuli and cellular redox status. In this review, we focus on the recent findings regarding the functions of TRP channels in relation to other ion channels, and other proteins which also go through redox modification of cysteine (Cys) residues. We aim to understand the structural and molecular basis of the redox-sensing mechanisms of TRP channels in exerting various functions under physiological conditions as well as pathological conditions such as cancer malignancy. Their future potential as drug targets will also be discussed.

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Introduction Living organisms including us humans sense physical and chemical changes in the environment and adapt to them in order to survive. Various protein molecules mediate signals of these adaptive responses, and one of such signal mediators is the second messenger Ca2+, known to form a large gradient between the inside and outside of the cell membrane. Na+ mediates the electrical signal by inducing changes in the voltage formed across the membrane. Once the influx of these cation occurs, it triggers various cellular responses. There are several cation channels that facilitate this cation influx, and Transient Receptor Potential (TRP) channels are one of them. TRP channels are a family of ion channels that were first identified in Drosophila in 1989 (1). They are highly conserved among species and are classified into 6 subfamilies and 28 members in mammals, including TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystic kidney disease), TRPML (mucolipin) and TRPA (ankyrin) subfamilies (Figure 1). TRPN homologs have been discovered in D. melanogaster, C. elegans, X. laevis, and D. rerio, but not in mammals. TRPC family was the first to be discovered among the members and is implicated to play a role in neuronal development and survival (2,3). TRPV family was identified as the receptor for capsaicin (4). It consists of 6 family members, most of them activated by heat. TRPM family consists of 8 members that are implicated in mechano-sensing (TRPM4, TRPM7) (5,6) or cold sensing (TRPM8) (7,8). TRPA1 is the only member of TRPA1 family in mammals and is activated by various modulators (9). However, these are just a few examples of the various and yet unknown properties of these channels. TRP proteins share the common structure that consists of six transmembrane

4

regions and a pore region in between 5th and 6th transmembrane domains, assembling into a tetramer to form a divalent cation channel (10). They are responsible for transmitting the information from the outer world and evoke necessary response. Other than the triggers mentioned above, many of the TRP channels are known to function as “biosensors” for the surrounding environment of our bodies (11), and of all of them, the best evidence is for TRPA1. TRPA1 can act as a sensor for cold temperature (12,13), heat (14), electrophiles (15-18), and molecular oxygen O2 (19), and also mediates pain sensation (13,20). Other examples include TRPV1, which is known to detect heat (4) and TRPM8, known to be activated by menthol (8). TRPM2 senses H2O2, one of the reactive oxygen species (ROS) (21). To date, extensive studies have been conducted to elucidate their properties and functions. In this review, we would like to focus on the recent findings regarding the roles of mainly TRP channels which detect changes in redox status in regulating cancer malignancy and/or other pathological conditions. The functions of these TRP channels as sensors in vivo will be discussed, along with their future potential as drug targets.

Redox-sensitive TRP channels activated by cysteine modification: Regulation by nitric oxide and other redox stimuli Cells in our bodies are regulated by various molecules and factors. Among them, Ca2+ is one of the most well-studied signaling molecules that controls diverse cellular functions. Another important signaling molecule is nitric oxide (NO), one of the reactive nitrogen species (RNS), which also plays a role in numerous physiological processes such as vasodilation. Furthermore, NO and Ca2+ together regulate diverse biological processes through closely coordinated mechanisms. Our group have

5

previously reported a novel activation mechanism in TRP channels mediated by cysteine (Cys) S-nitrosylation (22). Labeling and functional assays using Cys mutants demonstrated that Cys553 and nearby Cys558 are nitrosylation sites mediating NO sensitivity in mouse TRPC5. The responsive TRP proteins such as TRPC1, TRPC4, TRPV1, TRPV3, and TRPV4 have these Cys residues conserved on the same N-terminal side of the pore region. The nitrosylation of native TRPC5 upon G protein–coupled ATP receptor stimulation enhanced the entry of Ca2+ into endothelial cells. These findings suggested the functional coupling of Ca2+ and NO pathways in endothelial cells (22). Separately, Maddox et al. have reported that in chick retinal amacrine cells, TRPC5 is required for the NO-dependent increase in dendritic Ca2+ and GABA release (23,24). Here, the authors evaluated the ability of NO to elevate dendritic Ca2+ and to stimulate GABA release from cultured amacrine cells. Using inhibitors and the CRISPR/Cas9-mediated TRPC5 knockdown, both the NO-dependent Ca2+ elevations and the increase in GABA release were shown to be dependent on the expression of TRPC5. Other than NO, a recent study showed that increased glutathionylation and activation of TRPC5 by oxidative stress contributes to neuronal damage in the striatum. The TRPC5 blocker ML204 improved rearing behavior in Huntington's disease transgenic mice, suggesting the potential of TRPC5 as a drug target (25). It has also been reported that the extracellular reduced thioredoxin activates homomeric TRPC5 and heteromeric TRPC5–TRPC1 channels by breaking a disulfide bridge in the predicted extracellular loop next to the ion-selectivity filter of TRPC5 (26). Thioredoxin is an endogenous redox protein with established intracellular functions but its extracellular targets were largely unknown. Here, it has been shown that TRPC5 and

6

TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, whose extracellular concentrations of thioredoxin is high. The endogenous TRPC5–TRPC1 channels of the cells are activated by reduced thioredoxin, and the blockade of the channels enhances secretory activity and prevents the suppression of secretion by thioredoxin. These data suggest the TRPC5 activation mechanism that couples extracellular reducing molecule to cell function (26). Similar to TRPC5, some other TRP channels are also activated by oxidative stimuli. TRPA1 is modulated by noxious compounds through covalent modification of Cys residues (12). Hinman et al. have reported that the TRPA1 channel is activated by covalent, reversible modification (27). Here, thiol-reactive compounds of diverse structures activate TRPA1 in a manner that relies on covalent modification of Cys residues within the cytoplasmic N terminus of the channel. These findings suggest a mechanism where natural products activate a receptor through direct, reversible, and covalent protein modification. More recent study has shown that the activation of TRPV4 enhances oxidative stress by inhibiting catalase and glutathione peroxidase and increasing neuronal nitric oxide synthase (nNOS) activity (24). This TRPV4 activity induced neurotoxicity characterized by neuronal death and apoptosis in the hippocampal CA1 area. The authors suggest that a positive feedback loop is formed by TRPV4 and free radicals, which is involved in neuronal injury under pathological conditions, on the basis of previous reports indicating that TRPV4 is activated by NO and H2O2 (22,29). Thus, TRPV4 would also be an attractive target for neuronal protection. Another example is NO-activated Ca2+ signaling and calpain activation via TRPV1 (30), which is an extremely well-known sensor for noxious stimuli such as heat

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and for diverse inflammatory mediators such as oxidative stress to mediate nociception in a subset of sensory neurons. In macrophages, treatment with NO donors induced Liver X receptor α (LXRα) degradation and reduced the expression of ATP-binding cassette transporter A1 (ABCA1) and cholesterol efflux. NO induced Ca2+ influx into cells, increased calpain activity and promoted the formation of calpain-LXRα complex. Pharmacological

inhibition

S-nitroso-N-acetylpenicillamine

of

calpain

(SNAP)-induced

activity degradation

reversed of

the LXRα,

down-regulation of ABCA1, and impairment of cholesterol efflux in macrophages. Removal of extracellular Ca2+ by EGTA or pharmacological inhibition of TRPV1 channel activity diminished SNAP-induced increase in intracellular Ca2+, calpain activation, LXRα degradation, and impaired cholesterol efflux. These findings suggest that NO activates calpain via TRPV1 channel and regulate TRPV1-mediated Ca2+ signaling. A more recent report demonstrated that rats treated with NO donor showed a decrease in the area of acetic acid-induced gastric ulcer. This effect was abolished by TRPV1 antagonist, suggesting that NO donor alleviates acetic acid-induced gastric ulcer in rats via vagus nerve, and S-nitrosylation of TRPV1 may participate in this route. This finding revealed a new mechanism for vagal afferent activation, and a new potential anti-inflammatory target (31). Recently, our group have characterized the oxidative status of Cys residues in different redox environments and proposed a model of TRPV1 activation by oxidation (32). Cys oxidation has been considered as the principal mechanism of TRPV1 oxidation sensing. The formation of subunit dimers carrying a stable intersubunit disulfide bond between Cys258 and Cys742 of human TRPV1 (hTRPV1) was identified. C258S and C742S hTRPV1 mutants were shown to have a decreased protein half-life,

8

supporting the role of the intersubunit disulfide bond in channel stability. Interestingly, the C258S hTRPV1 mutant lost the response to oxidants. Mass spectrometric analysis of Cys residues of hTRPV1 treated with H2O2 showed that Cys258 is highly sensitive to oxidation. These results suggest that Cys258 residues are heterogeneously modified in the hTRPV1 tetrameric complex, involved both in oxidation sensing and in the disulfide bond for stable subunit dimerization. hTRPV1 channel has a heterogeneous subunit composition in terms of both redox status and function. Another good example of Ca2+ signaling regulation was reported for TRPC1. The [Ca2+]i oscillation stimulated by aromatic amino acids was selectively abolished by TRPC1 down-regulation using siRNAs that targeted different coding regions of TRPC1. Furthermore, this [Ca2+]i oscillation was also abolished by inhibition of TRPC1 function with an antibody that binds to the pore region of the channel. In addition, the aromatic amino acid-stimulated [Ca2+]i oscillation was shown to be inhibited by protein kinase C (PKC) inhibitors or siRNA-mediated PKCα down-regulation and impaired by either calmodulin antagonists or by the expression of a dominant-negative calmodulin mutant. A model was proposed for the generation of transient [Ca2+]i oscillation mediated by Ca2+-sensing receptors in which the stimulation of oscillation by aromatic amino acids is integrated with TRPC1 regulation via PKC and calmodulin (33).

Redox-sensitive TRP channels activated by cysteine modification: Physiological roles in cardiovascular function Although accumulating evidence support the interplay of Ca2+ and NO pathways, the signaling proteins that underlie this interplay remain unclear. TRP channels are candidates to play key roles in associating signal proteins. For example,

9

TRPC5 is known to be expressed in vascular endothelial cells (34). Our group have evaluated

the physical

and

functional

interaction

of the

receptor-activated

Ca2+-permeable TRPC5 channel with Ca2+-dependent endothelial NO synthase (eNOS) in endothelial cells. Activation of TRPC5 is induced by NO via nitrosylation, facilitating Ca2+ influx, which in turn enhances NO production by eNOS. This raises the hypothesis that upon stimulation of a G-protein-coupled ATP receptor, Ca2+ influx via receptor-activated TRPC5 channels elicits NO production by eNOS, which in turn induces secondary activation of TRPC5 channels via Cys S-nitrosylation in vascular endothelial cells (22,35,36) (Figure 2). On the other hand, some reports deny the direct effect of NO modification in TRPC5. One study has demonstrated that in HEK293 cells overexpressing mouse TRPC5 or human TRPC5, SNAP failed to stimulate TRPC5 measured by Ca2+ indicator or by recording membrane current under voltage clamp (37). In this study, it was suggested that NO is not a direct modulator of homomeric TRPC5 channels but may inhibit endogenous channels of bovine aortic endothelial cells that contain TRPC5. They have concluded that the discrepancy probably comes from the different measurement systems (37). The same group have later reported a systematic investigation of antioxidants and found that TRPC5 has a chemical-sensing feature including stimulation by endogenous H2O2 and relatively potent, but indirect, inhibition by the important dietary factor resveratrol (38). Another

recent

endothelium-dependent

work

reported

contraction.

In

that this

TRPC5 study,

is

essential

for

acetylcholine-induced

endothelium-dependent contraction in mouse carotid arteries was reduced in TRPC5 knockout (KO) mice than in wild-type (WT) mice. Clemizole and ML204, which are

10

TRPC5 inhibitors, also reduced the endothelium-dependent contractions, indicating the positive role of TRPC5 in vascular contraction (34). TRPC5 has also been demonstrated to be involved in endothelial cell angiogenesis and blood perfusion in an oxygen-induced retinopathy model and ischemia model. A regulatory link between nuclear factor of activated T cell (NFAT) isoform c3 and angiopoietin-1 that provides the mechanistic basis for the angiogenic function of TRPC5 is demonstrated. Treatment with riluzole which activates TRPC5 in endothelial cells improved the recovery from ischemia in mice, also supporting the notion that TRPC5 regulates angiogenesis (39). Using TRPC5 KO mice, the same group also demonstrated that TRPC5 is a key pressure transducer in the baroreceptors and plays an important role in maintaining blood pressure stability. Here, although the basal blood pressure is unaffected, the daily fluctuation of the blood pressure is larger in TRPC5 KO mice than the WT mice (40-42). This might reflect the disordered production of NO by eNOS, presumably due to the lack of precise regulation by TRPC5. Alternatively, the discrepancy between the results showing that TRPC5 is important for NO release, and the unaffected baseline blood pressure in TRPC5 KO mice might simply rise from the difference between the experimental systems, two-dimensional (2D) cell culture and in vivo analysis. For instance, it is possible that in TRPC5 KO mice, compensation pathways via TRPC1 and TRPC4 channels or other signaling molecules operate to maintain vascular responses. Also, in 2D culture, some physiological cellular arrangements are not preserved, the role of TRPC5 in NO production is enhanced compared to the endothelial cells in vivo. Although the outcome of the function seems controvertial, TRPC5 is undoubtedly playing an important role in the regulation of vascular endothelial system.

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As previously pointed out, these disagreements might come from the experimental differences such as cell line sources, procedures of cell isolation, or culture conditions, which may alter the expression profiles of certain cofactors (37). Further studies are awaited to clarify the full aspects of TRPC5 in physiological systems. Dysregulation of TRP channels other than TRPC5 have also been known to affect cardiovascular function under pathological stress. For example, cold stress is known to trigger cardiac hypertrophy by down-regulation of TRPV1. The phenotype caused by cold stress is mimicked by TRPV1 antagonist (43). In contrary with this protective effect of TRPV1, crotonaldehyde or acrolein, which are unsaturated aldehydes in cigarette smoke, are reported to trigger proinflammatory response and contractile disfunction via TRPV1 activation (44,45). In addition, systemic capsaicin pretreatment resulted in cardiac dysfunction characterized by elevation of left ventricular end-diastolic pressure (46). This might be related to the bidirectional functions of TRPV1 where it is activated at low dose of agonist and later becomes deactivated at higher concentration of capsaicin. In cultured neonatal rat cardiomyocytes, it has been observed that TRPC1 (47), TRPC3 (48), and TRPC7 (49) are up-regulated upon development of cardiac hypertrophy, induced by agonists such as endothelin-1 or angiotensin II. Using cultured rat myocytes, it has also been demonstrated that TRPC3 and TRPC6 mediate angiotensin II-induced NFAT translocation (50), which is the crucial step in cardiac hypertrophy (51). Collectively, these studies prove the important role of TRP channels in cardiovascular systems (52).

Redox-sensitive TRP channels activated by cysteine modification: Roles of TRPA1

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channel in cancer Aside from ROS and RNS, another reactive species in the biological system are the reactive disulfides with various redox potentials. These can modify certain proteins to activate signaling pathways. Our group have previously performed the systematic evaluation of TRP channels using reactive disulfides with various redox potentials and revealed the capability of TRPA1 to sense O2. This led to the discovery that the TRPA1 channel, which is activated both under hypoxia and hyperoxia conditions, contributes to O2-sensing mechanisms in mammals. This O2 sensing is based upon two distinct processes. Under normal oxygen environment (~20%), prolyl hydroxylases (PHDs) inhibit TRPA1 activity in an O2-dependent manner. Under hyperoxia, direct O2 action overwhelms this inhibition via the sensitivity of TRPA1 to Cys-mediated oxidation. TRPA1 is also activated through relief from the same PHD-mediated inhibition in hypoxia. In TRPA1 KO mice, hyperoxia- and hypoxia-induced cationic currents are abolished in vagal and sensory neurons. These results suggest a completely new O2-sensing mechanism mediated by TRPA1 (19). The roles of hypoxic environment are receiving more attention both in physiological and pathological conditions. For example, cancer environment is well known to be hypoxic due to the insufficient growth of matured blood vessels into the cancer as well as the high respiration of the malignant cells, and under hypoxic conditions, tumor cells undergo a series of adaptations that promote further evolution of aggressive tumor phenotype (53). Recently, Takahashi et al. have reported that TRPA1 is overexpressed in human cancer and mediates a noncanonical ROS defense program (Figure 3). TRPA1 promotes ROS tolerance via up-regulation of Ca2+-dependent anti-apoptotic pathways that are initiated by an oxidant-defense transcription factor

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nuclear factor erythroid 2-related factor 2 (Nrf2), which directly controls TRPA1 expression. TRPA1 inhibition suppresses xenograft tumor growth and enhances chemosensitivity. These findings reveal an oxidative-stress defense program involving TRPA1 that could be exploited for targeted cancer therapies (54). The interplay between Ca2+ and ROS signaling in cancer is described in detail in a recent review by Hempel et al. (55).

Redox-sensitive TRP channels activated by cysteine modification: 3D-Structural analysis To elucidate the mechanisms of protein functions and interactions in details, structural information is a powerful tool. However, solving the structures has been a challenge for TRP channels. Very recently, the cryo-electron microscopy (cryo EM) technique has dramatically expanded the resolution and thus the availability of the structural information of proteins (56), and the new era of the structural biology of TRP channels began with the report of the cryo-EM structure of TRPV1 at 3.4 Å resolution, breaking the side-chain resolution barrier for membrane proteins (57,58). The structure revealed the four-fold symmetry of TRPV1 around the central ion pathway formed by transmembrane segments 5–6 (S5–S6) and the intervening pore loop, which is flanked by S1–S4 voltage-sensor-like domains. The conserved TRP domain interacts with the S4–S5 linker, and the subunit organization is facilitated by interactions among cytoplasmic domains, including the ankyrin repeats. Furthermore, the authors utilized a peptide toxin and small vanilloid agonists as probes to determine the structures of two activated states of TRPV1. Channel opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as dilation of a

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hydrophobic constriction at the lower gate, suggesting a dual gating mechanism. They concluded that allosteric coupling between upper and lower gates may account for rich physiological modulation exhibited by TRPV1 and other TRP channels (57,58). The structures of TRPC4 were also reported very recently from two different groups due to the improvement of cryo-EM technology (59,60). One of them is a cryo-EM structure of TRPC4 from zebrafish in its unliganded, closed state with the resolution of 3.6 Å. This provided insights into the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate of TRPC4 (Figure 4) (59). The cytoplasmic domain contained hubs that have been shown to interact with modulating proteins. Around the same time, a cryo-EM structure of mouse TRPC4 also in its unliganded form was solved at the overall resolution of 3.3Å (60). The narrowest constriction of the ion conduction pathway was approximately 3.6 Å, suggesting that this structure represents the closed state. A disulfide bond is formed between Cys549 and Cys554 located near the pore helix which is likely to be stabilizing the pore loop and is critical to the redox sensitivity (Figure 4). The double mutation of these cysteines both to alanines lost the reactivity to DTT but retained sensitivity to englerin A. Englerin A was previously reported as an agonist of TRPC4 by electrophysiological analysis and genetic experiments, demonstrating that TRPC4 expression is both necessary and sufficient for englerin A-induced growth inhibition of renal cell carcinoma (61,62). This retained sensitivity suggests that the TRPC4 channel without the pore loop disulfide bond is still functional but lacks redox sensitivity. Strikingly, the single mutations of these two residues each to an alanine both lead to the loss of sensitivity to englerin A together with DTT sensitivity, implying that the pore loop architecture is severely disrupted in these mutants. Interestingly, Cys549 and Cys554 are conserved among TRPC1, 4, 5 channels,

15

and these residues are critical for the NO sensing in TRPC5 as mentioned earlier (22). We can present an interesting discussion on the functional nature of the disulfide bond observed in the cryo-EM structure of TRPC4 channel complexes. Notably, in cryo-EM experiment, although purified TRPC4 complexes in the solution containing reductants, such as tris(2-carboxyethyl)phosphine (59) or DTT (60), are directly subjected to the freezing procedure with nitrogen-cooled liquid ethane, the disulfide structure is clearly observed. This strongly suggests that formation of the disulfide bond between Cys549 and Cys554 is an extremely favored chemical process, in terms of energetics. Therefore, TRPC4 proteins with Cys549 and Cys554 linked by disulfide bond is likely in a highly stable "closed" state insusceptible to the attacks by either reductants or oxidants responsible for opening of the channel, rather than in an activatable "closed" state. Also, it is conceivable that freezing temperature during the cryo-procedure causes a conformational change that regulates the localization of the two Cys residues in the close vicinity to strongly induce the formation of a disulfide bond. Further structural comparisons with other TRP channels carrying the counterpart Cys residues would provide further insight into the general architecture and domain organization of TRP channels, facilitating the understandings of their function and pharmacological sensitivity (59,60). The cryo-EM structure of human TRPA1 was also reported, unveiling the full-length architecture at ~4 Å resolution. The key residues involved in detecting irritation were shown to be solvent accessible and suggest a structural basis of TRPA1 as a sensitive electrophile receptor. Other features such as coiled-coil assembly domain stabilized by polyphosphate co-factors were also revealed (63). Because this 3D TRPA1 structure was obtained for the reduced form of the N-terminus deletant without the

16

hydroxylated Pro, it is extremely interesting to analyze the 3D structure of the oxidized or electrophile-conjugated forms of the full length TRPA1. This information will provide insights into the mechanism of TRPA1 regulation and potential development of anti-inflammation or analgesic drugs.

TRPM2 channel activated by H2O2 independently of oxidative cysteine modification: TRPM2 channel acts as a biosensor for H2O2 Although NO is one of the most reactive molecules in our bodies, there are many other reactive species that also play important physiological roles. H2O2 is one of the reactive oxygen species (ROS) well known for exerting oxidative stress on cells comprising our body as an environmental stimulus. Other established roles of H2O2 include release by immune cells in response to microbial invasion or activation of caspases. Our group has previously shown that H2O2 activates TRPM2, a widely expressed Ca2+-permeable cation channel (21). Interestingly, other oxidants, NO, and Cys-targeting oxidants failed to activate TRPM2. This distinctive sensitivity of TRPM2 to H2O2 was at least in part attributable to an agonistic binding of ADP ribose (ADPR)/NAD+. Interestingly, in TRPM2, oxidative modification of Cys down-regulates the receptor signaling activity through binding of formyl peptide receptor 1 (FPR1) and its subsequent internalization (64). Knockdown experiments revealed physiological involvement of TRPM2 in H2O2-induced Ca2+ influx in different cells (21). Thus, TRPM2 represents an important mechanism that mediates Ca2+ and Na+ overload in response to disturbance of oxidative stresses in cell death. Another study has shown that TRPM2 plays a role in H2O2-induced cell apoptosis in endothelial cells. Here, TRPM2 overexpression aggravated the H2O2-induced apoptotic cell death. Downstream

17

pathways following TRPM2 activation was examined and caspase-8, caspase-9 and caspase-3 were found to be stimulated by TRPM2 activity, suggesting that TRPM2 channel mediates cellular Ca2+ overload in response to H2O2 and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells (65). More recent study has demonstrated that TRPM2 mediates mitochondria-dependent apoptosis in certain tissue under oxidative stress (66). In melanocytes, it was reported that the demethylation of the promoter region in TRPM2 gene occurs by H2O2. In fact, TRPM2 expression was enhanced by oxidative stress. Furthermore, TRPM2 caused mitochondria-dependent melanocyte apoptosis via Ca2+ influx. Both TRPM2 inhibitors and TRPM2 shRNA were able to suppress H2O2-induced apoptosis, mitochondrial ROS accumulation, and mitochondrial membrane potential loss. In addition, TRPM2 mediated the Ca2+ influx into the cytoplasm and the mitochondria of melanocytes exposed to H2O2, and a mitochondrial Ca2+ uptake inhibitor protected the melanocytes from apoptosis and mitochondrial damages caused by H2O2. Taken together, TRPM2 is a promising target for protecting melanocytes under oxidative stress (66,67). We have also demonstrated that TRPM2-mediated Ca2+ influx in response to ROS induces chemokine interleukin-8 (CXCL8) production in monocytes (68). This expression was significantly diminished in the monocytes from TRPM2 KO mice. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. These results strongly suggest that the TRPM2-mediated Ca2+ influx controls the ROS-induced signaling

cascade

responsible

for

chemokine

inflammation (68).

18

production,

which

aggravates

TRPM2 channel activated by H2O2 independently of oxidative cysteine modification: 3D-structual analysis Latest cryo-EM studies released from different groups provided structural information of TRPM2 (69-71), unveiling the interaction with ADPR and Ca2+. The comparison between human, zebra fish and sea anemone TRPM2 structure revealed the species-specific difference of ADPR binding and gating, and as well the overall features such as NUDT4 homology (NUDT9H) domain and TRPM homology region (MHR1/2/3) well conserved among species. In human TRPM2 (hsTRPM2), the NUDT9H domain is important for channel gating (70), while the ADPR binding at the MHR1/2 domain is functionally important in zebra fish TRPM2 (drTRPM2). However, mutagenesis analysis revealed that this MHR1/2 domain in hsTRPM2 does not substantially affect channel gating by ADPR, whose equivalent mutation in drTRPM2 nearly abolished the current (69). On the other hand, TRPM2 from the sea anemone (nvTRPM2) was shown to be gated independently of the NUDT9H domain (72). Collectively, these data imply that drTRPM2 and nvTRPM2 use MHR1/2 domain but hsTRPM2 use NUDT9H domain for ADPR sensing. Furthermore, Met191, which takes part in forming the ADPR-binding site of drTRPM2, has been proposed to “sensitize” TRPM2 by oxidization (73). This supports the idea that in drTRPM2, the MHR1/2 domain is a physiologically relevant ADPR binding site and implies the mechanism of redox sensitization, in which oxidation of this Met might mimic ADPR binding (69).

Comparison of TRP channels with other ion channels and proteins regulated by cysteine modifications Recently, ion channels other than TRP channels have also been reported to be

19

regulated by redox signaling. In the Kv4 channel, redox regulation derives from the Zn2+ binding site in the intracellular inter-subunit interface (74). When NO is applied internally, the channel activity is inhibited profoundly, which is reversed by reduced glutathione and suppressed by intracellular Zn2+. Biochemical evidence suggests that NO induces a disulfide bridge between Cys110 and Cys132 in intact cells. The authors proposed that the interfacial Zn2+ site of Kv4 channels acts as a Zn2+-dependent redox switch that may regulate the activity of neuronal and cardiac A-type K+ currents under physiological and pathological conditions. This interesting involvement of Zn2+ has not been reported for TRP channels, and should be examined as well. Another interesting example of NO modulation is the acid-sensing ion channels (ASICs). Whole-cell patch-clamp recordings were performed on neonatal rat cultured dorsal root ganglion (DRG) neurons and on recombinantly expressed ASIC isoforms. The NO donor SNAP potentiated proton-gated currents of ASICs. Through the studies performed through the skin of human volunteers, application of the NO donor significantly increased acid-evoked pain. ASICs may therefore play an important role in the pain associated with metabolic stress and inflammation (75). Later evaluation of the pH dependency of the NO-induced ASIC currents and subsequent cell death suggested that during mild and moderate acidosis, NO promotes cell death by potentiating ASICs, whereas this potentiation subsides in severe acidosis due to inhibition of NO synthase (76). It has also been reported that Pannexin 1 channel function is inhibited by S-nitrosylation (77), whereas homomeric ρ1 GABAC receptor function has been shown to be enhanced by S-nitrosylation (78). Oxidative stress by S-glutathionylation has been reported to modulate vascular K-ATP channel (79). Exposure of isolated mesenteric rings to H2O2 impaired the K-ATP

20

channel-mediated vascular dilation, and both whole-cell recordings and inside-out patches showed that H2O2 or diamide caused a strong inhibition of the vascular K-ATP channel in the presence of glutathione. This oxidant-mediated channel inhibition was reversed by the reducing agent DTT, suggesting that S-glutathionylation is an important mechanism for the vascular K-ATP channel modulation in oxidative stress (79). Interestingly, K-ATP is reported to be the modulator of microvascular O2 pressure in rat skeletal muscle at the onset of contractions (80), and controls the mismatch of O2 supply and utilization in muscle during contraction (81). Similarly, the importance of S-Glutathionylation has been reported in the modulation of the heteromeric Kir4.1-Kir5.1 channel, but not homomeric Kir4.1 (82). This suggests a novel Kir4.1-Kir5.1 channel modulation mechanism that is likely to occur in oxidative stress. Proteins other than ion channels are also modulated by NO. These proteins share regulatory mechanisms and physiological significance and may cross talk with TRP channels. For instance, mitochondria complex I is reversibly inhibited by S-nitrosylation, exerting a cardioprotective effect (83). Another example is the S-nitrosylation of G-protein-coupled receptor kinase 2 (GRK2) (84), which is S-nitrosylated by both NO synthases and S-nitrosothiols, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists in both cells and tissues. Thus, this study reveals a molecular mechanism through which GPCR signaling may be co-regulated with redox-sensitive TRP channels such as TRPC5 and TRPA1 known to be activated upon stimulation of GPCR. In the nerves system, peroxiredoxin 2 (Prx2) is S-nitrosylated by reaction with NO at two critical Cys residues (Cys51 and Cys172), preventing its reaction with peroxides. An increase of S-nitrosylated Prx2 has been observed in human Parkinson's

21

disease brains, and S-nitrosylation of Prx2 inhibited both its enzymatic activity and protective function from oxidative stress. The data provide a direct link between nitrosylation/oxidative stress and Parkinson's disease (85). Considering our report suggesting involvements of TRPM2 channels in neurodegenerative diseases, TRPM2 may interact with Prx2 (86,87). Separate study has performed a proteome assay of the S-nitrosylated protein in the spinal cord before and after nerve injury. The identified S-nitrosylated proteins are involved in mitochondrial function, protein folding and transport, synaptic signaling and redox control. The data show that NO mediated S-nitrosylation contributes to the nerve injury-evoked pathology in nociceptive signaling pathways (88). NO-mediated TRP channels are widely distributed in the nervous system (89), and may contribute to nerve injury. NF-κB-dependent expression of inducible nitric-oxide synthase (iNOS) gene in dystrophic skeletal myotubes was reported to be dependent on the resting levels of intracellular Ca2+ (90). Although the target is still unveiled, the enhanced level of NO produced by iNOS is likely to promote damage in dystrophic skeletal muscle cells. Recently, the same group have reported the benefits of whole-body periodic acceleration which regulates the expression of constitutive NO synthases targeting dystrophins, improving dystrophic neuronal functions (91,92). Because Ca2+ influx mediated by TRPM2 promotes NF-κB activation, TRPM2 can be a regulator of this mechanism.

Pharmacotherapy targeting TRP channels As discussed earlier, some TRP channels are directly associated with pain sensation and others regulate various signaling pathways, the malfunction of which

22

leads to serious illness. To name a few, TRPV3 has been implicated as a cause of Olmsted syndrome (93,94) and a mutation in TRPC6 has been identified as the cause of focal segmental glomerular sclerosis, a disease in kidney (95-97). We have previously shown that liver damage and cell death is induced by acetaminophen via multiple redox-activated TRP channels, especially TRPV1 and TRPC1 (29). Therefore, individual TRP channels are attractive targets for drug development. Numerous clinical trials are ongoing for TRP channels. For example, NEO6860, a TRPV1 inhibitor, is being developed for osteoarthritic pain. According to a recent publication, this compound does not have an effect on human body temperature, unlike other TRPV1 antagonists that cause hyperthermia (98). Contrarily, resiniferatoxin (RTX), a very potent TRPV1 activator, has been used to create permanent analgesia by destroying the signaling potential of susceptible sensory neurons (99). Dramatic reductions in pain behaviors after intrathecal treatment with RTX have been tested in various species, including dogs suffering from pain due to osteosarcoma (100). Taken together, these data support the clinical trials under way exploring the benefits of RTX in patients with intractable cancer pain (101). TRPM8 is a member of the melastatin subfamily which is activated by cold temperatures below 28 °C. Antagonists of TRPM8 would have the potential to treat cold-induced allodynia and hyperalgesia. On the other hand, TRPM8 has also been implicated in mammalian thermoregulation. Therefore, its antagonists have the potential to induce hypothermia in patients. Identification and optimization of various TRPM8 antagonists led to the development of PF-05105679 which went through clinical trials and proved efficacy in a cold pressor test comparable to oxycodone, an opioid medication commonly used for treatment of moderate to severe pain (102,103).

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TRPA1 is also an attractive target for pain treatment and several companies are conducting Phase 1 or 2 trials with TRPA1 inhibitors. One of them is GRC 17536, which is reported to reduce the pain from diabetic neuropathy (104). GRC 17536 has shown statistically significant data in a Phase 2a double-blind, placebo-controlled study conducted on 138 patients. GRC 17536 did not show evidence of drug-related side effects (101). Collectively, TRP channels are potential targets for various pharmaceutical interventions. Hopefully, the ongoing clinical trials would be successful, and the resultant products will be available in the near future.

Summary Evidence from different studies including our data support the notion that a variety of physiological responses are mediated by the signal cascade involving Ca2+ and ROS/RNS and oxidative stress. The Ca2+ influx mediated by TRP channels is the likely molecular basis that link Ca2+ and oxidative stress signaling. Due to the development of cryo-EM technique, the structural information of TRP channels is expected to expand dramatically in the next few years. Detailed analysis of these structures would further facilitate the understanding in the critical mechanisms of the activation and inhibition of these channels, and in the action of drugs. The functional inhibition of TRP channels will be a promising target for a new drug development for treating cancer and inflammatory diseases.

Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Oxygen biology: a new criterion for integrated understanding of life”

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(No. 26111004) of The Ministry of Education, Culture, Sports, Science and Technology, Japan.

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FIGURE LEGENDS Figure 1. Phylogenetic Tree of the TRP Superfamily. Dendrogram showing the relatedness of the TRP proteins. The tree includes all of the human TRPs, mouse TRPC2, zebrafish TRPN1, along with one Drosophila (yellow letters in filled box) and one C. elegans (white letters in filled box) member of each subfamily. The reported redox-sensitive members are indicated by red arrow heads. Modified from Montell C., Sci. STKE, 2005, re3 (2005). Reprinted with permission from AAAS.

Figure 2. Model for TRPC5-caveolin-1-eNOS signalplexes that coordinate interplay between Ca2+ and NO signals in endothelial cells. Caveolin-1 provides a scaffold for assembly of TRPC5 and eNOS into a protein complex. When Ca2+ influx via TRPC5 is induced, eNOS associates with Ca2+-calmodulin (CaM). This releases eNOS from caveolin-1 and allows eNOS to interact with TRPC5, enabling eNOS to produce NO in the vicinity of TRPC5 for amplified secondly Ca2+ signaling.

Figure 3. TRPA1 promotes ROS tolerance via induction of Ca2+-dependent anti-apoptotic pathways in cancer cells. Reproduced from Takahashi et al., Cancer Cell, 33, 985-1003 (2018) with permission from Elsevier.

Figure 4. Cryo-EM Structure of TRPC4. (A) Ribbon representation of the atomic model of TRPC4 from zebrafish with each protomer colored differently and shown as side, bottom, and top view. The overall structure of the homo-tetrameric TRPC4DR is similar to that of other TRP channels, with extended cytoplasmic domains that reaches ~80 Å into the cytosol. (B) Linear diagram depicting the major structural domains of

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the mouse TRPC4 monomer. The N-terminal cytosolic region consists of four ankyrin repeats (AR1–AR4) and seven α-helices (H1–H7), and the C-terminal cytosolic domain contains a connecting helix and a coiled-coil domain. The disulfide bond important for redox sensitivity is located in between S5 and pore helix. Figures reproduced from Vinayagam et al., eLife, 7, e36615 (2018) and Duan et al., J. Nat Commun 9, 3102 (2018) under the Creative Commons Attribution 4.0 International License. https://creativecommons.org/licenses/by/4.0/

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Highlights: TRP channels mediate acute signal transduction in response to environmental change. Structural basis of the channel’s redox sensing mechanism is starting to be unveiled. The disfunction of the channels lead to certain disease such as cancer. TRP channels are attractive drug targets and some are going through clinical trials.