TRPA1 and TRPV1 channels participate in atmospheric-pressure plasma-induced [Ca2+]i response

Despite successful clinical application of non-equilibrium atmospheric pressure plasma (APP), the details of the molecular mechanisms underlying APP-inducible biological responses remain ill-defined. We previously reported that exposure of 3T3L1 cells to APP-irradiated buffer raised the cytoplasmic free Ca2+ ([Ca2+]i) concentration by eliciting Ca2+ influx in a manner sensitive to transient receptor potential (TRP) channel inhibitors. However, the precise identity of the APP-responsive channel molecule(s) remains unclear. In the present study, we aimed to clarify channel molecule(s) responsible for indirect APP-responsive [Ca2+]i rises. siRNA-mediated silencing experiments revealed that TRPA1 and TRPV1 serve as the major APP-responsive Ca2+ channels in 3T3L1 cells. Conversely, ectopic expression of either TRPA1 or TRPV1 in APP-unresponsive C2C12 cells actually triggered [Ca2+]i elevation in response to indirect APP exposure. Desensitization experiments using 3T3L1 cells revealed APP responsiveness to be markedly suppressed after pretreatment with allyl isothiocyanate or capsaicin, TRPA1 and TRPV1 agonists, respectively. APP exposure also desensitized the cells to these chemical agonists, indicating the existence of a bi-directional heterologous desensitization property of APP-responsive [Ca2+]i transients mediated through these TRP channels. Mutational analyses of key cysteine residues in TRPA1 (Cys421, Cys621, Cys641, and Cys665) and in TRPV1 (Cys258, Cys363, and Cys742) have suggested that multiple reactive oxygen and nitrogen species are intricately involved in activation of the channels via a broad range of modifications involving these cysteine residues. Taken together, these observations allow us to conclude that both TRPA1 and TRPV1 channels play a pivotal role in evoking indirect APP-dependent [Ca2+]i responses.


Plasma irradiation system.
A non-equilibrium APP was generated as previously reported 11 . Briefly, helium was used as the working gas, with its flow rate (3 L/min.) through the dielectric tube regulated by a mass flow controller. High-voltage (V p-p ) was applied with a frequency of approximately 9 kHz between the two electrodes. The powered electrode was a 1.5-mm diameter tungsten rod and the other was a hot plate, allowing APP to be generated and flow from the nozzle of the quartz glass tube into ambient air. The distance between the bottom edge of the electrode and the edge of the nozzle was 23 mm. The APP was exposed to HEPES-buffered saline (HBS) containing 138 mM NaCl, 5 mM KCl, 0.3 mM KH 2 PO 4 , 4 mM NaHCO 3 , 2 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM HEPES (pH 7.4). The calculated mean power was 1 W for V p-p = 7.0 kV. Based on our previous reports, HBS was exposed to the APP for 30 sec., we then waited another 30 sec., and finally the APP-irradiated HBS was applied to the Fluo-4-loaded cells via infusion.
Cell culture. Mouse 3T3L1 fibroblasts (ATCC CL-173) and C2C12 myoblasts 13 were plated onto 6-well culture plates or glass-bottom dishes (no. 1 S; Matsunami-glass, Osaka, Japan) and cultured in DMEM containing either 10% calf serum (for 3T3L1 fibroblasts) or 10% FBS (for C2C12 myoblasts) at 37 °C under a 5% CO 2 atmosphere. 3T3L1 fibroblasts were transfected with Lipofectamine RNAiMax (Thermo Fischer Scientific) with 250 pmol siRNAs, and C2C12 myoblasts were transfected with Lipofectamine 3000 (Thermo Fischer Scientific) with 1 μg of plasmid DNAs, according to the manufacturer's instructions. For siRNA silencing experiments, the transfected 3T3L1 fibroblasts were subjected to experiments at approximately 40 hours after siRNA introduction. For exogenous expression experiments, the transfected C2C12 myoblasts were subjected to experiments at approximately 20 hours after transfection.

Cytoplasmic calcium ([Ca 2+
] i ) imaging. 3T3L1 fibroblasts were loaded with 5 μM fluo-4 acetoxymethyl ester (Fluo-4AM) (Invitrogen, Carlsbad, CA) and 0.03% Cremophor-EL (C5135, Sigma-Aldrich, St. Louis, MO, USA) in serum-free DMEM for 30 min at 37 °C in an atmosphere of 5% CO 2 14 . Imaging experiments were performed with an inverted epifluorescence microscope (IX81; Olympus, Tokyo, Japan) equipped with a CCD camera (CoolSNAP; Photometrics), a xenon lamp, and an objective lens (UPlanApo20X, NA0.8, Olympus). Fluo-4 measurements were carried out at room temperature. Fluo-4 fluorescence was acquired through UMNB2 filter cubes (Olympus) every 2 s using illumination periods between 100 and 250 ms in duration for 10-25 min with μManager (http://www.micro-manager.org). The cells were treated with APP-irradiated HBS at 5 and 15 min of acquisition, and then treated with 10 μM ionomycin at ~24 min. Image analysis was performed using Fiji 15 . After subtraction of background fluorescence, changes in [Ca 2+ ] i were expressed as (F − F0)/F0, where F, and F0 represent the fluorescence intensity of Fluo-4, and the averaged fluorescence intensity of the dye before stimulation with APP-irradiated HBS, respectively. In some experiments, after acquisition of Fluo-4 fluorescence, the cells expressing HaloTag proteins were stained wih 0.5 μM HaloTag TMR ligand (Promega) for 15 min on site and then a snapshot of TMR fluorescent image was acquired to specify HaloTag-positive cells. We stained the cells with HaloTag TMR ligand afterward to avoid undesirable interference of TMR fluorescence for measuring Fluo-4 fluorescent intensity during [Ca 2+ ] i imaging experiments. All experiments were performed at least three times using different batches of cells. -fused TRPA1 and TRPV1 with or without APP-HBS treatment. 3T3L1 fibroblasts expressing Halo-TRPA1 or TRPV1-Halo were stained with 0.5 μM HaloTag TMR ligand for 15 min, washed twice with serum-free medium and then incubated for 20 min to remove excess HaloTag TMR ligands. Finally, the cells were treated with or without APP-irradiated HBS for 5 min, fixed with PBS containing 2% paraformaldehyde, and observed with an Olympus FV1000 microscope.
Statistical analysis. The statistical analyses were performed using GraphPad Prism version 7 (GraphPad Software, Inc., La Jolla, CA, USA). All experimental data are presented as means ± S.E. The statistical significance of differences was determined by applying the Dunnett's multiple comparison test or the Mann-Whitney's U test. A p-value less than 0.05 was taken to indicate a statistically significant difference.

Results
[Ca 2+ ] i transients induced by indirect APP were significantly blunted by siRNA-mediated knockdown of TRPA1 and/or TRPV1 in 3T3L1 fibroblasts. Application of APP-irradiated HBS (APP- 2+ ] i in 50 to 70% of 3T3L1 fibroblasts (Fig. 1), but these rises were completely abolished in the presence of RR (data not shown), as we previously reported 11 . However, siRNA-mediated knockdown of TRPV1 and/or TRPA1 in 3T3L1 fibroblasts, which was confirmed by RT-PCR analysis (Fig. 1D), markedly inhibited the indirect APP-inducible [Ca 2+ ] i transients (Fig. 1A). All values of [Ca 2+ ] i transients were calculated using the area under the curve (AUC), and summarized graphically to clearly demonstrate the significant reductions in their indirect APP responsiveness (Fig. 1B). The siRNA-mediated suppression in 3T3L1 fibroblasts of either TRPA1 or TRPV1 expression alone significantly inhibited the indirect APP-dependent [Ca 2+ ] i responses. Although the TRPA1/TRPV1 double-knockdown cells exhibited the strongest suppressive response, this suppression did not reach the levels observed in the presence of RR. Pharmacological analyses using AITC (TRPA1 agonist) and capsaicin (TRPV1 agonist) confirmed the existence of functional TRPA1 and TRPV1 channels in 3T3L1 fibroblasts (Fig. 1C), and the agonist-dependent Ca 2+ influx was significantly inhibited by TRPA1 and TRPV1 double knockdown treatment in 3T3L1 fibroblasts (Fig. 1D). These observations indicate that TRPA1 and TRPV1 serve as the indirect APP-responsive Ca 2+ -permeable channels, both of which are expressed in 3T3L1 fibroblasts.

Heterologous desensitization of TRPA1 and TRPV1 channels by indirect APP in 3T3L1 fibroblasts.
To characterize the indirect APP-responsive regulation of TRP channels, heterologous desensitization experiments were performed by sequentially applying APP-HBS and TRP channel agonists (i.e. AITC for TRPA1 and capsaicin for TRPV1). Treatment with either AITC or capsaicin as the 1 st stimulus evoked [Ca 2+ ] i transients and the pretreatments markedly inhibited the [Ca 2+ ] i responses induced by APP-HBS as the 2 nd stimulus ( Fig. 2A). Furthermore, pretreatment with APP-HBS resulted in significantly compromised [Ca 2+ ] i transients in response to AITC as well as to capsaicin (Fig. 2B). These observations demonstrated the existence of bi-directional heterologous desensitization properties; i.e. chemical agonists, such as AITC or capsaicin, desensitize cells to APP-HBS, and APP-HBS exposure desensitizes the cells to these agonists.
In an attempt to elucidate the mechanism underlying the indirect APP-dependent desensitization, we examined possible changes in subcellular localizations of TRPA1 and TRPV1 channels after APP-HBS exposure. To address this issue, HaloTag-fused TRP channels (i.e. Halo-TRPA1 and TRPV1-Halo) were exogenously expressed and stained with TMR Halo ligands in 3T3L1 fibroblasts. As shown in Fig. 2C, both Halo-TRPA1 and TRPV1-Halo were mostly localized at the intracellular compartments with slight staining of the plasma membrane, and these localization patterns were not detectably changed by APP-HBS treatment, at least with 5-min exposure.

Exogeneous expression of TRPA1 or TRPV1 generates the indirect APP-responsiveness in C2C12 cells.
In order to directly address whether TRPA1 and TRPV1 molecules serve as the indirect APP-responsive Ca 2+ -permeable channel, we performed exogenous expression experiments employing Halo-TRPA1 and TRPV1-Halo by using plasma-unresponsive C2C12 myoblasts (Fig. 3). As we previously observed in a human breast cancer cell line, MCF-7 cells 16 , C2C12 myoblasts failed to respond to indirect APP treatment and no rise in [Ca 2+ ] i was observed even after APP-HBS administration (Fig. 3A, upper). In contrast, exogenous expression of either Halo-TRPA1 or TRPV1-Halo, alone or in combination, in C2C12 myoblasts Scientific RepoRtS | (2020) 10:9687 | https://doi.org/10.1038/s41598-020-66510-y www.nature.com/scientificreports www.nature.com/scientificreports/ consistently produced the indirect APP-responsive [Ca 2+ ] i transients, which were completely blunted in the presence of RR, a broad spectrum inhibitor for TRP channels 17 (Fig. 3A,B). Co-expression of TRPA1 and TRPV1 channels did not enhance [Ca 2+ ] i transient amplitudes, at least under the present experimental conditions using C2C12 myoblasts. It should be noted that due to the cytotoxicity of TRPV1 possibly due to a robust increase in mitochondrial calcium 18 , a low expression vector (pFC17K-CMVd3) had to be used for exogenous TRPV1 expression experiments using C2C12 myoblasts.

Mutational analysis of TRPA1 and TRPV1 channels and their indirect APP-responsiveness.
To further understand the indirect APP-responsive regulation of TRPA1 and TRPV1 channels, we generated site-directed mutations of several key cysteine residues in TRPA1 19,20 and TRPV1 21 channels and examined their impact on the indirect APP-dependent [Ca 2+ ] i responses in C2C12 myoblasts. As shown in Fig. 4, TRPA1/C421S,  (Fig. 4A), though TRPA1/C641S showed the most suppressed [Ca 2+ ] i responses. These TRPA1 mutants were all responsive to AITC, though TRPA1/C421S exhibited significantly impaired [Ca 2+ ] i responses to AITC. Like TRPA1 cysteine mutations, TRPV1/C258S, TRPV1/C363S, and TRPV1/C742S all exhibited reduced [Ca 2+ ] i transients in response to the indirect APP (Fig. 4B), and no further reduction was observed with its double and triple mutants (data not shown), though TRPV1/C258S and TRPV1/ C742S, but not TRPV1/C363S, exhibited impaired [Ca 2+ ] i transients in response to capsaicin.

Discussion
A central issue in understanding biological impacts of APP is elucidation of the molecular mechanisms allowing cells to decipher and respond to the physiologically-relevant, but not excessive and non-beneficial, levels of APP exposure. By employing cell-based [Ca 2+ ] i analyses of both siRNA-mediated silencing (Figs. 1 and 2) and exogenous expression strategies (Figs. 3 and 4), we demonstrated both TRPA1 and TRPV1 channels to be molecules  or TRPV1 (pink bar), alone or in combination (green bar), with or without 100 μM RR from 3 independent experiments. AUC were evaluated for 300 sec. in total after APP-HBS administration. For each experiment, more than 10 transfected cells (TMR-positive cells) from each glass-bottom dish were measured. Statistical significance was determined by applying the Dunnett's multiple comparison versus the control (parental C2C12 cells). The effects of exogenous expression of TRPA1 and TRPV1, alone or in combination, are indicated by *(P < 0.05), and the effects of RR are indicated by # (P < 0.05). (C) Quantification of agonist-dependent [Ca 2+ ] i transients in C2C12 myoblasts exogenously expressing either TRPA1 (blue bar) or TRPV1 (pink bar) channels from 3 independent experiments. AUC was evaluated from 100-500 sec., and results are expressed as the fold increase versus the AUC obtained from the parental C2C12 cells. For each experiment, more than (2020) 10:9687 | https://doi.org/10.1038/s41598-020-66510-y www.nature.com/scientificreports www.nature.com/scientificreports/ responsible for indirect APP-dependent [Ca 2+ ] i response. Our data also revealed that the indirect APP-responsive [Ca 2+ ] i transients exhibited a bi-directional heterologous desensitization property with either TRPA1 or TRPV1 activation by their agonists (Fig. 2). Furthermore, mutational analysis of key cysteine residues in TRPA1 (Cys421, Cys621, Cys641, and Cys665) and TRPV1 (Cys258, Cys363, and Cys742) exogenously expressed in C2C12 myoblasts strongly suggests that the cysteine residues of TRPA1 and TRPV1 channels play a crucial role in the indirect APP-responsive regulation of channel activation (Fig. 4). While it is possible that other types of channels responsive to APP may exist 22 , our findings provide pivotal insights into the molecular mechanisms that underlie the indirect APP-dependent [Ca 2+ ] i transients, thereby further extending the potential of plasma medicine based on APP technology.
Consistently with several reports showing that 3T3L1 fibroblasts endogenously express multiple TRP channel family members including TRPA1 and TRPV1 23,24 , we observed that 3T3L1 fibroblasts exhibit rises in [Ca 2+ ] i in response not only to APP-HBS exposure but also to either AITC or capsaicin, pharmacological activators of TRPA1 and TRPV1, respectively. Consequently, we confirmed all of these [Ca 2+ ] i responses induced by APP-HBS as well as by these chemical agonists to be significantly suppressed by siRNA-mediated knockdown of TRPA1 and/or TRPV1 (Fig. 1). It has now been well established that both TRPA1 and TRPV1 channels, in addition to their temperature-sensing property, serve as redox-sensitive TRP channels that can be activated by environmental and endogenous RONS 9,25 . In particular, TRPA1 and TRPV1 are reportedly activated by hydrogen peroxide (H 2 O 2 ), OH • , NO and ONOO − with varying sensitivities 9,26-29 . Considering the highly efficient capability of APP for producing OH • , O 2 •− , H 2 O 2 , ONOO − , • NO, and so on, in an APP-irradiated solution 4,30 , it is evident that multiple RONS generated in the APP-HBS, not only alone but also perhaps collectively, participate in exerting the entire range of APP-dependent [Ca 2+ ] i responses by modulating channel activities of both TRPA1 and TRPV1 in 3T3L1 fibroblasts.
We have not as yet defined the precise reactive species directly involved in the activation of TRPA1 and TRPV1 channels, but the findings of our previous study 11 strongly suggest the involvement of relatively short-lived reactive species (i.e. deactivated within approximately 10 min) generated in the APP-HBS in response to APP irradiation. In this regard, a recent report demonstrated that alginate solution and hydrogels show a capacity for sustained release of reactive species 31 . Furthermore, we have recently found that HEPES, an amine-based buffer compound with a piperazine skeleton, to play an important role in the cycling chain reaction of chemically reactive species, which is initiated by the APP-generated hydroxyl radicals, resulting in continuous release of O 2 •− / ONOO − in the APP-irradiated HBS which persists for several minutes, at least 32 . Thus, despite our general former understanding that O 2 •− , • NO, and ONOO − have very short half-lives (less than one second) in biological solutions, these highly potent reactive species, both biologically and chemically, may be directly involved in the activation processes of both TRPA1 and TRPV1 channels when certain compounds exist in the solution. Future work is necessary to clarify the precise molecular interactions between these APP-generated reactive species and TRPA1/TRPV1 channels under conditions wherein the aforementioned reactive species are experimentally manipulated in various ways allowing the roles of individual molecules to be demonstrated.
Given that exogenous expression of either TRPA1 or TRPV1, alone or in combination, actually endowed C2C12 cells with APP-responsiveness (Fig. 3), it is obvious that each molecule individually possesses a property that can be independently activated by indirect APP exposure. Intriguingly, however, siRNA-mediated knockdown of either TRPA1 alone or TRPV1 alone in 3T3L1 fibroblasts significantly suppressed indirect APP-responsive [Ca 2+ ] i transients, even though the highest degrees of suppression was observed when both TRPA1 and TRPV1 were depleted (Fig. 1). These results suggest that the [Ca 2+ ] i rises in response to APP-HBS could also be mediated through functional interactions between endogenous TRPA1 and TRPV1 channels in 3T3L1 fibroblasts. In this regard, several lines of evidence point to TRPA1 and TRPV1 being able to form physical and functional heteromeric channel complexes possessing synergistic activation properties [33][34][35] . For example, a combined application of AITC and capsaicin reportedly resulted in significantly longer durations of responses in isolated rat vagal pulmonary sensory neurons, which were dependent upon extracellular Ca 2+,36 . Moreover, potentiating effects were also reportedly generated when capsaicin was replaced by high temperature (>39 °C), a natural biological activator of TRPV1 36 . Furthermore, TRPA1/TRPV1-interacting proteins, such as Tmem100, AKAP and Toll-like receptor 4, have been reported to modulate TRPA1 and/or TRPV1 channels [37][38][39] . Thus, while the TRPA1 or TRPV1 channels can each be independently activated by administration of APP-HBS, it is likely that indirect APP-responsive [Ca 2+ ] i transients in 3T3L1 fibroblasts were induced by cooperative actions of TRPA1 and TRPV1 channels possibly along with the other aforementioned interacting protein(s) and their splicing variant forms 40,41 . These intricate regulatory mechanisms including the participation of such interacting proteins, depending on cellular contexts, might contribute to the observed differences in the profiles of the APP-responsive [Ca 2+ ] i transients between ectopically expressed C2C12 cells and naive 3T3L1 fibroblasts.
Consistently with our findings depicted in Fig. 2, heterologous desensitization of TRPA1 and TRPV1 by their pharmacological agonists including AITC and capsaicin has been reported in endogenous neurons and in heterologous expression systems [42][43][44][45] . In this regard, it is well-established that highly increased [Ca 2+ ] i levels in response to pretreatments with pharmacological agonists resulted in a Ca 2+ -dependent desensitization of the channel itself at the molecular level via multiple mechanisms involving protein kinase A, protein kinase C, Ca 2+ /Calmodulin-dependent kinase II and/or calcineurin 35,42,44 . Thus, relatively high increases in [Ca 2+ ] i levels upon application of the initial stimulus with either AITC or capsaicin may contribute to the desensitization 10 transfected cells (TMR-positive cells) from each glass-bottom dish were measured. Data are means ± SE., and statistical significance was determined by applying the Mann-Whitney's U test. The effects of exogenous expression of TRPA1 or TRPV1 channels are indicated by *(P < 0.05).

Scientific RepoRtS |
(2020) 10:9687 | https://doi.org/10.1038/s41598-020-66510-y www.nature.com/scientificreports www.nature.com/scientificreports/ phenomenon observed with subsequent APP-HBS exposure ( Fig. 2A). In addition, several lines of evidence indicate that endocytosis of TRPV1 channels might be involved in the capsaicin-dependent desensitization process [46][47][48] . As shown in Fig. 2C, given that no obvious alterations in subcellular localizations of both TRPA1 and TRPV1 upon APP-HBS exposure were observed, this appears to not be the case for APP-induced desensitization. It is also possible that the extrusion activity of [Ca 2+ ] i by Ca 2+ ATPase 49,50 could be impacted by this indirect APP treatment.
Thus, although the molecular mechanisms underlying the indirect APP-dependent cooperative activation of as well as the desensitization of TRPA1 and TRPV1 are not understood in detail, and the functional properties of the two channels in combination in response to APP-HBS treatment require further elucidation, our present data provide important insights into the mode of APP-responsive [Ca 2+ ] i rises through TRPA1 and TRPV1 channels in 3T3L1 fibroblasts.
Finally, mutational analyses of several key cysteine residues in TRPA1 and TRPV1 provide compelling evidence that cysteine residues play crucial roles in their channel activation in response to the APP-irradiated HBS containing various reactive species that have yet to be clarified (Fig. 4). Given that all cysteine mutants of either TRPA1 (C421S, C621S, C641S, C665S) or TRPV1 (C258S, C363S, C742S) clearly suppressed the indirect APP-inducible [Ca 2+ ] i transients, though there were slight variations among these mutants (e.g. TRPA1/C665S), the precise molecular details of their channel activation processes upon exposure to APP-irradiated HBS remain uncertain at present. Nevertheless, some mutant forms of TRPA1 (i.e. TRPA1/C421S) and TRPV1 (i.e. TRPV1/ C258S and TRPV1/C742S) also exhibited impaired agonist-dependent [Ca 2+ ] i transients.
It has been well established that modifications of several key cysteine residues of both TRPA1 and TRPV1 channels, including their oxidation, electrophilic interactions, and S-nitrosylation of sulfhydryls, are among the most common mechanisms for channel activations in response to a wide array of stimuli 51 . As an example, TRPA1, Cys633, Cys641, and Cys665 are reportedly crucial for activation by electrophiles 52,53 , and Cys641 and Cys665 have been shown to be involved in channel activation processes evoked by H 2 O 2 and NO 54,55 . Moreover, disulfide formation between Cys621 and the neighboring Cys633 as well as that between Cys651 and Cys665 have been shown to play important roles in the activation elicited by HNO, the one-electron reduced form of NO 56 . Several lines of evidence have also demonstrated a crucial role of Cys641 in the Pro394 hydroxylation-dependent channel modulation via inhibition of prolyl hydroxylase, which apparently sensitizes TRPA1 to reactive oxygen species 19 . For instance in TRPV1, Cys258 and C363 are reportedly involved in H 2 O 2 -dependent activation 21 . Furthermore, it has been reported that Cys258 and Cys742, which are located near each other, are heterogeneously modified to form disulfide bonds in the TRPV1 tetrameric complexes facilitating subunit dimerization 21 . Thus, while each cysteine residue appears to individually contribute to stimulus-specific responses to some extent, functions of these cysteine residues are related to each other in complex ways, even within either the TRPA1 or the TRPV1 molecule alone, obviously adding a further layer of complexity when both are expressed in a cell. Based on these considerations, our mutation analysis, displaying all examined cysteine mutants with phenotypes associated with suppressive actions in response to indirect APP, strongly suggested that multiple reactive species which vary over time are intricately involved in the activation processes of both TRPA1 and TRPV1 channels. This complex interplay presumably occurs via a broad range of modifications of these cysteine residues. Furthermore, our findings using these various mutants raised the clear possibility that APP-dependent desensitization (Fig. 2B) might be attributable to covalent modifications of these cysteine residues, which could ] i transients as well as the agonistresponsive [Ca 2+ ] i transients (grey bars) in C2C12 myoblasts expressing either TRPA1/its mutants (blue bars) or TRPV1/its mutants (pink bars) from 3 independent experiments. APP-HBS was applied at approximately 100 sec., followed by Fluo-4 monitoring for approximately for 10 min as described in Materials and Methods. AUC was evaluated from 100-500 sec. and the results are expressed as a percentage of the data obtained from C2C12 cells expressing wild-type channels. Transfected cells were identified by TMR-conjugated HaloTagligand staining after evaluation of [Ca 2+ ] i responses by Fluo-4 fluorescence. For each experiment, more than 10 transfected cells (TMR-positive cells) from each glass-bottom dish were measured. Statistical significance was determined by applying the Dunnett's multiple comparison versus the control (WT). The effects of mutations are indicated by *(P < 0.05).

Scientific RepoRtS |
(2020) 10:9687 | https://doi.org/10.1038/s41598-020-66510-y www.nature.com/scientificreports www.nature.com/scientificreports/ be promiscuous (non-physiological), by multiple RNOS including OH • , O 2 •− , H 2 O 2 , ONOO − , • NO, and their derivatives in the APP-irradiated solution. Indeed, desensitization of TRPA1 channels and their covalent modifications involving several cysteine residues by mastered oil, another reactive electrophilic chemical compound, reportedly occurred even in the absence of Ca 2+ 44,57 . Future studies employing more sophisticated approaches are clearly required to elucidate the mechanistic details of the APP-responsive activation as well as the desensitization of these channels.
In summary, we herein provide strong evidence that both TRPA1 and TRPV1 channels play a pivotal role in evoking indirect APP-dependent [Ca 2+ ] i responses. Since both TRPA1 and TRPV1 have received considerable attention as potential therapeutic targets for the treatment of several disorders including chronic pain, inflammation, respiratory diseases, and cancers 58-60 , further detailed mechanistic analyses of the APP-responsive activations of TRPA1 and TRPV1 channels is anticipated to provide new research insights for developing therapeutic interventions applicable to plasma medicine.