TRPA 1 and TRPV 1 contribute to iodine antiseptics‐associated pain and allergy
2016; Springer Nature; Volume: 17; Issue: 10 Linguagem: Inglês
10.15252/embr.201642349
ISSN1469-3178
AutoresDeyuan Su, Hong Zhao, Jinsheng Hu, Danling Tang, Jianmin Cui, Ming Zhou, Jian Yang, Shu Wang,
Tópico(s)Respiratory and Cough-Related Research
ResumoScientific Report26 August 2016free access Transparent process TRPA1 and TRPV1 contribute to iodine antiseptics-associated pain and allergy Deyuan Su Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China Search for more papers by this author Hong Zhao Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Search for more papers by this author Jinsheng Hu Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China Search for more papers by this author Dan Tang Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Search for more papers by this author Jianmin Cui Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Department of Biomedical Engineering, Center for the Investigation of Membrane Excitability Disorders, Cardiac Bioelectricity and Arrhythmia Center, Washington University, St. Louis, MO USA Search for more papers by this author Ming Zhou Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Jian Yang Corresponding Author [email protected] [email protected] orcid.org/0000-0003-0290-7688 Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Shu Wang Corresponding Author [email protected] Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China Search for more papers by this author Deyuan Su Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China Search for more papers by this author Hong Zhao Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Search for more papers by this author Jinsheng Hu Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China Search for more papers by this author Dan Tang Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Search for more papers by this author Jianmin Cui Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Department of Biomedical Engineering, Center for the Investigation of Membrane Excitability Disorders, Cardiac Bioelectricity and Arrhythmia Center, Washington University, St. Louis, MO USA Search for more papers by this author Ming Zhou Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Jian Yang Corresponding Author [email protected] [email protected] orcid.org/0000-0003-0290-7688 Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Shu Wang Corresponding Author [email protected] Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China Search for more papers by this author Author Information Deyuan Su1,2,‡, Hong Zhao1,‡, Jinsheng Hu1,2,‡, Dan Tang1, Jianmin Cui1,3, Ming Zhou1,4, Jian Yang *,*,1,5 and Shu Wang *,1,2 1Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China 2Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China 3Department of Biomedical Engineering, Center for the Investigation of Membrane Excitability Disorders, Cardiac Bioelectricity and Arrhythmia Center, Washington University, St. Louis, MO USA 4Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA 5Department of Biological Sciences, Columbia University, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 212 854 6161; E-mail: [email protected] or [email protected] *Corresponding author. Tel: +86 651 892 57; E-mail: [email protected] EMBO Rep (2016)17:1422-1430https://doi.org/10.15252/embr.201642349 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Iodine antiseptics exhibit superior antimicrobial efficacy and do not cause acquired microbial resistance. However, they are underused in comparison with antibiotics in infection treatments, partly because of their adverse effects such as pain and allergy. The cause of these noxious effects is not fully understood, and no specific molecular targets or mechanisms have been discovered. In this study, we show that iodine antiseptics cause pain and promote allergic contact dermatitis in mouse models, and iodine stimulates a subset of sensory neurons that express TRPA1 and TRPV1 channels. In vivo pharmacological inhibition or genetic ablation of these channels indicates that TRPA1 plays a major role in iodine antiseptics-induced pain and the adjuvant effect of iodine antiseptics on allergic contact dermatitis and that TRPV1 is also involved. We further demonstrate that iodine activates TRPA1 through a redox mechanism but has no direct effects on TRPV1. Our study improves the understanding of the adverse effects of iodine antiseptics and suggests a means to minimize their side effects through local inhibition of TRPA1 and TRPV1 channels. Synopsis This report identifies the cation channel TRPA1 as an endogenous molecular target of iodine in mammals. The iodine-dependent activation of TRPA1 and also TRPV1 is associated with major iodine antiseptics-induced adverse reactions such as pain and cutaneous allergy. TRPA1 is a major mediator of iodine antiseptics-induced pain. Iodine in the PVP-I iodophor promotes contact hypersensitivity mainly through a direct TRPA1-dependent mechanism. Also TRPV1 plays a role in the adverse effects of iodine antiseptics. Introduction The therapeutic effects of iodine on wounds were discovered almost 2,000 years ago 12. Elemental iodine was discovered in 1811. Shortly thereafter, iodine became widely used as an antiseptic and disinfectant worldwide, owing to its efficacy and low cost 1234. Iodine has an antimicrobial activity superior to that of other antiseptics and disinfectants 14. It is the only agent that is simultaneously active against Gram-positive and Gram-negative bacteria, spores, amebic cysts, virus, fungi, protozoa, and yeasts 12456. Importantly, despite more than 150 years of prolonged and extensive use, microbial resistance to iodine has not been observed in a clinical setting to date 1256. Antimicrobial resistance has become a serious threat to global public health. The overuse of antibiotics is a key factor contributing to antibiotic resistance 7; thus, iodine antiseptics have regained attention 56. As Alexander Fleming stated in 1919, in estimating the value of an antiseptic, it is necessary to study its effect on the tissues more than its effect on bacteria 8. The adverse effects of iodine antiseptics are major factors limiting their clinical use, but their underlying mechanisms are largely unclear. Several forms of iodine antiseptics exist and have varying adverse effects. Lugol's solution and iodine tincture, which typically contain 2–7% iodine, cause substantial pain and irritation to wounds, skin, and mucosa 2346. To overcome this limitation, iodophors, the complexes of iodine and iodine-releasing agents, have been developed since the middle of the last century. Currently, povidone–iodine (PVP-I) is the most commonly used iodophor 12346. Iodophors release only very low concentrations of free iodine, thus dramatically reducing its noxious effects 12346. However, even iodophors are associated with burning or stinging sensations, local irritation, contact dermatitis, and deleterious effects on wound healing in patients 236910111213. These adverse effects are believed to result from iodine's chemical or immunogenic properties, but specific molecular targets and mechanisms are not known. TRPA1 and TRPV1 are structurally related, non-selective, Ca2+-permeable cation channels that belong to the transient receptor potential (TRP) ion channel superfamily 14. The majority of TRPA1 is co-expressed with TRPV1 in a subset of C-fiber nociceptors that express the neuropeptides substance P, neurokinin A, and CGRP 1415. Both channels are activated by a variety of noxious stimuli and play critical roles in pain and inflammatory disorders, making them key molecular targets for drug development 1415. In this work, we demonstrate that iodine can cause pain and promote allergic contact hypersensitivity induced by an experimental allergen in mice. Both effects are largely dependent on the nociceptor ion channel TRPA1, which is directly activated by iodine. Another pain-sensing ion channel TRPV1 is not directly activated by iodine but plays a minor role in iodine-induced pain and iodine-enhanced allergy. Results and Discussion Iodine-induced pain in mice is mediated by TRPA1 and TRPV1 We first examined whether iodine could cause pain in mice as it does in humans. Intraplantar injection of 500 ppm (parts per million) (500 ppm is equal to 0.05%) iodine in aqueous solution (25 μl volume) into the mouse hindpaw produced substantial nociceptive behavior, including licking and lifting of the injected hindpaw (Fig 1A). The same dose of iodine in a smaller volume (1,250 ppm in 10 μl) had a similar effect in mice (Appendix Fig S1). In addition, intraplantar injection of a 50% alcohol solution with 1,000 ppm iodine also produced a much stronger pain behavior than did the alcohol solution alone (Appendix Fig S2). These concentrations are far below that experienced by patients treated with local applications of Lugol's solution or iodine tincture, which contain 2–7% (i.e. 20,000–70,000 ppm) iodine 13. Because pain is initially detected by sensory neurons, we tested the hypothesis that iodine directly stimulates pain-sensing neurons. Iodine at a concentration of 0.25 ppm induced Ca2+ influx in a subset of dorsal root ganglion (DRG) neurons in adult mice (Fig 1B). All iodine-sensitive neurons responded to capsaicin, and most of them (93%) also responded to allyl isothiocyanate (AITC). Capsaicin is a specific agonist of TRPV1, and AITC is a potent agonist of TRPA1 but also activates TRPV1 with a low potency 16. Therefore, we examined whether TRPV1 or TRPA1 is involved in iodine-induced pain in mice. We performed an intraperitoneal injection of the TRPA1-specific antagonist HC030031 or the TRPV1-specific antagonist AMG517 1 h prior to iodine injection. These agents substantially suppressed the nociceptive responses in mice, by ~93 and ~46%, respectively (Fig 1C and D). Moreover, in TRPA1−/− mice, iodine-induced nociceptive responses were also substantially attenuated by ~78% (Fig 1E). These data indicate that TRPA1 is a major mediator of iodine-induced pain. The nociceptive reactions induced by iodine in TRPA1−/− mice were almost completely inhibited by the TRPV1-specific antagonist AMG517 (Fig 1F). In contrast to the partial analgesic effect of AMG517 in wild-type (WT) mice (Fig 1D), this result suggests that TRPV1 accounts for the remainder of the iodine-induced pain. Thus, TRPA1 and TRPV1 are specifically responsible for the iodine-induced pain in vivo. Figure 1. TRPA1 and TRPV1 mediate iodine-induced pain in mice A. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine. In this and subsequent similar figures, the number of mice is indicated. B. Averaged intracellular Ca2+ signals in cultured mouse DRG neurons in response to consecutive applications of 0.25 ppm iodine, 30 μM AITC, and 2 μM capsaicin. All of the iodine responsive neurons (n = 332) from 6 adult mice were included in the analysis. C, D. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of HC030031 (HC) (C), AMG517 (D), or vehicle. E. Quantification of the nociceptive responses within 5 min after intraplantar injection of control saline in WT mice or 500 ppm iodine in WT and TRPA1−/− mice. F. Quantification of the nociceptive responses in TRPA1−/− mice within 5 min after intraplantar injection of control saline or 1,000 ppm iodine, following intraperitoneal injection of AMG517 or vehicle. Data information: Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Download figure Download PowerPoint Iodine in PVP-I promotes contact hypersensitivity in mice through TRPA1 and TRPV1 PVP-I, a complex of povidone (polyvinylpyrrolidone) and iodine, is the most widely used iodophor and does not cause serious pain, owing to the low concentration of iodine (0.2–10 ppm free iodine) 34111213. Indeed, intraplantar injection of 5% PVP-I caused much less nociceptive behavior in mice than did the injection of 500 ppm iodine (compare Fig 1A and Appendix Fig S3). However, numerous clinical case reports and studies from the past 30 years have shown that PVP-I is associated with allergic contact dermatitis 910111213. The reported prevalence of PVP-I allergy in clinics is highly variable (between 0.7 and 41%) 2917, and the effects of PVP-I on allergic contact dermatitis have never been experimentally verified and are mechanistically unclear. Furthermore, whether and how iodine plays a role in allergic reactions is controversial 1819. We therefore explored whether PVP-I could promote skin allergy in mice. Topical application of 5% clinical PVP-I solution on mouse skin twice a day for 2 days had no significant effects (Appendix Fig S4). However, the same treatment substantially promoted a delayed-type cutaneous allergy in a modified mouse model of allergic contact dermatitis (Fig 2A). In this model, an oxazolone derivative (Oxa) was used as an allergen to sensitize the mouse through topical application to the shaved abdomen, and 50% more amount of Oxa was used in TRPA1−/− mice than in wild type in order to compensate the lower Oxa sensitivity of TRPA1−/− mice 20. Six days later, the mouse ear was challenged with relative low concentrations of Oxa to elicit delayed-type cutaneous allergies, as measured by the ear thickness change. In this model, Oxa elicits similar allergic responses in wild-type and TRPA1−/− mice (Fig EV1). At a subthreshold concentration of 0.15%, Oxa did not elicit significant allergic reactions in Oxa-sensitized mice (Figs 2B and EV1). However, the concomitant application of 5% PVP-I with 0.15% Oxa induced marked ear swelling in Oxa-sensitized mice, and the reaction was substantially diminished in TRPA1−/− mice (Fig 2A). In addition, 5% povidone, one of the major components of PVP-I, was co-applied with 0.15% Oxa. This combination had no significant effect on Oxa-sensitized mice (Fig 2A). In contrast, iodine alone with 0.15% Oxa caused substantial delayed-type cutaneous reactions in a dose-dependent manner (Fig 2B). The iodine effect was observed only in Oxa-sensitized mice (Fig 2B), suggesting that the reaction is an Oxa allergy, and iodine acts as an adjuvant. In order to examine whether the adjuvant effect of iodine came from its nonspecific impairment of the skin, we showed in a control experiment that mechanical polish of the mouse ear skin with a fine abrasive paper did not cause a significant potentiation of the allergenic effect of Oxa (Appendix Fig S5). This result indicates that slight damages of the skin have no obvious effect on 0.15% Oxa-induced cutaneous allergy. However, the iodine effect was attenuated by more than 70% in TRPA1−/− mice (Fig 2C), and this effect was anatomically evident (Fig 2D). Further pharmacological inhibition of TRPV1 by AMG 517 suppressed the retained allergic inflammation in TRPA1−/− mice (Fig 2E). The effect of iodine in this animal model mimics a situation in which PVP-I triggers allergic contact dermatitis by aggravating pre-existing sensitization in patients with asymptomatic contact allergy 21. Our study does not explain all types of allergy correlated with iodine antiseptics, but it demonstrates that iodine promotes cutaneous allergy in some conditions through a TRPA1- and TRPV1-dependent mechanism. These data may also explain why the prevalence of PVP-I-dependent allergy varies considerably in patients. If PVP-I acts as an adjuvant, the apparent prevalence of “PVP-I allergy” would be determined by multiple factors, including the concentrations of true allergens and patients' sensitivity to the allergens. In the animal model described above, the true allergen is Oxa. Figure 2. Iodine in PVP-I promotes contact hypersensitivity in mice mainly through TRPA1 and TRPV1 Left, time course of ear swelling elicited by topical application of 5% PVP-I solution in 0.15% Oxa-challenged WT and TRPA1−/− mice, or 5% povidone solution in Oxa-challenged WT mice. In this and subsequent similar figures, the number of mice is indicated. Right, bar graph highlighting the response at 24 h shown at left. Time course of ear swelling elicited by topical application of the indicated concentrations of iodine in 0.15% Oxa-challenged mice and a bar graph highlighting the response at 24 h. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged WT and TRPA1−/− mice and bar graph highlighting the response at 24 h. Hematoxylin- and eosin-stained tissue sections of iodine-treated ear in 0.15% Oxa-challenged WT and TRPA1−/− mice (n ≥ 3). Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged TRPA1−/− mice, following intraperitoneal injection of AMG517 or vehicle, and bar graph highlighting the response at 24 h. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged mice, following intraperitoneal injection of RP67580 or vehicle, and bar graph highlighting the response at 24 h. Data information: Data are presented as mean ± s.e.m. Statistical significance was evaluated using two-tailed Student's t-test (for all two-group comparisons) or one-way analysis of variance (ANOVA) followed by Tukey's test (for multi-group comparisons). *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Time course of ear swelling caused by the indicated concentrations of Oxa in Oxa-sensitized wild-type and TRPA1−/− mice The number of mice is indicated. Data are presented as mean ± s.e.m. Download figure Download PowerPoint Activation of TRPA1 and TRPV1 causes release of neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), which are critical for allergic inflammation in the dermatitis, colitis, and asthma 15202223. We thus examined the downstream signaling pathways by testing the effects of SP receptor NK1 or CGRP receptor antagonists on iodine-promoted cutaneous allergies. Intraperitoneal injection of NK1 antagonist RP67580 significantly suppressed the effects of iodine on Oxa-sensitized mice (Fig 2F). In contrast, the CGRP receptor antagonist BIBN4096 was ineffective (Fig EV2A), although in a positive control experiment, BIBN4096 was shown to effectively attenuate complete Freund's adjuvant (CFA)-induced mechanical hypersensitivity in mice (Fig EV2B) 24. These data suggest that only the SP signaling pathway is involved in the adjuvant effect of iodine on cutaneous allergy in mice. Click here to expand this figure. Figure EV2. CGRP signaling pathway does not significantly contribute to the iodine effect on cutaneous allergy in mice Left, time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged mice, following intraperitoneal injection of CGRP receptor antagonist BIBN4096 or vehicle. Right, bar graph highlighting the response at 24 h in the left. About 50% paw withdrawal thresholds were measured with von Frey hair filaments at 24 h after injecting mouse hindpaw with CFA, following intraperitoneal injection of BIBN4096 or vehicle. Data information: The number of mice is indicated. Data are presented as mean ± s.e.m. *P < 0.05, ***P < 0.001 (two-tailed Student's t-test). NS indicates no significant difference. Download figure Download PowerPoint Iodine directly activates TRPA1 but not TRPV1 We then sought to determine whether iodine directly acts on TRPA1 and TRPV1 to cause these adverse effects. We examined the effects of iodine on TRPA1 and TRPV1 activity. Iodine increased the intracellular Ca2+ concentration in HEK 293 cells expressing recombinant human TRPA1 (hTRPA1) in a dose-dependent manner, with an EC50 of 0.25 ppm (Fig 3A and B). However, these effects did not occur in mock-transfected cells or in cells expressing TRPV1 (Fig EV3A and B). Iodine also elicited membrane currents in hTRPA1-expressing Xenopus oocytes with an EC50 of 0.19 ppm at +80 mV (Fig 3C and D), and the currents were completely inhibited by the TRPA1 antagonist HC030031 or the non-specific TRP channel antagonist ruthenium red (Figs 3C and EV3C). In addition, iodine-induced HC030031-sensitive macroscopic currents in an inside-out membrane patch from hTRPA1-expressing HEK 293 cells (Fig 3E) or whole-cell currents in cells expressing mouse TRPA1 (Fig EV3D). In contrast, iodine had no effects on HEK 293 cells and Xenopus oocytes lacking TRPA1 expression (Fig EV3E and F). Notably, the EC50 values of hTRPA1 activation by iodine derived from the calcium imaging or electrophysiological experiments were far below the concentrations of iodine in most iodine antiseptics, such as Lugol's solution (2–5%), iodine tincture (2–7%), and 10% PVP-I solution (~10 ppm free iodine) 13111213. Figure 3. Iodine activates TRPA1 but not TRPV1 A. Representative intracellular Ca2+ signals in hTRPA1-expressing HEK 293 cells in response to different concentrations of iodine. 2-APB, a TRPA1 agonist, was subsequently applied to fully activate TRPA1. RFU: relative fluorescence unit. B. Concentration–response relationships of iodine-induced intracellular Ca2+ increase in HEK 293 cells expressing WT or mutant TRPA1 channels. Data are presented as mean ± s.e.m. n ≥ 8 for each construct at each concentration. The smooth curves are fits to the Hill equation. C. Time course of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. HC: HC030031. D. Concentration–response relationship of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. Data are presented as mean ± s.e.m. n ≥ 8 for each concentration. The smooth curve is a fit to the Hill equation. E. Time course of intracellular iodine-induced macroscopic currents in an inside-out patch from hTRPA1-expressing HEK 293 cell (n = 3). F. Time course of iodine-induced currents in hTRPA1-expressing Xenopus oocytes, which presents the current reduction upon DTT treatment (n = 3). G, H. Comparisons of the iodine-induced whole-cell current in HEK 293 cells expressing WT hTRPA1 (G) or the triple mutant channel (C421S/K710R/C856S) (H) (n ≥ 4). I. Representative whole-cell currents in HEK 293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin (n = 3). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Iodine specifically activates TRPA1 in heterologous expression systems and native neurons A, B. Representative intracellular Ca2+ signals in HEK 293 cells transfected with the empty vector (A) or in cells expressing TRPV1 (B) in response to iodine and subsequently applied a Ca2+ ionophore ionomycin (A) or TRPV1 agonist capsaicin (B), respectively (n ≥ 3). C. Time course of currents in hTRPA1-expressing Xenopus oocytes in response to iodine and subsequently applied non-specific TRP channel antagonist ruthenium red (RR) (n = 4). D. Time course of iodine-induced whole-cell currents in HEK 293 cell expressing mouse TRPA1 (n = 3). E, F. Iodine does not elicit membrane currents in HEK 293 cells (E) and Xenopus oocytes (F) without TRPA1 expression (n ≥ 3). G, H. Representative whole-cell currents induced by iodine (n = 4) (G) or capsaicin (n = 6) (H) in cultured DRG neurons. The holding potential was −60 mV. Download figure Download PowerPoint We then sought to understand how iodine activates TRPA1. Iodine's microbicidal effect is dependent on its reactions with amino acid residues such as cysteine and lysine, resulting in lethal changes to bacterial protein structures (Appendix Fig S6A), and reactions with unsaturated fatty acids in the bacteria membrane (Appendix Fig S6B), changing the chemico-physical properties of the membrane 317. Previous studies have demonstrated that electrophilic compounds activate TRPA1 through a mechanism of covalent modification of cysteine and lysine 2526. Therefore, we test the hypothesis that reactive amino acid residues may be involved in the activation of TRPA1 by iodine. Iodine-induced hTRPA1 currents were not reversible by washout (Fig 3C), but 3 mM dithiothreitol (DTT), a cell-permeable reducing agent, completely reversed the sustained hTRPA1 activation (Fig 3F). In contrast, DTT did not significantl
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