Structure of the TRPA1 ion channel suggests regulatory mechanisms

Article metrics

  • A Corrigendum to this article was published on 22 July 2015

Abstract

The TRPA1 ion channel (also known as the wasabi receptor) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here we use single-particle electron cryo- microscopy to determine the structure of full-length human TRPA1 to 4 Å resolution in the presence of pharmacophores, including a potent antagonist. Several unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted transient receptor potential (TRP)-like allosteric domain. These findings provide new insights into the mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: 3D reconstruction of human TRPA1.
Figure 2: Structural details of a single TRPA1 subunit.
Figure 3: C-terminal coiled-coil mediates cytosolic interactions and polyphosphate association.
Figure 4: Cytoplasmic domains form an integrated nexus.
Figure 5: Structural integration of the ARD.
Figure 6: The ion permeation pathway and antagonist binding site.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The 3D cryo-EM density maps of TRPA1 complexes without low-pass filter and amplitude modification have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-6267 (TRPA1-AITC), EMD-6268 (TRPA1-HC030031/A967079) and EMD-6269 (TRPA1-HC030031). Particle images related to this entry are available for download at http://www.ebi.ac.uk/pdbe/emdb/empiar/ with identification number EMPIAR-10024. Atomic coordinates for the atomic model of TRPA1 have been deposited in the Protein Data Bank under the accession number 3J9P.

References

  1. 1

    Julius, D. TRP channels and pain. Annu. Rev. Cell Dev. Biol. 29, 355–384 (2013)

  2. 2

    Wang, H. & Woolf, C. J. Pain TRPs. Neuron 46, 9–12 (2005)

  3. 3

    Bandell, M. et al. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 41, 849–857 (2004)

  4. 4

    Bautista, D. M. et al. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 124, 1269–1282 (2006)

  5. 5

    Bautista, D. M. et al. Pungent products from garlic activate the sensory ion channel TRPA1. Proc. Natl Acad. Sci. USA 102, 12248–12252 (2005)

  6. 6

    Jordt, S. E. et al. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 427, 260–265 (2004)

  7. 7

    McNamara, C. R. et al. TRPA1 mediates formalin-induced pain. Proc. Natl Acad. Sci. USA 104, 13525–13530 (2007)

  8. 8

    Taylor-Clark, T. E. et al. Prostaglandin-induced activation of nociceptive neurons via direct interaction with transient receptor potential A1 (TRPA1). Mol. Pharmacol. 73, 274–281 (2008)

  9. 9

    Trevisani, M. et al. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc. Natl Acad. Sci. USA 104, 13519–13524 (2007)

  10. 10

    Caspani, O. & Heppenstall, P. A. TRPA1 and cold transduction: an unresolved issue? J. Gen. Physiol. 133, 245–249 (2009)

  11. 11

    Wilson, S. R. et al. TRPA1 is required for histamine-independent, Mas-related G protein-coupled receptor-mediated itch. Nature Neurosci. 14, 595–602 (2011)

  12. 12

    Andrade, E. L., Meotti, F. C. & Calixto, J. B. TRPA1 antagonists as potential analgesic drugs. Pharmacol. Ther. 133, 189–204 (2012)

  13. 13

    Kremeyer, B. et al. A gain-of-function mutation in TRPA1 causes familial episodic pain syndrome. Neuron 66, 671–680 (2010)

  14. 14

    Hinman, A., Chuang, H. H., Bautista, D. M. & Julius, D. TRP channel activation by reversible covalent modification. Proc. Natl Acad. Sci. USA 103, 19564–19568 (2006)

  15. 15

    Macpherson, L. J. et al. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 445, 541–545 (2007)

  16. 16

    Kim, D. & Cavanaugh, E. J. Requirement of a soluble intracellular factor for activation of transient receptor potential A1 by pungent chemicals: role of inorganic polyphosphates. J. Neurosci. 27, 6500–6509 (2007)

  17. 17

    Nilius, B., Prenen, J. & Owsianik, G. Irritating channels: the case of TRPA1. J. Physiol. (Lond.) 589, 1543–1549 (2011)

  18. 18

    Wang, Y. Y., Chang, R. B., Waters, H. N., McKemy, D. D. & Liman, E. R. The nociceptor ion channel TRPA1 is potentiated and inactivated by permeating calcium ions. J. Biol. Chem. 283, 32691–32703 (2008)

  19. 19

    Cvetkov, T. L., Huynh, K. W., Cohen, M. R. & Moiseenkova-Bell, V. Y. Molecular architecture and subunit organization of TRPA1 ion channel revealed by electron microscopy. J. Biol. Chem. 286, 38168–38176 (2011)

  20. 20

    Cao, E., Liao, M., Cheng, Y. & Julius, D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504, 113–118 (2013)

  21. 21

    Liao, M., Cao, E., Julius, D. & Cheng, Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107–112 (2013)

  22. 22

    Samad, A. et al. The C-terminal basic residues contribute to the chemical- and voltage-dependent activation of TRPA1. Biochem. J. 433, 197–204 (2011)

  23. 23

    Woolfson, D. N. The design of coiled-coil structures and assemblies. Adv. Protein Chem. 70, 79–112 (2005)

  24. 24

    Macbeth, M. R. et al. Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309, 1534–1539 (2005)

  25. 25

    Gray, M. J. et al. Polyphosphate is a primordial chaperone. Mol. Cell 53, 689–699 (2014)

  26. 26

    Rohacs, T. Phosphoinositide regulation of TRP channels. Handb. Exp. Pharmacol. 223, 1143–1176 (2014)

  27. 27

    Paulsen, C. E. & Carroll, K. S. Cysteine-mediated redox signaling: chemistry, biology, and tools for discovery. Chem. Rev. 113, 4633–4679 (2013)

  28. 28

    Chen, J. et al. Molecular determinants of species-specific activation or blockade of TRPA1 channels. J. Neurosci. 28, 5063–5071 (2008)

  29. 29

    Moparthi, L. et al. Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain. Proc. Natl Acad. Sci. USA 111, 16901–16906 (2014)

  30. 30

    Jaquemar, D., Schenker, T. & Trueb, B. An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts. J. Biol. Chem. 274, 7325–7333 (1999)

  31. 31

    Zayats, V. et al. Regulation of the transient receptor potential channel TRPA1 by its N-terminal ankyrin repeat domain. J. Mol. Model. 19, 4689–4700 (2013)

  32. 32

    Gracheva, E. O. et al. Molecular basis of infrared detection by snakes. Nature 464, 1006–1011 (2010)

  33. 33

    Sokabe, T., Tsujiuchi, S., Kadowaki, T. & Tominaga, M. Drosophila painless is a Ca2+-requiring channel activated by noxious heat. J. Neurosci. 28, 9929–9938 (2008)

  34. 34

    Viswanath, V. et al. Opposite thermosensor in fruitfly and mouse. Nature 423, 822–823 (2003)

  35. 35

    Zhong, L. et al. Thermosensory and nonthermosensory isoforms of Drosophila melanogaster TRPA1 reveal heat-sensor domains of a thermoTRP Channel. Cell Rep 1, 43–55 (2012)

  36. 36

    Cordero-Morales, J. F., Gracheva, E. O. & Julius, D. Cytoplasmic ankyrin repeats of transient receptor potential A1 (TRPA1) dictate sensitivity to thermal and chemical stimuli. Proc. Natl Acad. Sci. USA 108, E1184–E1191 (2011)

  37. 37

    Jabba, S. et al. Directionality of temperature activation in mouse TRPA1 ion channel can be inverted by single-point mutations in ankyrin repeat six. Neuron 82, 1017–1031 (2014)

  38. 38

    Payandeh, J., Scheuer, T., Zheng, N. & Catterall, W. A. The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011)

  39. 39

    Long, S. B., Campbell, E. B. & Mackinnon, R. Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309, 897–903 (2005)

  40. 40

    Susankova, K., Ettrich, R., Vyklicky, L., Teisinger, J. & Vlachova, V. Contribution of the putative inner-pore region to the gating of the transient receptor potential vanilloid subtype 1 channel (TRPV1). J. Neurosci. 27, 7578–7585 (2007)

  41. 41

    Voets, T., Janssens, A., Droogmans, G. & Nilius, B. Outer pore architecture of a Ca2+-selective TRP channel. J. Biol. Chem. 279, 15223–15230 (2004)

  42. 42

    McGaraughty, S. et al. TRPA1 modulation of spontaneous and mechanically evoked firing of spinal neurons in uninjured, osteoarthritic, and inflamed rats. Mol. Pain 6, 14 (2010)

  43. 43

    Petrus, M. et al. A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition. Mol. Pain 3, 40 (2007)

  44. 44

    Banzawa, N. et al. Molecular basis determining inhibition/activation of nociceptive receptor TRPA1: a single amino acid dictates species-specific actions of the most potent mammalian trpa1 antagonists. J. Biol. Chem. 289, 31927–31939 (2014)

  45. 45

    Klement, G. et al. Characterization of a ligand binding site in the human transient receptor potential ankyrin 1 pore. Biophys. J. 104, 798–806 (2013)

  46. 46

    Nakatsuka, K. et al. Identification of molecular determinants for a potent mammalian TRPA1 antagonist by utilizing species differences. J. Mol. Neurosci. 51, 754–762 (2013)

  47. 47

    Xiao, B. et al. Identification of transmembrane domain 5 as a critical molecular determinant of menthol sensitivity in mammalian TRPA1 channels. J. Neurosci. 28, 9640–9651 (2008)

  48. 48

    Bagnéris, C. et al. Prokaryotic NavMs channel as a structural and functional model for eukaryotic sodium channel antagonism. Proc. Natl Acad. Sci. USA 111, 8428–8433 (2014)

  49. 49

    Catterall, W. A. Structure and function of voltage-gated sodium channels at atomic resolution. Exp. Physiol. 99, 35–51 (2014)

  50. 50

    Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)

  51. 51

    Chae, P. S. et al. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nature Methods 7, 1003–1008 (2010)

  52. 52

    Ohi, M., Li, Y., Cheng, Y. & Walz, T. Negative staining and image classification - powerful tools in modern electron microscopy. Biol. Proced. Online 6, 23–34 (2004)

  53. 53

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013)

  54. 54

    Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)

  55. 55

    Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996)

  56. 56

    Elmlund, H., Elmlund, D. & Bengio, S. PRIME: probabilistic initial 3D model generation for single-particle cryo-electron microscopy. Structure 21, 1299–1306 (2013)

  57. 57

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

  58. 58

    Scheres, S. H. Beam-induced motion correction for sub-megadalton cryo-EM particles. Elife 3, e03665 (2014)

  59. 59

    Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nature Methods 9, 853–854 (2012)

  60. 60

    Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nature Methods 11, 63–65 (2014)

  61. 61

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  62. 62

    Söding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005)

  63. 63

    Jones, D. T. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195–202 (1999)

  64. 64

    Gruber, M., Soding, J. & Lupas, A. N. REPPER–repeats and their periodicities in fibrous proteins. Nucleic Acids Res. 33, W239–W243 (2005)

  65. 65

    Gaudet, R. A primer on ankyrin repeat function in TRP channels and beyond. Mol. Biosyst. 4, 372–379 (2008)

  66. 66

    Penczek, P., Ban, N., Grassucci, R. A., Agrawal, R. K. & Frank, J. Haloarcula marismortui 50S subunit-complementarity of electron microscopy and X-Ray crystallographic information. J. Struct. Biol. 128, 44–50 (1999)

  67. 67

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

  68. 68

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

  69. 69

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

  70. 70

    Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014)

  71. 71

    Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

  72. 72

    Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360, 376(1996)

Download references

Acknowledgements

We thank M. Liao for initial electron microscopy analysis of vampire bat TRPA1, and S. Wu and M. Zhao for help with refining the atomic model. This work was supported by grants from the National Institutes of Health (R01NS055299 to D.J. and R01GM098672 to Y.C.) and the UCSF Program for Breakthrough Biomedical Research (Y.C.). C.E.P. was supported by a T32 Postdoctoral Training Grant from the UCSF CVRI, and is currently a HHMI Fellow of the Helen Hay Whitney Foundation.

Author information

C.E.P. expressed and purified protein samples, determined conditions to enhance protein stability, and performed functional studies. Y.G. carried out initial negative-stain analysis and characterization of cryo-EM conditions. J.-P.A. carried out detailed cryo-EM experiments, including data acquisition and processing. C.E.P. and J.-P.A. built the atomic model on the basis of cryo-EM maps. All authors contributed to experimental design, data analysis and manuscript preparation.

Correspondence to Yifan Cheng or David Julius.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Pre-cryo-EM screening of TRPA1 species orthologues and purification of human TRPA1.

a, FSEC traces from eGFP–TRPA1 fusion proteins. Void volume and peak corresponding to tetrameric channels are indicated. b, Representative section of negative-stain micrographs showing typical structure of tetrameric MBP-tagged TRPA1 from various species, as indicated (text colour matches traces in a). Particles from species orthologues exhibited highly similar shapes, except rattlesnake TRPA1, which were not homogenous and tended to aggregate. The human TRPA1 orthologue was chosen after negative-stain screening owing to exemplary homogeneity of individual particles. c, Cartoon diagram of MBP-tagged construct used for single-particle cryo-EM studies. d, MBP-tagged TRPA1 construct is active when transduced in HEK293T cells as assessed by calcium imaging (scale bar indicates relative calcium levels: low (blue) to high (red)). e, Gel filtration profile (Superose 6) of MBP-tagged TRPA1 after detergent solubilization, purification on amylose affinity resin, followed by exchange into PMAL-C8. Peaks correspond to void (1), tetrameric MBP–TRPA1 (2), and excess PMAL-C8 (3). f, Material from peak 2 migrates as a single, homogenous band (173 kDa) on SDS–PAGE (4–12% gradient gel, Coomassie stain). g, PMAL-C8-stabilized MBP–TRPA1 appears as homogenous particles with a clear crescent density by negative-stain imaging.

Extended Data Figure 2 Initial single-particle cryo-EM study of TRPA1.

a, Raw micrograph of MBP–TRPA1 recorded using a scintillator-based CMOS camera. b, 2D class averages of MBP–TRPA1 particles. c, Euler angle distribution of initial 3D reconstruction. d, FSC curve of final 3D reconstruction. e, Final 3D reconstruction of MBP–TRPA1 at 28 Å resolution. This 3D reconstruction was used as the initial model for subsequent cryo-EM studies of TRPA1 using a direct electron detection camera.

Extended Data Figure 3 Single-particle cryo-EM studies of TRPA1 with agonist (AITC).

a, Raw micrograph of MBP–TRPA1 with agonist (AITC) recorded using K2 Summit operated in super-resolution counting mode. b, Gallery of 2D class averages. c, Euler angle distribution of all particles included in calculating the final 3D reconstruction. The size of the ball is proportional to the number of particles in this specific orientation. d, Selected slice views of the unsharpened 3D density map. The views are oriented in parallel with the membrane plane. The numbers of slices are marked. e, Two views of TRPA1 density map filtered to 6 Å resolution and displayed in two different isosurface levels (high in yellow and low in grey). At low isosurface level, density contributed by PMAL-C8 is visible. f, FSC curves between two independently refined half maps (red) and between the final combined density map and the map calculated from atomic model (blue). g, Voxel histogram corresponding to local resolution. There are significant numbers of voxels with higher than 4 Å local resolution. h, Final 3D reconstruction coloured with local resolution. i, Cryo-EM densities of the S4, S4–S5 linker, pore helices, S6, TRP-like domain, and coiled-coil in longitudinal cross sections are superimposed on an atomic model. Only two diagonally opposed subunits are shown for clarity. Dashed ovals indicate regions highlighted at sides.

Extended Data Figure 4 Single-particle cryo-EM studies of TRPA1 with antagonist (HC-030031).

a, Raw micrograph of MBP–TRPA1 with single antagonist HC-030031 recorded using K2 Summit operated in super-resolution counting mode. b, Gallery of 2D class averages. c, Euler angle distribution of all particles included in calculating the final 3D reconstruction. The size of the ball is proportional to the number of particles in this specific orientation. d, FSC curve between two independently refined half maps. e, Three different views of the final density map. f, Voxel histogram corresponding to local resolution. g, Final 3D reconstruction coloured with local resolution.

Extended Data Figure 5 Single-particle cryo-EM studies of TRPA1 with double antagonist (HC-030031 and A-967079).

a, Raw micrograph of MBP–TRPA1 with double antagonists recorded using K2 Summit operated in super-resolution counting mode. b, Gallery of 2D class averages. c, Euler angle distribution of all particles included in calculating the final 3D reconstruction. The size of the ball is proportional to the number of particles in this specific orientation. d, FSC curve between two independently refined half maps. e, Three different views of the final density map. f, Voxel histogram corresponding to local resolution. g, Final 3D reconstruction coloured with local resolution.

Extended Data Figure 6 Refinement of de novo atomic model of TRPA1 determined from cryo-EM density maps.

a, Statistics of atomic model refinement. b, FSC curves between the density map calculated from the refined model and half map 1 (work, green curve), half map 2 (free, red curve) and summed map (blue).

Extended Data Figure 7 Detailed views of unique structural features in TRPA1.

a, Density map showing the location of a poorly resolved α-helix within the S1–S2 linker that extends into the extracellular space. b, Density map and α-carbon trace for an α-helix in the inner membrane leaflet located within a flexible loop connecting the third β-strand to the C-terminal coiled-coil. c, Cross section of the density map corresponding to Fig. 3d. d, Cross section of the density map corresponding to Fig. 3c. InsP6 density is depicted in orange. e, Size of the density corresponding to InsP6 (yellow) is consistent with an InsP6 molecule. f, g, Cryo-EM densities of Asp 915 (f), and Ile 957 and Val 961 (g) along the pore are superimposed on the atomic model; both panels represent views along the four-fold axis, showing residues from each subunit of the homotetrameric channel. h, i, Density maps and ribbon diagrams of atomic models showing the location of Phe 909 in AITC (h) and double antagonist (i) samples. Density of A-967079 is indicated in the latter. j, Size of the density corresponding to A-967079 (yellow) is consistent with a A-967079 molecule. The resolution of these ligand densities (>6 Å) is insufficient to propose a precise model for ligand binding. The positioning of coordinates for ligands represents only the scale-context and does not present any proposed mode of interaction with the channel.

Extended Data Figure 8 Distal N terminus contains an ankyrin-repeat-rich region that forms a crescent-shaped density surrounding the main body of the particle.

a, Sequence alignment indicates that the N terminus of human TRPA1 contains at least 16 ankyrin repeats. The last five can be modelled into all human TRPA1 density maps. b, 2D class averages of negatively stained MBP–TRPA1 in PMAL-C8. c, Three selected 2D class averages indicating dimension of the crescent-shaped density. d, A homology model for the first 11 predicted ankyrin repeats spanning a dimension of 100 Å, suggesting that the crescent-shaped density can accommodate at least 11 ankyrin repeats. e, f, Two models for the extended ankyrin repeats are superimposed on the human TRPA1 core atomic model determined by single-particle cryo-EM. Resolution of the crescent is insufficient to determine confidently extended ARD orientation, but which could assemble as a propeller (e) or independent wings (f). On the basis of the combined movement of the crescent density in distinct negative-stain particles (b), we favour a propeller orientation.

Extended Data Figure 9 Characterization of human TRPA1 Phe909Thr sensitivity to A-967079.

a, b, Ratiometric calcium imaging of HEK293 cells transiently transfected with wild-type (a) or Phe909Thr mutant (b) human TRPA1. Cells were stimulated with AITC (250 μM) with (right) or without (left) pre-application of A-967079 (10 μM). ch, Representative recordings from oocytes expressing wild-type (ce) or Phe909Thr mutant (fh) human TRPA1 activated with AITC (200 μM) before co-application of A-967079 (10 μM) (c and f), HC-030031 (100 μM) (d and g), or ruthenium red (10 μM) (e and h). i, Chemical structures and molecular masses of compounds used in this study.

Extended Data Table 1 Summary of human TRA1 structure determinations by single-particle cryo-EM

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Paulsen, C., Armache, J., Gao, Y. et al. Structure of the TRPA1 ion channel suggests regulatory mechanisms. Nature 520, 511–517 (2015) doi:10.1038/nature14367

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.