The transient receptor potential ion channel TRPA1 is expressed by primary afferent nerve fibres, in which it functions as a low-threshold sensor for structurally diverse electrophilic irritants, including small volatile environmental toxicants and endogenous algogenic lipids1. TRPA1 is also a ‘receptor-operated’ channel whose activation downstream of metabotropic receptors elicits inflammatory pain or itch, making it an attractive target for novel analgesic therapies2. However, the mechanisms by which TRPA1 recognizes and responds to electrophiles or cytoplasmic second messengers remain unknown. Here we use strutural studies and electrophysiology to show that electrophiles act through a two-step process in which modification of a highly reactive cysteine residue (C621) promotes reorientation of a cytoplasmic loop to enhance nucleophilicity and modification of a nearby cysteine (C665), thereby stabilizing the loop in an activating configuration. These actions modulate two restrictions controlling ion permeation, including widening of the selectivity filter to enhance calcium permeability and opening of a canonical gate at the cytoplasmic end of the pore. We propose a model to explain functional coupling between electrophile action and these control points. We also characterize a calcium-binding pocket that is highly conserved across TRP channel subtypes and accounts for all aspects of calcium-dependent TRPA1 regulation, including potentiation, desensitization and activation by metabotropic receptors. These findings provide a structural framework for understanding how a broad-spectrum irritant receptor is controlled by endogenous and exogenous agents that elicit or exacerbate pain and itch.
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Some data for this study were collected at the Toronto High-Resolution High-Throughput cryo-EM facility, supported by the Canada Foundation for Innovation and Ontario Research Fund. This work was supported by an American Heart Association Postdoctoral Fellowship (J.Z.), a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research (J.Z.), an NSF Graduate Research Fellowship (No. 1650113 to J.V.L.K.), a UCSF Chuan-Lyu Discovery Fellowship (J.V.L.K.), a Helen Hay Whitney Foundation Postdoctoral Fellowship (C.E.P.) and grants from the NIH (R35 NS105038 to D.J; R01 GM098672, S10 OD021741, and S10 OD020054 to Y.C.; T32 HL007731 to C.E.P.; and T32 GM007449 to J.V.L.K.). Y.C. is an Investigator of the Howard Hughes Medical Institute.
The authors declare no competing interests.
Peer review information Nature thanks Thomas Taylor-Clark and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
a, All points histograms depicting the change in open probability (P(o)) in a single TRPA1 channel in response to IA application. Data represent n = 9 independent excised inside-out patches. Vhold = −40 mV. b, SDS–PAGE showing MBP–TRPA1 (arrowhead) after pull-down and elution from amylose beads. c, Cryo-EM image of MBP–TRPA1. Scale bar, 20 nm. d, Two-dimensional classification of cryo-EM particle images showing TRPA1 in different orientations. Scale bar, 25 Å. e, Pharmacological agents used in this study.
Extended Data Fig. 2 Fourier shell correlation of cryo-EM maps, orientation distribution of particle image views, and local resolution of TRPA1 cryo-EM maps.
a, Fourier shell correlation and 1D directional Fourier shell correlation plots. TRPA1 (PMAL) + A-96 class 2 denotes the structure derived from 3D classification of antagonist-treated samples in PMAL and repre\sents the open state channel without antagonist bound. b, Three-dimensional representations of the directional Fourier shell correlation. c, Fourier space covered, based on dFSC at 0.143. d, Orientation distribution of particle image refinement angles. e, The A-loop is lower resolution than surrounding map regions, indicating its dynamic nature. In the activated (TRPA1 + iodoacetamide) and open (TRPA1 + A967079 PMAL-C8 class 2) state conformations, the bottom of S6 is lower resolution than surrounding regions, indicating structural flexibility at the level of the lower gate. Scale bars, 5 Å.
Electrostatic potential maps were calculated in APBS and are displayed at ±10 kT e−1. In silico mutations of D915 were modelled and experimentally determined relative permeability ratios for these mutations sourced from ref. 13. Scale bar, 30 Å.
a, Strong density is observed for iodoacetamide bound to C621. Weaker density is observed next to C665, which indicate that some of the channels may be modified by agonist at this site. Map threshold: σ = 4. b, Clear density for BIA is observed bound to C621. No additional density is observed next to C665 in this case. Map threshold: σ = 6. c, Segmented map densities and atomic models for TRPA1 + BIA (LMNG). Scale bars, 3 Å. d, Map density of the A-loop in different states: undefined (TRPA1 + A-96, PMAL-C8), down (TRPA1 agonist-free, LMNG), and up (TRPA1 + BIA, LMNG). Densities are shown at two different thresholds (σ = 4 and 6). Scale bars, 5 Å.
a, IA (100 μM) activates TRPA1 through covalent modification of cysteines; AITC (250 or 1,000 μM). Data represent n = 6 (WT) or 5 (3C) independent experiments. **P = 0.002, two-tailed Mann–Whitney test; Vhold = −80 mV. b, c, No single cysteine is sufficient for TRPA1 activation by IA. WT, data represent n = 9 independent experiments; C621S/C641S n = 3; C621S/C665S, n = 3; and C641S/C665S, n = 4. Data were acquired in whole-cell patch-clamp mode and reflect the results of 500-ms test pulse (80 mV). Vhold = −80 mV. Doses: IA, 100 μM; A-96, 10 μM; AITC, 250 or 1,000 μM. Scale bars, x, 50 ms; y, 100 pA. I = 0, dashed line. d, Quantification of double cysteine mutant data. Left, WT, n = 6 independent experiments; C621S/C641S n = 3; C621S/C665S, n = 3; and C641S/C665S, n = 3. Vhold = −80 mV. Right, WT, data represent n = 9 independent experiments; C621S/C641S n = 3; C621S/C665S, n = 3; and C641S/C665S, n = 4. Doses: IA, 100 μM; A-96, 10 μM; AITC, 250 or 1,000 μM. *P = 0.02; **P = 0.007, Kruskal–Wallis test with post hoc Dunn’s test to correct for multiple comparisons. e, f, C621S displays complete loss of IA sensitivity while C641S retains full sensitivity. Data represent n = 5 independent experiments/construct. Data were acquired in whole-cell patch-clamp mode and reflect the results of 500-ms test pulses from −80 to 80 mV. Vhold = −80 mV. Doses: IA, 100 μM; A-96, 10 μM. Scale bars, x, 25 ms; y, 100 pA. g, Binding of BIA to TRPA1 C641S/C665S double mutant (C621*) is similar to wild type. Statistical significance is represented as the results of one-way ANOVA with post hoc Holm–Sidak correction for multiple comparisons; *P = 0.03; n = 3 independent experiments per construct. h, TRPA1 cysteine pKa values and deduced proportion of thiolate in the agonist-free state (PDB ID: 6V9W), and IA-bound (‘activated’, PDB ID: 6V9V) state in the presence or absence of covalent modification at C621. Data are mean ± s.e.m.
a, Scaled averaged basal (WT, n = 10 independent experiments; C665S, n = 6), IA (100 μM; WT, n = 5; C665S, n = 5), or BIA (100 μM; C665S, n = 6)-evoked tail currents for TRPA1 WT and C665S mutant channels. Mean deactivation time constants (τ) are shown with 95% CI in parentheses. Scale bar, x, 5 ms; y, arbitrary units. Data were acquired in whole-cell patch-clamp mode after a 500-ms pre-pulses (−80 to 80 mV in 10 mV increments) followed by a 250-ms test pulse (−120 mV). Vhold = −80 mV. b, c, Quantification of changes in IA (b) and BIA (c) -evoked TRPA1 tail-current decay time constants in WT and C665S TRPA1. Statistical significance is represented as the results of a ratio paired two-tailed Student’s t-test; in b, *P = 0.01; in c, *P = 0.02, **P = 0.009.
a, The TRP helix forms an electric dipole with electro-positive K969 at the N terminus and electronegative carbonyl oxygens at the C terminus. b, When the A-loop is oriented in the up position, K671 is coordinated by the carbonyl oxygens at the C terminus of the TRP helix and increases its dipole moment to enhance the positive electrostatic potential at the N terminus. c, The C-terminal carbonyl oxygens of the TRP helix form a pocket that is unoccupied in the agonist-free channel. d, Coordination of K671 with the carbonyl oxygens at the TRP helix C terminus increases the positive electrostatic potential at the TRP helix N terminus. In silico substitution of K671 with glutamate decreases the electrostatic potential of the TRP helix. e, Conformational changes associated with pore dilation further increase the positive electrostatic potential of the TRP domain. f, Multiple sequence alignment of TRPA1 orthologues.
a, Calcium is bound in both agonist-free (σ = 4) and agonist-treated (σ = 8) samples in LMNG detergent, with E788 and N805 displaying the most robust densities coordinating calcium. No density for calcium is observed for the channel in amphipol (grey, σ = 4; blue, σ = 8). b, Carbachol (Cbc., 100 μM) evokes intracellular Ca2+-release through activation of the M1 muscarinic acetylcholine receptor. Cbc. was applied in Ca2+-free Ringer’s solution with 1 mM EGTA to isolate intracellular responses. n = 16 (M1), 18 (M1 + Thg.), 33 (Mock), or 44 (TRPA1) cells. Each graph represent n = 3 (M1, M1 + Thg.), 4 (Mock), or 5 (TRPA1) independent experiments. Iono., ionomycin 1 μM; thapsigargin, 1 μM; AITC, 50 μM. Grey traces represent individual cells and black traces the average of all cells in a given experiment. c, Quantification of Ca2+-imaging experiments. The ratio evoked by Cbc. was normalized to the ionomycin-evoked response, or in TRPA1-transfected cells, the AITC-evoked response. *P < 0.01, Kruskal–Wallis test with post hoc Dunn’s test to correct for multiple comparisons; n = 3 (M1, M1 + Thg.), 4 (Mock), or 5 (TRPA1) independent experiments. ND, response not detected. Data are mean ± s.e.m.
a, The overall architecture of agonist-free and antagonist-bound TRPA1 is similar, representing a closed state. b, A-96 binds at the elbow of S5, sandwiched between S6 and P1. c, Binding of A-96 results in a slight shift in S5 and repositioning of F877. d, The antagonist is in an ideal position to block the straightening of the S5 elbow and inhibit channel gating. e, Two-step model of electrophile action on TRPA1. Attachment of a small electrophile to C621 results in A-loop rearrangement to the up position, bringing C665 into the reactive pocket. Modification of C665 by a second small electrophile stabilizes the A-loop in the up conformation and positions K671 at the C terminus of the TRP helix, enhancing the electric dipole of this region. f, Attachment of a large electrophile to C621 is sufficient to stabilize the A-loop in the up conformation and activate the channel. g, Increased positive electrostatic potential and charge repulsion at N termini of adjacent TRP helices initiates conformational changes associated with dilation of the lower gate. These movements are coupled to widening of the upper gate and selectivity filter through straightening of the S5 helix. The antagonist A-96 binds to the bent elbow region of S5, inhibiting straightening of the α-helix required for channel gating.
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Zhao, J., Lin King, J.V., Paulsen, C.E. et al. Irritant-evoked activation and calcium modulation of the TRPA1 receptor. Nature 585, 141–145 (2020). https://doi.org/10.1038/s41586-020-2480-9
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