Irritant-evoked activation and calcium modulation of the TRPA1 receptor

Abstract

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.

Extended Data Fig. 1.. Pharmacology and cryo-EM data collection and processing for TRPA1.

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. V_hold = −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, 2D 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-967079 class 2 denotes the structure derived from 3D classification of antagonist-treated samples in PMAL and represents the open state channel without antagonist bound. b, 3D 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 compared to surrounding map regions, indicating its dynamic nature. In the activated (TRPA1+iodoacetamide) and open state (TRPA1 + A967079 PMAL-C8 class 2) conformations, the bottom of S6 is lower resolution compared to surrounding regions, indicating structural flexibility at the level of the lower gate. Scale bars: 5Å.

Extended Data Fig. 3.. Surface charge distribution of TRPA1's extracellular face.

Electrostatic potential maps were calculated in APBS and are displayed at ± 10 kT/e⁻. In silico mutations of D915 were modeled and experimentally determined relative permeability ratios for these mutations sourced from Wang et al. (ref. 13). Scale bar: 30Å.

Extended Data Fig. 4.. Map densities of agonists and transmembrane α-helices.

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 BODIPY-iodoacetamide (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: 3Å. 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: 5Å.

Extended Data Fig. 5.. Characterization of TRPA1 activation by IA and BIA.

a, IA (100 μM) activates TRPA1 through covalent modification of cysteines; AITC (250 or 1000 μM). Data represent n = 6 (WT) or 5 (3C) independent experiments. Statistical significance is reported as the result of an unpaired two-tailed Mann-Whitney test; **, p = 0.002; V_hold = −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 msec. test pulse (80 mV). V_hold = −80 mV. Doses: IA, 100 μM; A-96,10 μM; AITC, 250 or 1000 μM. Scale bars: x, 50 msec.; 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. V_hold = −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 1000 μM. Statistical significance is reported as the result of a Kruskal-Wallis test with post-hoc Dunn’s test to correct for multiple comparisons; *, p = 0.02; **, p = 0.007. e,f C621S display 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 msec. test pulses from −80 to 80 mV. V_hold = −80 mV. Doses: IA, 100 μM; A-96, 10 μM. Scale bars: x, 25 msec.; y, 100 pA. g, Binding of BIA to TRPA1 C641S/C665S double mutant (C621*) is similar to wildtype. 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/construct. h, TRPA1 cysteine pK_a 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. All summary data are displayed as the mean ± S.E.M..

Extended Data Fig. 6.. Analysis of TRPA1 tail currents.

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 msec., y = arbitrary units. Data were acquired in whole-cell patch clamp mode after a 500 msec. pre-pulses (−80 to 80 mV in 10 mV increments) followed by a 250 msec test pulse (−120 mV). V_hold = −80 mV. b, Quantification of changes in IA- and c, BIA-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; (panel b) *, p = 0.01; (panel c) *, p = 0.02, **, p = 0.009.

Extended Data Fig. 7.. Positive electrostatic potential below the lower gate.

a, The TRP helix forms an electric dipole with electro-positive K969 at the N terminus and electro-negative 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 orthologs.

Extended Data Fig. 8.. Calcium map densities and calcium-imaging of Ca²⁺ modulation.

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 (gray, 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; Thg., Thapsigargin, 1 μM; AITC, 50 μM. Gray 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 Iono.- or, in TRPA1-transfected cells, 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 represent the mean ± S.E.M..

Extended Data Fig. 9.. Binding of A-967079 to TRPA1 and 2-step model of electrophile action.

a, The overall architecture of agonist-free and antagonist-bound TRPA1 is similar, representing a closed state. b, A-967079 binds at the elbow of S5, sandwiched between S6 and P1. c, Binding of A-967079 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, 2-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-967079 binds to the bent elbow region of S5, inhibiting straightening of the α-helix required for channel gating.

Fig. 1.. Dynamic equilibrium between closed and activated conformations

a, Closed and open states of TRPA1 were captured by incubation with an irreversible electrophilic agonist (Iodoacetamide, IA) or an antagonist (A-967079, A-96) before or after membrane solubilization, respectively. Representative inside-out patch recordings show spontaneous (basal) and IA (100 μM)-evoked (persistent) TRPA1 channel openings (V_hold = −40 mV; scale bars: x = 10 msec., y = 2 pA, n = 9 independent experiments. b, Cryo-EM density maps of TRPA1 bound to A-96 or IA in closed or activated state, respectively. A-96 binds to the membrane domain while iodoacetamide binds to the allosteric nexus. c, Comparison between subunits in closed and activated conformations showing ~15° rotation of voltage sensor-like domain (VSLD) and twisting and translation of pore domain. Ankyrin repeat domain remains stationary. d, VSLD rotates around the cytoplasmic base of transmembrane α-helices S1 and S2 in a near-rigid-body movement. e, Pore domain twists and translates upward towards the extracellular milieu, concomitant with a shift in the S6 π-helix by one helical turn. f, Conformational changes in the upper pore region are coupled to widening of the lower gate through straightening of the S5 α-helix, enabling movement of S6 to facilitate gating. Scale bars: 10Å.

Fig. 2.. Coupled dilation of upper pore region and lower gate

a, Activation of TRPA1 involves concerted dilation of both upper and lower gates with upward shift of the selectivity filter. b, Lower gate is formed by residues I957 and V961, while upper gate is formed by D915 and backbone carbonyl of G914. c, Pore widening is associated with counter-clockwise rotation of transmembrane α-helices (viewed from extracellular face to cytoplasmic side). d, Acidic residues lining the upper pore create a highly negatively charged surface, especially in the activated conformation, likely facilitating calcium selectivity. Scale bars: 5Å. Electrostatic surface charge distribution of TRPA1’s cytoplasmic face in apo- and activated states were calculated in APBS and displayed at ±10 kT/e⁻. Scale bars: 25Å.

Fig. 3.. Activation by electrophiles occurs through two-step mechanism

a, A dynamic activation loop (A-loop) adopts a 'down' conformation in the agonist-free channel, partially occluding a reactive pocket containing C621. b, Following attachment of BODIPY-iodoacetamide (BIA) to C621, the A-loop transitions to an 'up' conformation, bringing C665 into the reactive pocket and repositioning K671 to coordinate backbone carbonyl oxygens at the TRP domain C-terminus. Scale bars: 5Å. c, IA (100 μM)-evoked currents for WT and C665S mutant TRPA1 channels (n = 9 and 5 independent experiments, respectively), followed by inhibition with A-96 (10 μM). d, Same for BIA (100 μM)-evoked currents (n = 5 for WT and C665S mutant). 500 msec. voltage steps from −80 to 80 mV, 0 mV colored red. Scale bars: x = 25 msec., y = 100 pA. e, (left) Quantification of IA-evoked currents for WT or mutant TRPA1 channels at 80 mV (n = 9 and 5 independent experiments/construct, respectively). (right) Normalized IA-evoked currents (TRPA1 WT, n = 6; C665S, n = 3; K671A, n = 3) and BIA-evoked currents (TRPA1 WT n = 5; C665S, n =4). *, p = 0.05; ** p = 0.007, Kruskal-Wallis test with post-hoc Dunn’s correction for multiple comparisons. Data displayed as ± S.E.M. f, AITC dose-response curves for WT (EC₅₀ = 37; 95% CI: 30 – 46 nM) and K671A (EC₅₀ = 344; 95% CI: 313 – 381 nM) TRPA1 channels. EC₅₀ values determined by nonlinear Poisson regression and statistically significant difference confirmed with extra sum-of-squares F test (p < 0.0001). n = 3 cells/dose/construct except K671A 50 mM (n = 6) and 100 mM (n = 4). Data are displayed as mean ± S.E.M. g, IA (100 μM) and AITC (1 mM)-evoked whole cell currents for WT and K671A channels (V_hold = −80 mV) blocked by A-96 (10 μM). n = 3 independent experiments/construct.

Fig. 4.. One site subserves three distinct modes of calcium

a, A calcium ion binds at the cytoplasmic end of S2-S3 helices in the VSD, as seen in TRPM4 and TRPM8. Scale bars = 5Å. b, Sequence alignment reveals conservation of calcium-coordinating residues in TRPA1, TRPM4 and TRPM8. c, Normalized whole-cell currents from TRPA1 WT (n = 6 independent experiments), E788S (n = 8), and E788S, Q791S, N80S triple mutant (n = 10) following bath application of AITC (50 μM) and Ca²⁺ (2 mM). Data were extracted from 500 msec. voltage ramps (−80 to 80 mV). Scale bars: x = 1 sec, y = arbitrary units. d, Quantification of Ca²⁺ potentiation (I-transient, I_trans, measured 5 secs post-Ca²⁺ application) and desensitization (I-sustained, I_sus, measured 60 sec post-Ca²⁺ application). WT, n = 6 independent experiments; E788S, n = 8; E788S, Q791S, N805S triple mutant, n = 10. *, p = 0.04; **, p = 0.005; ****, p < 0.0001 one-way mixed-effects analysis ANOVA with post-hoc Holm-Sidak correction for multiple comparisons; Data displayed as mean ± S.E.M. e-f, Normalized whole cell recordings for TRPA1 channels co-expressed with M1 muscarinic acetylcholine receptor in response to bath applied carbachol (Cbc, 100 μM; data represent n = 7 independent experiments for WT and n = 4 for E788S mutant), with or without Thapsigargin (Thg, 1 μM; n = 5) pre-treatment or inclusion of rapid intracellular Ca²⁺ chelators EGTA and BAPTA (1 mM each; n = 5). AITC, 50 μM and A-96,10 μM. Scale bars: x = 1 sec, y = arbitrary units. g, Quantification of responses in e-f. ** p = 0.002 for WT vs. +Thg and +Chel; p = 0.004 for WT vs E788S; Kruskal-Wallis test with post-hoc Dunn’s correction for multiple comparisons. Data displayed as mean ± S.E.M.