Journal: Nature Communications

Article Title: Molecular mechanism of hyperactivation conferred by a truncation of TRPA1

doi: 10.1038/s41467-023-38542-1

Figure Lengend Snippet: a Cartoon schematic of a full-length hTRPA1 monomeric subunit, with relevant structural features denoted. Regions retained in R919* hTRPA1 are indicated in teal and regions truncated are indicated in pink. Ribbon diagrams of WT hTRPA1 atomic model for residues K446-T1078 from a single subunit ( b ) and the homotetrameric channel ( c ). Color scheme and relevant structural features denoted as in a . In c , only one subunit is colored for clarity. Allosteric nexus indicated with brackets. Models built with the human TRPA1 Cryo-EM structure (PDB: 6V9W) in UCSF Chimera. d Ratiometric calcium imaging of HEK293T cells transfected with empty vector (mock), WT, R919*, or N855S hTRPA1. Cells were stimulated with 100 µM AITC. Images are representative of three independent experiments. Scale bars indicate 100 µm. e Quantification of AITC-evoked change in Fura-2 ratio (arbitrary units, arb. units) for WT (black), R919* (deep pink), or N855S (yellow) hTRPA1 variants. Data represent mean ± SEM. n = 4 independent experiments, n ≥ 90 cells per transfection condition per experiment. **** p < 0.0001, one-way ANOVA with Bonferroni’s post hoc analysis. f Western blot of lysates from cells expressing 3×FLAG-tagged hTRPA1 variants, probed using HRP-conjugated anti-FLAG antibody. Tubulin was the loading control. Results were verified in four independent experiments. g Representative voltage ramp (−80 mV to +80 mV) current-voltage (I-V) relationships from Xenopus laevis oocytes expressing WT (black), N855S (yellow), R919* (deep pink), or Δ934–1119 (pink) hTRPA1 variants. Currents evoked by 250 µM AITC. Extracellular solution contained no calcium. h Quantification of AITC-evoked peak current amplitudes at −80 mV and +80 mV. Colors as indicated in g . Data represent mean ± SEM. n = 5 (R919* and Δ934–1119 hTRPA1), 6 (N855S hTRPA1), or 7 (WT hTRPA1) independent oocytes. **** p < 0.0001, *** p < 0.001, * p < 0.05, one-way ANOVA with Bonferroni’s post hoc analysis. i Western blot of lysates from representative Xenopus oocytes used for recordings in g expressing 3×FLAG-tagged hTRPA1 variants, probed using HRP-conjugated anti-FLAG antibody. f , i Full blots are included in Supplementary Fig. . e , g , h Source data are provided as a Source Data file.

Article Snippet: For calcium imaging experiments, untagged human TRPA1 in a combined mammalian/oocyte expression vector pMO (modified from pcDNA3 - obtained from David Julius) or 3×FLAG-tagged TRPA1, TRPV1, or TRPM8 in p3×FLAG-eYFP-CMV-7.1 (Addgene #34582) vector were used.

Techniques: Cryo-EM Sample Prep, Imaging, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Nature Communications

Article Title: Molecular mechanism of hyperactivation conferred by a truncation of TRPA1

doi: 10.1038/s41467-023-38542-1

Figure Lengend Snippet: Representative time-traces at −80 and +80 mV holding potentials (above) and the corresponding current-voltage relationships from timepoints indicated by o, i and ii (boxed below) from oocytes expressing WT TRPA1 ( a , black), WT and R919* TRPA1 ( b , deep pink), or WT and Δ934–1119 hTRPA1 ( c , pink). Currents in the current-voltage relationships are raw, unadjusted values. Baseline currents (black o) and currents evoked with 150 µM AITC in the absence (orange i) and presence (blue ii) of 1.8 mM extracellular calcium are shown. d Quantification of peak current amplitudes from Xenopus oocytes used in a – c . Colors as indicated in a – c . Data represent mean ± SEM. **** p < 0.0001, *** p < 0.001, ** p < 0.01,* p < 0.05, n.s. not significant. n = 10 (WT with Δ934–1119 hTRPA1), 12 (WT with R919* hTRPA1), or 15 (WT hTRPA1) oocytes per condition, one-way ANOVA with Tukey’s post hoc analysis. Representative time-traces at −80 mV holding potential ( f ) and peak current amplitude quantification ( g ) from Xenopus oocytes expressing WT (black) or WT and R919* (deep pink) hTRPA1 with NDMG + as the predominant monovalent extracellular cation. Currents evoked with 150 µM AITC in the absence (iii) and presence (iv) of 1.8 mM extracellular Ca 2+ . Data represent mean ± SEM. *** p < 0.001. n = 12 oocytes per condition, two-tailed Student’s t -test. a – c , f Dashed line denotes 0 µA current. Protocol of condition application indicated above. e , h Western blot of lysates from Xenopus oocytes used for representative recordings in a – c ( e ) and f ( h ) expressing 3×FLAG-tagged hTRPA1 variants, probed using HRP-conjugated anti-FLAG antibody. Blots are representative of one oocyte per injection type (WT hTRPA1 ( n = 15 ( d ) and 12 ( f )), WT with R919* hTRPA1 ( n = 12 ( d , f )), and WT with Δ934–1119 hTRPA1 ( n -10)). Full blots are included in Supplementary Fig. . ( a – d , f and g ) Source data are provided as a Source Data file.

Article Snippet: For calcium imaging experiments, untagged human TRPA1 in a combined mammalian/oocyte expression vector pMO (modified from pcDNA3 - obtained from David Julius) or 3×FLAG-tagged TRPA1, TRPV1, or TRPM8 in p3×FLAG-eYFP-CMV-7.1 (Addgene #34582) vector were used.

Techniques: Expressing, Two Tailed Test, Western Blot, Injection

Journal: Nature Communications

Article Title: Molecular mechanism of hyperactivation conferred by a truncation of TRPA1

doi: 10.1038/s41467-023-38542-1

Figure Lengend Snippet: Quantification of sub-saturating agonist-evoked Fura-2 ratio relative to a saturating agonist response for ( a ) mouse TRPA1 variants (10 µM AITC relative to 100 µM AITC), ( b ) zebrafish TRPA1 variants (100 µM AITC relative to 1 mM AITC), ( c ) human TRPV1 variants (1 nM Capsaicin relative to 1 µM capsaicin), and ( d ) rat TRPM8 variants (10 µM Menthol relative to 200 µM Menthol). Colors indicate ( a ) WT (black) or WT with R922* (orange) mouse TRPA1, ( b ) WT (black) or WT with Q914* (light orange) zebrafish TRPA1, ( c ) WT (black) or WT with F656* (grey) human TRPV1, and ( d ) WT (black) or WT with I955* (green) rat TRPM8. Data represent mean ± SEM. * p < 0.05, n.s. not significant. n = 4 ( a , b ) or 3 ( c , d ) independent experiments, n ≥ 90 cells per transfection condition per experiment, two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: For calcium imaging experiments, untagged human TRPA1 in a combined mammalian/oocyte expression vector pMO (modified from pcDNA3 - obtained from David Julius) or 3×FLAG-tagged TRPA1, TRPV1, or TRPM8 in p3×FLAG-eYFP-CMV-7.1 (Addgene #34582) vector were used.

Techniques: Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Molecular mechanism of hyperactivation conferred by a truncation of TRPA1

doi: 10.1038/s41467-023-38542-1

Figure Lengend Snippet: a R919* TRPA1 patients are heterozygotes and carry WT and R919* TRPA1 copies. WT and R919* hTRPA1 protein subunits can assemble into separate homomeric complexes that are active and inactive, respectively. WT and R919* hTRPA1 subunits may also co-assemble into hyperactive hetero-tetrameric channels of four possible subunit stoichiometries and configurations. We propose these heteromeric channels confer gain-of-function. b In WT TRPA1 channels, electrophile agonist-evoked gating involves rearrangements in the cytoplasmic domains including conformational flipping of the activation loop, contraction of the coiled coil and adjacent ankyrin repeat domain (ARD), and a sliding rotation of the TRP domain towards the central channel axis (light green arrows). These conformational changes occur with concerted dilation of the upper and lower gates in the S6 transmembrane helix and selectivity filter, which are coupled through straightening of the S5 transmembrane helix (dark green arrows). Loss of the cytoplasmic C-terminus and S6 transmembrane helix in the R919* mutant contribute to conferred hyperactivity in WT-R919* heteromer channels to different degrees (indicated by gradations of pink). Electrophile modification (orange star) of reactive cysteines (yellow circle) in R919* subunits may communicate channel activation to the pore through associated WT subunits via contraction of the ARD and coiled coil domains (purple arrow 1) that could propagate up to the allosteric nexus (purple arrow 2). Two subunits are shown for clarity.

Article Snippet: For calcium imaging experiments, untagged human TRPA1 in a combined mammalian/oocyte expression vector pMO (modified from pcDNA3 - obtained from David Julius) or 3×FLAG-tagged TRPA1, TRPV1, or TRPM8 in p3×FLAG-eYFP-CMV-7.1 (Addgene #34582) vector were used.

Techniques: Activation Assay, Mutagenesis, Modification