Fig. 1: The R919* mutant is a nonfunctional TRPA1 natural variant.
From: Molecular mechanism of hyperactivation conferred by a truncation of TRPA1
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. 14. e, g, h Source data are provided as a Source Data file.
