Fig. 1: Calmodulin (CaM) C-lobe binding regulates TRPA1 activity.

a Cartoon schematic of a full-length hTRPA1 monomeric subunit with relevant structural features, a previously identified Calmodulin binding domain (CaMBD, pink), and the CaM binding element discovered here (DCTCaMBE, yellow) denoted. Dashes and transparencies indicate structurally unresolved regions. Arrows indicate truncations used in this study. b Ribbon diagram of hTRPA1 atomic model for residues S448-T1078 (PDB: 6V9W). Each subunit colored differently for clarity. Features denoted as in (a). c WT hTRPA1 interacts with CaM in a Ca²⁺-dependent manner. Immunoblotting analysis of 3xFLAG-WT hTRPA1 after CaM-agarose pulldown at the indicated Ca²⁺ concentrations from lysates of HEK293T cells transfected with 3xFLAG-WT hTRPA1 or empty vector (mock). Tubulin from whole cell lysates (10%, inputs) was the loading control. d Quantification of CaM-agarose binding assays as in (c) at the indicated Ca²⁺ concentrations relative to the maximum enrichment within each replicate. n = 11. e Ratiometric Ca²⁺ imaging of HEK293T cells transfected with empty vector (mock) or WT hTRPA1 and empty vector (mock), WT CaM, or CaM₁₂₃₄. Cells were stimulated with 100 µM AITC. Scale bars indicate 50 µm. f Quantification of Fura-2 ratio data as from (e). n = 3 independent replicates. g Immunoblotting analysis of 3xFLAG-WT hTRPA1, WT CaM or CaM₁₂₃₄ protein expression in biotin-labeled plasma membranes from transfected cells. CaM was  the negative control for plasma membrane localization. h Quantification of the plasma membrane localization of WT hTRPA1 relative to Tubulin inputs from data as in (g). n = 4, two-tailed Student’s t-test. i Co-expression with a functional CaM C-lobe suppresses WT TRPA1 activity. Ratiometric Ca²⁺ imaging of HEK293T cells transfected with WT hTRPA1 and WT or the indicated CaM mutants. Cells were stimulated with 100 µM AITC. Scale bars indicate 50 µm. j Quantification of 100 µM AITC-evoked change in Fura-2 ratio of data from (i). n = 3 (WT CaM, CaM₁₂, CaM₃₄, and CaM₁₂₃₄) or 4 (CaM C-lobe and CaM₃₄ C-lobe) independent replicates. d, f, h, j Data represent mean ± SEM. P-values indicated in figure panels. d, f, j One-way ANOVA with Tukey’s post hoc analysis.