Figure 4. H₂O₂ induces TRPA1-dependent influx of Ca²⁺ and ionic currents in mustard oil–responsive sensory neurons.

(A) Responses of cultured DRG neurons from littermate Trpa1^+/+ and Trpa1^–/– mice to 5 mM H₂O₂, followed by 3 μM capsaicin, as measured by Fura-2 imaging. Trpa1^–/– neurons showed no [Ca²⁺]_i increase after H₂O₂ exposure, but were activated by capsaicin. Pseudocolors denote 0–3 μM [Ca²⁺]_i. (B) Activation of Ca²⁺ influx by H₂O₂ into DRG neurons plotted against time. Average [Ca²⁺]_i concentration of neurons activated by application of H₂O₂ followed by mustard oil, capsaicin, and 65 mM KCl. Thick and thin lines denote mean and ± SEM, respectively. Neurons (n = 189 [Trpa1^+/+]; 146 [Trpa1^–/–]) were analyzed at ×10 magnification. (C) Activation of DRG neurons (n = 161) by 5 mM H₂O₂, NaOCl (24 ppm), 100 μM mustard oil, 5 μM capsaicin, and 65 mM KCl. Neurons were considered activated when [Ca²⁺]_i exceeded 500 nM. Values denote activated KCl-sensitive cells. (D) Activation of DRG neurons (n = 130) by NaOCl (24 ppm), 5 mM H₂O₂, 100 μM mustard oil, 5 μM capsaicin, and 65 mM KCl. (E) Kinetics of H₂O₂-activated cationic currents and ruthenium red–induced block in a cultured murine sensory neuron. H₂O₂ was superfused after 50-s initiation of whole-cell configuration, after which ruthenium red was coapplied at 210 s. Currents were measured using a ±80 mV voltage ramp protocol over 100 ms at 0.5-Hz intervals (0 mV holding potential throughout). Intracellular Cs-based solution contained 10 mM EGTA. (F) Representative current-voltage relationships of currents recorded from a DRG neuron before application of H₂O₂ (black), during maximal activation by H₂O₂ (green), and after application of 20 μM ruthenium red (red). Currents were measured as in C. Error bars represent SEM.