Fig. 1. Irritant volatile anesthetics activate heterologously expressed transient receptor potential (TRP)-A1 in a dose-dependent manner. (A ) Representative traces of fura-2-fluorescence ratios indicating increases in intracellular calcium concentration [Ca²⁺]ⁱinduced by allylisothiocyanate (AITC, 20 μm) in human embryonic kidney (HEK) cells after tetracycline-induced (tet) TRPA1 expression (TRPA1 + tet). Control traces show cells without tetracycline induction of TRPA1 expression (TRPA1 – tet) and TRPA1 expressing cells in the absence of extracellular calcium. The horizontal line indicates application of AITC. (B ) Typical traces of isoflurane-induced increase in [Ca²⁺]ⁱin TRPA1-expressing HEK cells. Same controls as in (A ). The horizontal line indicates application of isoflurane (300 μm). (C ) Dose–response curve for the changes in intracellular calcium (change in fura-2-fluorescence ratio) induced by isoflurane in TRPA1-expressing HEK cells. N = 3–5 coverslips per data point. The calculated EC⁵⁰is 534 μm (∼1.6 MAC, E⁰= 0.52, E^max= 2.58). (D ) Comparison of the change in [Ca²⁺]ⁱinduced by equianesthetic concentrations of the four most commonly used volatile anesthetics. Bars represent mean ± SEM, numbers in parentheses indicate the number of observations per group. All intergroup comparisons are statistically significant* (one-way ANOVA followed by Student–Newman–Keuls post hoc  test for all intergroup comparisons). (E ) Comparison of the concentration–response relationships for the four most commonly used volatile anesthetics. N = 3–5 coverslips per data point. Parameters calculated for the concentration–response curve of desflurane: EC⁵⁰= 1150 mm, E⁰= 0.38, E^max= 3.45. (F ) Inward current responses induced by AITC (50 μm) and isoflurane (300 μm) in TRPA1-expressing HEK cells. Responses were measured by whole cell patch clamp in a voltage clamp configuration with a holding potential of −60 mV.