An environmental pollutant, 9,10‐phenanthrenequinone, activates human TRPA1 via critical cysteines 621 and 665

Transient receptor potential ankyrin 1 ( TRPA 1) is activated by noxious cold, mechanical stimulation, and irritant chemicals. In our recent study, 9, 10‐phenanthrenequinone (9,10‐ PQ ) is the most potent irritant for activation of NRF 2 among 1395 cigarette smoke components and it may be, therefore, important to find its additional targets. Here, we show that 9,10‐ PQ functions as an activator of TRPA 1 in human embryonic kidney ( HEK ) cells expressing human wild‐type TRPA 1 ( HEK ‐ wTRPA 1) and human alveolar A549 (A549) cells. Application of 9,10‐ PQ at 0.1–10  μ mol/L induced a concentration‐dependent Ca 2+ response as well as inward currents at −50  mV in HEK ‐ wTRPA 1 cells. The current response was blocked by TRPA 1 antagonists, HC ‐030031 ( HC ) and A‐967079. To test whether 9,10‐ PQ affects the cysteine residues of TRPA 1, we expressed mutant TRPA 1 channels in HEK cells ( HEK ‐mu TRPA 1) in which six different cysteine residues were replaced with serine. Among them, a mutation of cysteine 621 (C621S) abolished the 9,10‐ PQ ‐induced Ca 2+ and current responses. The channel activity induced by 9,10‐ PQ was also abolished in excised inside‐out patches isolated from HEK ‐mu TRPA 1 cells with the C621S substitution. Although a mutation of cysteine 665 (C665S) reduced the 9,10‐ PQ ‐induced response, channel sensitization by pretreatment with Cu 2+ plus 1,10‐phenanthroline and by internal dialysis of 3  μ mol/L Ca 2+ restored the response. However, a double mutant with C621S and C665S substitutions had little response to 9,10‐ PQ , even when sensitized by Ca 2+ dialysis. In A549 cells, 9,10‐ PQ induced an HC ‐sensitive Ca 2+ response. Our findings demonstrate that 9,10‐ PQ activation of human TRA1 is dependent on cysteine residues 621 and 665.