Figure 5. Agonist‐induced sensitisation of TRPA1 is independent of ion flux through the channel and is long lasting .

A, sensitisation of TRPA1 in HEK293t cells increases with agonist exposure even when the channel is blocked. Responses 5–8 to carvacrol (100 μm; 15 s) were blocked by the specific TRPA1 antagonist HC030031 (50 μm; 155 s) (see inset) and the measured inward currents (filled squares) are compared to control recordings without antagonist (open triangles). Responses 9–12 were comparable in amplitude to recordings where no antagonist had been applied (P ≥ 0.458; n = 7 for HC030031‐treated, n = 21 for control). B, TRPA1 is significantly sensitised by agonist exposure during channel block (P < 0.001; n = 7). C, TRPA1 sensitisation in HEK293t cells is long lasting. Gap periods of 30 s, 120 s or 240 s (dashed lines) were left between applications of carvacrol (100 μm; 120 s; continuous line). Sensitisation does not recover even with 240 s rest period. Sequence of gaps was changed at random between each cell. Arrows indicate time points for calculating the recovery ratio displayed in D. Inset: prolonged stimulation with carvacrol (100 μm; 150 s) induces two phases of current increase (average ± SEM trace; n = 16). D, the recovery ratio was calculated by measuring the early current at the end of the first steep increase (I _early) of each response and comparing it to the peak current (I _peak) of the previous response. None of the calculated recovery ratios were significantly different from 1 (P ≥ 0.24; n = 7–8).