DHA activates rat TRPA1 in transfected HEK293 cells.
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(A) Representative I–V relationship for DHA (100 µM) and AITC (1 mM) in a voltage-clamped TRPA1-expressing HEK293 cell. (B&C) N-acetyl-L-cysteine (NAC, 15 mM) prevents the activation of TRPA1 current by AITC (1 mM) but not DHA (100 µM). (D) Normalized EPA-evoked currents (% of 1 mM AITC) in control buffer (n = 4), NAC (15 mM, n = 4), or Ascorbic Acid (15 mM, n = 4). (E) DHA activates TRPA1 currents in a dose- and voltage-dependent manner (n = 3–5; holding potential −60 mV or +120 mV). The smooth lines show the best-fits to a Hill equation yielding an Emax of 49±6%, EC50 of 41±5 µM and Hill Coefficient of 3.8±1.3 for −60 mV, and Emax of 100%, EC50 of 13.2±1.8 µM and Hill Coefficient of 1.0±0.1 for +120 mV. (F) Normalized increase in intracellular [Ca2+] evoked by AITC in TRPA1-expressing HEK293 cells with or without pre-treatment with DHA (100 µM, 10 min, n = 40–50). DHA was washed out for 2 min prior to AITC challenge. (G) Representative currents evoked by repeated application of DHA (100 µM) and AITC (500 µM) in a voltage-clamped TRPA1-expressing HEK293 cell.