Abstract
BACKGROUND AND PURPOSE: Chemical 2, 4-dinitrofluorobenzene (DNFB),
commonly called as Sanger’s reagent, is well known as skin sensitizer to
cause dermatitis. However, how the DNFB causes skin inflammation remains
unknown. In this study we aimed at identifying the molecular target that
DNFB acts on. EXPERIMENTAL APPROACH: We used a fluorescent calcium
imaging plate reader as an initial screening assay and patch-clamp
recordings for validation. Molecular docking in combination with
site-directed mutagenesis was carried out to investigate DNFB binding
sites in TRPA1 ion channel. KEY RESULTS: We found the chemical DNFB that
selectively activates TRPA1 channel with EC50 of 2.36 ± 0.26 µM.
Single-channel recording reveals that DNFB increases the channel open
probability and acts on three residues C621, Y658 and E625 critical for
DNFB-mediated TRPA1 activation. CONCLUSION AND IMPLICATIONS: Our
findings not only explain a molecular mechanism underlying the
dermatitis and pruritus caused by chemical DNFB, but also provides a
molecular tool that is 7.5-time more potent than current AITC molecule
and can be used for elucidating TRPA1 channel pharmacology and
pathology.