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.