Fig 1: TRPA1 single channels were inhibited by PHL-B. (A) Basal single-channel activity (black traces) in the cell-attached configuration at −60 and +60 mV was low under vehicle conditions. After the addition of 100 μM AITC to the bath solution (orange traces), channel activity was clearly elevated. Exposure of the cells to 5 μM PHL-B in the presence of AITC (green traces) substantially reduced the open probability of TRPA1 channels. (B) Histogram plots of single-channel counts vs current amplitude, where the inhibitory effect of PHL-B can be clearly visualized.
Fig 2: Identification of PHL-B as an antagonist of TRPA1 channels. (A) Relative fluorescence intensity (F/F0) of GCaMP6s induced by 100 μM AITC after treatment with Vehicle (0.1% DMSO), A967079 (1 μM), and 27 natural compounds at a concentration of 10 μM. (B) A scatter plot showing the percentage inhibition for all the compounds tested. A967079 (red circle) and Vehicle (blue circle) were used as the positive and negative controls, respectively. The cutoff value for a hit molecule was set at 50%, and only PHL-B (green circle) was identified. (C) Normalized fluorescence (F/F0) was plotted against drug concentration and fitted with the Hill equation, yielding an IC50 of 1.35 ± 0.3 μM. All data are expressed as means ± SEM, N = 3–8 independent experiments.
Fig 3: A library of natural products was used for the primary screening assay on TRPA1 channels.
Fig 4: PHL-B inhibited TRPA1 channels in whole-cell recordings. (A) Representative current recordings from HEK293T cells transiently expressing TRPA1 in response to slow voltage ramps from −100 mV to +50 mV. Cells were stimulated with 100 μM AITC and then exposed to AITC + 5 μM PHL-B before washout. A967079 in the presence of AITC was applied at the end of the experiment. (B) Comparison of mean current density (pA/pF) recorded at −100 mV and +50 mV under the same conditions. (C) Time course of recovery of PHL-B inhibition, estimated at a single potential of −60 mV applied in 5-s intervals. Dashed lines represent single exponential fits to the data. Data are expressed as means ± SEM and were analyzed using ordinary one-way ANOVA followed by Dunnett’s post hoc test. N = 4 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to AITC.
Supplier Page from DNASU for TRPA1 (Homo sapiens) in pENTR223.1 (Gateway donor/master vector)