Fig. 2.
sTRP1 channel acts as a natural repellent receptor. (A) Amino acid sequence alignments of sTRP1 and mouse TRPC1-7 within pore region, TM domain 6 (TM6), and TRP helix. Residues that are identical or similar among the sequences are shaded in blue and brown, respectively. (B) Distribution of sTRP1-mCherry protein (red) in transfected HEK 293T cells assessed by confocal microscopy. Na+/K+-ATPase (green) was labeled by antibodies to indicate where the cell membrane is, and nucleus (blue) was stained by DAPI. (C) Representative whole-cell recordings from sTRP1-expressing HEK 293T cells evoked by repeated applications of citronellol (0.1 mM) or citronellal (0.5 mM). Vh = −60 mV. (D) Statistical plot of relative current of peak response over repetitive stimulation. Data were normalized to the last pulse response (n = 11 for citronellol, n = 7 for citronellal). (E) Representative whole-cell recordings of sTRP1-expressing HEK 293T cells that responded to variable repellents after full sensitization. (F) Summary plot of relative response. Currents were normalized to the current elicited by 2 mM citronellal (n = 9). (G) Concentration-response curves of citronellal, citronellol, and camphor activation of sTRP1 channels. Solid lines indicate fits to the Hill equation with EC50 = 0.56 ± 0.01 mM, nH = 3.3 ± 0.5 for citronellal (n = 9); EC50 = 0.30 ± 0.01 mM, nH = 3.6 ± 0.3 for citronellol (n = 10), and 3.3 ± 0.2 mM, nH = 2.4 ± 0.4 for camphor (n = 6). (H) Dose-response curves of inhibitory effects of RR and 2-APB on sTRP1 in the presence of 2 mM citronellal after sensitization. The solid lines correspond to fits by the Hill equation with IC50 = 3.12 ± 0.29 μM, nH = 1.6 ± 0.2 for RR (n = 6); and IC50 = 48.32 ± 5.58 μM, nH = 1.3 ± 0.1 for 2-APB (n = 8). The dotted line indicates zero current level. Error bars represent SEM.