Fig. 2
Conformational changes near the selectivity filter revealed by ANAP fluorescence. a Representative images of negative control cells (for which the pANAP vector was not added) and positive cells expressing ANAP-incorporated TRPV1. YFP (yellow fluorescent protein) fluorescence was readily detected in positive cells but not in negative controls. Negative control cells were imaged with longer exposure time to ensure that no genuine fluorescent signal was missed. Pseudo colors for ANAP and YFP were used. Scale bar: 10 μm. b Representative emission spectra (black, bath solution; red, solution containing 10 µM capsaicin) of ANAP incorporated at different sites. For K240 site, the emission peak around 512 nm likely represented YFP emission due to FRET with ANAP11. c ANAP emission peak values in the absence or presence of capsaicin. Data points were shown as circles. Two-sided t-test: **, p < 0.01; ***, p < 0.001; N.S., not significant; n = 5–7. d Correlation between shift in ANAP emission peak (y axis) and changes in SASA measured from cryo-EM structures (x axis). Residues in the first and third quadrants (shaded in green) exhibited matched shift in emission and change in SASA, whereas T651 in the second quadrant (shaded in red) showed a discrepancy between these two measurements; n = 5–7. All statistical data are given as mean ± s.e.m
