Fig. 5: Menthol-induced conformational rearrangements revealed by ANAP imaging.

a The selectivity filer of TRPM8 was missing in its cryo-EM structure (PDB ID: 6BPQ). It is expected to locate within the dashed circle in red. b The models of selectivity filter after nine rounds of KIC loop modeling exhibited a funnel-shaped distribution of total energy calculated by Rosetta (REU, Rosetta energy unit). c Distribution of pore radii in TRPM8 with the selectivity filter modeled. The regions in red are too narrow to allow a water molecule to pass. The regions in green or blue are sites allowing single or multiple water molecules and ions to pass, respectively. Pore radii were calculated by the HOLE program. SF selectivity filter. d Chemical structure of ANAP and representative images of negative control cells where the pANAP vector was not added, and positive cells expressing ANAP-incorporated TRPM8. Pseudocolors for ANAP were used. Scale bar: 10 μm. e Representative emission spectra (black, bath solution; red, solution containing 0.5 mM menthol) of ANAP-incorporated TRPM8 mutants. f Summary of shifts in ANAP emission peak at different incorporation sites (for each ANAP-incorporation site, n = 4–6). Right and left shifts larger than 2 nm were colored in red and blue, respectively. g Shifts in ANAP emission peak mapped onto the TRPM8 model with a worm representation, where the worm radius was proportional to the amplitude of shifts. Right and left shifts were colored in red and blue, respectively. Color scale is in the unit of nanometer. All statistical data are given as mean ± s.e.m. Source data are provided as a Source Data file.