Fig. 2: ATP induces conformational changes in IP-HD as revealed by fluorescent unnatural amino acids (flUAA) incorporation and voltage-clamp fluorometry (VCF).

a Chemical structure of ANAP and strategy for incorporating L-ANAP into the P2X3 receptor. Plasmids containing ANAP tRNA synthetase and the corresponding tRNAs, as well as plasmids carrying the genes encoding the receptor, were transiently transfected into HEK293T cells, and then cultured in ANAP-containing medium for at least 24 h to ensure that ANAP was integrated into the proper location of the receptor. b Representative images of negative control cells (no pANAP vector added) and positive cells (ANAP-integrated P2X3). Fluorescence of yellow fluorescent protein (YFP) was detected only when cells were transfected with both pANAP and P2X3 receptor plasmids. Pseudo-color was used in ANAP and YFP (scale bar, 20 μm). The experiment was repeated thrice with similar results. c Location of selected sites in the IP-HD of P2X3 and the negative control (upper panel), their responses to saturating ATP (lower panel), and a summary of the shift of the peak of ANAP emission wavelengths after ATP administration (middle), Each circle represents an independent cell; n = 3 (T134ANAP/Y37A, I149ANAP, and E156ANAP), 4 (E111ANAP and Y114ANAP), 5 (V143ANAP, G131ANAP, E156ANAP/Y37A, and R281ANAP), 6 (WT), 10 (L297ANAP) or 11 (E111ANAP/Y37A). Data are expressed as mean ± SEM. P value was calculated from a paired, two-tailed t test. d Representative emission spectra of cells that expressed WT P2X3 and some of its ANAP-incorporated mutants (black, bath solution; red, solution containing 10 μM ATP). Source data are provided as a Source Data file.