Fig. 3: NaK2K SF conformational changes in cellular membranes.

A Chemical structure of ANAP inserted using the amber codon at positions V45 (TM1), A53 (PH) and G76 (TM2) in NaK and NaK2K (PBD 3E8H and 3OUF, respectively, with C-terminal FCYENE sequence motif expressed in CosM6 cells (left) and representative (for at least n = 3 vesicles in each case) images of negative control giant plasma membrane vesicles (GPMVs) where the pANAP vector was not added, and positive GPMVs expressing ANAP-incorporated NaK (right). Pseudocolors for ANAP were used. B Representative emission spectra (red, 150 mM NaCl bath solution; blue, solution containing 150 mM KCl) of ANAP-incorporated NaK and NaK2K mutants. C Summary of shifts in ANAP emission peak at different incorporation sites (for each ANAP-incorporation site, n patches are indicated in each condition) in 150 mM Na⁺ or K⁺. Error bars indicate s.e.m. *p < 0.05 vs. Na⁺ and **p < 0.01 vs. Na⁺. Comparisons between Na and K analyzed by unpaired t-test. K⁺ compared with Na⁺ ANAP emission peaks (NaK2K V45ANAP p = 0.0492; NaK2K A53ANAP p = 0.3589; NaK2K G76ANAP p = 0.0081; NaK V45ANAP p = 0.0558; NaK A53ANAP p = 0.7479; NaK G76ANAP p = 0.1863). Source data are provided as a Source Data file.