Fig. 3: Alternative tmFRET pairs yielded similar intermediate distances in the hERG-R531Q channel.
From: Voltage sensor conformations induced by LQTS-associated mutations in hERG potassium channels
a Structural cartoon illustrating the four different tmFRET pairs, L523TAG as the donor and A430TAA, T436TAA, G439TAA, P440TAA as acceptors. b Representative phasor analysis showing the different FRET efficiencies comparing the L523TAG/T436TAA and L523TAG/P440TAA pairs. Cells are imaged in the 120 mM KCl depolarizing condition. c Summary of donor–acceptor distance estimates calculated from measured tmFRET of the four pairs in the depolarizing (high KCl) condition, n = 5 cells for A430, n = 3 cells for T436, n = 4 cells for G439, and n = 5 cells for A440. Data are presented as mean ± s.e.m. One-way ANOVA (no adjustment) showed statistical significance between all pair-wise comparisons, particularly p = 4e-3 (A430 vs T436), p = 5e-4 (A430 vs G439), p = 4e-3 (G439 vs P440). d Representative phasor-plot analysis of the L523/P440 tmFRET from individual cells for hERGtmFRET (control) and hERGtmFRET-R531Q channels under various conditions. Summary data showing FRET efficiencies of hERGtmFRET-L523/P440 pair (e), estimated donor–acceptor distances (f), and the fraction of the high FRET state (g). Data shown are presented as mean ± s.e.m. For FRET efficiencies and estimated distances of R531Q, p = 0.025 (NMDG vs dPBS), p = 1.3e-9 (NMDG vs KCl), and p = 2.1e-8 (dPBS vs KCl), one-way ANOVA (no adjustment). For the percentage of FRET state of hERGtmFRET-L523/P440, p = 0.024 (NMDG vs dPBS), p = 7.8e-6 (NMDG vs KCl), and p = 1e-4 (dPBS vs KCl), one-way ANOVA (no adjustment). Sample sizes: hERGtmFRET: NMDG (n = 3 cells), dPBS (n = 4 cells), KCl (n = 5 cells); hERGtmFRET-R531Q: NMDG (n = 6), dPBS (n = 6), KCl (n = 5). Source data are provided as a Source Data file.
