Fig. 6: Phasor FLIM-FRET measurements of hHCN2 channel localization and voltage sensor rearrangements.

a, b Representative phasor plots showing FRET between L10-CFP and hHCN2-YFP expressed in small DRG neurons before and after 10 nM paclitaxel treatment. c Summary data of the effects of the β-CD and paclitaxel on the FRET between L10-CFP and hHCN2-YFP. Data shown are mean ± s.e.m., n = 7 for control and β-CD conditions, n = 8 for paclitaxel conditions, **p = 1e-6 for β-CD and **p = 4e-6 for paclitaxel compared with the control. One-way ANOVA (no adjustment) was used. d Cartoon showing the design of the FRET sites in the VSD of human HCN2 channels and the representative phasor plot showing FRET between donor AF-488 labeled to the HCN2-L323TAG site and the FRET acceptor AF-555 labeled to the HCN2-T240TAA site. e Representative phasor plot of the same FRET pair as in panel (d) following acute 5 mM β-cyclodextrin treatment. f Summary data of the effect of the β-CD on the FRET efficiency of hHCN2-L323TAG/T240TAA channels. Data shown are mean ± s.e.m., n = 5 cells for the control and n = 4 cells after β-CD, **p = 0.002 using two-sided student’s t-test. g Representative phasor plot of the same FRET pair as in panels (d) and (e), following acute 5 mM β-cyclodextrin treatment with 120 mM KCl in the bath. h Summary data of the effect of the β-CD on the FRET efficiency as shown in panel (g). Data shown are mean ± s.e.m., n = 7 cells for the control and n = 6 cells with β-CD, p = 0.054 using two-sided student’s t-test.