Fig. 4: Amber/ochre dual stop-codon suppression enables fluorescence labeling for FLIM-FRET in living cells.
From: Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization
a Strategies for incorporating and labeling two different amino acids orthogonally using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), along with the IEDDA. b Linear correlation of the labeled AF488 versus AF555 fluorescence using the hHCN4 L374TAG/N291TAA construct. c Cartoon showing the design of the FRET pairs at the VSD of HCN channels. FRET sites were chosen for the HCN4 channel. d Phasor-plot presentation for the measured lifetimes of both the FRET donor AF-488 labeled to the HCN4-I382 site and the FRET acceptor AF-555 labeled to the HCN4-N291TAA site. e and f FRET was detected after CuAAC for the construct containing the ochre site; FRET in the hHCN4-L374TAG/N291TAA pair was greater than that in the hHCN4-I382TAG/N291TAA pair. g FRET was not detected after CuAAC for the construct without the ochre site, hHCN4-L374TAG-mCherry. h Effects of the 5 mM β-cyclodextrin on the measured FRET using the phasor FLIM approach in the specified FRET pairs in the VSD of hHCN1 and hHCN4 channels. Data shown are mean ± s.e.m. For the HCN1 L254/T171: n = 6 cells for both conditions; for the HCN1 I262/T171: n = 5 cells for both conditions; for the HCN4 L374/N291: n = 11 cells for the control and n = 7 cells after β-CD; for the HCN4 I382/N291: n = 5 cells for the control and n = 6 cells after β-CD. One-way ANOVA, followed by Tukey’s post hoc tests and pairwise comparisons (no adjustment), was used as the statistical analysis method; the highlighted statistical significance is p = 7.1E−05 for HCN4 L374/N291 and p = 0.035 for HCN4 I382/N291; *p < 0.05, **p < 0.01.
