Fig. 3: Probing the change of the lifetime of L-Anap incorporated to the key serine residue in the voltage-sensing S4 helix of HCN channels.
From: Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization
a Sequence alignment of the S4 helix voltage-sensor region of human HCN1, HCN2, and HCN4 channels. The numbers correspond to specific residues of HCN4. b Phasor FLIM imaging of L-Anap incorporated in the middle of the S4 helix of the HCN4 channel (S392 site) expressed in tsA201 cells versus the free L-Anap in PBS buffer, and in ethanol. Application of the PBS buffer with 120 mM KCl decreased the lifetime. c Effects of the 120 mM KCl and/or 5 mM β-cyclodextrin on the lifetime of L-Anap for hHCN4-S392Anap (n = 5 cells for the control, n = 7 cells for the 120 mM KCl condition, n = 5 cells for the condition after β-CD, and n = 6 cells for the condition of 120 mM KCl after β-CD), as well as the effect of the 120 mM KCl on the lifetime of L-Anap for hHCN1-S272Anap (n = 6 cells for the control and n = 5 cells for the 120 mM KCl condition). Data shown are mean ± s.e.m. One-way ANOVA, followed by Tukey’s post hoc tests and pairwise comparisons (no adjustment), was used for these six groups as the statistical method. Key comparisons are highlighted as p = 3.3E−04 between the control and the 120 mM KCl for hHCN4-S392Anap, p = 0.56 between groups with and without 120 mM KCl, and in the presence of β-CD for hHCN4-S392Anap, p = 0.37 between the control and the 120 mM KCl for hHCN1-S272Anap; *p < 0.05, **p < 0.01.
