Fig. 5: Activation-dependent affinities in K464 mutant and mHCN2 wild-type channels.

a Representative confocal images for mHCN2 and K464E. Upper panels show recordings at a non-activating voltage of −30 mV, lower panels at activating voltage of −130 mV. The tip of a patch pipette carrying a membrane patch expressing either mHCN2 or K464E is shown. The green signal is caused by 0.5 µM 8-Cy3B-AHT-cAMP binding to the channels, the red signal staining the background is caused by 5 µM Dy647, a reference dye required for subtracting the background signal of unbound 8-Cy3B-AHT-cAMP. b Time courses of simultaneously measured fluorescence increase (green) and current increase (black) following an activating voltage pulse from −30 to −130 mV. c Concentration-binding relationship for mHCN2 and K464E. The Hill function (Eq. (3)) is approximated to the averaged data (n = 3 to 6) yielding the concentration of half-maximum binding, BC₅₀, and the Hill coefficient, H. Error bars indicate SEM. d Isolated CL-CNBD shown as cartoon. Residues that behave significantly different to apo wild-type HCN2 are shown as spheres (C_α atoms only) and colored according to the residue-wise average ΔRMSF (see color bar on the right; see also Eq. (4); n = 80 independent replicas). Residues colored in blue are significantly more mobile in the apo wild-type channel (p < 0.05; p value by t-test). Residues colored in red are significantly more mobile in the cAMP-bound wild-type channel (top panel) or apo K464E channel (lower panel) (p < 0.05; p value by t-test). cAMP and K464 are shown as sticks.