Fig. 9: Activation-induced affinity increase in HCN2 and ∆DE measured by confocal patch-clamp fluorometry (cPCF).

a Representative confocal images of pipette tips carrying excised macropatches from X. laevis oocytes. Either full-length mHCN2 (left) or ∆DE (right) channels were expressed. The green fluorescence signal of the patch is caused by binding of 0.25 µM f1cAMP to the binding sites of the functional channels. The red signal in the background is caused by the reference dye Dy647 (5 µM), used to subtract the background intensity of unbound f1cAMP. The scale bar is 10 μm. The microscope settings were similar throughout all wildtype and ∆DE recordings. For the sake of a better visibility of the ΔDE patch in exported images, the brightness of the green channel was increased during image processing, letting the original data unchanged. b Simultaneously measured current (black) and fluorescence traces (green) for full-length mHCN2 (left) and ∆DE (right). The voltage protocol is shown above. Fluorescence intensities at −30 mV were normalized to 1.