Extracellular Cs⁺ blocks the ionic conductance but has no effect on cAMP binding. (A) A schematic model showing the pore region and the CNBD in HCN channels. (B) Voltage protocol (top), timing of image collection (middle), and ionic current traces (bottom). The black line indicates control condition without Cs⁺; the red indicates 2 mM Cs⁺ added to the pipette (extracellular) solution. (C) Brightfield (BF) images of the membrane patch (indicated by arrows) and the corresponding fluorescence images collected before (C) or near the end of the hyperpolarizing voltage step (V). (D) Comparison of current amplitude (top) and fluorescence intensity (bottom) between control (black) and with Cs⁺ (red) in the pipette solution. The difference in the current amplitude between control and Cs⁺ was significant (independent samples t test: t = 3.57, P < 0.01). No significant difference in the fluorescence intensity was found (independent samples t test: t = 0.05, P = 0.96). Error bars indicate standard error.