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	Figure 1. Time course of fcAMP binding and unbinding to HCN2 channels. (A) Ligand binding and current activation in channels preactivated by a voltage pulse from −30 to −130 mV in a representative patch. Application of fcAMP generated a time course of ligand binding and unbinding that consisted of a rapid and a slow component (blue trace, top) and an extra current component (black trace, bottom). The time course for binding (F) was normalized with respect to the steady-state binding at −130 mV and 15 μM fcAMP (Fmax). (B) Respective time course of ligand binding and unbinding for nonactivated channels at −30 mV in a representative patch. In the absence of current activation, fcAMP binding and unbinding are rapid. Same normalization as in (A). (C) Superimposition of normalized averaged time courses of binding and unbinding at −30 mV (n = 5) and −130 mV (n = 12).
	Figure 2. Binding of fcAMP to nonactivated HCN2 channels at five concentrations. (A) Averaged time courses of binding and unbinding. The number of individual traces, n, included in each averaged trace is indicated. Each individual trace was obtained from a different patch. The traces are normalized with respect to the binding at −130 mV and 15 μM fcAMP (F/Fmax). (B) Steady-state concentration-binding relationship at −130 and −30 mV. Fit of the relationships with Eq. 1 (continuous curves) yielded −130 mV, BC50 = 0.61 μM, and H = 1.56 and −30 mV, BC50 = 2.46 μM, and H = 1.28. (C) Fit of the time courses (red curves) of fcAMP binding and unbinding with a single exponential using Eqs. 2a and 2b, respectively.
	Figure 3. Global fit with a C-C model assuming independently operating subunits. (A) Scheme of model 1 (Table 1). L denotes a ligand. (B) Normalized and averaged traces of binding and unbinding at five fcAMP concentrations with superimposed curves (red) as best fit. (C) Fit of steady-state binding (F/Fmax) obtained by the same global fit as in (B). Fmax was determined at −130 mV and 15 μM fcAMP. Parameters: a = 8.58 × 105 M−1 s−1, b = 2.20 s−1, χr2 = 15.86.
	Figure 4. Global fit with a model assuming binding and flipping of independent subunits. (A) Scheme of the model containing a ligand (L) binding step and a subsequent flip of only the same subunit (model 2 in Table 1). (B and C) Analog to Fig. 3. Parameters: a = 1.32 × 106 M−1 s−1, b = 4.00 s−1, c = 3.14 × 10−1 s−1, d = 5.54 × 10−2s−1, and χr2 = 12.63.
	Figure 5. Global fit with a model containing four binding steps and one concerted flip. (A) Scheme of the model containing four ligand (L) binding steps and a concerted flip of all four subunits (model 5 in Table 1). (B and C) Analog to Fig. 3. The parameters are provided by Table 2.
	Figure 6. Microscopic binding affinity for fcAMP in nonactivated and activated channels. The values of the equilibrium association constants KA1–KA4 obtained for model 5 (Table 1) are plotted for nonactivated channels (−30 mV) and activated channels (−130 mV). The values for the activated channels were obtained from a previous study (29). The values for the second and the third binding step at −30 mV were set equal.

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