Figure 1. A mathematical model using experimentally derived binding information describes the concentration–response curve for the effect of cAMP on the HCN2 channel with varying numbers of functional cAMP binding sites. (A) A diagram of the model used for the facilitation of channel opening by cAMP binding to the HCN2 channel. The colored parentheses on the right show the part of the model used to simulate the concentration–response curves for one, two, three, and four cAMP binding sites, which are shown in D. (B) Plots of heat produced upon progressive injections of cAMP to 200 μM of the wild-type HCN2 C-terminus, measured by ITC. The inflections in the top plot arise from injections of cAMP, where each inverted peak shows the heat difference between the sample and reference compartment. The peaks decrease in magnitude as binding sites become saturated. The lower plot shows values determined by integration of the area under the peaks from the upper plot versus the ratio of injected ligand to protein. The solid line through the values represents a two-site independent binding site model, which yielded values for affinity and energetics (ΔG, ΔH, and ΔS). (C) Bar graphs of high- and low-affinity binding values that arise from the fitting of the heat values with a two-site independent binding model for the binding of cAMP to the wild-type HCN2 C-terminus. Values represent means ± SEM. Each mean was determined from independent ITC binding experiments. Values for binding affinity are also given in Table 1. (D) Concentration–response data and simulated curves for the shift in gating produced by cAMP. The data points represent data from for the shifts produced by cAMP on a channel with one functional binding site (black squares), two functional binding sites (red squares), three binding sites (blue squares), and four binding sites (purple squares). Tetrameric HCN2 channels with one and three functional sites were formed by tandem tetramers, whereas channels with two and four binding sites were formed with tandem dimers; a highly conserved arginine residue in the PBC in the CNBD was substituted with glutamate residue to prevent binding. The data points shown are reproduced from Ulens and Siegelbaum (2003). The solid lines are theoretical curves produced by the mathematical model for channels containing the corresponding number of binding sites (shown in A).
Figure 1.

A mathematical model using experimentally derived binding information describes the concentration–response curve for the effect of cAMP on the HCN2 channel with varying numbers of functional cAMP binding sites. (A) A diagram of the model used for the facilitation of channel opening by cAMP binding to the HCN2 channel. The colored parentheses on the right show the part of the model used to simulate the concentration–response curves for one, two, three, and four cAMP binding sites, which are shown in D. (B) Plots of heat produced upon progressive injections of cAMP to 200 μM of the wild-type HCN2 C-terminus, measured by ITC. The inflections in the top plot arise from injections of cAMP, where each inverted peak shows the heat difference between the sample and reference compartment. The peaks decrease in magnitude as binding sites become saturated. The lower plot shows values determined by integration of the area under the peaks from the upper plot versus the ratio of injected ligand to protein. The solid line through the values represents a two-site independent binding site model, which yielded values for affinity and energetics (ΔG, ΔH, and ΔS). (C) Bar graphs of high- and low-affinity binding values that arise from the fitting of the heat values with a two-site independent binding model for the binding of cAMP to the wild-type HCN2 C-terminus. Values represent means ± SEM. Each mean was determined from independent ITC binding experiments. Values for binding affinity are also given in Table 1. (D) Concentration–response data and simulated curves for the shift in gating produced by cAMP. The data points represent data from for the shifts produced by cAMP on a channel with one functional binding site (black squares), two functional binding sites (red squares), three binding sites (blue squares), and four binding sites (purple squares). Tetrameric HCN2 channels with one and three functional sites were formed by tandem tetramers, whereas channels with two and four binding sites were formed with tandem dimers; a highly conserved arginine residue in the PBC in the CNBD was substituted with glutamate residue to prevent binding. The data points shown are reproduced from Ulens and Siegelbaum (2003). The solid lines are theoretical curves produced by the mathematical model for channels containing the corresponding number of binding sites (shown in A).

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