Figure 5. The greater potency of cAMP versus cGMP is due to greater binding affinity and stronger effects on channel opening. (A) A ribbon diagram showing an overlay of the cAMP-bound and cGMP-bound structures of the wild-type HCN2 C-terminus. Note that cAMP and cGMP are bound in anti and syn configurations, respectively. Residues distal to R635 are not resolved in the cGMP-bound wild-type structure. PDB accession no. 1Q50 (cAMP bound) and PDB accession no. 1QE3 (cGMP bound). (B) Plots of heat produced upon progressive injections of cGMP to 200 μM HCN2 C-terminus, measured by ITC. The inflections in the top plot arise from injections of cGMP, 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 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 and cGMP to the wild-type HCN2 C-terminus. Values for binding affinity are given in Table 1. (D) Bar graphs of values for thermodynamics that arise from the fitting of the heat values with a two-site independent binding model for the binding of cAMP and cGMP to the wild-type HCN2 C-terminus. Values in C and D represent means ± SEM. Each mean was determined from independent ITC binding experiments. (E) Concentration–response data and simulated curves for the shift in gating produced by cAMP and cGMP. The solid red line is produced by the mathematical model using the experimental binding affinities for cAMP obtained by ITC for the wild-type protein and fitted to the concentration–response data for cAMP facilitation of the wild-type HCN2 channel. The solid blue line is produced by the mathematical model using the experimental binding affinities obtained for cGMP binding to wild-type protein by ITC but using the gating parameters that were obtained from the fit of the model to the cAMP concentration–response data. Note that this blue line does not fit the cGMP concentration–response data (purple squares) for the wild-type HCN2 channel. For the model to fit the cGMP concentration–response data (purple line), changes in the transitions between the closed and open pore were necessary in addition to using the binding affinities obtained by ITC from cGMP binding to the C-terminus protein.
Figure 5.

The greater potency of cAMP versus cGMP is due to greater binding affinity and stronger effects on channel opening. (A) A ribbon diagram showing an overlay of the cAMP-bound and cGMP-bound structures of the wild-type HCN2 C-terminus. Note that cAMP and cGMP are bound in anti and syn configurations, respectively. Residues distal to R635 are not resolved in the cGMP-bound wild-type structure. PDB accession no. 1Q50 (cAMP bound) and PDB accession no. 1QE3 (cGMP bound). (B) Plots of heat produced upon progressive injections of cGMP to 200 μM HCN2 C-terminus, measured by ITC. The inflections in the top plot arise from injections of cGMP, 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 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 and cGMP to the wild-type HCN2 C-terminus. Values for binding affinity are given in Table 1. (D) Bar graphs of values for thermodynamics that arise from the fitting of the heat values with a two-site independent binding model for the binding of cAMP and cGMP to the wild-type HCN2 C-terminus. Values in C and D represent means ± SEM. Each mean was determined from independent ITC binding experiments. (E) Concentration–response data and simulated curves for the shift in gating produced by cAMP and cGMP. The solid red line is produced by the mathematical model using the experimental binding affinities for cAMP obtained by ITC for the wild-type protein and fitted to the concentration–response data for cAMP facilitation of the wild-type HCN2 channel. The solid blue line is produced by the mathematical model using the experimental binding affinities obtained for cGMP binding to wild-type protein by ITC but using the gating parameters that were obtained from the fit of the model to the cAMP concentration–response data. Note that this blue line does not fit the cGMP concentration–response data (purple squares) for the wild-type HCN2 channel. For the model to fit the cGMP concentration–response data (purple line), changes in the transitions between the closed and open pore were necessary in addition to using the binding affinities obtained by ITC from cGMP binding to the C-terminus protein.

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