Figure 6. Isoleucine 636 in the C-helix of the HCN2 C-terminus contributes to cAMP selectivity over cGMP. (A) Plots of heat produced by progressive injections of cAMP or cGMP to the 200 μM mutant HCN2 C-terminus. The inflections in the top plot arise from injections of cyclic nucleotide, where the inverted peaks show the heat difference between the sample and reference compartment. The peaks decrease in magnitude as binding sites become saturated. The lower plots show 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). (B) A ribbon diagram showing the CNBD of either wild type (I636, dark teal) or mutant (I636D, light green) bound to cGMP. Note that the aspartate residue forms a contact with the guanine ring of cGMP in the mutant, but not wild-type, structure. Residues distal to R635 (including I636) are not resolved in the cGMP-bound wild-type structure, whereas residues distal to D636 are not resolved in the cGMP-bound mutant structure. PDB accession no. 1Q3E (cGMP bound to wild type) and PDB accession no. 2Q0A (cGMP bound to mutant). The new hydrogen bond made by the aspartate side chain with 2-NH2 of cGMP is shown in magenta. (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, cGMP binding to the I636A mutant, and cAMP and cGMP binding to the I636D mutant. 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, cGMP binding to the I636A mutant, and cAMP and cGMP binding to the I636D mutant. Values in C and D represent means ± SEM. Each mean was determined from independent ITC binding experiments. (E) Concentration–response data and theoretical curves for the shift in gating produced by cAMP or cGMP to the 200-μM wild-type and mutant HCN2 C-terminus. The solid red line in the left and middle panels is the theoretical curve produced by the model that is fitted through the cGMP wild-type concentration–response data (from Fig. 6). The solid red line in the right panel is the theoretical curve produced by the model that is fitted through the cAMP wild-type concentration–response data (from Fig. 2). The solid blue lines are theoretical curves produced by the model using the experimental binding data obtained by ITC for the corresponding HCN2 mutant and using the gating parameters for the effect of cGMP (left and middle panels) or for cAMP (right panel) on the wild-type HCN2 channel. The purple triangles represent the concentration–response data for the effect of cGMP (left and middle panel) or the effect of cAMP (right panel) on the shift in the activation curve of the corresponding full-length mutant HCN2 channel. Note that the blue lines overlie closely with the experimental data (purple triangles), which are shifted to the left of the wild-type concentration–response data for cGMP (left and middle panel) and to the right of the wild-type concentration–response data for cAMP (right panel). The solid purple line represents a fit of the model to the mutant concentration–response data (purple triangles), which was made by adjusting the closed-to-open gating transitions.
Figure 6.

Isoleucine 636 in the C-helix of the HCN2 C-terminus contributes to cAMP selectivity over cGMP. (A) Plots of heat produced by progressive injections of cAMP or cGMP to the 200 μM mutant HCN2 C-terminus. The inflections in the top plot arise from injections of cyclic nucleotide, where the inverted peaks show the heat difference between the sample and reference compartment. The peaks decrease in magnitude as binding sites become saturated. The lower plots show 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). (B) A ribbon diagram showing the CNBD of either wild type (I636, dark teal) or mutant (I636D, light green) bound to cGMP. Note that the aspartate residue forms a contact with the guanine ring of cGMP in the mutant, but not wild-type, structure. Residues distal to R635 (including I636) are not resolved in the cGMP-bound wild-type structure, whereas residues distal to D636 are not resolved in the cGMP-bound mutant structure. PDB accession no. 1Q3E (cGMP bound to wild type) and PDB accession no. 2Q0A (cGMP bound to mutant). The new hydrogen bond made by the aspartate side chain with 2-NH2 of cGMP is shown in magenta. (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, cGMP binding to the I636A mutant, and cAMP and cGMP binding to the I636D mutant. 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, cGMP binding to the I636A mutant, and cAMP and cGMP binding to the I636D mutant. Values in C and D represent means ± SEM. Each mean was determined from independent ITC binding experiments. (E) Concentration–response data and theoretical curves for the shift in gating produced by cAMP or cGMP to the 200-μM wild-type and mutant HCN2 C-terminus. The solid red line in the left and middle panels is the theoretical curve produced by the model that is fitted through the cGMP wild-type concentration–response data (from Fig. 6). The solid red line in the right panel is the theoretical curve produced by the model that is fitted through the cAMP wild-type concentration–response data (from Fig. 2). The solid blue lines are theoretical curves produced by the model using the experimental binding data obtained by ITC for the corresponding HCN2 mutant and using the gating parameters for the effect of cGMP (left and middle panels) or for cAMP (right panel) on the wild-type HCN2 channel. The purple triangles represent the concentration–response data for the effect of cGMP (left and middle panel) or the effect of cAMP (right panel) on the shift in the activation curve of the corresponding full-length mutant HCN2 channel. Note that the blue lines overlie closely with the experimental data (purple triangles), which are shifted to the left of the wild-type concentration–response data for cGMP (left and middle panel) and to the right of the wild-type concentration–response data for cAMP (right panel). The solid purple line represents a fit of the model to the mutant concentration–response data (purple triangles), which was made by adjusting the closed-to-open gating transitions.

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