Figure 3. Residues of the C-helix contribute to ligand binding affinity and variably couple binding to channel opening. (A) A ribbon diagram showing the position of R635, I636, and K638, which are located in the C-helix at the distal region of the CNBD. PDB accession no. 1Q50. (B) Plots of heat produced upon progressive injections of cAMP to 200 μM of R635A, I636A, and K638A 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). Values for binding affinity are shown in Table 1. (C) Bar graphs of high-affinity (left) and low-affinity (middle) binding values and thermodynamics (right) that arise from the fitting of the heat values with a two-site independent binding model for the binding of cAMP to the mutants (as in B) and 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 theoretical curves for the shift in activation gating produced by cAMP. The solid red lines are produced by the mathematical model using the experimental binding affinities obtained by ITC for the wild-type protein and fitted to the concentration–response data for the wild-type channel. The solid blue lines are produced by the mathematical model using the experimental binding affinities obtained for cAMP binding to the corresponding mutant proteins but using the gating parameters that were obtained from the fit of the model to the wild-type data. Note that the blue line for the K638A channel fits the concentration–response data (blue squares) very well (middle graph). However, the blue lines for the R635A and I636A channels do not fit the concentration–response data (purple squares) very well; the open and closing transitions were therefore also changed to produce theoretical curves (purple lines) that fit the concentration–response data for those mutants appropriately. The concentration–response data were reproduced from Zhou and Siegelbaum (2007).
Figure 3.

Residues of the C-helix contribute to ligand binding affinity and variably couple binding to channel opening. (A) A ribbon diagram showing the position of R635, I636, and K638, which are located in the C-helix at the distal region of the CNBD. PDB accession no. 1Q50. (B) Plots of heat produced upon progressive injections of cAMP to 200 μM of R635A, I636A, and K638A 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). Values for binding affinity are shown in Table 1. (C) Bar graphs of high-affinity (left) and low-affinity (middle) binding values and thermodynamics (right) that arise from the fitting of the heat values with a two-site independent binding model for the binding of cAMP to the mutants (as in B) and 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 theoretical curves for the shift in activation gating produced by cAMP. The solid red lines are produced by the mathematical model using the experimental binding affinities obtained by ITC for the wild-type protein and fitted to the concentration–response data for the wild-type channel. The solid blue lines are produced by the mathematical model using the experimental binding affinities obtained for cAMP binding to the corresponding mutant proteins but using the gating parameters that were obtained from the fit of the model to the wild-type data. Note that the blue line for the K638A channel fits the concentration–response data (blue squares) very well (middle graph). However, the blue lines for the R635A and I636A channels do not fit the concentration–response data (purple squares) very well; the open and closing transitions were therefore also changed to produce theoretical curves (purple lines) that fit the concentration–response data for those mutants appropriately. The concentration–response data were reproduced from Zhou and Siegelbaum (2007).

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