Distinct effects of disease-associated mutations in the proximal C-linker on negatively cooperative binding of cyclic nucleotides to the human HCN4 channel

The direct binding of cAMP to the hyperpolarization-activated cyclic nucleotide-gated channel form 4 (HCN4) protein contributes to autonomic modulation of heart rate. Here, we determine affinity for binding of cyclic nucleotides directly to the cytosolic C-terminus of the HCN4 channel of three disease-linked mutations that are found in the C-linker of the C-terminus, which attaches the more distal binding domain to the pore. Two of these mutations, one in the A’ helix (K530N) and another in the B’-helix (D553N), reduced binding affinity but did not alter the negatively cooperative pattern of binding found in the wild type channel. Finally, a third mutation in the A’-helix (R524Q) did not alter either the pattern or affinity of binding to the cyclic nucleotides despite a dramatic, and previously reported, increase in cAMP potency in the full channel. Our data may be explained by location of each mutation and its interactions with associated domains external to this region. The data support negatively cooperative binding of cAMP and cGMP to HCN4 and help to identify distinct structural contributions to ligand binding and gating, both of which determine potency.