Lipid bilayers determine allostery but not intrinsic affinity of cAMP to pacemaker channels.

The binding of cyclic adenosine monophosphate (cAMP) to hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels regulates cardiac pacemaking but key aspects of the mechanism of ligand-dependent regulation remain unresolved. Here, we examine the role of the lipid environment by reconstituting purified human HCN channels into lipid nanodiscs and measuring successive cAMP binding to single HCN channels using nanophotonic waveguides. Regardless of nanodisc size or lipid composition, cAMP molecules bind cooperatively to HCN channels in lipid bilayers, unlike channels solubilized in detergents. The affinity of the first ligand remains unchanged across conditions, indicating that the bilayer selectively alters higher-order ligation states. Cryo-EM structures of apo- and holo-HCN channels reveal additional lipid densities that are weak or absent in detergent-solubilized preparations. Together, these findings show that the lipid bilayer is both necessary and sufficient to induce cooperative ligand binding in HCN channels, thereby enhancing their sensitivity to gating stimuli.