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

In the human heart, the binding of cyclic adenosine monophosphate (cAMP), a second messenger, to hyperpolarization and cyclic nucleotide-gated (HCN) regulates the automaticity of pacemaker cells. Recent single-molecule binding studies show that cAMP bound to each subunit of purified tetrameric HCN channels independently, in contrast to findings in cells. To explore the lipid membrane’s role in cAMP regulation, we reconstituted purified human HCN channels in various lipid nanodiscs and resolved single molecule ligand-binding dynamics. Our measurements show that lipid membranes promote cooperative binding of cAMP to HCN channels without altering the intrinsic binding affinity. Cryo-EM structures of HCN channels in lipid nanodiscs reveal enhanced inter-subunit interactions, underscoring the fundamental role of lipid membranes in amplifying the gating sensitivity of ion channels.