cAMP binding to closed pacemaker ion channels is cooperative

Most of receptor proteins in biomembranes are oligomers of either homologue or identical subunits, that govern a cooperative activation process. HCN ion channels, generating electric rhythmicity in specialized brain neurons and cardiomyocytes1, are tetramers activated by hyperpolarizing voltage and modulated by the intracellular binding of the second messenger cAMP2,3. The molecular processes underlying the cooperative action of the subunits4-6 by the binding are not fully understood, in particular if they are necessarily associated with channel activation7 or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides8. Here we show positive cooperativity in ligand binding to resting HCN2 channels in native cell membranes by analyzing the binding of fluorescence-labelled single cAMP derivatives to the channels. Kinetic modelling reveals, that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in resting HCN2 channels is already cooperative prior to channel activation. This shows that single-molecule binding studies can quantify cooperativity in ligand binding to receptors in native membranes based on equilibrium measurements.