cAMP binding to closed pacemaker ion channels is cooperative

Abstract

Activation of tetrameric hyperpolarization activated cyclic nucleotide-modulated (HCN) ion channels by the binding of cyclic nucleotides is a complex cooperative process. Here we address the cooperativity in the binding process of resting murine mHCN2 channels at the single-molecule level by monitoring the binding of fluorescently labeled cAMP to the four binding sites. The channels were tagged with the fluorescent protein eGFP and were expressed in native cell membranes. We show that the ligand binding to resting mHCN2 channels is positively cooperative. A model-based analysis revealed that ligand binding promotes a transition to a flip state and that both ligand affinity and flip state population increase with the degree of occupation of the binding sites. We conclude that in mHCN2 channels embedded in natural cell membranes subunit cooperativity is not only associated with channel activation but appears already in closed channels if more than one ligand is bound.