cAMP binding to closed pacemaker ion channels is non-cooperative

Electrical activity in the brain and heart depends on rhythmic generation of action potentials by pacemaker ion channels (HCN) whose activity is regulated by cAMP binding 1 . Previous work has uncovered evidence for both positive and negative cooperativity in cAMP binding 2 , 3 , but such bulk measurements suffer from limited parameter resolution. Efforts to eliminate this ambiguity using single-molecule techniques have been hampered by the inability to directly monitor binding of individual ligand molecules to membrane receptors at physiological concentrations. Here we overcome these challenges using nanophotonic zero-mode waveguides 4 to directly resolve binding dynamics of individual ligands to multimeric HCN1 and HCN2 ion channels. We show that cAMP binds independently to all four subunits when the pore is closed, despite a subsequent conformational isomerization to a flip state at each site. The different dynamics in binding and isomerization are likely to underlie physiologically distinct responses of each isoform to cAMP 5 and provide direct validation of the ligand-induced flip-state model 6 – 9 . This approach for observing stepwise binding in multimeric proteins at physiologically relevant concentrations can directly probe binding allostery at single-molecule resolution in other intact membrane proteins and receptors. Direct monitoring of individual cAMP molecules binding to HCN ion channels reveals the binding dynamics underlying the distinct physiological responses of ion channel isoforms.