Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels are pacemaker channels, generating rhythmicity in neurons and cardiomyocytes. They are activated by hyperpolarizing voltages and modulated by the binding of cAMP. HCN2 channels are tetramers composed of four structurally identical subunits. The molecular mechanism underlying the subunit interaction is still unknown. To gain further insight into the molecular processes leading to channel gating, we measured ligand binding and channel activation simultaneously using confocal patch-clamp fluorometry in combination with a piezo-driven solution switch for applying fast ligand-concentration jumps, thereby using a fluorescent ligand, fcAMP. We compared the ligand binding and subsequent channel activation following concentration jumps to three different fcAMP concentrations and back to zero at -130 mV, a voltage generating channel activation, and at -30 mV, a voltage generating no channel activation. In channels activated maximally by voltage and saturating fcAMP, two of the four subunits bind their ligands very tightly in the sense of a trapping, releasing them only very slowly when removing the ligand in the bath. The other two subunits release their ligands rapidly. In the absence of voltage-induced activation, maximally one ligand per channel is tightly bound. The trapping is also demonstrated for the natural ligand cAMP. Furthermore, the slow component of the time courses of binding and unbinding matches that of current activation and deactivation, respectively, indicating that these processes are closely related. The data were globally fitted by a Markovian state model which provided detailed insight into the gating kinetics of each binding step and each closed-open isomerisation.