HCN channels are nonselective tetrameric cation channels that are activated by hyperpolarizing voltages and modulated by the ligand cAMP. They generate spontaneous rhythmic activity in cells of the heart and brain. Ligand binding to the intracellular cyclic nucleotide-binding site accelerates the activation kinetics, shifts the steady-state activation to more positive voltages, and increases the open probability. Though it is relatively simple to determine an apparent affinity for the ligand action, it is not so simple to determine the true ligand affinity during channel activation because, according to the principle of reciprocity, ligand binding and efficacy depend on each other, i.e. the affinity of the binding sites must increase when the channel opens. To learn more about channel activation it is an attractive idea to measure the ligand binding and gating simultaneously.

Activation of homotetrameric HCN2 channels was studied in excised inside-out macropatches. Ligand binding was simultaneously monitored by means of patch-clamp fluorometry, using a fluorescent cAMP derivative (fcAMP) that activates the channels in a similar manner as cAMP. As a result the binding of the ligand to the open channels exceeded that to the closed channels. The slowness of the activation time course allowed us to record the ligand binding during the activation process. As predicted, the activation time course was accompanied by an increase of bound ligands. The closely related time courses of depolarization-induced deactivation and ligand unbinding show that it is indeed channel activation that enhances ligand binding, but not the voltage itself. The conclusion is that activation gating indeed increases the binding affinity for the ligands, quite as predicted by the principle of reciprocity.