Ligand Binding and Gating in HCN2 Channels

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 heart and brain. Ligand binding to the intracellular cyclic nucleotide-binding site accelerates 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. It is therefore important to determine the binding of the ligands to the channels and channel activation simultaneously.Activation of homotetrameric HCN2 channels was studied in inside-out macropatches simultaneously with ligand binding by means of patch-clamp fluorometry, using a fluorescent cAMP (fcAMP) that activates the channels in a similar manner as cAMP. With 1 μM fcAMP the binding of the ligand to the open channels exceeded that to closed channels. The slowness of the activation time course of HCN2 channels allowed us to monitor the ligand binding during the activation process. As predicted, the slow activation time course was accompanied by an increase of ligand binding. Moreover, the increase of binding was exponential whereas activation obeyed the typical sigmoidal time course. Hence, in the superimposed normalized time courses, the initial binding preceded activation whereas at later times activation preceeded binding. These results show that activation gating indeed increases the binding affinity for the ligands, quite as predicted by the principle of reciprocity, and that the gating of the first of the four subunits, which does not lead to channel opening, is associated with ligand binding.