Voltage- and Camp-Dependent Gating in Heterotetrameric HCN2/4-Pacemaker Channels

Abstract

HCN pacemaker channels play an important role in generating and regulating rhythmicity of special neurons and cardiac cells. They are activated by hyperpolarizing voltages and modulated by the binding of cyclic nucleotides. Four isoforms, HCN1-HCN4, have been identified. HCN2 and HCN4, expressed in cardiac sinoatrial node and ventricular cells, build functional homotetrameric and heterotetrameric channels in various heterologous cell systems. Heterotetrameric HCN2/4 channels in Xenopus oocytes show two changes compared to each of the homotetramers: the voltage of half maximum activation is shifted to more depolarized voltages and activation kinetics are faster (Zhang et al., Biochim Biophys Acta, 2009). However, little is known about the ligand dependence of these channels. Herein, we studied the differences in activation gating of HCN2/4 channels in comparison to the respective homotetramers, thereby focusing on the effect of cAMP. We monitored activation under both steady-state and non steady-state conditions in the presence and absence of cAMP, as well as the ligand-dependent activation kinetics after ligand jumps. We found (1) that in HCN2/4 the apparent affinity for cAMP was between that of the two homotetramers, whereas the Hill coefficient was lowest, (2) that cAMP accelerates voltage-induced activation in HCN2/4 only slightly (factor ∼2), resembling HCN4, whereas in HCN2 it accelerates activation in a voltage-dependent manner by a factor of up to ∼12, (3) that the activation kinetics following a cAMP jump to channels pre-activated by voltage was fastest in HCN2/4, whereas the increase of current amplitude by a concentration jump was similar to homotetramers. Our results confirm that coinjection of the HCN2 and HCN4 isoforms in Xenopus oocytes leads to heterotetrameric channels. They suggest that in native heart cells the formation of heterotetramers leads to pacemaker channels with specific characteristics, thereby fine-tuning the process of pacemaking.