Gating Mechanism of Hyperpolarization-Activated HCN Pacemaker Channels

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential for rhythmic activity in the heart and brain, and mutations in HCN channels are linked to heart arrhythmia and epilepsy. HCN channels belong to the family of voltage-gated K+ (Kv) channels. However, why HCN channels are activated by hyperpolarization (i.e. when the membrane potential becomes more negative) whereas Kv channels are activated by depolarization (i.e. when the membrane potential becomes more positive) is not clear. Hyperpolarization-activated HCN channels and depolarization-activated Kv10.1 (EAG) channels have very similar tetrameric structures with six transmembrane segments (S1-S6) per subunit: S1-S4 form the voltage-sensing domain (VSD) and S5-S6 form the pore domain (PD). In both Kv and HCN channels, S4 is the positively charged voltage sensor and the C -terminal part of S6 forms the gate. Here we find that the most C-terminal region of S4 is required for both S4 movement and the coupling between S4 and the gate in HCN channels. We reverse the voltage dependence of HCN channels by mutating only two residues located at the interface between the C-terminal region of S4 and the PD such that the channels now open upon depolarization instead of hyperpolarization. Because the S4 movement in the mutant depolarization-activated HCN channels is similar to that in hyperpolarization-activated HCN channels, we suggest that these mutations reverse the coupling between S4 and the gate. Our data indicate that what determines whether an ion channel opens by hyperpolarizations or depolarizations are small differences in free energy between the closed and open states, due to different interactions between S4 and the pore in the different channels. Understanding how HCN channels gate will aid in development of better anti-arrhythmic and anti-epileptic drugs targeting HCN channels.