S5-S6 interactions important for the electromechanical coupling in HCN channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to the rhythmic firing of pacemaker neurons and cardiomyocytes. Mutations in HCN channels are associated with cardiac arrhythmia and epilepsy. HCN channels belong to the superfamily of voltage-gated K+ channels, most of which are activated by depolarizations. HCN channels, however, are activated by hyperpolarizations and the mechanism underlying the electromechanical coupling in HCN channels is not fully clear yet. The electromechanical coupling is thought to be mainly contributed by non-covalent interactions between S4 in the voltage-sensing domain (VSD) and S5 in the pore domain (PD). Little attention has been paid to the interactions between S5 and S6 within the PD. This study seeks to identify the role of the S5-S6 interactions in the electromechanical coupling of HCN channels. Voltage clamp fluorometry, patch clamp, and molecular modeling were conducted on mutations in S5 and S6 in the PD. Here we show that some mutations in S5 and S6 change the gating of HCN channels without affecting the S4 movement. These interactions between S5 and S6 are important for closing HCN channels. Our results suggest that in addition to canonical S4-S5 interactions, S5-S6 interactions are crucial for the electromechanical coupling in HCN channels.