Conserved Residues at the Interface between the S4 and S5 Segments are Critical for Normal Gating of HCN Channels

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have the unique characteristic of being activated at negative membrane potentials, but the mechanism of HCN reverse voltage-dependence is not known. HCN channels share a similar tetrameric architecture with depolarization-activated Kv channels, having six transmembrane domains, where S1-S4 form the voltage-sensing domain (VSD) and S5-S6 form the pore domain (PD). As opposed to Kv channels, HCN channels have a non-domain swapped architecture with a short S4-S5 linker that covalently binds the VSD to the PD. The non-domain swapped architecture implies that the S4 from one subunit directly interacts with the S5 from the same subunit. As well as in depolarization-activated channels, S4 is the positively charged voltage sensor of HCN channels and moves across the membrane in response to changes in the membrane potential. It has been shown before that cutting the S4-S5 linker of HCN channels and other non-domain swapped channels does not impair their typical gating. Therefore, the coupling between the VSD and the PD in HCN channels may involve residues located at the interface between the S4 and S5 segments. The interface between S4 and S5 of HCN channels possesses several residues that are absolutely conserved in the HCN family and that therefore, are potentially important for their unique gating. We individually mutated several of these conserved residues at the S4-S5 interface in the sea urchin HCN (spHCN) channel and expressed them in Xenopus laevis oocytes to measure the effects of each mutation using Voltage Clamp Fluorometry. Our results show that some residues at the S4-S5 interface are critical for normal S4 movement, while others are important to stabilize the gate or are involved in the coupling between S4 and the gate.