The Role of HCN Domain in the Function of HCN Channels

Abstract

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels generate funny currents (If) in the heart and hyperpolarization-activated currents (Ih) in the brain. HCN channels are assembled from four subunits, with each subunit containing six transmembrane segments (S1-S6) and a cyclic-nucleotide binding (CNB) domain in the C-terminal region linked to the pore-forming transmembrane segment with a C-linker. Recent cryo-EM structure of HCN1 channels (Lee et al., Cell, 2017, 168:111-120) identified a stretch of 45 amino acids directly preceding the S1 transmembrane segment as a 3-alpha-helical structural motif conserved in all four mammalian HCN channel isoforms. The 3-alpha-helical domain, dubbed HCN domain, forms direct interactions with the S4 transmembrane segment in the voltage sensor from the same subunit and the C-linker/CNB domain from the adjacent subunit. Here we explored the role of the HCN domain in the function of HCN channels. Deletion of the HCN domain abolished currents from HCN channels expressed in Xenopus laevis oocytes. Mutant HCN channels with alanines substituted for the interacting residues between the HCN domain and the C-linker/CNB domain gave rise to hyperpolarization-activated currents with the half-maximal activation voltage (V1/2) similar to the V1/2 of the wild-type HCN channels. Taken together our observations point towards the role of the HCN domain in the functional assembly of HCN channels.