HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

Abstract

Hyperpolarization-activated cyclic nucleotidegated (HCN) channels are major regulators of synaptic plasticity, and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs).In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD) and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotidebinding domain (CNBD) connected to the poreforming S6 transmembrane domain via the Clinker. Previous functional analysis of HCN channels suggested a direct or allosteric coupling between the voltage-and cNMPdependent activation mechanisms. However, the specifics of the coupling were unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed HCN domain (HCND), forms a direct structural link between the VSD and Clinker/CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified R237 and G239 residues on the S2 of the VSD that form direct interactions with I135 on the HCND. Disrupting these interactions abolished HCN2 currents. We then identified three residues on the C-linker/CNBD (E478, Q382 and H559) that form direct interactions with residues R154 and S158 on the HCND. Disrupting these interactions affected both voltage-and cAMPdependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the surface expression of HCN channels, and provides a functional link between the voltage-and cAMP-dependent mechanisms of HCN channel gating.Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are peculiar members of the voltage-gated potassium channel superfamily. Unlike other potassium channels, which are depolarization-activated and highly selective for K + , HCN channels are activated by membrane hyperpolarization and permeate both Na + and K + (1,2). The opening of HCN channels is further facilitated by cyclic nucleotides (1-3).