The HCN domain couples voltage gating and cAMP response in hyperpolarization-activated cyclic nucleotide-gated channels.

Alessandro Porro,Anna Moroni,Gerhard Thiel,Matteo Pisoni,Daniel Bauer,Andrea Saponaro,Christine Gross,Kay Hamacher,Bina Santoro,Gerardo Abbandonato,Federica Gasparri

doi:10.7554/elife.49672

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltage-dependent gating in three HCN isoforms.

Highlights

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (Gauss et al, 1998; Ludwig et al, 1998; Santoro et al, 1997; Santoro et al, 1998) are the molecular determinants of the If/Ih current, a mixed Na+/K+ current that controls pacemaking in cardiac and neuronal cells (DiFrancesco, 1993; Pape, 1996)
HCNa lies parallel to the plane of the membrane, HCNb faces the C-linker ring, and HCNc ends in a loop which penetrates the membrane plane and connects the HCND to S1, the first transmembrane domains (TM) helix of the voltage sensing domain (VSD)
By inspecting the HCN1 structural model and density map, we found that the HCND appears to directly contact both the VSD and the C-linker

Summary

Introduction

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (Gauss et al, 1998; Ludwig et al, 1998; Santoro et al, 1997; Santoro et al, 1998) are the molecular determinants of the If/Ih current, a mixed Na+/K+ current that controls pacemaking in cardiac and neuronal cells (DiFrancesco, 1993; Pape, 1996). Voltage is both necessary and sufficient to open the channel, as HCN1 mutants lacking the cytosolic C-linker/CNBD domain are fully regulated by voltage (Wainger et al, 2001) This indicates that the VSD does not require the rotation of the C-linker gating ring in order to open the pore, but presumably acts via a membrane-delimited pathway. In the presence of cAMP, the gating ring only marginally rotates by about 1 Aand the displacement of S6 is correspondingly minimal (Lee and MacKinnon, 2017) These functional and structural observations suggest that the gating ring movement must be under the control of voltage, implying the existence of a direct physical contact between the VSD and the C-linker. We uncover a second important role of the HCND in gating, which is to set the range of voltages at which the channel opens, ensuring channel operation at physiological relevant voltages only

Results

Discussion

Materials and methods

Funding Funder Fondazione Cariplo