Mechanical transduction of cytoplasmic-to-transmembrane-domain movements in a hyperpolarization-activated cyclic nucleotide–gated cation channel

Abstract

Hyperpolarization-activated cyclic nucleotide–gated cation (HCN) channels play a critical role in the control of pacemaking in the heart and repetitive firing in neurons. In HCN channels, the intracellular cyclic nucleotide–binding domain (CNBD) is connected to the transmembrane portion of the channel (TMPC) through a helical domain, the C-linker. Although this domain is critical for mechanical signal transduction, the conformational dynamics in the C-linker that transmit the nucleotide-binding signal to the HCN channel pore are unknown. Here, we use linear response theory to analyze conformational changes in the C-linker of the human HCN1 protein, which couple cAMP binding in the CNBD with gating in the TMPC. By applying a force to the tip of the so-called “elbow” of the C-linker, the coarse-grained calculations recapitulate the same conformational changes triggered by cAMP binding in experimental studies. Furthermore, in our simulations, a displacement of the C-linker parallel to the membrane plane (i.e. horizontally) induced a rotational movement resulting in a distinct tilting of the transmembrane helices. This movement, in turn, increased the distance between the voltage-sensing S4 domain and the surrounding transmembrane domains and led to a widening of the intracellular channel gate. In conclusion, our computational approach, combined with experimental data, thus provides a more detailed understanding of how cAMP binding is mechanically coupled over long distances to promote voltage-dependent opening of HCN channels.

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

“…Compared to molecular dynamics simulations, this coarse-grained technique requires much shorter computational time and allows insights into conformational changes on larger timescales. In our previous study (Gross et al, 2018) we have already successfully simulated binding of cAMP to the CNBD in HCN1 by applying an external force on the ‘elbow’ of the C-linker. Here we used the HCN1 anisotropic network model to test if C-linker is mechanically connected to HCND and VSD.…”

“…The four C-linkers are a continuation of the S6 pore helices and tetramerize to form a disc-like structure, called the ‘gating ring’. Binding of cAMP to the CNBD induces an iris-like rotation in the gating ring, which is thought to help unwrap the bundle of S6 helices and open the pore (Gross et al, 2018; Lee and MacKinnon, 2017; Marchesi et al, 2018; Shin et al, 2004; Weißgraeber et al, 2017).…”

Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels

“…This elbow region is of particular interest as previous studies already suggested an essential role in channel activation via coupling the conformational changes in the CNBD to the channel pore (10,(30)(31)(32)(33). It has been suggested that channel opening is related to a rotation of the CL around the central channel axis, due to the disruption of stabilizing interactions (34)(35)(36).…”

“…Indeed, by using the full-length hHCN1 structure, Gross and coworkers proposed a model in which, upon cAMP binding, the elbow moves to the shoulder of the adjacent subunit in an overall centrifugal motion away from the central axis of the channel pore, which might lead to a widening of the channel pore (33). However, the authors also stated that with their approach, they could not gauge whether the widening is sufficient to open the intracellular channel gate.…”

cyclic AMP Regulation and Its Command in the Pacemaker Channel HCN4

“…The comparison of the available HCN1 structures, obtained in the cAMP-free (apo) and -bound (holo) form, provides little mechanistic information on how pore gating may be modulated by cAMP. Minimal differences in the conformation of the C-linker are observed between the apo and holo structures of HCN1 ( Lee and Mackinnon, 2017 ), despite a large body of literature arguing that movements of the CNBD transfer force to the TMD portion of the channel through a rearrangement in the C-linker, leading to a rotation of the elbow ( Craven and Zagotta, 2004 ; Craven et al, 2008 ; Weissgraeber et al, 2017 ; Gross et al, 2018 ). This finding is somehow not surprising considering the known minimal response of HCN1 to cAMP ( Porro et al, 2019 ), and highlights the expectation for other structures of HCN isoforms with a larger cAMP response to come.…”