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.

Keywords: HCN1 channel; anisotropic network model; cAMP dependent gating; computational biology; cyclic AMP (cAMP); linear response theory; potassium channel; protein conformation; protein dynamic.

Figure 1.

LRT null model of cAMP-free HCN1 channel with clustering of different perturbation directions. a, clustering of the different perturbation directions imposed on Ala-425 at the tip of the elbow (light blue) based on the displacement of the shoulder (orange). Both elements are part of the C-linker, which connects the CNBD to the S6 domain of the channel. For clarity, a single subunit of HCN1 is shown. Perturbation directions on a sphere around Ala-425 that belong to the same cluster are represented in the same color (red, blue, yellow, or green). b, clustering of the perturbation directions shown for all four subunits of HCN1. To illustrate contacts between individual subunits, the four monomers are shown in different colors. The dark gray color of the subunit in b corresponds to the same color in a.

Figure 2.

Perturbation of the ANM from HCN1 in the cAMP-free form. Perturbations at the tip of the elbow from four different clusters lead to distinct horizontal and vertical displacements of the C-linker. Displacements of HCN1 after perturbation at Ala-425 from one representative direction (a–d) for each cluster correspond to the coloring in Fig. 1. For clarity, the displacements are only shown for one subunit, which is highlighted in cartoon representation. The direction of force application is illustrated in the insets. The other subunits are illustrated in transparent surface representation. The thin arrows demonstrate the displacement of each residue. As in Fig. 1, the elbow is highlighted in light blue, and the shoulder is highlighted in orange.

Figure 3.

Comparison of predicted and measured displacements of the C-linker in cAMP-free and cAMP-bound HCN1 channel. Displacements of the C-linker of HCN1 after perturbation at Ala-425 are from one representative direction (a–d) for each cluster. Coloring corresponds to clusters in Figs. 1 and 2. e, experimentally observed displacement of the C-linker after superposition of the full-length cAMP-free and cAMP-bound HCN1 structures. Because the displacements are very small in the experimental structures, the arrows shown represent an arbitrary multiple of the actual displacement so as to allow to see the directions properly and compare them with the LRT displacements. f, superposition of the cAMP-free (colored) and cAMP-bound (black) HCN1 structures showing the actual displacements. In all visualizations the proteins are shown in top view from an extracellular perspective, so that the movements of all four subunits can be seen. The elbow again is highlighted in blue, and the shoulder is highlighted in orange. The remaining residues are colored in gray.

Figure 4.

Comparison between predicted and measured displacements of CNBD in cAMP-free and cAMP-bound HCN1 channel. a, cAMP-bound HCN1 structure (residue 401–608, cytosolic domain) showing the orientation of cAMP within the binding pocket. The HCN1 structure is shown in cartoon representation with elbow and shoulder of the C-linker colored according to Fig. 1. The cAMP molecule is highlighted by sphere representation. b–d, comparison of the displacements in the CNBD (residues 480–586) after perturbing the elbow from the most realistic direction (b, yellow arrows) to the experimentally resolved displacement between the cAMP-free (gray) and cAMP-bound (black) HCN1 structure (c, black arrows; d, superposition). The length of the arrows was chosen such that the length in b and c are similar; the absolute length has no quantitative meaning. The red and blue arrows highlight exemplary displacements, which are similar or different between the computational model and the experimental data, respectively. The experimental data ±cAMP are from Ref. . For clarity, only one subunit of HCN1 is shown.

Figure 5.

Simulation predicts that the C-linker and the S4–S5 linker move closer together after cAMP binding. a, visualization of the distance between Tyr-289 of one subunit (gray) and Lys-412 of the neighboring subunit (lime green). b, computed distance between the CA positions of Tyr-289 and Lys-412 of two neighboring subunits after perturbation at Ala-425 with forces of increasing strengths (yellow cluster in Figs. 2 and 3). Both the forces as well as the distances are given in arbitrary units (a.u.) and thus can only show a trend.

Figure 6.

Simulation predicts tilting movements of transmembrane helices S1–S6 after cAMP binding. Displacements of S1 to S6 helices of the TMPC (a–f) after perturbing the elbow of the C-linker from the most realistic perturbation direction. The corresponding helix is highlighted and labeled in lime green, and the displacement is visualized as yellow arrows. For clarity, only one subunit (residue 94–402) is shown from the front and side views.

Figure 7.

cAMP binding predicts a local increase in the distances between S4 and surrounding transmembrane domains. a, HCN1 monomer with reference amino acids in S3, S4, and S5. b, computed distances between these amino acids in the same subunit after perturbation of Ala-425 with forces of increasing strengths (yellow cluster in Figs. 2 and 3). The colors in b cross-reference with colors in a. Forces as well as the distances are again given in arbitrary units (a.u.).

Figure 8.

Only a horizontal displacement of the elbow causes widening of inner channel gate. a, location of inner gate (inset) in the global structure of HCN1. b, development of minimal inner gate radii in the region from Val-390 to Gln-398 with forces of increasing strengths acting on Ala-425. Force direction is represented according to the color coding used in Figs. 2 and 3. Both the forces applied and the minimal radii are given in arbitrary units (a.u.) as the LRT model only provides a trend for protein movements, in a qualitative manner but not quantitatively. Upon perturbations from directions of the yellow cluster, a widening of the inner gate can be observed, whereas for perturbations from other directions, the inner gate becomes narrower.