Gating movements and ion permeation in HCN4 pacemaker channels

Figure 1. HCN4 structure and its comparison with that of HCN1 (A) Structure of the HCN4 channel tetramer in cAMP-bound state (holo), in a cross-membrane view. For clarity, only two subunits are shown in full (tan and blue), while for the other two subunits (black and green), only the cytosolic C-linker/CNBD domains are presented. (B) Protomer of HCN4 holo with structural elements indicated. (C) HCN4 comparison with HCN1. Only two subunits in the tetramer are shown in full, while for the other two subunits the cytosolic C-linker/CNBD domains are shown. HCN4 structure color-coded (gray-to-red) according to the local deviations observed for the Cα atoms of HCN4 versus HCN1 holo (PDB: 5U6P). Gray color represents 0.5-Å displacements, and red color represents displacements up to 5 Å, as shown by the bar.

Figure 2. Structural, biochemical, and functional evidence for the tetrad, connecting the transmembrane and cytosolic domains in HCN4 (A) Comparison of the S4-S5 linker of HCN1 (yellow) and HCN4 (blue). Backbones of I405 in HCN4 and the corresponding I284 in HCN1 are shown in red. (B) Ribbon representation of S4-S5 linker of one subunit (blue) and underlying C-linker “elbow” (A′–B′ helices) of adjacent subunit (green) of HCN4. Four residues forming the ion coordination site (tetrad) in HCN4 are shown as sticks and labeled (H407, D411, H553, E557). Ion coordinated by the tetrad is represented as a purple sphere. (C and D) Thermostability assay based on fluorescence-detection size-exclusion chromatography (fSEC-TS) was employed to quantify the change in melting temperature of purified protein in absence or presence of Mg2+ (see STAR Methods). (C) Representative melting curves for holo HCN4 protein purified in LMNG/CHS. Normalized fluorescence intensity (FI) plotted over pre-conditioning temperature (°C). The protein was incubated without (black) or with 10 mM EDTA (red), or, after EDTA removal, with 10 mM MgCl2 (blue). Wild-type protein (HCN4 WT) is shown on the left; double-mutant…

Figure 3. cAMP-dependent rearrangement of the cytosolic region of HCN4 channels (A) Ribbon representation of HCN4 apo/LC dimer in gray, along with C-linker/CNBD of a third subunit in the tetramer shown in orange. (B) Ribbon representation of S4-S5 linker of one subunit and C-linker “elbow” (A′-B′ helices) of adjacent subunit of apo/LC HCN4. Density map of tetrad region is shown as gray mesh. Only one of the four residues forming the ion coordination site in HCN4 holo (tetrad) was modeled and is shown as sticks and labeled H553. (C) Superimposition of C-linker “elbow” (A′, B′ helices) of HCN4 apo/LC (orange) and HCN4 holo (blue) shown in two orientations, rotated by 45 degrees. Dotted black lines indicate displacement of 2.9 Å in Cα of S540 (at the tip of the elbow) and of 3.8 Å in Cα of H558 at end of B′ helix; black arrows show direction of movement induced by cAMP binding to underlying CNBD. (D) Superimposition of CNBDs of HCN4 in the apo (orange, apo/LC) and holo (blue) configurations. The structural elements are labeled (helices E′, F′, beta roll, helices A–C). Arrows indicate direction of movements induced by cAMP binding, downward (in this figure orientation) in beta roll and upward…

Figure 4. Transmembrane domain rearrangements in the three HCN4 structures (A) Ribbon representation of S4, S5, and S6 TM helices in HCN4 holo (blue) in a cross-membrane view. For clarity, only two subunits are shown. Putative lipid densities contacting S4 (green mesh) and S5 (red and yellow mesh) are shown. Residues involved in polar interaction (dotted line) between S4 (E403) and S5 (N421) are shown as sticks and labeled on one subunit. TM helices S4, S5, and S6 are also labeled on one subunit. (B and C) Ribbon representation of pore of HCN1 holo (yellow, PDB: 5U6P) (B) and HCN4 holo (blue) (C). For ease of view, only two opposite subunits in the assembled tetramer are shown. The pore diameter is shown as a gray surface. Residues facing pore inner cavity and residue of SF facing extracellular side (A363 in HCN1 and R484 in HCN4) are shown as sticks and labeled. The atoms of side chain of R484 whose density is not resolved are colored in gray. (D) Ribbon representation of S4, S5, and S6 TM helices in HCN4 apo/LC (orange) in a cross-membrane view. For clarity, only two subunits of tetramer are shown. Lipids are absent, and polar interaction between E403 and N421 is lost. Inset: in…

Figure 5. Pore hydration and ivabradine docking in HCN4 structures (A and B) Representative snapshot from unrestrained MD simulation of PD of HCN4 structure with narrow cytosolic entrance (blue, holo) (A) and with wider cytosolic entrance (B) (green, apo/AM; identical results were obtained with apo/LC). Two subunits of tetramer are shown for clarity. In (A), Y507, I511, T515, and Q519 side chains occupy cavity and do not allow passage of water molecules (red and white sticks and balls) and K+ ions (blue spheres) through bundle crossing. In (B), a different conformation of S6 allows water and K+ ions to pass through bundle crossing and enter inner pore cavity. (C and D) Docking of ivabradine in two PDs (colored as above, with orange mesh representing ivabradine density). In (C), the narrow constriction created by Q519 prevents ivabradine entry into cavity. In (D), ivabradine enters in cavity and explores several positions, including one (ivabradine shown in orange sticks) in close proximity with Y507 and I511 (highlighted in red).

Figure 6. Mechanism of ion permeation in HCN channels (A) Comparison of selectivity filters of an open HCN4 pore (apo/LC) and HCN1. The sequence 479CIGYG481 (HCN4 numbering) is marked. (B) Conduction trajectory for individual K+ ions traversing selectivity filter in a MD simulation of HCN4 in KCl solution. The positions occupied by the ion are identified by dotted lines, labeled a–c, and cross-referenced in (C). K+ ions entering filter at subsequent times are shown in different shades of blue. Positions of the carbonyl plane of C479 and I480 are indicated by black arrows. (C) Snapshots of MD simulation cross-referenced by arrowheads in (B). The ion entering from extracellular side of SF (dark blue) occupies position a, half coordinated by water molecules from top and by carbonyl oxygen atoms of I480 at the bottom. Occupancy of position a is compatible with a second ion (light blue) in filter at position c, coordinated by carbonyl oxygen atoms of I480 from the top and by water molecules from the bottom (two-ion state). Next, the entering ion moves to position b, at plane of carbonyl oxygen atoms of C479, and the second ion moves out of the filter, into the cavity (one-ion state). Entrance…