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 of a new K⁺ ion in position a (purple) moves the second ion from position b to c and resets initial two-ion state.

(D) Conduction trajectory of Na⁺ (yellow) and K⁺ ions (blue) in a MD simulation with equimolar NaCl and KCl solution. Labeling as in (B) with ion positions cross-referenced in (E). K⁺ and Na⁺ ions entering the filter at subsequent times are shown in different shades of blue and orange, respectively.

(E) Snapshots of MD simulation cross-referenced by arrowheads in (D). A Na⁺ ion stably occupies position b, coordinated by carbonyl oxygen atoms of C479 (one-ion state) until entry of a K⁺ ion displaces it, with no appreciable resting time of ions in (A) or (C). Several K⁺ conduction events follow as described in (B) and (C) until the next Na⁺ ion enters (see Figure S13C for full trajectory and density plot).

(F) Density profiles of distance between opposite carbonyl oxygens of Cys 479 (tetramer subunits A and C, solid line; B and D, dotted line) in SF of HCN4 in MD simulation with pure KCl solution. Solid and dashed lines correspond to measured width between subunits A/C and B/D, respectively. Histograms were calculated over time frame indicated in Figure S16A. Inset: cartoon representation of C479 carbonyl distortion (and increase in width) when K⁺ ion enters in (B).

(G) Density profiles (as per F) in MD simulation with equimolar KCl:NaCl, calculated over time frame indicated in Figure S16C. Inset: entry of a Na⁺ in (b) does not induce changes in carbonyl plane.

cav, cavity; ext, extracellular side.