Gating mechanism of hyperpolarization-activated HCN pacemaker channels

Fig. 1. Deleting QWE at the C-terminal S4 leads to an always open channel with an immobilized S4. a S4 sequence alignment of spHCN, hHCN1, and hERG channels. The conserved QWE sequence is shown in bold. R332 is shown with an *. b spHCN homology model showing the position of the QWE residues. c, Representative traces of the current (black) and fluorescence (red) from c wt* and d ΔQWE* channels. Scale bars: 0.1 s, 5 µA, and 2% ΔF/F. e Normalized G(V) (black) and F(V) (red) curves of wt* and ΔQWE* channels. f Tetramethyl-rhodamine C5-maleimide labeling of uninjected oocytes and oocytes expressing the R332C (wt) and R332C/ΔQWE (ΔQWE) channels in high K+ and NMDG solutions. Labeling rates (k): 0.25 ± 0.06 (wt in high K+), 0.11 ± 0.01 (p = 0.05) (wt in NMDG), 0.05 ± 0.01 (p = 0.01) (ΔQWE in high K+), 0.07 ± 0.02 (p = 0.001) (ΔQWE in NMDG). Mean ± SEM. p values indicate statistical difference from wt in high K+ solution. Source data are provided as a Source Data file.

Fig. 2. W355N*, N370W*, and F216A* single mutants do not close at positive voltages. a spHCN homology model showing the position of W355, N370, and F216 with a closed gate and S4 up. b–d Representative current (black) and fluorescence (red) from b W355N*, c N370W*, and d F216A* channels. Scale bars: 0.1 s, 1 µA, and 1% ΔF/F. e Normalized G(V) and F(V) curves of W355N*, N370W*, and F216A* channels. G(V) and F(V) of wt* channels are shown for comparison (dashed lines). Mean ± SEM. Source data are provided as a Source Data file.

Fig. 3. Hydrophobic N370 mutants have wt-like S4 movement but do not close completely. Representative current (black) and fluorescence (red) from a N370G*, b N370A*, c N370V*, d N370I*, and e N370E* channels. Scale bars: 0.1 s, 5 µA, and 0.5% ΔF/F. f, g Normalized f G(V) and g F(V) curves of wt*, N370G*, N370A*, N370V*, N370I*, N370D*, and N370E* channels. Mean ± SEM. Source data are provided as a Source Data file.

Fig. 4. W355N–N370W switches the polarity of voltage activation. a, b Currents (black) and fluorescence (red) from W355N–N370W* and wt spHCN* channels using holding voltages of a −80 mV and b −10 mV. Scale bars: 0.1 s, 5 µA, and 1% ΔF/F. c G(V) and F(V) curves of wt* and W355N–N370W* channels. Mean ± SEM. d W355-F216, N370-E356, and R367–D471 interactions hold wt* channels closed7,21. Downward S4 movement breaks these interactions and exposes N370 to water-filled crevice. e W355N–N370W removes stabilizing interactions in closed state with S4 up and destabilize water-filled crevice in open state with S4 down. See Supplementary Figs. 3–5 and Supplementary Movies 1–2 for more details. Source data are provided as a Source Data file.