A second S4 movement opens hyperpolarization-activated HCN channels

Rhythmic activity in pacemaker cells, as in the sino-atrial node in the heart, depends on the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. As in depolarization-activated K+ channels, the fourth transmembrane segment S4 functions as the voltage sensor in hyperpolarization-activated HCN channels. But how the inward movement of S4 in HCN channels at hyperpolarized voltages couples to channel opening is not understood. Using voltage clamp fluorometry, we found here that S4 in HCN channels moves in two steps in response to hyperpolarizations and that the second S4 step correlates with gate opening. We found a mutation in sea urchin HCN channels that separate the two S4 steps in voltage dependence. The E356A mutation in S4 shifts the main S4 movement to positive voltages, but channel opening remains at negative voltages. In addition, E356A reveals a second S4 movement at negative voltages that correlates with gate opening. Cysteine accessibility and molecular models suggest that the second S4 movement opens up an intracellular crevice between S4 and S5 that would allow radial movement of the intracellular ends of S5 and S6 to open HCN channels.

Automated summary

Rhythmic activity in pacemaker cells relies on the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, with recent research suggesting that S4 movement in HCN channels occurs in two steps in response to hyperpolarizations, with the second step correlating with gate opening. Mutations in sea urchin HCN channels have helped separate the two S4 steps in voltage dependence, shedding light on the mechanism by which S4 movement leads to HCN channel opening.