Fig. 5.

Two-step S4 movement opens HCN channels. (Upper Right) In the closed state with S4 outward, there is a E356-N370 hydrogen bond, which stabilizes S4 outward. We and others have previously shown that F216-W355 and R367-D471 interactions stabilize a closed gate (28). (Upper Left) Closed state with S4 intermediate. Upon hyperpolarization S4 moves inward to the intermediate state, here shown as an initial inward S4 movement (red 1 arrows). The inward S4 movement breaks the E356-N370 hydrogen bond. (Lower Left) Open state with S4 inward. The initial inward S4 movement is followed by a subsequent tilting S4 movement (red 2 arrows), which separates S4 from S5, opening a hydrophilic crevice around both segments and allowing the gate to open at hyperpolarized voltages. The opening of the crevice during the second S4 movement exposes L340 to the intracellular solution. We have previously shown that a hydrophilic residue at N370 stabilizes an open gate (28).