Hyperpolarization and cyclic-nucleotide (HCN) activated ion channels play a critical role in generating self-propagating action potentials in pacemaking and rhythmic electrical circuits in the human body. Unlike most voltage-gated ion channels, the HCN channels activate upon membrane hyperpolarization, but the structural mechanisms underlying this gating behavior remain unclear. Here, we present cryo-electron microscopy structures of human HCN1 in Closed, Intermediate, and Open states. Our structures reveal that the inward motion of two gating charges past the charge transfer center (CTC) and concomitant tilting of the S5 helix drives the opening of the central pore. In the intermediate state structure, a single gating charge is positioned below the CTC and the pore appears closed, whereas in the open state structure, both charges move past CTC and the pore is fully open. Remarkably, the downward motion of the voltage sensor is accompanied by progressive unwinding of the inner end of S4 and S5 helices disrupting the tight gating interface that stabilizes the Closed state structure. This "melting" transition at the intracellular gating interface leads to a concerted iris-like displacement of S5 and S6 helices, resulting in pore opening. These findings reveal key structural features that are likely to underlie reversed voltage-dependence of HCN channels.

Structural Basis for Hyperpolarization-dependent Opening of the Human HCN1 Channel