The extracellular turret as a structural hub for voltage sensor-pore coupling in HCN channels

Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential for rhythmic electrical activity in cardiac and neuronal cells, and their dysfunction underlies disorders such as arrhythmia and epilepsy. Although HCN channels share a conserved six-transmembrane architecture with voltage-gated K⁺ (Kv) channels, how voltage-sensing domains (VSDs) are coupled to pore opening remains incompletely understood. Here, we identify an extracellular pathway that couples voltage-sensor movement to pore opening via the extracellular turret helix, which acts as a critical determinant of voltage-dependent gating in HCN channels. Alanine-scanning mutagenesis of HCN4 revealed six turret residues whose substitution abolished hyperpolarization-evoked currents. Voltage-clamp fluorometry showed that these mutations preserved S4 activation while selectively disrupting turret conformational changes, thereby uncoupling VSD movement from pore opening. Structural and functional analyses further identified an extracellular interaction network linking the turret to the S1, S5, and S6 segments that is essential for hyperpolarization-dependent gating. Collectively, these findings identify the extracellular turret as an active gating hub that coordinates concerted extracellular rearrangements to transmit voltage-sensor displacement to the pore. On the basis of these results, we propose a hand-in-hand coupling model, in which turret rearrangements act as a key downstream relay in the voltage-dependent opening of HCN channels.