Helix Breaking Transition in the S4 of HCN Channel is Critical for Hyperpolarization-Dependent Gating

Abstract

HCN channels, unlike other members of the VGIC superfamily, open upon hyperpolarization rather than depolarization. Structural and functional analyses reveal that the voltage-sensor of these channels are unique but the mechanisms that lead to inward rectification remain poorly understood. Here, using all-atom molecular dynamics simulations under hyperpolarization conditions, we report that the S4 voltage-sensor of HCN channels initially moves downward but, upon transfer of the last gating charge, breaks into two sub-helices with the lower sub-helix becoming parallel to the membrane plane. Substitution of the serine at the breakpoint with a hydrophobic residue favors outward rectification but this effect can be reversed by inserting a serine in an adjacent position. We find that the gating polarity strongly correlates with hydrophilicity and helical turn propensity of the substituents at the breakpoint. Our studies reveal an unexpected mechanism of inward rectification involving a linker sub-helix emerging from HCN S4 during hyperpolarization.