Refining HCN1 channel closed and open state models using cysteine bridge simulations.

Abstract

The hyperpolarization-activated cyclic nucleotide (HCN) gated channel underlies the control of rhythmic activity in cardiac and neuronal pacemaker cells at heart, brain, and kidney. Unlike most of the Kv channels that are activated by membrane depolarization, HCN channels are activated by membrane hyperpolarization. Knowledge of multiple conformational states of ion channels is a prerequisite to understand their function. Atomistic models of spHCN channels in three different states (up-closed, up-closed, down-open) were created using existing hHCN1 cryo-EM structures and the open hERG cryo-EM structure and equilibrated using 1.5 to 3.0 μs all-atom MD simulations. While all three models were stable, they have not satisfied all known experimental derived structural constraints at open and closed states. In this work, we used non-interacting molecular fragments approach to refine our up-closed and down-open spHCN1 models by integrating multiple experimentally determined cysteine-cysteine Cd2+ bridges simultaneously during MD simulations. A total of 28 cysteine bridges were generated for each model. The force constants for the harmonic restraints were gradually turned on. We found that for most of the cysteine bridges between S4-S5 linker and C-linker, an upward movement of C linker was sufficient to satisfy the distance criteria without large conformational re-arrangement from its starting configuration. These refined models will be further validated using new cysteine bridge experiments. These models will help reveal the mechanism of voltage sensor-to-gate coupling and lead to deeper understanding of disease mutations on HCN channel at the molecular level.