The wild-type hERG cryo-EM structure fails to support conduction and evolve toward an inactivated-like selectivity filter conformation in molecular dynamics simulations

Abstract

The atomic structure of the human Ether-à-go-go–Related Gene (hERG) K+ channel has recently been resolved by cryo-electron microscopy (cryo-EM) under both high- and low-K+ conditions, in order to obtain information on the mechanism of the K+-sensitive, very rapid C-type inactivation typical of this channel. Although the currently available high-K+ structures have been widely interpreted as representing the conductive, active state, whether they correspond to a dynamically stable conductive conformation remains unresolved. Here, we used extensive all-atom molecular dynamics (MD) simulations, with and without Electronic Continuum Correction (ECC), to investigate selectivity filter (SF) dynamics and ion permeation in wild-type (WT) hERG and the non-inactivating N629D mutant. Across all membrane potentials tested, WT hERG failed to support K+ permeation and instead spontaneously evolved towards a non-conductive SF conformation characterized by extracellular dilation, localized inner constriction, depletion of the outer ion-binding sites, and persistent trapping of K+ ions within the central binding sites of the filter, closely resembling the inactivated SF of Shaker channels recently resolved by cryo-EM. By contrast, N629D maintained a stable conductive SF architecture, analogous to the conductive filters of canonical K+ channels such as KcsA, while exhibiting robust voltage-dependent K+ permeation. ECC enhanced ion permeation in N629D mutant, but failed to support conduction in WT hERG, which remained structurally and functionally non-conductive. Together, these findings challenge the prevailing interpretation of the high-K+ WT cryo-EM structure as a stable conductive state and identify SF remodeling as the structural mechanism underlying hERG C-type inactivation.