Leonardi, M. V. | bioRxiv Science

bioRxiv2026

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

The atomic structure of the human Ether-a-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.

biophysics↗

bioRxiv2024

Building predictive Markov State Models of ion channel permeation from Molecular Dynamics

Molecular dynamics (MD) simulation of biological processes has always been a very challenging task due to the long timescales of the processes involved and the challenges associated with handling the large amount of output data. Markov State Models (MSMs) have been recently introduced as a powerful tool in this area of research, as they provide a mechanistically comprehensible synthesis of the large amount of MD data and, at the same time, can be used to estimate experimental properties of biological processes. Of the many studies on protein simulation and the MSM-assisted approach, only a few have addressed ion channel permeation and, more importantly, none of these have tried to build a model capable to predict the currents passing through the channels, which are ultimately crucial for comparing simulations with experimental results. Herein, we propose a method for building an MSM of ion channel permeation that correctly evaluates the current flowing through the channel. This was done by including in the model the definition of a flux matrix carrying information on the charge moving across the channel, suitably built to be used in conjunction with the transition matrix to predict the ion current. The proposed method is also able to drastically reduce the number of states so to obtain an MSM simple enough to be easily understood. Finally, we applied the method to the KcsA channel, obtaining a four-state MSM capable of accurately reproducing the single channel ion current from microseconds MD trajectories.

biophysics↗