Distinct mechanisms of inhibition of Kv2 potassium channels by tetraethylammonium and RY785

Reviewer #1 (Public review):

Summary:

This study seeks to identify a molecular mechanism whereby the small molecule RY785 selectively inhibits Kv2.1 channels. Specifically, it sought to explain some of the functional differences that RY785 exhibits in experimental electrophysiology experiments as compared to other Kv inhibitors, namely the charged and non-specific inhibitor tetraethylammonium (TEA). This study used a recently published cryo-EM Kv2.1 channel structure in the open activated state and performed a series of multi-microsecond-long all-atom molecular dynamics simulations to study Kv2.1 channel conduction under the applied membrane voltage with and without RY785 or TEA present. While TEA directly blocks K+ permeation by occluding ion permeation pathway, RY785 binds to multiple non-polar residues near the hydrophobic gate of the channel driving it to a semi-closed non-conductive state. This mechanism was confirmed using an additional set of simulations and used to explain experimental electrophysiology data,

Strengths:

The total length of simulation time is impressive, totaling many tens of microseconds. The study develops forcefield parameters for the RY785 molecule based on extensive QM-based parameterization. The computed permeation rate of K+ ions through the channel observed under applied voltage conditions is in reasonable agreement with experimental estimates of the single-channel conductance. The study performed extensive simulations with the apo channel as well as both TEA and RY785. The simulations with TEA reasonably demonstrate that TEA directly blocks K+ permeation by binding in the center of the Kv2.1 channel cavity, preventing K+ ions from reaching the SCav site. The conclusion is that RY785 likely stabilizes a partially closed conformation of the Kv2.1 channel and thereby inhibits the K+ current. This conclusion is plausible given that RY785 makes stable contact with multiple hydrophobic residues in the S6 helix. This further provides a possible mechanism for the experimental observations that RY785 speeds up the deactivation kinetics of Kv2 channels from a previous experimental electrophysiology study.

Weaknesses:

The study, however, did not produce this semi-closed channel conformation and acknowledges that more direct simulation evidence would require extensive enhanced-sampling simulations. The study has not estimated the effect of RY785 binding on the protein-based hydrophobic pore constriction, which may further substantiate their proposed mechanism. And while the study quantified K+ permeation, it does not make any estimates of the ligand binding affinities or rates, which could have been potentially compared to the experiment and used to validate the models.

Reviewer #3 (Public review):

Summary:

In this manuscript, Zhang et al. investigate the conductivity and inhibition mechanisms of the Kv2.1 channel, focusing on the distinct effects of TEA and RY785 on Kv2 potassium channels. The study employs microsecond-scale molecular dynamics simulations to characterize K+ ion permeation and compound binding inhibition in the central pore.

Strengths:

The findings reveal a unique inhibition mechanism for RY785, which binds to the channel walls in the open structure while allowing reduced K+ flow. The study also proposes a long-range allosteric coupling between RY785 binding in the central pore and its effects on voltage-sensing domain dynamics. Overall, this well-organized paper presents a high-quality study with robust simulation and analysis methods, offering novel insights into voltage-gated ion channel inhibition that could prove valuable for future drug design efforts.

Weaknesses:

(1) The study neglects to consider the possibility of multiple binding sites for RY785, particularly given its impact on voltage sensors and gating currents. Specifically, there is potential for allosteric binding sites in the voltage-sensing domain (VSD), as some allosteric modulators with thiazole moieties are known to bind VSD domains in multiple voltage-gated sodium channels (Ahuja et al., 2015; Li et al., 2022; McCormack et al., 2013; Mulcahy et al., 2019).

(2) The study describes RY785 as a selective inhibitor of Kv2 channels and characterizes its binding residues through MD simulations. However, it is not clear whether the identified RY785-binding residues are indeed unique to Kv2 channels.

(3) The study does not clarify the details, rationale, and ramifications of a biasing potential to dihedral angles.

(4) The observation that the Kv2.1 central pore remains partially permeable to K+ ions when RY785 is bound is intriguing, yet it was not revealed whether polar groups of RY785 always interact with K+ ions.