Mironenko, A. | Search bioRxiv

bioRxiv2025

Lipid gating of BK channels and mechanism of activation by negatively charged lipids

BK channels are a class of K+ channels that possess an unusually high conductance and are synergistically gated by intracellular Ca2+ and voltage. Despite the significant array of experimental and computational data, many aspects of their function and dynamics remain unclear - such as how ion permeation is halted in the closed state of the channel. Available CryoEM structures obtained in deactivating conditions capture the channel with a wide, unobstructed pore, in contrast to e.g. a helix bundle crossing observed in some K+ channels. Several hypotheses of BK closure were proposed, including selectivity filter and hydrophobic gating. In this work, we expand on the model of hydrophobic gating and focus on the role of lipids. Using atomistic molecular dynamics simulations with applied voltage, we directly investigate the ability of the full length BK channel in various CryoEM states to permeate ions, and propose lipid entrance into the pore - either with lipid tails or entire lipid molecules - through the membrane-facing fenestrations to be a critical determinant of BK conductivity. Furthermore, we elucidate the mechanism of BK activation by negatively charged lipids via a combination of atomistic & coarse-grained simulations, and suggest that they act by a multi-modal mechanism, which encompasses lipid entry reduction, increase of the K+ occupancy of the pore, and stabilization of the channels open-state structure - in broad agreement with experimental data.

biophysics↗

bioRxiv2023

Selectivity filter mutations shift ion permeation mechanism in potassium channels

Potassium (K+) channels combine high conductance with high ion selectivity. To explain this efficiency, two molecular mechanisms have been proposed. The direct knock-on mechanism is defined by water-free K+ permeation and formation of direct ion-ion contacts in the highly conserved selectivity filter (SF). The soft knock-on mechanism involves co-permeation of water and separation of K+ by water molecules. With the aim to distinguish between these mechanisms, crystal structures of the KcsA channel with mutations in two SF residues - G77 and T75 - were published, where the arrangements of K+ ions and water display canonical soft knock-on configurations. These data were interpreted as evidence of the soft knock-on mechanism in wild-type channels (C. Tilegenova, et al., Structure, function, and ion-binding properties of a K+ channel stabilized in the 2,4-ion-bound configuration. Proceedings of the National Academy of Sciences 116, 16829-16834 (2019)). Here, we test this interpretation using molecular dynamics simulations of KcsA and its mutants. We show that, while a strictly water-free direct knock-on permeation is observed in the wild-type, conformational changes induced by these mutations lead to distinct ion permeation mechanisms, characterized by co-permeation of K+ and water. These mechanisms are characterized by reduced conductance and impaired potassium selectivity, supporting the importance of full dehydration of potassium ions for the hallmark high conductance and selectivity of K+ channels. In general, we present a case where mutations introduced at the critical points of the permeation pathway in an ion channel drastically change its permeation mechanism in a non-intuitive manner. Significance statementPotassium (K+) channels conduct K+ with high permeation rates and ion selectivity. An ongoing debate in the field has been focused on the molecular mechanisms underlying this remarkable efficiency. Here, we performed molecular dynamics simulations of two selectivity filter mutants of a model K+ channel to investigate this question. These mutations led to a substantial decrease in conductance and ion selectivity, while accompanied by a shift from water-free K+ permeation to co-permeation of water and K+. Our findings not only provide a fundamental example of how single point mutations in the selectivity filter can alter the ion permeation mechanism, but also reinforce the notion that water exclusion underlies the remarkable efficiency of K+ channels.

biophysics↗