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.