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. 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 nonintuitive manner.