Selectivity filter mutations shift ion permeation mechanism in potassium channels

Abstract

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.