Selectivity filter mutations shift ion permeation mechanism in potassium channels

Abstract

AbstractPotassium (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 two SF mutants in the KcsA channel - 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 Sciences116, 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 a 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 exhibit exceptional efficiency and selectivity in conducting potassium ions. Elucidating the molecular mechanisms that govern ion conduction is crucial for understanding K+ channels. In this study, we investigate the effects of two mutations introduced in the highly conserved functional core - the selectivity filter - of a model K+ channel KcsA using molecular dynamics simulations. We demonstrate that these mutations lead to a substantial decrease in conductance and compromised ion selectivity, which is accompanied by a shift from water-free K+ permeation to co-permeation with water molecules. Our findings provide a fundamental example of how single point mutations in the selectivity filter alter the permeation mechanism, and reinforce the notion that water exclusion underlies the remarkable efficiency of K+ channels.