Structure, function, and ion-binding properties of a K⁺ channel stabilized in the 2,4-ion-bound configuration

Here, we present the atomic resolution crystallographic structure, the function, and the ion-binding properties of the KcsA mutants, G77A and G77C, that stabilize the 2,4-ion-bound configuration (i.e., water, K⁺, water, K⁺-ion-bound configuration) of the K⁺ channel's selectivity filter. A full functional and thermodynamic characterization of the G77A mutant revealed wild-type-like ion selectivity and apparent K⁺-binding affinity, in addition to showing a lack of C-type inactivation gating and a marked reduction in its single-channel conductance. These structures validate, from a structural point of view, the notion that 2 isoenergetic ion-bound configurations coexist within a K⁺ channel's selectivity filter, which fully agrees with the water-K⁺-ion-coupled transport detected by streaming potential measurements.