Novel insights into K⁺ selectivity from high-resolution structures of an open K⁺ channel pore

K⁺ channels are highly selective for K⁺ over Na ⁺. Here we present several crystal structures of the MthK K ⁺ channel pore at up to 1.45-Åresolution. The MthK selectivity filter maintains a conductive conformation even in the absence of K⁺, allowing the channel to conduct Na⁺. The high-resolution structures, along with single-channel recordings, allow for an accurate analysis of how K⁺ competes with Na⁺ in a conductive selectivity filter. At high K⁺ concentrations, two K⁺ ions equivalently occupy the four sites in the selectivity filter, whereas at low K⁺/high Na⁺ concentrations, a single K⁺ ion remains bound in the selectivity filter, preferably at site 1 or site 3. This single K⁺ binding at low concentration effectively blocks the permeation of Na ⁺, providing a structural basis for the anomalous mole-fraction effect, a key property of multi-ion pores.