Selective exclusion and selective binding both contribute to ion selectivity in KcsA, a model potassium channel

The selectivity filter in potassium channels, a main component of the ion permeation pathway, configures a stack of binding sites (sites S1–S4) to which K+ and other cations may bind. Specific ion binding to such sites induces changes in the filter conformation, which play a key role in defining both selectivity and permeation. Here, using the potassium channel KcsA as a model, we contribute new evidence to reinforce this assertion. First, ion binding to KcsA blocked by tetrabutylammonium at the most cytoplasmic site in the selectivity filter (S4) suggests that such a site, when in the nonconductive filter conformation, has a higher affinity for cation binding than the most extracellular S1 site. This filter asymmetry, along with differences in intracellular and extracellular concentrations of K+ versus Na+ under physiological conditions, should strengthen selection of the permeant K+ by the channel. Second, we used different K+ concentrations to shift the equilibrium between nonconductive and conductive states of the selectivity filter in which to test competitive binding of Na+. These experiments disclosed a marked decrease in the affinity of Na+ to bind the channel when the conformational equilibrium shifts toward the conductive state. This finding suggested that in addition to the selective binding of K+ and other permeant species over Na+, there is a selective exclusion of nonpermeant species from binding the channel filter, once it reaches a fully conductive conformation. We conclude that selective binding and selective exclusion of permeant and nonpermeant cations, respectively, are important determinants of ion channel selectivity.This work was funded in part by Grants BFU2012-31359 and BFU2015-66612-P from the Spanish MINECO/FEDER