Ba2+, a doubly charged analogue of K+, specifically blocks K+ channels by virtue of electrostatic stabilization in the permeation pathway. Ba2+ block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K+, the block-time distribution is double exponential, marking at least two Ba2+ sites in the selectivity filter, in accord with a Ba2+-containing crystal structure of KcsA. By analyzing block as a function of extracellular K+, we determined the equilibrium dissociation constant of K+ and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb+ (3 μM) Cs+ (23 μM) K+ (29 μM) NH4+ (440 μM) Na+ and Li+ (1 M). This represents an unusually high selectivity for K+ over Na+, with |ΔΔG| of at least 7 kcal mol?1. These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions.
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