Potassium-selective block of barium permeation through single KcsA channels

摘要

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 |ΔΔG⁰| 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.