KcsA Ion Affinity at an External Site Probed by Barium Block

Abstract

Block by Ba2+ is a distinctive property of K+ channels since Ba2+, a doubly charged analog for K+, is electrostatically stabilized in the permeation pathway. Ba2+ block was used in BK channels as a tool to determine the equilibrium binding affinity for various ions at specific sites in the selectivity filter. In this work, we applied this approach to discrete block of single E71A KcsA channels, a non-inactivating mutant, in order to determine a thermodynamic measure of selectivity in a channel with abundant high-resolution structural information. We find at high concentrations of external K+ the block time distribution is described by two distinct populations of Ba2+ block events. This argues there are at least two Ba2+ sites in the selectivity filter, fitting well with the published Ba2+ containing structure of KcsA where a Ba2+ ion resides approximately in S2 and S4. Utilizing a kinetic analysis of the blocking events as a function of external K+, we determined the equilibrium dissociation constant of K+ and other monovalent cations in an extracellular site, presumably S1, to arrive at a selectivity sequence for this particular site: Rb+ (1 M) > K+ (19 M) >> Na+ (>1M). This represents an unusually high selectivity for K+ over Na+ with a ΔΔG0 of at least −7 kcal mol−1. We are currently determining affinities for Li+, NH4+, and Cs+ at this site. The results fit well with other kinetic measurements of selectivity as well as with the many structures in various ionic conditions.