Abstract
Block by Ba2+ is a distinctive property of K+ channels, and in a few cases this block can be used as a tool to determine the affinity for various ions at specific sites in the selectivity filter. We measure the discrete block of single E71A KcsA channels, a non-inactivating mutant, with micromolar concentrations of internal Ba2+ and find at high concentrations of external K+ the block time distribution is described by a double exponential. This suggests there are 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, we can determine the occupancy of K+ and other cationic monovalent ions in an extracellular site, presumably S1, and thus determine selectivity for this particular site. Our kinetic data has shown KcsA has an unusually high selectivity for K+ over Na+ with a ddG0 of −5.6 kcal mol−1.
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