A computational study of barium blockades in the KcsA potassium channel based on multi-ion potential of mean force calculations and free energy perturbation

Electrophysiological studies have established that the permeation of Ba2+ ions through the KcsA K+-channel is impeded by the presence of K+ ions in the external solution, while no effect is observed for external Na+ ions. This Ba2+ lock-in effect suggests that at least one of the external binding sites of the KcsA channel is thermodynamically selective for K+. We used molecular dynamics simulations to interpret these lock-in experiments in the context of the crystallographic structure of KcsA. Assuming that the Ba2+ is bound in site S-2 in the dominant blocked state, we examine the conditions that could impede its translocation and cause the observed lock-in effect. Although the binding of a K+ ion to site S-1 when site S-2 is occupied by Ba2+ is prohibitively high in energy (>10 kcal/mol), binding to site S-0 appears to be more plausible (Delta G >4 kcal/mol). The 2D potential of mean force (PMF) for the simultaneous translocation of Ba2+ from site S-2 to site S-1 and of a K+ ion on the extracellular side shows a barrier that is consistent with the concept of external lock-in. The barrier opposing the movement of Ba2+ is very high when a cation is in site S-0, and considerably smaller when the site is unoccupied. Furthermore, free energy perturbation calculations show that site S-0 is selective for K+ by 1.8 kcal/mol when S-2 is occupied by Ba2+. However, the same site S-0 is nonselective when site S-2 is occupied by K+, which shows that the presence of Ba2+ affects the selectivity of the pore. A theoretical framework within classical rate theory is presented to incorporate the concentration dependence of the external ions on the lock-in effect.