Free energy landscapes of KcsA inactivation

The bacterial ion channel KcsA has become a useful model of complex K+-ion channels thanks to its single pore domain structure whose sequence shares many similarities with eukaryotic channels. Like many physiologically-relevant ion channels, KcsA inactivates after prolonged exposure to stimuli (in this case, a lowered pH). The inactivation mechanism has been heavily investigated, using structural, functional and simulations methods, but the molecular basis underlying the energetics of the process remains actively debated. In this work, we use the "string method with swarms of trajectories" enhanced sampling technique to characterize the free energy landscape lining the KcsA inactivation process. After channel opening following a pH drop, KcsA presents metastable open states leading to an inactivated state. The final inactivation step consists of a constriction of the selectivty filter and entry of three water molecules into binding sites behind each selectivity filter subunit. Based our simulations, we propose a key role for residue L81 in opening a gateway for water molecules to enter their buried sites, rather than for Y82 which has previously been suggested to act as a lid. In addition, since we found the energetically favored inactivation mechanism to be dependent on the force field, our results also address the importance of parameter choice for this type of mechanism. In particular, inactivation involves passing through the fully-open state only when using the AMBER force field. In contrast, using CHARMM, selectivity filter constriction proceeds directly from the partially open state. Finally, our simulations suggest that removing the co-purifying lipids stabilizes the partially open states, rationalizing their importance for the proper inactivation of the channel.