Recovery from slow inactivation in K+ channels is controlled by water molecules

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Abstract
A series of long molecular dynamics simulations shows that the K+ channel is sterically locked in the inactive conformation by buried water molecules bound behind the selectivity filter; a kinetic model deduced from the simulations shows how releasing the buried waters can elongate the timescale of the recovery period, and this hypothesis is confirmed using ‘wet’ biophysical experiments. After a stimulus has caused the gate of an ion channel to open, the channel conducts until its selectivity filter inactivates. A recovery phase follows, involving conformational transition of the closed channel from an inactive to a conductive state. The bacterial K+ channel KcsA provides an excellent model system to study the mechanisms by which slow (C-type) inactivation of the channel and recovery from the inactivated state occur. In this manuscript, the authors used a series of long molecular dynamics simulations to show that the selectivity filter is sterically locked in the inactive conformation by buried water molecules bound behind the selectivity filter. A kinetic model deduced from the simulations shows how releasing the buried waters can elongate the timescale of the recovery period, and this model is probed using 'wet' biophysical experiments. Application of a specific stimulus opens the intracellular gate of a K+ channel (activation), yielding a transient period of ion conduction until the selectivity filter spontaneously undergoes a conformational change towards a non-conductive state (inactivation). Removal of the stimulus closes the gate and allows the selectivity filter to interconvert back to its conductive conformation (recovery). Given that the structural differences between the conductive and inactivated filter are very small, it is unclear why the recovery process can take up to several seconds. The bacterial K+ channel KcsA from Streptomyces lividans can be used to help elucidate questions about channel inactivation and recovery at the atomic level. Although KcsA contains only a pore domain, without voltage-sensing machinery, it has the structural elements necessary for ion conduction, activation and inactivation1,2,3,4,5,6,7. Here we reveal, by means of a series of long molecular dynamics simulations, how the selectivity filter is sterically locked in the inactive conformation by buried water molecules bound behind the selectivity filter. Potential of mean force calculations show how the recovery process is affected by the buried water molecules and the rebinding of an external K+ ion. A kinetic model deduced from the simulations shows how releasing the buried water molecules can stretch the timescale of recovery to seconds. This leads to the prediction that reducing the occupancy of the buried water molecules by imposing a high osmotic stress should accelerate the rate of recovery, which was verified experimentally by measuring the recovery rate in the presence of a 2-molar sucrose concentration.