Rate of Recovery from Slow Inactivation in K+ Channels Controlled by Buried Water Molecules

Abstract

The transition of the selectivity filter of K+ channels between its two known important functional states, namely the conductive state and the slow (c-type) inactivated state, is coupled to the opening and closing of the activation (intracellular) gate. Opening the activation gate by applying an external stimulus results in a transient current before the selectivity filter undergoes a spontaneous transition toward the non-conductive inactivated conformation that blocks the passage of ionic current through the channel. Only after closing the activation gate by removing the external stimulus will the selectivity filter return back to its original conductive conformation, resetting the filter so that it may once again pass current. While it is thought that recovery from the non-conductive inactivated state involves subtle conformational changes of the selectivity filter, the reason why this process can take up to several seconds, which is extremely slow on the molecular timescale, is not understood. Our results from a series of MD simulations reveal the selectivity filter is sterically locked in the inactive conformation for more than 15 microseconds by 12 buried water molecules, 3 for each subunit, that are strongly bound behind the filter. Even the presence of a few of these buried waters appears to lock the selectivity filter in the inactive conformation, blocking the filter from returning to a conductive conformation until the buried waters spontaneously vacate each subunit. Such an event would be rare, stretching the process of recovery to the timescale of seconds. To validate this mechanism, experiments were conducted where an osmotic stress was applied on the extracellular side of the channel to decrease the probability of waters occupying the cavities located behind the filter. As predicted, this accelerated the rate of recovery from slow inactivation.