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

Abstract

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.

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Figure 1: Gating, inactivation and recovery in KcsA channels.
Figure 2: Molecular dynamics simulations reveal mechanism of recovery from inactivation.
Figure 3: Two-dimensional free energy landscape of the recovery process.
Figure 4: Impact of water molecules on recovery process.

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Acknowledgements

This work was supported by the National Institute of Health through grant R01-GM062342 (J.O. and B.R) and R01-GM57846 (S.C. and E.P.). R. Hulse and C. Palka provided purified KcsA. J.O. is a student in the Graduate Program in Computational Neuroscience at the University of Chicago. This research used resources of the Oak Ridge Leadership Computing Facility located in the Oak Ridge National Laboratory, which is supported by the Office of Science of the Department of Energy under Contract DE-AC05-00OR22725. Anton computer time was provided by the National Resource for Biomedical Supercomputing and the Pittsburgh Supercomputing Center (PSC) through Grant RC2GM093307 from the National Institutes of Health and from a generous loan from D. E. Shaw. We are most grateful for the opportunity to use Anton20 and for the support from R. Roskies and M. Dittrich at the PSC.

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J.O. carried out all the final molecular dynamics simulations and 2D-PMF calculations and the computational analysis; A.C.P. initiated the molecular dynamics simulations and the 2D-PMF calculations; B.R. designed and simulated the kinetic models; S.C. and E.P. planned, carried out and analysed the experiments; B.R. conceived and supervised the entire project. All authors contributed to writing the manuscript.

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Correspondence to Benoît Roux.

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The authors declare no competing financial interests.

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Ostmeyer, J., Chakrapani, S., Pan, A. et al. Recovery from slow inactivation in K+ channels is controlled by water molecules. Nature 501, 121–124 (2013). https://doi.org/10.1038/nature12395

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