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Potassium channel selectivity filter dynamics revealed by single-molecule FRET

Abstract

Potassium (K) channels exhibit exquisite selectivity for conduction of K+ ions over other cations, particularly Na+. High-resolution structures reveal an archetypal selectivity filter (SF) conformation in which dehydrated K+ ions, but not Na+ ions, are perfectly coordinated. Using single-molecule FRET (smFRET), we show that the SF-forming loop (SF-loop) in KirBac1.1 transitions between constrained and dilated conformations as a function of ion concentration. The constrained conformation, essential for selective K+ permeability, is stabilized by K+ but not Na+ ions. Mutations that render channels nonselective result in dilated and dynamically unstable conformations, independent of the permeant ion. Further, while wild-type KirBac1.1 channels are K+ selective in physiological conditions, Na+ permeates in the absence of K+. Moreover, whereas K+ gradients preferentially support 86Rb+ fluxes, Na+ gradients preferentially support 22Na+ fluxes. This suggests differential ion selectivity in constrained versus dilated states, potentially providing a structural basis for this anomalous mole fraction effect.

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Fig. 1: The SF-loop conformation is dependent on ion occupancies.
Fig. 2: Permeant ion-dependent kinetics of SF-loop conformational dynamics.
Fig. 3: Quaternary ammonium ions and impermeant NMDG stabilize low FRET states.
Fig. 4: Nonselective KirBac1.1 mutants exhibit dynamic conformations that are not stabilized by either K or Na.
Fig. 5: Permeant ion induced conductance in KirBac1.

Code availability

The script that converts .tif movie files to .pma files for later data processing is available upon request. IDL scripts developed by the Ha group (fully described in refs. 48,49) were used for movie data processing (.pma files), and are available for download at https://cplc.illinois.edu/software/. Subsequent analysis was carried out using Clampfit v7.0 and in Microsoft Excel.

Data availability

All data generated or analyzed during this study are included in this article (and the Supplementary Information files) or are available from the corresponding authors upon reasonable request.

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Acknowledgements

The work was funded by NIH grant R35 HL140024 to C.G.N.

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S.W. and C.G.N. conceived and designed the studies; S.W. performed smFRET experiments and fluorescence flux assays with help from C.Z.; S.-J.L. performed radioactive rubidium flux assays; S.W. and S.-J.L. analyzed data with help from X.F., G.M. and C.G.N. S.W. and C.G.N. prepared the manuscript with editing input from other authors.

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Correspondence to Shizhen Wang or Colin G. Nichols.

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Wang, S., Lee, SJ., Maksaev, G. et al. Potassium channel selectivity filter dynamics revealed by single-molecule FRET. Nat Chem Biol 15, 377–383 (2019). https://doi.org/10.1038/s41589-019-0240-7

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