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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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.
References
Hille, B. I on channels of excitable membranes, xviii (Sinauer, Sunderland, Mass., 2001).
Doyle, D. A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998).
Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0A resolution. Nature 414, 43–48 (2001).
Varma, S., Rogers, D. M., Pratt, L. R. & Rempe, S. B. Perspectives on: ion selectivity: design principles for K+ selectivity in membrane transport. J. Gen. Physiol. 137, 479–488 (2011).
Roux, B. Ion conduction and selectivity in K+ channels. Annu. Rev. Biophys. Biomol. Struct. 34, 153–171 (2005).
Bernèche, S. & Roux, B. Energetics of ion conduction through the K+ channel. Nature 414, 73–77 (2001).
Aqvist, J. & Luzhkov, V. Ion permeation mechanism of the potassium channel. Nature 404, 881–884 (2000).
Noskov, S. Y., Bernèche, S. & Roux, B. Control of ion selectivity in potassium channels by electrostatic and dynamic properties of carbonyl ligands. Nature 431, 830–834 (2004).
Shrivastava, I. H., Tieleman, D. P., Biggin, P. C. & Sansom, M. S. K. K+ versus Na+ ions in a K channel selectivity filter: a simulation study. Biophys. J. 83, 633–645 (2002).
Wang, W. & MacKinnon, R. Cryo-EM structure of the open human ether-a-go-go-related K+ channel hERG. Cell 169, 422–430.e10 (2017).
Cuello, L. G., Jogini, V., Cortes, D. M. & Perozo, E. Structural mechanism of C-type inactivation in K+ channels. Nature 466, 203–208 (2010).
Labro, A. J., Cortes, D. M., Tilegenova, C. & Cuello, L. G. Inverted allosteric coupling between activation and inactivation gates in K+ channels. Proc. Natl. Acad. Sci. USA. 115, 5426–5431 (2018).
Wang, S., Vafabakhsh, R., Borschel, W. F., Ha, T. & Nichols, C. G. Structural dynamics of potassium-channel gating revealed by single-molecule FRET. Nat. Struct. Mol. Biol. 23, 31–36 (2016).
Zhou, Y. & MacKinnon, R. The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates. J. Mol. Biol. 333, 965–975 (2003).
Krishnan, M. N., Bingham, J. P., Lee, S. H., Trombley, P. & Moczydlowski, E. Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel. J. Gen. Physiol. 126, 271–283 (2005).
Lockless, S. W., Zhou, M. & MacKinnon, R. Structural and thermodynamic properties of selective ion binding in a K+ channel. PLoS Biol. 5, e121 (2007).
Blanco, M. & Walter, N. G. Analysis of complex single-molecule FRET time trajectories. Methods Enzymol. 472, 153–178 (2010).
Guo, D., Ramu, Y., Klem, A. M. & Lu, Z. Mechanism of rectification in inward-rectifier K+ channels. J. Gen. Physiol. 121, 261–275 (2003).
Guo, D. & Lu, Z. Kinetics of inward-rectifier K+ channel block by quaternary alkylammonium ions. dimension and properties of the inner pore. J. Gen. Physiol. 117, 395–406 (2001).
Lenaeus, M. J., Burdette, D., Wagner, T., Focia, P. J. & Gross, A. Structures of KcsA in complex with symmetrical quaternary ammonium compounds reveal a hydrophobic binding site. Biochemistry 53, 5365–5373 (2014).
Lenaeus, M. J., Vamvouka, M., Focia, P. J. & Gross, A. Structural basis of TEA blockade in a model potassium channel. Nat. Struct. Mol. Biol. 12, 454–459 (2005).
Thompson, J. & Begenisich, T. Affinity and location of an internal K+ ion binding site in shaker K channels. J. Gen. Physiol. 117, 373–384 (2001).
Thompson, A. N. et al. Mechanism of potassium-channel selectivity revealed by Na+ and Li+ binding sites within the KcsA pore. Nat. Struct. Mol. Biol. 16, 1317–1324 (2009).
Sauer, D. B., Zeng, W., Canty, J., Lam, Y. & Jiang, Y. Sodium and potassium competition in potassium-selective and non-selective channels. Nat. Commun. 4, 2721 (2013).
McCoy, J. G. & Nimigean, C. M. Structural correlates of selectivity and inactivation in potassium channels. Biochim. Biophys. Acta 1818, 272–285 (2012).
Heginbotham, L., Lu, Z., Abramson, T. & MacKinnon, R. Mutations in the K+ channel signature sequence. Biophys. J. 66, 1061–1067 (1994).
Wang, S., Alimi, Y., Tong, A., Nichols, C. G. & Enkvetchakul, D. Differential roles of blocking ions in KirBac1.1 tetramer stability. J. Biol. Chem. 284, 2854–2860 (2009).
Enkvetchakul, D. et al. Functional characterization of a prokaryotic Kir channel. J. Biol. Chem. 279, 47076–47080 (2004).
Cheng, W. W., Enkvetchakul, D. & Nichols, C. G. KirBac1.1: it’s an inward rectifying potassium channel. J. Gen. Physiol. 133, 295–305 (2009).
Lange, A. et al. Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR. Nature 440, 959–962 (2006).
Bhate, M. P., Wylie, B. J., Tian, L. & McDermott, A. E. Conformational dynamics in the selectivity filter of KcsA in response to potassium ion concentration. J. Mol. Biol. 401, 155–166 (2010).
Krishnan, M. N., Trombley, P. & Moczydlowski, E. G. Thermal stability of the K+ channel tetramer: cation interactions and the conserved threonine residue at the innermost site (S4) of the KcsA selectivity filter. Biochemistry 47, 5354–5367 (2008).
Roux, B. et al. Ion selectivity in channels and transporters. J. Gen. Physiol. 137, 415–426 (2011).
Roux, B. Ion channels and ion selectivity. Essays Biochem. 61, 201–209 (2017).
Nimigean, C. M. & Allen, T. W. Origins of ion selectivity in potassium channels from the perspective of channel block. J. Gen. Physiol. 137, 405–413 (2011).
Dixit, P. D. & Asthagiri, D. Thermodynamics of ion selectivity in the KcsA K+ channel. J. Gen. Physiol. 137, 427–433 (2011).
Alam, A. & Jiang, Y. Structural studies of ion selectivity in tetrameric cation channels. J. Gen. Physiol. 137, 397–403 (2011).
Andersen, O. S. Perspectives on: ion selectivity. J. Gen. Physiol. 137, 393–395 (2011).
Ye, S., Li, Y. & Jiang, Y. Novel insights into K+ selectivity from high-resolution structures of an open K+ channel pore. Nat. Struct. Mol. Biol. 17, 1019–1023 (2010).
Kalinin, S. et al. A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Methods 9, 1218–1225 (2012).
Lee, C. H. & MacKinnon, R. Structures of the human HCN1 hyperpolarization-activated channel. Cell 168, 111–120.e11 (2017).
Kiss, L., Immke, D., LoTurco, J. & Korn, S. J. The interaction of Na+ and K+ in voltage-gated potassium channels. Evidence for cation binding sites of different affinity. J. Gen. Physiol. 111, 195–206 (1998).
Almers, W. & McCleskey, E. W. Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore. J. Physiol. (Lond.) 353, 585–608 (1984).
Joo, C. & Ha, T. Preparing sample chambers for single-molecule FRET. Cold Spring Harb. Protoc. 2012, 1104–1108 (2012).
Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008).
Dave, R., Terry, D. S., Munro, J. B. & Blanchard, S. C. Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging. Biophys. J. 96, 2371–2381 (2009).
Fan, J. S. & Palade, P. Perforated patch recording with β-escin. Pflugers Arch. 436, 1021–1023 (1998).
Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).
Joo, C. & Ha, T. Single-molecule FRET with total internal reflection microscopy. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.top072058 (2012).
Wang, S., Brettmann, J.B. & Nichols, C.G. in Potassium Channels (eds. Shyng, S.L., Valiyaveetil, F. & Whorton, M.) 163–180 (Springer, 2018).
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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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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DOI: https://doi.org/10.1038/s41589-019-0240-7
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