Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Calcium-gated potassium channel blockade via membrane-facing fenestrations

Abstract

Quaternary ammonium blockers were previously shown to bind in the pore to block both open and closed conformations of large-conductance calcium-activated potassium (BK and MthK) channels. Because blocker entry was assumed through the intracellular entryway (bundle crossing), closed-pore access suggested that the gate was not at the bundle crossing. Structures of closed MthK, a Methanobacterium thermoautotrophicum homolog of BK channels, revealed a tightly constricted intracellular gate, leading us to investigate the membrane-facing fenestrations as alternative pathways for blocker access directly from the membrane. Atomistic free energy simulations showed that intracellular blockers indeed access the pore through the fenestrations, and a mutant channel with narrower fenestrations displayed no closed-state TPeA block at concentrations that blocked the wild-type channel. Apo BK channels display similar fenestrations, suggesting that blockers may use them as access paths into closed channels. Thus, membrane fenestrations represent a non-canonical pathway for selective targeting of specific channel conformations, opening novel ways to selectively drug BK channels.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Alternative entry pathways for intracellular QA blockers in the closed MthK and BK pores.
Fig. 2: TPeA and bbTBA bind below the selectivity filter and change the K+ occupancy in closed MthK structures.
Fig. 3: Fenestration and lipid binding in MthK closed structure.
Fig. 4: Free energetics of TPeA entry to the closed MthK pore.
Fig. 5: TPeA entry is impeded through the narrower fenestrations of A90L MthK closed.

Similar content being viewed by others

Data availability

The maps have been deposited in the Electron Microscopy Data Bank (EMDB) under the following accession codes: blocker-free closed MthK: 9405; TPeA-bound closed MthK: 9406; bbTBA-bound closed MthK: 9407; blocker-free closed A90L MthK: 27459; and TPeA-bound closed A88F MthK: 29605. Atomic coordinates for the three structures have been deposited in the Protein Data Bank (PDB) with accession codes 5BKI, 5BKJ, 5BKK, 8DJB and 8FZ7, respectively. Figure 5 and Extended Data Fig. 8 have raw data associated with them. Raw stopped-flow fluorescence quenching traces are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

References

  1. Hille, B. Ion Channels of Excitable Membranes 3rd edn (Sinauer Associates, Inc., 2001).

  2. Latorre, R., Oberhauser, A., Labarca, P. & Alvarez, O. Varieties of calcium-activated potassium channels. Annu. Rev. Physiol. 51, 385–399 (1989).

    Article  CAS  PubMed  Google Scholar 

  3. McManus, O. B. Calcium-activated potassium channels: regulation by calcium. J. Bioenerg. Biomembr. 23, 537–560 (1991).

    Article  CAS  PubMed  Google Scholar 

  4. Bailey, C. S., Moldenhauer, H. J., Park, S. M., Keros, S. & Meredith, A. L. KCNMA1-linked channelopathy. J. Gen. Physiol. 151, 1173–1189 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hite, R. K., Tao, X. & MacKinnon, R. Structural basis for gating the high-conductance Ca2+-activated K+ channel. Nature 541, 52–57 (2017).

    Article  CAS  PubMed  Google Scholar 

  6. Tao, X., Hite, R. K. & MacKinnon, R. Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel. Nature 541, 46–51 (2017).

    Article  CAS  PubMed  Google Scholar 

  7. Tao, X. & MacKinnon, R. Molecular structures of the human Slo1 K. channel in complex with β4.eLife 8, e51409 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Li, W. & Aldrich, R. W. State-dependent block of BK channels by synthesized Shaker ball peptides. J. Gen. Physiol. 128, 423–441 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhou, Y., Yang, H., Cui, J. & Lingle, C. J. Threading the biophysics of mammalian Slo1 channels onto structures of an invertebrate Slo1 channel. J. Gen. Physiol. 149, 985–1007 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tang, Q. Y., Zeng, X. H. & Lingle, C. J. Closed-channel block of BK potassium channels by bbTBA requires partial activation. J. Gen. Physiol. 134, 409–436 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wilkens, C. M. & Aldrich, R. W. State-independent block of BK channels by an intracellular quaternary ammonium. J. Gen. Physiol. 128, 347–364 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fan, C. et al. Ball-and-chain inactivation in a calcium-gated potassium channel. Nature 580, 288–293 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jiang, Y. et al. The open pore conformation of potassium channels. Nature 417, 523–526 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Jiang, Y. et al. Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Jiang, Y., Pico, A., Cadene, M., Chait, B. T. & MacKinnon, R. Structure of the RCK domain from the E. coli K+ channel and demonstration of its presence in the human BK channel. Neuron 29, 593–601 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Yuan, P., Leonetti, M. D., Pico, A. R., Hsiung, Y. & MacKinnon, R. Structure of the human BK channel Ca2+-activation apparatus at 3.0 Å resolution. Science 329, 182–186 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Dong, J., Shi, N., Berke, I., Chen, L. & Jiang, Y. Structures of the MthK RCK domain and the effect of Ca2+ on gating ring stability. J. Biol. Chem. 280, 41716–41724 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Wu, Y., Yang, Y., Ye, S. & Jiang, Y. Structure of the gating ring from the human large-conductance Ca2+-gated K+ channel. Nature 466, 393–397 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Posson, D. J., McCoy, J. G. & Nimigean, C. M. The voltage-dependent gate in MthK potassium channels is located at the selectivity filter. Nat. Struct. Mol. Biol. 20, 159–166 (2013).

    Article  CAS  PubMed  Google Scholar 

  21. Posson, D. J., Rusinova, R., Andersen, O. S. & Nimigean, C. M. Calcium ions open a selectivity filter gate during activation of the MthK potassium channel. Nat. Commun. 6, 8342 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Choi, K. L., Mossman, C., Aube, J. & Yellen, G. The internal quaternary ammonium receptor site of Shaker potassium channels. Neuron 10, 533–541 (1993).

    Article  CAS  PubMed  Google Scholar 

  23. Armstrong, C. M. Time course of TEA+-induced anomalous rectification in squid giant axons. J. Gen. Physiol. 50, 491–503 (1966).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Armstrong, C. M. Inactivation of the potassium conductance and related phenomena caused by quaternary ammonium ion injection in squid axons. J. Gen. Physiol. 54, 553–575 (1969).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Armstrong, C. M. Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J. Gen. Physiol. 58, 413–437 (1971).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Armstrong, C. M. & Hille, B. The inner quaternary ammonium ion receptor in potassium channels of the node of Ranvier. J. Gen. Physiol. 59, 388–400 (1972).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Choi, K. L., Aldrich, R. W. & Yellen, G. Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. Proc. Natl Acad. Sci. USA 88, 5092–5095 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Baukrowitz, T. & Yellen, G. Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K+ channels. Proc. Natl Acad. Sci. USA 93, 13357–13361 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Baukrowitz, T. & Yellen, G. Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K+ channel. Science 271, 653–656 (1996).

    Article  CAS  PubMed  Google Scholar 

  30. Li, W. & Aldrich, R. W. Unique inner pore properties of BK channels revealed by quaternary ammonium block. J. Gen. Physiol. 124, 43–57 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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).

    Article  CAS  PubMed  Google Scholar 

  32. Faraldo-Gomez, J. D. et al. Mechanism of intracellular block of the KcsA K+ channel by tetrabutylammonium: insights from X-ray crystallography, electrophysiology and replica-exchange molecular dynamics simulations. J. Mol. Biol. 365, 649–662 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. 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).

    Article  CAS  PubMed  Google Scholar 

  34. 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).

    Article  CAS  PubMed  Google Scholar 

  35. Cuello, L. G. et al. Structural basis for the coupling between activation and inactivation gates in K+ channels. Nature 466, 272–275 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Cuello, L. G., Jogini, V., Cortes, D. M. & Perozo, E. Structural mechanism of C-type inactivation in K+ channels. Nature 466, 203–208 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bouchard, G., Carrupt, P. A., Testa, B., Gobry, V. & Girault, H. H. The apparent lipophilicity of quaternary ammonium ions is influenced by galvani potential difference, not ion-pairing: a cyclic voltammetry study. Pharm. Res. 18, 702–708 (2001).

    Article  CAS  PubMed  Google Scholar 

  38. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  39. Posson, D. J., Rusinova, R., Andersen, O. S. & Nimigean, C. M. Stopped-flow fluorometric ion flux assay for ligand-gated ion channel studies. Methods Mol. Biol. 1684, 223–235 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Shi, N., Zeng, W., Ye, S., Li, Y. & Jiang, Y. Crucial points within the pore as determinants of K+ channel conductance and gating. J. Mol. Biol. 411, 27–35 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Anishkin, A., Akitake, B., Kamaraju, K., Chiang, C. S. & Sukharev, S. Hydration properties of mechanosensitive channel pores define the energetics of gating. J. Phys. Condens. Matter 22, 454120 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Beckstein, O. & Sansom, M. S. Liquid–vapor oscillations of water in hydrophobic nanopores. Proc. Natl Acad. Sci. USA 100, 7063–7068 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Beckstein, O. & Sansom, M. S. A hydrophobic gate in an ion channel: the closed state of the nicotinic acetylcholine receptor. Phys. Biol. 3, 147–159 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. del Camino, D. & Yellen, G. Tight steric closure at the intracellular activation gate of a voltage-gated K+ channel. Neuron 32, 649–656 (2001).

    Article  PubMed  Google Scholar 

  45. Liu, Y., Holmgren, M., Jurman, M. E. & Yellen, G. Gated access to the pore of a voltage-dependent K+ channel. Neuron 19, 175–184 (1997).

    Article  PubMed  Google Scholar 

  46. Holmgren, M., Smith, P. L. & Yellen, G. Trapping of organic blockers by closing of voltage-dependent K+ channels: evidence for a trap door mechanism of activation gating. J. Gen. Physiol. 109, 527–535 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Jia, Z., Yazdani, M., Zhang, G., Cui, J. & Chen, J. Hydrophobic gating in BK channels. Nat. Commun. 9, 3408 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Dopico, A. M. & Bukiya, A. N. Lipid regulation of BK channel function. Front. Physiol. 5, 312 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Tian, Y. et al. Atomic determinants of BK channel activation by polyunsaturated fatty acids. Proc. Natl Acad. Sci. USA 113, 13905–13910 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Yuan, C., O’Connell, R. J., Jacob, R. F., Mason, R. P. & Treistman, S. N. Regulation of the gating of BKCa channel by lipid bilayer thickness. J. Biol. Chem. 282, 7276–7286 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Vaithianathan, T. et al. Direct regulation of BK channels by phosphatidylinositol 4,5-bisphosphate as a novel signaling pathway. J. Gen. Physiol. 132, 13–28 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Yohannan, S., Hu, Y. & Zhou, Y. Crystallographic study of the tetrabutylammonium block to the KcsA K+ channel. J. Mol. Biol. 366, 806–814 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Boiteux, C., Posson, D. J., Allen, T. W. & Nimigean, C. M. Selectivity filter ion binding affinity determines inactivation in a potassium channel. Proc. Natl Acad. Sci. USA 117, 29968–29978 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Gamal El-Din, T. M., Lenaeus, M. J., Zheng, N. & Catterall, W. A. Fenestrations control resting-state block of a voltage-gated sodium channel. Proc. Natl Acad. Sci. USA 115, 13111–13116 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Yan, Z. et al. Structure of the Nav1.4-β1 complex from electric eel. Cell 170, 470–482 (2017).

    Article  CAS  PubMed  Google Scholar 

  56. Boiteux, C. et al. Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel. Proc. Natl Acad. Sci. USA 111, 13057–13062 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Zhao, Y. et al. Molecular basis for ligand modulation of a mammalian voltage-gated Ca2+ channel. Cell 177, 1495–1506 (2019).

    Article  CAS  PubMed  Google Scholar 

  58. Wu, J. et al. Structure of the voltage-gated calcium channel Cav1.1 complex. Science 350, aad2395 (2015).

    Article  PubMed  Google Scholar 

  59. Dong, Y. Y. et al. K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac. Science 347, 1256–1259 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Chen, Y. et al. Structure of the STRA6 receptor for retinol uptake. Science 353, aad8266 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Jorgensen, C. et al. Lateral fenestrations in K+-channels explored using molecular dynamics simulations. Mol. Pharm. 13, 2263–2273 (2016).

    Article  CAS  PubMed  Google Scholar 

  62. Smith, F. J., Pau, V. P., Cingolani, G. & Rothberg, B. S. Structural basis of allosteric interactions among Ca2+-binding sites in a K+ channel RCK domain. Nat. Commun. 4, 2621 (2013).

    Article  PubMed  Google Scholar 

  63. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    Article  CAS  PubMed  Google Scholar 

  64. Zivanov, J. et al. RELION-3: new tools for automated high-resolution cryo-EM structure determination. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  67. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  68. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).

    Article  CAS  Google Scholar 

  70. Brooks, B. R. et al. CHARMM: the biomolecular simulation program. J. Comput. Chem. 30, 1545–1614 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Shaw, D. E. et al. Anton 2: raising the bar for performance and programmability in a special-purpose molecular dynamics supercomputer. Proc. Int. Conf. for High Performance Computing, Networking, Storage and Analysis 41–53 (IEEE, 2014).

  73. Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. MacKerell, A. D. Jr et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998).

    Article  CAS  PubMed  Google Scholar 

  75. MacKerell, A. D., Feig, M. & Brooks, C. L. Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput. Chem. 25, 1400–1415 (2004).

    Article  CAS  PubMed  Google Scholar 

  76. Noskov, S. Y., Berneche, S. & Roux, B. Control of ion selectivity in potassium channels by electrostatic and dynamic properties of carbonyl ligands. Nature 431, 830–834 (2004).

    Article  CAS  PubMed  Google Scholar 

  77. Vanommeslaeghe, K. et al. CHARMM general force field: a force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J. Comput. Chem. 31, 671–690 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Andersen, H. C. Molecular dynamics simulations at constant pressure and/or temperature. J. Chem. Phys. 72, 2384–2393 (1980).

    Article  CAS  Google Scholar 

  79. Feller, S. E., Zhang, Y., Pastor, R. W. & Brooks, B. R. Constant pressure molecular dynamics simulation: the Langevin piston method. J. Chem. Phys. 103, 4613–4621 (1995).

    Article  CAS  Google Scholar 

  80. Martyna, G. J., Tobias, D. J. & Klein, M. L. Constant pressure molecular dynamics algorithms. J. Chem. Phys. 101, 4177–4189 (1994).

    Article  CAS  Google Scholar 

  81. Nosé, S. A unified formulation of the constant temperature molecular dynamics methods. J. Chem. Phys. 81, 511–519 (1984).

    Article  Google Scholar 

  82. Hoover, W. G. Canonical dynamics: equilibrium phase-space distributions. Phys. Rev. A Gen. Phys. 31, 1695–1697 (1985).

    Article  PubMed  Google Scholar 

  83. Andersen, H. C. Rattle: a ‘velocity’ version of the shake algorithm for molecular dynamics calculations. J. Comput. Phys. 52, 24–34 (1983).

    Article  CAS  Google Scholar 

  84. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: an N•log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089 (1993).

    Article  CAS  Google Scholar 

  85. Shan, Y., Klepeis, J. L., Eastwood, M. P., Dror, R. O. & Shaw, D. E. Gaussian split Ewald: a fast Ewald mesh method for molecular simulation. J. Chem. Phys. 122, 054101 (2005).

    Article  Google Scholar 

  86. Torrie, G. M. & Valleau, J. P. Nonphysical sampling distributions in Monte Carlo free-energy estimation: umbrella sampling. J. Comput. Phys. 23, 187–199 (1977).

    Article  Google Scholar 

  87. Kumar, S., Rosenberg, J. M., Bouzida, D., Swendsen, R. H. & Kollman, P. A. The weighted histogram analysis method for free-energy calculations on biomolecules. I. The method. J. Comput. Chem. 13, 1011–1021 (1992).

    Article  CAS  Google Scholar 

  88. Allen, T. W., Andersen, O. S. & Roux, B. The structure of gramicidin A in a lipid bilayer environment determined using molecular dynamics simulations and solid-state NMR data. J. Am. Chem. Soc. 125, 9868–9877 (2003).

    Article  CAS  PubMed  Google Scholar 

  89. Crouzy, S., Woolf, T. B. & Roux, B. A molecular dynamics study of gating in dioxolane-linked gramicidin A channels. Biophys. J. 67, 1370–1386 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Allen, T. W., Andersen, O. S. & Roux, B. Energetics of ion conduction through the gramicidin channel. Proc. Nat. Acad. Sci. USA 101, 117–122 (2004).

    Article  CAS  PubMed  Google Scholar 

  91. Roux, B., Andersen, O. S. & Allen, T. W. Comment on ‘Free energy simulations of single and double ion occupancy in gramicidin A’ [J. Chem. Phys. 126, 105103 (2007)]. J. Chem. Phys. 128, 227101 (2008).

  92. Hummer, G. Position-dependent diffusion coefficients and free energies from Bayesian analysis of equilibrium and replica molecular dynamics simulations. New J. Phys. 7, 34 (2005).

    Article  Google Scholar 

  93. Trabuco, L. G., Villa, E., Mitra, K., Frank, J. & Schulten, K. Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure 16, 673–683 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank L. Yen, M. Kopylov and E. Eng for their support during data collection at the Simons Electron Microscopy Center and the National Resource for Automated Molecular Microscopy, located at the New York Structural Biology Center, which is supported by grants from the Simons Foundation (349247), NYSTAR and the National Institutes of Health (NIH) National Institute of General Medical Sciences (GM103310). We also thank W. Rice and B. Wang for data collection at NYU Langone Health’s Cryo-Electron Microscopy Laboratory (RRID: SCR_019202). The work presented here was sponsored, in part, by NIH GM088352 to C.N., the Australian Research Council (DP210102405 and DP2201035501) to T.W.A. and the National Health and Medical Research Council (APP1141974), the National Computational Initiative (dd7), the LIEF HPC-GPGPU Facility (LE170100200), DE Shaw Anton 2 (PSCA17045P via NIH RC2GM093307) and the Medical Advances Without Animals Trust to T.W.A. and E.F.

Author information

Authors and Affiliations

Authors

Contributions

C.F., C.N. and T.A. designed the study. C.F. and S.A. prepared cryo-EM samples and collected and analyzed cryo-EM data. N.S. and C.F. performed mutant screening and stopped-flow assays and analyzed the data. E.F. and T.A. performed and analyzed the MD simulations. C.F., C.N. and T.A. assembled the manuscript and wrote the paper, with input from all authors.

Corresponding authors

Correspondence to Toby W. Allen or Crina M. Nimigean.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Structures of closed MthK with and without blockers.

Cryo-EM map and atomic model of blocker-free (a), TPeA-bound (b), and bbTBA-bound (c) closed MthK viewed parallel to the membrane. Each subunit is in a different color. The lipids bound to the fenestration were colored red. d. Lipid density in the structures of MthK with TPeA (left) and bbTBA (right). The lipid is in yellow (for carbon atoms) and red (for oxygen atoms) sticks. The lipid density is an overlayed mesh. The pore region of two adjacent MthK subunits shown as red and blue ribbons cartoon. e. Side chains of F87 adopt different conformations in each of the four MthK subunits (red, blue, green, yellow). Density shown as mesh, bbTBA as gold stick and K+ as purple sphere.

Extended Data Fig. 2 Cryo-EM characterization of closed MthK with and without blockers.

Representative micrographs of nanodisc-reconstituted (a) blocker-free, (f) TPeA-bound, (k) bbTBA-bound and (p) blocker-free A90L MthK. Calibration bar is 100 nm. b, g, l, q. Selected 2D class averages. c, h, m, r. Cryo-EM final maps colored by local resolution. d, i, n, s. FSC curves for the gold-standard method in black, and for the atomic model with the cryo-EM map in red. e, j, o, t. Angular distribution of the particles used for the reconstruction.

Extended Data Fig. 3 Cryo-EM data processing workflows.

a. MthK TPeA dataset b. MthK-bbTBA dataset. The TPeA (a) and bbTBA (b) densities are shown separately as mesh, with the atomic models as sticks.

Extended Data Fig. 4 Blocker binding site in MthK and other K+ channels.

a. Sequence alignment of the pore regions of MthK, Human BK and KcsA channels. Residues interacting with blockers (boxed red) are conserved. Identity and homology are indicated as dark and light purple, respectively. b. Modeling of TPeA binding in MthK open state. I84 and F87 side chains are shown as sticks. Note that the side chain of F87 has rotated and no longer contributes to TPeA interaction. c. Modeling of TPeA binding in BK channel. The side chains of L312 and F315 are shown as labeled sticks. d. Overlay of MthK-TPeA and KcsA-TBA (PDB 2JK5) structures. The pore region of two opposing subunits are shown as red and blue ribbons cartoon. TPeA and TBA are shown as sticks.

Extended Data Fig. 5 Membrane fenestrations in BK and other K+ channels.

a. Fenestrations in human BK channel in EDTA (PDB 6V3G), lipid yellow ball and stick. Part of the fenestration was blocked from view by the VSD. b. No fenestration observed in human Ca2+-bound BK channel (PDB 6V38), where fenestration was filled by the bent TM6 helix. c. Left: Small fenestration in closed KcsA (PDB 1K4C). Right: No fenestration in locked-open KcsA (PDB 5VK6). d. No fenestrations in closed (PDB 3SPC) or open (PDB 3SPI) Kir2.2, respectively. e. Left: Size of fenestration (dashed line) in BK with EDTA(PDB 6V3G). Protein rendered in surface representation. The VSDs and part of the S5 helix of the red subunit are removed for clarity. The surrounding residues are labeled sticks. Fenestration is formed by S6 helices from 2 adjacent subunits. Middle: lipid (mesh) binding in BK fenestration. Dashed rectangle indicated the region shown in the right panel, where surrounding residues are labeled sticks.

Extended Data Fig. 6 MD simulation of TPeA binding and membrane partitioning.

a. Attempted unbiased ‘flooding’ snapshot of MD simulation for TPeA+ entering the closed MthK channel (after 4 μs). b. The free energy for TPeA for the first 2 μs (left) and the last 2 μs (right) of the simulation. c. TPeA entering MthK though the fenestration based on 1D US simulation (note the different free energy scales in each panel). The free energy profile for the TPeA molecule is flat in the membrane, as TPeA comes closer to the channel there is a gradual slope and a minimum of −7 kcal/mol at x = 13 Å. The TPeA molecule then encounters a barrier with the discontinuity at x ~ 10 Å, due to lack of sampling, where the TPeA molecule is trying to enter in between the two TM2 helices, motivating the need for enhanced sampling methods in Fig.4. There is a minimum at x < 7 Å where TPeA is inside the MthK pore. Error bars are standard error of means based on 5 blocks (n = 5) following equilibration (see Methods). d. Free energy profile for membrane partitioning using 1D US reveals a free energy minimum of −4.9 ± 0.7 kcal/mol relative to bulk water, extending deep inside the bilayer, reaching to ~12 Å from the center with only ~1 kcal/mol penalty. Simulations used 71 independent simulations, with error bars based on asymmetry of the free energy in left and right leaflets (n = 2) following equilibration (see Methods). Insets show TPeA positioning in the membrane at the free energy well (left) and membrane deformation due to interactions between the TPeA ion and water and lipid head groups when TPeA moves closer to the membrane center (right).

Extended Data Fig. 7 Coordination numbers for TPeA+ ions and lipid-TPeA interactions during entry to the pore.

a. Coordination number for TPeA entering though the gate. As the TPeA molecule enters through the gate the nitrogen dehydrates progressively (blue line). It can also be seen to interact with backbone carbonyl oxygens and carboxylate oxygens (red line). b. Mean number of interactions between the TPeA N and water molecules. When TPeA crosses though the fenestration it is forced to dehydrate, before rehydrating in the pore. c. Mean number of interactions between the TPeA N and protein oxygen atoms. The crossing of the fenestration is helped by interactions between the TPeA N and the protein. d-e describe lipid interactions with TPeA from 1D US simulations for TPeA entering though the gate: d. Mean number of lipid C -TPeA C interactions for each window; and e. number of lipid C -TPeA C interactions over time for TPeA at 1 Å ≤ z ≤ 8 Å. Insets below show typical lipid-TPeA interactions at z = 3 Å. f-g describe lipid interactions with TPeA in 2D US simulations for TPeA entering though the fenestration: f. Mean number of lipid C -TPeA C interactions; and g. number of lipid C -TPeA C interactions over time for windows with TPeA at x = 0 Å and 1 Å ≤ z ≤ 8 Å. Insets below show lipid-TPeA interactions at x = 0 Å and z = 6 Å.

Extended Data Fig. 8 Flux rates and TPeA block in MthK channel mutants.

a. Tl+ flux rates of wild-type, A90L, A88F and V91F MthK after activation by 17.2 mM Ca2+ for 100 ms. Data are mean ± S.D. of 2-3 biological repeats. b. TPeA block dose-response curves of closed (left) and open (right) V91F MthK after 10 s and 100 ms incubation, respectively (IC50closed = 2.05 ± 0.5 μM, IC50open = 4.55 ± 0.1 μM). For comparison, the graphs for MthK WT in dotted lines after the same incubation time are overlayed, from Posson et al. (2015)21. Symbols are mean ± S.D of n = 3 biological repeats. c. Fluorescence quench traces after incubating MthK V91F (in 0 Ca2+) with 3 μM TPeA for 0.1 (black) and 10 (cyan) s and no blocker control (green) (lines include 6 repeats each, see Methods). d. TPeA equilibration plots for closed (left) and open (right) V91F MthK with 3 μM TPeA from data as in c. Solid lines are fits with a first-order exponential decay function, with τclosed = 1.86 ± 0.40 s, and τopen = 83 ± 0.66 ms. The dashed lines are the equilibration plots for 3 μM TPeA with WT MthK, from Posson et al. (2015))21. Symbols are mean ± S.D of n = 3 biological repeats.

Source data

Extended Data Fig. 9 Structural features of MthK channel mutants.

a. Cryo-EM structure of A88F MthK closed state displays lateral fenestrations between two TM2 helices from adjacent subunits, rendered as in Fig. 3a to be compared with WT (left) and to highlight fenestration size and the residues that line it (right). The introduced F88 faces towards the TM2 of neighboring subunit and F87 changes orientation so that the fenestration is now closer towards the extracellular side and similar in size to WT. b. Fenestration calculated using the program HOLE indicates a ~3.2 A radius for A88F. Plots of the fenestration cavity radius for WT, A90L, and A88F, as indicated. c. Density (grey mesh) detail of the fenestration area for WT, A90L, and A88F with 2 adjacent subunits (shown in cartoons of different colors). Side chains are indicated and those of L90 and F88 are in yellow sticks for emphasis. d. HOLE plots for the pores of A90L (left) and A88F (right). Only two opposing subunits shown. e. Pore radii as a function of the distance along the pore calculated from d. Selectivity filter (SF), L95, and I99 indicated.

Extended Data Fig. 10 MD simulations of MthK WT, A90L and V91F.

a. Representative structures from MD simulations of WT MthK (left), A90L-MthK (middle) and V91F-MthK (right) with side (top row) and top (bottom row) view. The sidechains of residues 90 and 91 are shown as colored spheres in cyan for unmutated residues, purple for L90 and red for F91. b. An example snapshot of WT MthK with 4 lipid tails (beige spheres) sticking into the fenestration. c. Distribution plots of the fenestration radii for the different channels analyzed with HOLE, as described in Methods. d. Time series of the number of lipid tails in the pore during the simulations for WT MthK (left), A90L-MthK and V91F-MthK (right).

Supplementary information

Supplementary Information

Supplementary Table 1 and Supplementary Figs. 1–5

Reporting Summary

Supplementary Video 1

Simulation of TPeA entry into the MthK pore through the membrane-facing fenestrations

Source data

Source Data Fig. 5

Statistical source data

Source Data Extended Data Fig. 8

Statistical source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fan, C., Flood, E., Sukomon, N. et al. Calcium-gated potassium channel blockade via membrane-facing fenestrations. Nat Chem Biol 20, 52–61 (2024). https://doi.org/10.1038/s41589-023-01406-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-023-01406-2

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing