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Three-dimensional architecture and gating mechanism of a K+ channel studied by EPR spectroscopy

Nature Structural Biology volume 5, pages 459–469 (1998)Cite this article

Abstract

The transmembrane organization of a potassium channel from Streptomyces lividans has been studied using site directed spin labeling techniques and electron paramagnetic resonance spectroscopy. In the tetrameric channel complex, two α-helices were identified per monomer and assigned to the amino acid sequence. Probe mobility and accessibility data clearly establish that the first helix (TM1) is located in the perimeter of the channel, showing extensive protein–lipid contacts, while the second helix (TM2) is closer to the four-fold symmetric axis of the channel, lining the intracellular vestibule. A large conformational change in the C-terminal end of TM2 was measured when comparing conditions that favor either the open or closed states. The present data suggest that the diameter of the internal vestibule increases with channel opening.

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References

  1. Hille, B. Ion channels of excitable membranes, (Sinauer, Sunderland, Massachusetts; (1992).

    Google Scholar 

  2. Miller, C. 1990: annus mirabilis of potassium channels. Science 252, 1092–1096 (1991).

    Article  CAS  Google Scholar 

  3. Jan, L.Y. & Jan, Y.N. Potassium channels and their evolving gates. Nature 371, 119–122 (1994).

    Article  CAS  Google Scholar 

  4. Jan, L.Y. & Jan, Y.N. Cloned potassium channels from eukaryotes and prokaryotes. Annu. Rev. Neurosci. 20, 91–123 (1997).

    Article  CAS  Google Scholar 

  5. Schrempf, H. et al. A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans. EMBO J. 14, 5170–5178 (1995).

    Article  CAS  Google Scholar 

  6. Heginbotham, L., Abramson, T. & MacKinnon, R. A functional connection between the pores of distantly related ion channels as revealed by mutant K+ channels. Science 258, 1152–1155 (1992).

    Article  CAS  Google Scholar 

  7. Heginbotham, L., Odessey, E. and Miller, C. Tetrameric stoichiometry of a prokariotic K+ channel. Biochemistry 36. 10335–10342 (1997).

    Article  CAS  Google Scholar 

  8. Cortes, D.M. & Perozo, E. Structural dynamics of the Streptomyces lividans K+ channel (SKC1): Oligomeric stoichiometry and stability. Biochemistry 36, 10343–10352 (1997).

    Article  CAS  Google Scholar 

  9. Tatulian, S.A., Cortes, D.M. & Perozo, E. Structural dynamics of the Streptomyces lividans K+ channel (SKC1): Secondary structure characterization from FTIR spectroscopy. FEBS Lett. 423, 205–212 (1998).

    Article  CAS  Google Scholar 

  10. Cuello, L.G., Romero, J.G., Cortes, D.M. & Perozo, E. pH-Dependent gating in the Streptomyces lividans K+ channel. Biochemistry 37, 3229–3236 (1998).

    Article  CAS  Google Scholar 

  11. Doyle, D.A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998).

    Article  CAS  Google Scholar 

  12. Hubbell, W.L. & Altenbach, C. Site-directed spin labeling of membrane proteins, (Oxford University Press, New York; 1994).

    Book  Google Scholar 

  13. Hubbell, W.L., McHaourab, H.S., Altenbach, C. & Lietzow, M.A. Watching proteins move using site-directed spin labeling. Structure 4, 779–783 (1996).

    Article  CAS  Google Scholar 

  14. Falke, J.J. et al. Structure of a bacterial sensory receptor. A site-directed sulfhydryl study. J. Biol. Chem. 263, 14850–14858 (1988).

    CAS  PubMed  Google Scholar 

  15. Rabenstein, M.D. & Shin, Y.K. Determination of the distance between two spin labels attached to a macromolecule. Proc. Natl. Acad. Sci. USA 92, 8239–8243 (1995).

    Article  CAS  Google Scholar 

  16. Hustedt, E.J., Smirnov, A.I., Laub, C.F., Cobb, C.E. & Beth, A.H. Molecular distances from dipolar coupled spin-labels—the global analysis of multifrequency continuous wave electron paramagnetic resonance data. Biophys. J. 72, 1861–1877 (1997).

    Article  CAS  Google Scholar 

  17. Mchaourab, H.S., Oh, K.J., Fang, C.J. & Hubbell, W.L. Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling. Biochemistry 36, 307–316 (1997).

    Article  CAS  Google Scholar 

  18. Altenbach, C., Marthi, T., Khorana, H.G. & Hubbell, W.L. Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants. Science 248, 1088–1092 (1990).

    Article  CAS  Google Scholar 

  19. Shin, Y.K., Levinthal, C., Levinthal, F. & Hubbell, W.L. Colicin E1 binding to membranes: time-resolved studies of spin-labeled mutants. Science 259, 960–963 (1993).

    Article  CAS  Google Scholar 

  20. Steinhoff, H.J. et al. Time-resolved detection of structural changes during the photocycle of spin-labeled bacteriorhodopsin. Science 266, 105–107 (1994).

    Article  CAS  Google Scholar 

  21. Oh, K.J. et al. Organization of diphtheria toxin T domain in bilayers: a site-directed spin labeling study. Science 273, 810–812 (1996).

    Article  CAS  Google Scholar 

  22. Farrens, D.L., Altenbach, C., Yang, K., Hubbell, W.L. & Khorana, H.G. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 274, 768–770 (1996).

    Article  CAS  Google Scholar 

  23. Subczynski, W.K. & Hyde, J.S. The diffusion-concentration product of oxygen in lipid bilayers using the spin-label T1 method. Biochim. Biophys. Acta 643, 283–291 (1981).

    Article  CAS  Google Scholar 

  24. Altenbach, C., Froncisz, W., Hyde, J.S. & Hubbell, W.L. Conformation of spin-labeled melittin at membrane surfaces investigated by pulse saturation recovery and continuous wave power saturation electron paramagnetic resonance. Biophys. J. 56, 1183–1191 (1989).

    Article  CAS  Google Scholar 

  25. Mchaourab, H.S., Lietzow, M.A., Hideg, K. & Hubbell, W.L. Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. Biochemistry 35, 7692–7704 (1996).

    Article  CAS  Google Scholar 

  26. Eisenberg, D., Weiss, R.M. & Terwifliger, T.C. The hydrophobic moment detects periodicity in protein hydrophobicity. Proc. Natl. Acad. Sci. USA 81, 140–144 (1984).

    Article  CAS  Google Scholar 

  27. Cornette, J.L. et al. Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins. J. Moi. Biol. 195, 659–685 (1987).

    Article  CAS  Google Scholar 

  28. Altenbach, C., Greenhalgh, D.A., Khorana, H.G. & Hubbell, W.L. A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin. Proc. Natl. Acad. Sci. USA 91, 1667–1671 (1994).

    Article  CAS  Google Scholar 

  29. Kulikov, A.V., Likhtenshtein, G.I., Rozantsev, E.G. & Suskina, V.I. Possibility of determining the distances between the functional groups of proteins by the method of paramagnetic labels. Biofizika 17, 42–48 (1972).

    CAS  PubMed  Google Scholar 

  30. 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. Phys. 109, 527–535 (1997).

    Article  CAS  Google Scholar 

  31. 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  Google Scholar 

  32. Walther, D., Eisenhaber, F. & Argos, P. Principles of helix-helix packing in proteins - the helical lattice superposition model. J. Mol. Biol. 255, 536–553 (1996).

    Article  CAS  Google Scholar 

  33. Bowie, J.U. Helix packing angle preferences. Nature Struct. Biol. 4, 915–917 (1997).

    Article  CAS  Google Scholar 

  34. Mingarro, I., Elofsson, A. & Vonheijne, G. Helix-helix packing in a membrane-like environment. J. Mol. Biol. 272, 633–641 (1997).

    Article  CAS  Google Scholar 

  35. Connolly, M.L. Solvent-accessible surfaces of proteins and nucleic acids. Science 221, 709–713 (1993).

    Article  Google Scholar 

  36. Lopez, G.A., Jan, Y.N. & Jan, L.Y. Evidence that the 56 segment of the Shaker voltage-gated K+ channel comprises part of the pore. Nature 367, 179–182 (1994).

    Article  CAS  Google Scholar 

  37. 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  Google Scholar 

  38. Shieh, C.C. & Kirsch, G.E. Mutational analysis of ion conduction and drug binding sites in the inner mouth of voltage-gated K+ channels. Biophys. J. 67, 2316–2325 (1994).

    Article  CAS  Google Scholar 

  39. Lu, Z. & MacKinnon, R. Electrostatic tuning of Mg2+ affinity in an inward-rectifier K+ channel. Nature 371, 243–246 (1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Armstrong, C.M., Swenson, R.P., & Taylor, S.R. Block of squid axon K channels by internally and externally applied barium ions. J. Gen. Phys. 80, 663–682 (1982).

    Article  CAS  Google Scholar 

  42. Miller, C., Latorre, R. & Reisin, I. Coupling of voltage-dependent gating and Ba++ block in the high-conductance, Ca++-activated K+ channel. J. Gen. Phys. 90, 427–449 (1987).

    Article  CAS  Google Scholar 

  43. Grissmer, S. & Cahalan, M.D. Divalent ion trapping inside potassium channels of human T lymphocytes. J. Gen. Phys. 93, 609–630 (1989).

    Article  CAS  Google Scholar 

  44. Sun, Z.P., Akabas, M.H., Goulding, E.H., Karlin, A. & Siegelbaum, S.A. Exposure of residues in the cyclic nucleotide-gated channel pore: P region structure and function in gating. Neuron 16, 141–149 (1996).

    Article  CAS  Google Scholar 

  45. Garty, H., Rudy, B. & Karlish, S.J. A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogenous populations of membrane vesicles. J. Biol. Chem. 258, 13094–13099 (1983).

    CAS  PubMed  Google Scholar 

  46. Becktel, W.J. & Schellman, J.A. Protein stability curves. Biopolymers 26, 1859–1877 (1987).

    Article  CAS  Google Scholar 

  47. Farahbakhsh, Z.T., Altenbach, C. & Hubbell, W.L Spin labeled cysteines as sensors for protein-lipid interaction and conformation in rhodopsin. Photochemistry & Photobiology 56, 1019–1033 (1992).

    Article  CAS  Google Scholar 

  48. Donnelly, D.M, Overington, J.P. & Blundell, T.L. The prediction and orientation of alpha-helices from sequence alignments: the combined use of environment-dependent substitution tables, Fourier transform methods and helix capping rules. Prot. Engng. 7, 645–653 (1994).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Molecular Physiology and Biological Physics and Center for Structural Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia, 22906-0011, USA

    Eduardo Perozo, D. Marien Cortes & Luis G. Cuello

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  1. Eduardo Perozo
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  2. D. Marien Cortes
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  3. Luis G. Cuello
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Correspondence to Eduardo Perozo.

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Perozo, E., Cortes, D. & Cuello, L. Three-dimensional architecture and gating mechanism of a K+ channel studied by EPR spectroscopy. Nat Struct Mol Biol 5, 459–469 (1998). https://doi.org/10.1038/nsb0698-459

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