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Energetic role of the paddle motif in voltage gating of Shaker K+ channels

Nature Structural & Molecular Biology volume 20, pages 574–581 (2013)Cite this article

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Abstract

Voltage-gated ion channels underlie rapid electric signaling in excitable cells. Electrophysiological studies have established that the N-terminal half of the fourth transmembrane segment (NTS4) of these channels is the primary voltage sensor, whereas crystallographic studies have shown that NTS4 is not located within a proteinaceous pore. Rather, NTS4 and the C-terminal half of S3 (CTS3 or S3b) form a helix-turn-helix motif, termed the voltage-sensor paddle. This unexpected structural finding raises two fundamental questions: does the paddle motif also exist in voltage-gated channels in a biological membrane, and, if so, what is its function in voltage gating? Here, we provide evidence that the paddle motif exists in the open state of Drosophila Shaker voltage-gated K+ channels expressed in Xenopus oocytes and that CTS3 acts as an extracellular hydrophobic 'stabilizer' for NTS4, thus biasing the gating chemical equilibrium toward the open state.

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Figure 1: Simultaneous replacement of ten residues in CTS3 with a single residue type.
Figure 2: Deletion analysis of CTS3.
Figure 3: Stepwise deletions in CTS3 and the S3–S4 linker.
Figure 4: Deletion analysis of CTS3 through NTS4.
Figure 5: Cysteine point mutations of CTS3 in the presence of a hexacysteine mutation in NTS4.
Figure 6: Cysteine mutation of individual hydrophobic residues in NTS4 in the presence of I325C in CTS3.
Figure 7: Cysteine pairs between CTS3 and NTS4 that lock the channels in the open state.
Figure 8: Biochemical examination of disulfide-bond formation between cysteine pairs in the paddle motif.
Figure 9: Partial structures of Kv1.2-2.1.

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References

  1. Armstrong, C.M. & Bezanilla, F. Currents related to movement of the gating particles of the sodium channels. Nature 242, 459–461 (1973).

    Article  CAS  Google Scholar 

  2. Noda, M. et al. Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature 312, 121–127 (1984).

    Article  CAS  Google Scholar 

  3. Catterall, W.A. Molecular properties of voltage-sensitive sodium channels. Annu. Rev. Biochem. 55, 953–985 (1986).

    Article  CAS  Google Scholar 

  4. Yang, N., George, A.L. Jr. & Horn, R. Molecular basis of charge movement in voltage-gated sodium channels. Neuron 16, 113–122 (1996).

    Article  Google Scholar 

  5. Larsson, H.P., Baker, O.S., Dhillon, D.S. & Isacoff, E.Y. Transmembrane movement of the shaker K+ channel S4. Neuron 16, 387–397 (1996).

    Article  CAS  Google Scholar 

  6. Mannuzzu, L.M., Moronne, M.M. & Isacoff, E.Y. Direct physical measure of conformational rearrangement underlying potassium channel gating. Science 271, 213–216 (1996).

    Article  CAS  Google Scholar 

  7. Stühmer, W. et al. Structural parts involved in activation and inactivation of the sodium channel. Nature 339, 597–603 (1989).

    Article  Google Scholar 

  8. Papazian, D.M., Timpe, L.C., Jan, Y.N. & Jan, L.Y. Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence. Nature 349, 305–310 (1991).

    Article  CAS  Google Scholar 

  9. Papazian, D.M. et al. Electrostatic interactions of S4 voltage sensor in Shaker K+ channel. Neuron 14, 1293–1301 (1995).

    Article  CAS  Google Scholar 

  10. Liman, E.R., Hess, P., Weaver, F. & Koren, G. Voltage-sensing residues in the S4 region of a mammalian K+ channel. Nature 353, 752–756 (1991).

    Article  CAS  Google Scholar 

  11. Aggarwal, S.K. & MacKinnon, R. Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron 16, 1169–1177 (1996).

    Article  CAS  Google Scholar 

  12. Seoh, S.A., Sigg, D., Papazian, D.M. & Bezanilla, F. Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron 16, 1159–1167 (1996).

    Article  CAS  Google Scholar 

  13. Schoppa, N.E., McCormack, K., Tanouye, M.A. & Sigworth, F.J. The size of gating charge in wild-type and mutant Shaker potassium channels. Science 255, 1712–1715 (1992).

    Article  CAS  Google Scholar 

  14. Islas, L.D. & Sigworth, F.J. Voltage sensitivity and gating charge in Shaker and Shab family potassium channels. J. Gen. Physiol. 114, 723–742 (1999).

    Article  CAS  Google Scholar 

  15. Zagotta, W.N., Hoshi, T., Dittman, J. & Aldrich, R.W. Shaker potassium channel gating. II: transitions in the activation pathway. J. Gen. Physiol. 103, 279–319 (1994).

    Article  CAS  Google Scholar 

  16. Long, S.B., Tao, X., Campbell, E.B. & MacKinnon, R. Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature 450, 376–382 (2007).

    Article  CAS  Google Scholar 

  17. Pless, S.A., Galpin, J.D., Niciforovic, A.P. & Ahern, C.A. Contributions of counter-charge in a potassium channel voltage-sensor domain. Nat. Chem. Biol. 7, 617–623 (2011).

    Article  CAS  Google Scholar 

  18. Starace, D.M. & Bezanilla, F. A proton pore in a potassium channel voltage sensor reveals a focused electric field. Nature 427, 548–553 (2004).

    Article  CAS  Google Scholar 

  19. Ahern, C.A. & Horn, R. Focused electric field across the voltage sensor of potassium channels. Neuron 48, 25–29 (2005).

    Article  CAS  Google Scholar 

  20. Tao, X., Lee, A., Limapichat, W., Dougherty, D.A. & MacKinnon, R. A gating charge transfer center in voltage sensors. Science 328, 67–73 (2010).

    Article  CAS  Google Scholar 

  21. 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 

  22. Hackos, D.H., Chang, T.H. & Swartz, K.J. Scanning the intracellular S6 activation gate in the shaker K+ channel. J. Gen. Physiol. 119, 521–532 (2002).

    Article  CAS  Google Scholar 

  23. 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 

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

    Article  CAS  Google Scholar 

  25. Lu, Z., Klem, A.M. & Ramu, Y. Ion conduction pore is conserved among potassium channels. Nature 413, 809–813 (2001).

    Article  CAS  Google Scholar 

  26. Lu, Z., Klem, A.M. & Ramu, Y. Coupling between voltage sensors and activation gate in voltage-gated K+ channels. J. Gen. Physiol. 120, 663–676 (2002).

    Article  CAS  Google Scholar 

  27. Tristani-Firouzi, M., Chen, J. & Sanguinetti, M.C. Interactions between S4–S5 linker and S6 transmembrane domain modulate gating of HERG K+ channels. J. Biol. Chem. 277, 18994–19000 (2002).

    Article  CAS  Google Scholar 

  28. Long, S.B., Campbell, E.B. & MacKinnon, R. Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309, 903–908 (2005).

    Article  CAS  Google Scholar 

  29. Jiang, Y. et al. X-ray structure of a voltage-dependent K+ channel. Nature 423, 33–41 (2003).

    Article  CAS  Google Scholar 

  30. Long, S.B., Campbell, E.B. & MacKinnon, R. Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309, 897–903 (2005).

    Article  CAS  Google Scholar 

  31. Payandeh, J., Scheuer, T., Zheng, N. & Catterall, W.A. The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011).

    Article  CAS  Google Scholar 

  32. Payandeh, J., Gamal El-Din, T.M., Scheuer, T., Zheng, N. & Catterall, W.A. Crystal structure of a voltage-gated sodium channel in two potentially inactivated states. Nature 486, 135–139 (2012).

    Article  CAS  Google Scholar 

  33. Zhang, X. et al. Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel. Nature 486, 130–134 (2012).

    Article  CAS  Google Scholar 

  34. Xu, Y., Ramu, Y. & Lu, Z. A Shaker K+ channel with a miniature engineered voltage sensor. Cell 142, 580–589 (2010).

    Article  CAS  Google Scholar 

  35. Alabi, A.A., Bahamonde, M.I., Jung, H.J., Kim, J.I. & Swartz, K.J. Portability of paddle motif function and pharmacology in voltage sensors. Nature 450, 370–375 (2007).

    Article  CAS  Google Scholar 

  36. Broomand, A. & Elinder, F. Large-scale movement within the voltage-sensor paddle of a potassium channel-support for a helical-screw motion. Neuron 59, 770–777 (2008).

    Article  CAS  Google Scholar 

  37. Henrion, U. et al. Tracking a complete voltage-sensor cycle with metal-ion bridges. Proc. Natl. Acad. Sci. USA 109, 8552–8557 (2012).

    Article  CAS  Google Scholar 

  38. Aziz, Q.H., Partridge, C.J., Munsey, T.S. & Sivaprasadarao, A. Depolarization induces intersubunit cross-linking in a S4 cysteine mutant of the Shaker potassium channel. J. Biol. Chem. 277, 42719–42725 (2002).

    Article  CAS  Google Scholar 

  39. Lainé, M. et al. Atomic proximity between S4 segment and pore domain in Shaker potassium channels. Neuron 39, 467–481 (2003).

    Article  Google Scholar 

  40. Elliott, D.J. et al. Molecular mechanism of voltage sensor movements in a potassium channel. EMBO J. 23, 4717–4726 (2004).

    Article  CAS  Google Scholar 

  41. Phillips, L.R. et al. Voltage-sensor activation with a tarantula toxin as cargo. Nature 436, 857–860 (2005).

    Article  CAS  Google Scholar 

  42. Ruta, V., Chen, J. & MacKinnon, R. Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel. Cell 123, 463–475 (2005).

    Article  CAS  Google Scholar 

  43. Campos, F.V., Chanda, B., Roux, B. & Bezanilla, F. Two atomic constraints unambiguously position the S4 segment relative to S1 and S2 segments in the closed state of Shaker K channel. Proc. Natl. Acad. Sci. USA 104, 7904–7909 (2007).

    Article  CAS  Google Scholar 

  44. Grabe, M., Lai, H.C., Jain, M., Jan, Y.N. & Jan, L.Y. Structure prediction for the down state of a potassium channel voltage sensor. Nature 445, 550–553 (2007).

    Article  CAS  Google Scholar 

  45. Posson, D.J. & Selvin, P.R. Extent of voltage sensor movement during gating of shaker K+ channels. Neuron 59, 98–109 (2008).

    Article  CAS  Google Scholar 

  46. Chakrapani, S., Sompornpisut, P., Intharathep, P., Roux, B. & Perozo, E. The activated state of a sodium channel voltage sensor in a membrane environment. Proc. Natl. Acad. Sci. USA 107, 5435–5440 (2010).

    Article  CAS  Google Scholar 

  47. DeCaen, P.G., Yarov-Yarovoy, V., Scheuer, T. & Catterall, W.A. Gating charge interactions with the S1 segment during activation of a Na+ channel voltage sensor. Proc. Natl. Acad. Sci. USA 108, 18825–18830 (2011).

    Article  CAS  Google Scholar 

  48. Lin, M.C., Hsieh, J.Y., Mock, A.F. & Papazian, D.M. R1 in the Shaker S4 occupies the gating charge transfer center in the resting state. J. Gen. Physiol. 138, 155–163 (2011).

    Article  CAS  Google Scholar 

  49. Hoshi, T. & Armstrong, C.M. Initial steps in the opening of a Shaker potassium channel. Proc. Natl. Acad. Sci. USA 109, 12800–12804 (2012).

    Article  CAS  Google Scholar 

  50. Jensen, M.Ø. et al. Mechanism of voltage gating in potassium channels. Science 336, 229–233 (2012).

    Article  CAS  Google Scholar 

  51. Yarov-Yarovoy, V. et al. Structural basis for gating charge movement in the voltage sensor of a sodium channel. Proc. Natl. Acad. Sci. USA 109, E93–E102 (2012).

    Article  CAS  Google Scholar 

  52. Armstrong, C.M. Sodium channels and gating currents. Physiol. Rev. 61, 644–683 (1981).

    Article  CAS  Google Scholar 

  53. Schmidt, D., Jiang, Q.X. & MacKinnon, R. Phospholipids and the origin of cationic gating charges in voltage sensors. Nature 444, 775–779 (2006).

    Article  CAS  Google Scholar 

  54. Xu, Y., Ramu, Y. & Lu, Z. Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels. Nature 451, 826–829 (2008).

    Article  CAS  Google Scholar 

  55. Woolfson, D.N. The design of coiled-coil structures and assemblies. Adv. Protein Chem. 70, 79–112 (2005).

    Article  CAS  Google Scholar 

  56. Lopez, G.A., Jan, Y.N. & Jan, L.Y. Hydrophobic substitution mutations in the S4 sequence alter voltage-dependent gating in Shaker K+ channels. Neuron 7, 327–336 (1991).

    Article  CAS  Google Scholar 

  57. Smith-Maxwell, C.J., Ledwell, J.L. & Aldrich, R.W. Uncharged S4 residues and cooperativity in voltage-dependent potassium channel activation. J. Gen. Physiol. 111, 421–439 (1998).

    Article  CAS  Google Scholar 

  58. Li-Smerin, Y., Hackos, D.H. & Swartz, K.J. A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel. Neuron 25, 411–423 (2000).

    Article  CAS  Google Scholar 

  59. Soler-Llavina, G.J., Chang, T.H. & Swartz, K.J. Functional interactions at the interface between voltage-sensing and pore domains in the Shaker Kv channel. Neuron 52, 623–634 (2006).

    Article  CAS  Google Scholar 

  60. Hessa, T. et al. Molecular code for transmembrane-helix recognition by the Sec61 translocon. Nature 450, 1026–1030 (2007).

    Article  CAS  Google Scholar 

  61. Tempel, B.L., Papazian, D.M., Schwarz, T.L., Jan, Y.N. & Jan, L.Y. Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila. Science 237, 770–775 (1987).

    Article  CAS  Google Scholar 

  62. Timpe, L.C. et al. Expression of functional potassium channels from Shaker cDNA in Xenopus oocytes. Nature 331, 143–145 (1988).

    Article  CAS  Google Scholar 

  63. Pongs, O. et al. Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila. EMBO J. 7, 1087–1096 (1988).

    Article  CAS  Google Scholar 

  64. Kamb, A., Tseng-Crank, J. & Tanouye, M.A. Multiple products of the Drosophila Shaker gene may contribute to potassium channel diversity. Neuron 1, 421–430 (1988).

    Article  CAS  Google Scholar 

  65. Hoshi, T., Zagotta, W.N. & Aldrich, R.W. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250, 533–538 (1990).

    Article  CAS  Google Scholar 

  66. Liman, E.R., Tytgat, J. & Hess, P. Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 9, 861–871 (1992).

    Article  CAS  Google Scholar 

  67. Spassova, M. & Lu, Z. Coupled ion movement underlies rectification in an inward-rectifier K+ channel. J. Gen. Physiol. 112, 211–221 (1998).

    Article  CAS  Google Scholar 

  68. Garcia, M.L., Garcia-Calvo, M., Hidalgo, P., Lee, A. & MacKinnon, R. Purification and characterization of three inhibitors of voltage-dependent K+ channels from Leiurus quinquestriatus var. hebraeus venom. Biochemistry 33, 6834–6839 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank P. De Weer (University of Pennsylvania) for critical review of our manuscript. This study was supported by grant GM55560 from the US National Institutes of Health. Z.L. is supported as an investigator of the Howard Hughes Medical Institute.

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  1. Yanping Xu and Yajamana Ramu: These authors contributed equally to this work.

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  1. Department of Physiology, Howard Hughes Medical Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA

    Yanping Xu, Yajamana Ramu, Hyeon-Gyu Shin, Jayden Yamakaze & Zhe Lu

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All authors designed research and performed experiments; Y.X., Y.R., H.-G.S. and J.Y. analyzed data; Y.X., Y.R., J.Y. and Z.L. wrote the manuscript.

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Correspondence to Zhe Lu.

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Xu, Y., Ramu, Y., Shin, HG. et al. Energetic role of the paddle motif in voltage gating of Shaker K+ channels. Nat Struct Mol Biol 20, 574–581 (2013). https://doi.org/10.1038/nsmb.2535

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