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.

  • Letter
  • Published:

Voltage-sensor activation with a tarantula toxin as cargo

Abstract

The opening and closing of voltage-activated Na+, Ca2+ and K+ (Kv) channels underlies electrical and chemical signalling throughout biology, yet the structural basis of voltage sensing is unknown. Hanatoxin is a tarantula toxin that inhibits Kv channels by binding to voltage-sensor paddles1,2,3,4,5, crucial helix-turn-helix motifs within the voltage-sensing domains that are composed of S3b and S4 helices6. The active surface of the toxin is amphipathic7,8, and related toxins have been shown to partition into membranes9,10,11,12, raising the possibility that the toxin is concentrated in the membrane and interacts only weakly and transiently with the voltage sensors. Here we examine the kinetics and state dependence of the toxin–channel interaction and the physical location of the toxin in the membrane. We find that hanatoxin forms a strong and stable complex with the voltage sensors, far outlasting fluctuations of the voltage sensors between resting (closed) conformations at negative voltages and activated (open) conformations at positive voltages. Toxin affinity is reduced by voltage-sensor activation, explaining why the toxin stabilizes the resting conformation. We also find that when hanatoxin partitions into membranes it is localized to an interfacial region, with Trp 30 positioned about 8.5 Å from the centre of the bilayer. These results demonstrate that voltage-sensor paddles activate with a toxin as cargo, and suggest that the paddles traverse no more than the outer half of the bilayer during activation.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Voltage-sensor paddle mutations affecting hanatoxin affinity and recovery kinetics.
Figure 2: Voltage-dependent unbinding of hanatoxin from F274A channels.
Figure 3: Rebinding of hanatoxin to F274A channels.
Figure 4: Location of hanatoxin in lipid membranes.

Similar content being viewed by others

References

  1. Lee, H. C., Wang, J. M. & Swartz, K. J. Interaction between extracellular Hanatoxin and the resting conformation of the voltage-sensor paddle in Kv channels. Neuron 40, 527–536 (2003)

    Article  CAS  Google Scholar 

  2. Swartz, K. J. & MacKinnon, R. Hanatoxin modifies the gating of a voltage-dependent K+ channel through multiple binding sites. Neuron 18, 665–673 (1997)

    Article  CAS  Google Scholar 

  3. Swartz, K. J. & MacKinnon, R. Mapping the receptor site for hanatoxin, a gating modifier of voltage-dependent K+ channels. Neuron 18, 675–682 (1997)

    Article  CAS  Google Scholar 

  4. Li-Smerin, Y. & Swartz, K. J. Localization and molecular determinants of the hanatoxin receptors on the voltage-sensing domain of a K+ channel. J. Gen. Physiol. 115, 673–684 (2000)

    Article  CAS  Google Scholar 

  5. Li-Smerin, Y. & Swartz, K. J. Helical structure of the COOH terminus of S3 and its contribution to the gating modifier toxin receptor in voltage-gated ion channels. J. Gen. Physiol. 117, 205–218 (2001)

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  7. Takahashi, H. et al. Solution structure of hanatoxin1, a gating modifier of voltage-dependent K+ channels: common surface features of gating modifier toxins. J. Mol. Biol. 297, 771–780 (2000)

    Article  CAS  Google Scholar 

  8. Wang, J. M. et al. Molecular surface of tarantula toxins interacting with voltage sensors in Kv channels. J. Gen. Physiol. 123, 455–467 (2004)

    Article  CAS  Google Scholar 

  9. Lee, S. Y. & MacKinnon, R. A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom. Nature 430, 232–235 (2004)

    Article  CAS  ADS  Google Scholar 

  10. Suchyna, T. M. et al. Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers. Nature 430, 235–240 (2004)

    Article  CAS  ADS  Google Scholar 

  11. Smith, J. J., Alphy, S., Seibert, A. L. & Blumenthal, K. M. Differential phospholipid binding by site 3 and site 4 toxins: Implications for structural variability between voltage-sensitive sodium channel domains. J. Biol. Chem. 280, 11127–11133 (2005)

    Article  CAS  Google Scholar 

  12. Jung, H. J. et al. Solution structure and lipid membrane partitioning of VSTx1, an inhibitor of the KvAP potassium channel. Biochemistry 44, 6015–6023 (2005)

    Article  CAS  Google Scholar 

  13. Rogers, J. C., Qu, Y., Tanada, T. N., Scheuer, T. & Catterall, W. A. Molecular determinants of high affinity binding of α-scorpion toxin and sea anemone toxin in the S3–S4 extracellular loop in domain IV of the Na+ channel α-subunit. J. Biol. Chem. 271, 15950–15962 (1996)

    Article  CAS  Google Scholar 

  14. Catterall, W. A. Membrane potential-dependent binding of scorpion toxin to the action potential Na+ ionophore. Studies with a toxin derivative prepared by lactoperoxidase-catalyzed iodination. J. Biol. Chem. 252, 8660–8668 (1977)

    CAS  PubMed  Google Scholar 

  15. Catterall, W. A. Binding of scorpion toxin to receptor sites associated with sodium channels in frog muscle. Correlation of voltage-dependent binding with activation. J. Gen. Physiol. 74, 375–391 (1979)

    Article  CAS  Google Scholar 

  16. McDonough, S. I., Lampe, R. A., Keith, R. A. & Bean, B. P. Voltage-dependent inhibition of N- and P-type calcium channels by the peptide toxin omega-grammotoxin-SIA. Mol. Pharmacol. 52, 1095–1104 (1997)

    Article  CAS  Google Scholar 

  17. McIntosh, T. J. & Holloway, P. W. Determination of the depth of bromine atoms in bilayers formed from bromolipid probes. Biochemistry 26, 1783–1788 (1987)

    Article  CAS  Google Scholar 

  18. Ladokhin, A. S. Evaluation of lipid exposure of tryptophan residues in membrane peptides and proteins. Anal. Biochem. 276, 65–71 (1999)

    Article  CAS  Google Scholar 

  19. Ladokhin, A. S., Jayasinghe, S. & White, S. H. How to measure and analyse tryptophan fluorescence in membranes properly, and why bother? Anal. Biochem. 285, 235–245 (2000)

    Article  CAS  Google Scholar 

  20. Jiang, Y., Ruta, V., Chen, J., Lee, A. & MacKinnon, R. The principle of gating charge movement in a voltage-dependent K+ channel. Nature 423, 42–48 (2003)

    Article  CAS  ADS  Google Scholar 

  21. Cuello, L., Cortes, D. M. & Perozo, E. Molecular architecture of the KvAP voltage-dependent K+ channel in a lipid bilayer. Science 306, 491–495 (2004)

    Article  CAS  ADS  Google Scholar 

  22. 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 

  23. 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 

  24. 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  ADS  Google Scholar 

  25. Ahern, C. A. & Horn, R. Specificity of charge-carrying residues in the voltage sensor of potassium channels. J. Gen. Physiol. 123, 205–216 (2004)

    Article  CAS  Google Scholar 

  26. Swartz, K. J. Towards a structural view of gating in potassium channels. Nature Rev. Neurosci. 5, 905–916 (2004)

    Article  CAS  Google Scholar 

  27. Islas, L. D. & Sigworth, F. J. Electrostatics and the gating pore of Shaker potassium channels. J. Gen. Physiol. 117, 69–89 (2001)

    Article  CAS  Google Scholar 

  28. Swartz, K. J. & MacKinnon, R. An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula. Neuron 15, 941–949 (1995)

    Article  CAS  Google Scholar 

  29. Ladokhin, A. S. Analysis of protein and peptide penetration into membranes by depth-dependent fluorescence quenching: theoretical considerations. Biophys. J. 76, 946–955 (1999)

    Article  CAS  ADS  Google Scholar 

  30. Abrams, F. S. & London, E. Calibration of the parallax fluorescence quenching method for determination of membrane penetration depth: refinement and comparison of quenching by spin-labelled and brominated lipids. Biochemistry 31, 5312–5322 (1992)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Diamond, L. Milescu, S. Silberberg and members of the Swartz laboratory for discussions, and the NINDS DNA sequencing facility for DNA sequencing.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kenton J. Swartz.

Ethics declarations

Competing interests

Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Revell Phillips, L., Milescu, M., Li-Smerin, Y. et al. Voltage-sensor activation with a tarantula toxin as cargo. Nature 436, 857–860 (2005). https://doi.org/10.1038/nature03873

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature03873

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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