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Evolutionary diversification of TTX-resistant sodium channels in a predator–prey interaction


Understanding the molecular genetic basis of adaptations provides incomparable insight into the genetic mechanisms by which evolutionary diversification takes place. Whether the evolution of common traits in different lineages proceeds by similar or unique mutations, and the degree to which phenotypic evolution is controlled by changes in gene regulation as opposed to gene function, are fundamental questions in evolutionary biology that require such an understanding of genetic mechanisms1,2,3. Here we identify novel changes in the molecular structure of a sodium channel expressed in snake skeletal muscle, tsNaV1.4, that are responsible for differences in tetrodotoxin (TTX) resistance among garter snake populations coevolving with toxic newts4. By the functional expression of tsNaV1.4, we show how differences in the amino-acid sequence of the channel affect TTX binding and impart different levels of resistance in four snake populations. These results indicate that the evolution of a physiological trait has occurred through a series of unique functional changes in a gene that is otherwise highly conserved among vertebrates.

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Figure 1: Amino-acid sequence differences for four snake populations.
Figure 2: The effect of different TTX concentrations on tsNaV1.4 and snake–human chimaeric channels.
Figure 3: TTX binding affinity of cloned channels compared with skeletal muscle in four snake populations.


  1. Rausher, M. D., Miller, R. E. & Tiffin, P. Patterns of evolutionary rate variation among genes of the anthocyanin biosynthetic pathway. Mol. Biol. Evol. 16, 266–274 (1999)

    CAS  Article  Google Scholar 

  2. Schluter, D., Clifford, E. A., Nemethy, M. & McKinnon, J. S. Parallel evolution and inheritance of quantitative traits. Am. Nat. 163, 809–822 (2004)

    Article  Google Scholar 

  3. Stern, D. L. Evolutionary developmental biology and the problem of variation. Evolution 54, 1079–1091 (2000)

    CAS  Article  Google Scholar 

  4. Brodie, E. D. Jr, Ridenhour, B. J. & Brodie, E. D. III The evolutionary response of predators to dangerous prey: hotspots and coldspots in the geographic mosaic of coevolution between garter snakes and newts. Evolution 56, 2067–2082 (2002)

    Article  Google Scholar 

  5. Beldade, P., Brakefield, P. M. & Long, A. D. Contribution of Distal-less to quantitative variation in butterfly eyespots. Nature 415, 315–318 (2002)

    ADS  CAS  Article  Google Scholar 

  6. Abzhanov, A., Protas, M., Grant, B. R., Grant, P. R. & Tabin, C. J. Bmp4 and morphological variation of beaks in Darwin's finches. Science 305, 1462–1465 (2004)

    ADS  CAS  Article  Google Scholar 

  7. Shapiro, M. D. et al. Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks. Nature 428, 717–723 (2004)

    ADS  CAS  Article  Google Scholar 

  8. Yamamoto, Y., Stock, D. W. & Jeffery, W. R. Hedgehog signalling controls eye degeneration in blind cavefish. Nature 431, 844–847 (2004)

    ADS  CAS  Article  Google Scholar 

  9. Hoekstra, H. E. & Nachman, M. W. Different genes underlie adaptive melanism in different populations of rock pocket mice. Mol. Ecol. 12, 1185–1194 (2003)

    CAS  Article  Google Scholar 

  10. ffrench-Constant, R. H., Daborn, P. J. & Le Goff, G. The genetics and genomics of insecticide resistance. Trends Genet. 20, 163–170 (2004)

    CAS  Article  Google Scholar 

  11. Hille, B. Ion Channels of Excitable Membranes (Sinauer, Sunderland, Massachusetts, 2001)

    Google Scholar 

  12. Brodie, E. D. III & Brodie, E. D. Jr Predator–prey arms races: asymmetrical selection on predators and prey may be reduced when prey are dangerous. Bioscience 49, 557–568 (1999)

    Article  Google Scholar 

  13. Janzen, F. J., Krenz, J. G., Haselkorn, T. S., Brodie, E. D. & Brodie, E. D. Molecular phylogeography of common garter snakes (Thamnophis sirtalis) in western North America: implications for regional historical forces. Mol. Ecol. 11, 1739–1751 (2002)

    CAS  Article  Google Scholar 

  14. Geffeney, S., Brodie, E. D. Jr, Ruben, P. C. & Brodie, E. D. III Mechanisms of adaptation in a predator-prey arms race: TTX-resistant sodium channels. Science 297, 1336–1339 (2002)

    ADS  CAS  Article  Google Scholar 

  15. Satin, J. et al. A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. Science 256, 1202–1205 (1992)

    ADS  CAS  Article  Google Scholar 

  16. Kaneko, Y., Matsumoto, G. & Hanyu, Y. TTX resistivity of Na+ channel in newt retinal neuron. Biochem. Biophys. Res. Commun. 240, 651–656 (1997)

    CAS  Article  Google Scholar 

  17. Yotsu-Yamashita, M. et al. Binding properties of 3H-PbTx-3 and 3H-saxitoxin to brain membranes and to skeletal muscle membranes of puffer fish Fugu pardalis and the primary structure of a voltage-gated Na+ channel alpha-subunit (fMNa1) from skeletal muscle of F. pardalis. Biochem. Biophys. Res. Commun. 267, 403–412 (2000)

    CAS  Article  Google Scholar 

  18. Terlau, H. et al. Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. FEBS Lett. 293, 93–96 (1991)

    CAS  Article  Google Scholar 

  19. Yamagishi, T., Li, R. A., Hsu, K., Marban, E. & Tomaselli, G. F. Molecular architecture of the voltage-dependent Na channel: functional evidence for alpha helices in the pore. J. Gen. Phys. 118, 171–181 (2001)

    CAS  Article  Google Scholar 

  20. Kallen, R. G. et al. Primary structure and expression of a sodium channel characteristic of denervated and immature rat skeletal muscle. Neuron 4, 233–242 (1990)

    CAS  Article  Google Scholar 

  21. Trimmer, J. S., Cooperman, S. S., Agnew, W. S. & Mandel, G. Regulation of muscle sodium channel transcripts during development and in response to denervation. Dev. Biol. 142, 360–367 (1990)

    CAS  Article  Google Scholar 

  22. Huey, R. B. & Moody, W. J. Neuroscience and evolution. Snake sodium channels resist TTX arrest. Science 297, 1289–1290 (2002)

    CAS  Article  Google Scholar 

  23. Zakon, H. H. Convergent evolution on the molecular level. Brain Behav. Evol. 59, 250–261 (2002)

    Article  Google Scholar 

  24. Lipkind, G. M. & Fozzard, H. A. KcsA crystal structure as framework for a molecular model of the Na+ channel pore. Biochemistry 39, 8161–8170 (2000)

    CAS  Article  Google Scholar 

  25. Plummer, N. W. & Meisler, M. H. Evolution and diversity of mammalian sodium channel genes. Genomics 57, 323–331 (1999)

    CAS  Article  Google Scholar 

  26. Raymond, C. K. et al. Expression of alternatively spliced sodium channel alpha-subnit genes: Unique splicing patterns are observed in dorsal root ganglia. J. Biol. Chem. 279, 46234–46241 (2004)

    CAS  Article  Google Scholar 

  27. Goldin, A. L. Resurgence of sodium channel research. Annu. Rev. Physiol. 63, 871–894 (2001)

    CAS  Article  Google Scholar 

  28. Choudhary, G., Yotsu-Yamashita, M., Shang, L., Yasumoto, T. & Dudley, S. C. Jr Interactions of the C-11 hydroxyl of tetrodotoxin with the sodium channel outer vestibule. Biophys. J. 84, 287–294 (2003)

    ADS  CAS  Article  Google Scholar 

  29. Penzotti, J. L., Fozzard, H. A., Lipkind, G. M. & Dudley, S. C. Jr Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule. Biophys. J. 75, 2647–2657 (1998)

    CAS  Article  Google Scholar 

  30. Stefani, E. & Bezanilla, F. Cut-open oocyte voltage-clamp technique. Methods Enzymol. 293, 300–318 (1998)

    CAS  Article  Google Scholar 

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We thank A. Correa for advice regarding the cut-open oocyte voltage clamp; S. Durham for advice regarding the statistical analysis; C. Feldman and M. Pfrender for advice regarding the phylogenetic analysis; J. Caldwell for primers; A. Goldin for sharing his sodium channel sequence alignment; and C. Hanifin and the USU herpetology group for comments that improved the manuscript. This work was supported by research grants from the National Institute of Health (P.C.R.) and from the National Science Foundation (E.D.B. Jr and E.D.B. III).

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Correspondence to Shana L. Geffeney.

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Supplementary information

Supplementary Figure S1

This figure shows a phylogenetic tree of voltage-gated sodium channels from a maximum-likelihood based, Bayesian analysis. (PDF 73 kb)

Supplementary Figure S2

This figure shows a schematic of the human/snake chimeric channel. (PDF 173 kb)

Supplementary Figure Legends

This file contains the figure legends for Supplementary Figure S1 and Supplementary Figure S2. (DOC 27 kb)

Supplementary Notes

This file contains Supplementary Methods and additional references references. (DOC 52 kb)

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Geffeney, S., Fujimoto, E., Brodie, E. et al. Evolutionary diversification of TTX-resistant sodium channels in a predator–prey interaction. Nature 434, 759–763 (2005).

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