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Sodium Channel Mutation Leading to Saxitoxin Resistance in Clams Increases Risk of PSP

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Sodium Channel Mutation Leading to Saxitoxin Resistance in Clams Increases Risk of PSP letters to nature 15. Satin, J. et al. A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. for inter-population variation in PSP resistance within a species, Science 256, 1202–1205 (1992). 16. Kaneko, Y., Matsumoto, G. & Hanyu, Y. TTX resistivity of Na+ channel in newt retinal neuron. consistent with genetic adaptation to PSTs. Softshell clams (Mya Biochem. Biophys. Res. Commun. 240, 651–656 (1997). arenaria) from areas exposed to ‘red tides’ are more resistant to 17. Yotsu-Yamashita, M. et al. Binding properties of 3H-PbTx-3 and 3H-saxitoxin to brain membranes PSTs, as demonstrated by whole-nerve assays, and accumulate and to skeletal muscle membranes of puffer fish Fugu pardalis and the primary structure of a voltage- toxins at greater rates than sensitive clams from unexposed areas. gated Na+ channel alpha-subunit (fMNa1) from skeletal muscle of F. pardalis. Biochem. Biophys. Res. Commun. 267, 403–412 (2000). PSTs lead to selective mortality of sensitive clams. Resistance is 18. Terlau, H. et al. Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. FEBS caused by natural mutation of a single amino acid residue, which Lett. 293, 93–96 (1991). causes a 1,000-fold decrease in affinity at the saxitoxin-binding 19. Yamagishi, T., Li, R. A., Hsu, K., Marban, E. & Tomaselli, G. F. Molecular architecture of the voltage- site in the sodium channel pore of resistant, but not sensitive, dependent Na channel: functional evidence for alpha helices in the pore. J. Gen. Phys. 118, 171–181 (2001). clams. Thus PSTs might act as potent natural selection agents, 20. Kallen, R. G. et al. Primary structure and expression of a sodium channel characteristic of denervated leading to greater toxin resistance in clam populations and and immature rat skeletal muscle. Neuron 4, 233–242 (1990). increased risk of PSP in humans. Furthermore, global expansion 21. Trimmer, J. S., Cooperman, S. S., Agnew, W. S. & Mandel, G. Regulation of muscle sodium channel 6 transcripts during development and in response to denervation. Dev. Biol. 142, 360–367 (1990). of PSP to previously unaffected coastal areas might result in 22. Huey, R. B. & Moody, W. J. Neuroscience and evolution. Snake sodium channels resist TTX arrest. long-term changes to communities and ecosystems. Science 297, 1289–1290 (2002). PSP,caused by human consumption of shellfish that feed on toxic 23. Zakon, H. H. Convergent evolution on the molecular level. Brain Behav. Evol. 59, 250–261 (2002). algae, is a public health hazard and causes severe economic losses 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). globally due to bans on harvesting of contaminated shellfish and the 25. Plummer, N. W. & Meisler, M. H. Evolution and diversity of mammalian sodium channel genes. need for costly monitoring programmes. PSP-producing dino- Genomics 57, 323–331 (1999). flagellates (for example Alexandrium spp.) cause toxic blooms 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). (‘red tides’) in North America and worldwide. PSTs block conduc- 27. Goldin, A. L. Resurgence of sodium channel research. Annu. Rev. Physiol. 63, 871–894 (2001). tion of the nerve impulse by interfering with the voltage-dependent 28. Choudhary, G., Yotsu-Yamashita, M., Shang, L., Yasumoto, T. & Dudley, S. C. Jr Interactions of the increase in sodium-ion conductance that generates the action C-11 hydroxyl of tetrodotoxin with the sodium channel outer vestibule. Biophys. J. 84, 287–294 potential in nerve and muscle fibres4,5, leading to neuromuscular (2003). 29. Penzotti, J. L., Fozzard, H. A., Lipkind, G. M. & Dudley, S. C. Jr Differences in saxitoxin and paralysis. Large differences in PST accumulation between bivalve 1 tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule. Biophys. J. 75, species in vivo have been associated with in vitro differences in 2647–2657 (1998). sensitivity of isolated nerves to saxitoxin (STX), the most potent 30. Stefani, E. & Bezanilla, F. Cut-open oocyte voltage-clamp technique. Methods Enzymol. 293, 300–318 PST, and the related tetrodotoxin (TTX)2,3. STX and TTX bind to a (1998). single site in the outer pore of the Naþ channel, formed by the Supplementary Information accompanies the paper on www.nature.com/nature. amino-acid residues in the outer-pore loops located between the S5 and S6 segments of each of the four homologous domains (I–IV) of Acknowledgements We thank A. Correa for advice regarding the cut-open oocyte voltage clamp; the a-subunit7,8. Could differences in amino acid sequence in the S. Durham for advice regarding the statistical analysis; C. Feldman and M. Pfrender for advice receptor site for STX and TTX in Naþ channels cause differences 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). Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to S.L.G. ([email protected]). Sequences described in this paper have been deposited in GenBank under accession numbers AY851743, AY851744, AY851745 and AY851746. .............................................................. Sodium channel mutation leading to saxitoxin resistance in clams increases risk of PSP V. Monica Bricelj1, Laurie Connell2, Keiichi Konoki3, Scott P. MacQuarrie1, Todd Scheuer3, William A. Catterall3 & Vera L. Trainer4 1Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia B3H 3Z1, Canada 2School of Marine Sciences, University of Maine, Orono, Maine 04469, USA 3Department of Pharmacology, University of Washington, Seattle, Washington Figure 1 Responses to PSTs in two M. arenaria populations. a, Percentage of clams that 98195, USA 4NOAA Fisheries, Northwest Fisheries Science Center, Seattle, Washington 98112, burrowed after 24 h of exposure to A. tamarense (strains PR18b or CCMP115) or USA I. galbana (T-Iso) (n ¼ 2 tanks). Map shows the study sites BF (filled circle) and LE (open ............................................................................................................................................................................. circle); the PSP-affected coastline is in red. b, Mortality after 16 days of toxification with Bivalve molluscs, the primary vectors of paralytic shellfish strain PR18b (determined by visual inspection (dead clams removed) (tank 1) and by poisoning (PSP) in humans, show marked inter-species variation removal of all clams every 2 days (live clams reintroduced in sediment) (tank 2)). In a and in their capacity to accumulate PSP toxins (PSTs)1 which has a b, filled columns are results for BF clams, and open columns for LE clams. c, Tissue neural basis2,3. PSTs cause human fatalities by blocking sodium toxicity of live clams after 16 days of toxification of resistant BF and sensitive LE clams conductance in nerve fibres4,5. Here we identify a molecular basis (burrowers and non-burrowers at 24-h, respectively). All results are means ^ s.e.m. NATURE | VOL 434 | 7 APRIL 2005 | www.nature.com/nature 763 © 2005 Nature Publishing Group letters to nature in toxin sensitivity and accumulation in shellfish and thereby contribute to the risk of PSP? We have probed this question with a combination of behavioural, physiological and molecular biological approaches. Mya arenaria is a commercially important native species with wide latitudinal distribution in Atlantic North America, from the Gulf of St Lawrence to Chesapeake Bay. We collected clams from two sites with contrasting histories of PSP (Fig. 1a inset): Lepreau Basin, Bay of Fundy (BF), where annual, recurrent toxic blooms of Alexandrium spp. occur in summer9, and the Lawrencetown estuary (LE), Nova Scotia, an area with no record of PSP.Burrowing activity provided an index of susceptibility to toxins in clams10.After exposure to toxic A. tamarense cells for 24 h, most clams (a mean of 86%) from LE were unable to re-burrow after deployment at the sediment surface, whereas only 10% of clams from the BF popu- lation were compromised (Fig. 1a). Burrowing was not affected by exposure to non-toxic Isochrysis galbana (T-Iso) or to A. tamarense strain CCMP115, which is of negligible toxicity. Therefore burrow- ing incapacitation in susceptible clams is toxin-induced and is attributed to muscle paralysis. PSTs also prevented siphon retrac- tion in susceptible clams11. Sublethal effects of PSTs on burrowing and siphon retraction in a high-energy, intertidal habitat, where clams can become exposed at the sediment surface, may cause indirect mortalities through desiccation and predation. Longer-term laboratory exposure to toxic A. tamarense cells resulted in high differential mortalities between the two populations Figure 2 Nerve response of toxin-free, individual clams to STX in vitro. a, Percentage (Fig. 1b). Mortalities of LE clams were consistently higher (26–42% decrease in the compound action potential in isolated nerve preparations exposed to after 16 days of toxin exposure)
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