Skip to main content

Advertisement

SpringerLink
Log in

Biophysical costs associated with tetrodotoxin resistance in the sodium channel pore of the garter snake, Thamnophis sirtalis

  • Original Paper
  • Published:
Journal of Comparative Physiology A Aims and scope Submit manuscript

Abstract

Tetrodotoxin (TTX) is a potent toxin that specifically binds to voltage-gated sodium channels (NaV). TTX binding physically blocks the flow of sodium ions through NaV, thereby preventing action potential generation and propagation. TTX has different binding affinities for different NaV isoforms. These differences are imparted by amino acid substitutions in positions within, or proximal to, the TTX-binding site in the channel pore. These substitutions confer TTX-resistance to a variety of species. The garter snake Thamnophis sirtalis has evolved TTX-resistance over the course of an arms race, allowing some populations of snakes to feed on tetrodotoxic newts, including Taricha granulosa. Different populations of the garter snake have different degrees of TTX-resistance, which is closely related to the number of amino acid substitutions. We tested the biophysical properties and ion selectivity of NaV of three garter snake populations from Bear Lake, Idaho; Warrenton, Oregon; and Willow Creek, California. We observed changes in gating properties of TTX-resistant (TTXr) NaV. In addition, ion selectivity of TTXr NaV was significantly different from that of TTX-sensitive NaV. These results suggest TTX-resistance comes at a cost to performance caused by changes in the biophysical properties and ion selectivity of TTXr NaV.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

NaV:

Sodium channel

TTX:

Tetrodotoxin

TTXr:

Tetrodotoxin-resistant

TTXs:

Tetrodotoxin-sensitive

P-region:

Sodium channel pore

DI-DIV:

Domains I–IV

S1-6:

Transmembrane segments 1–6

WT:

Wild type

BL:

Bear Lake (TTX-sensitive)

WA:

Warrenton (TTX-resistant)

WC:

Willow Creek (highly TTX-resistant)

mV:

Millivolts

References

  • Armstrong CM (2006) Na channel inactivation from open and closed states. Proc Natl Acad Sci USA 103:17991–17996

    Article  CAS  PubMed  Google Scholar 

  • Benitah JP, Ranjan R, Yamagishi T, Janecki M, Tomaselli GF, Marban E (1997) Molecular motions within the pore of voltage-dependent sodium channels. Biophys J 73:603–613

    Article  CAS  PubMed  Google Scholar 

  • Benitah JP, Chen Z, Balser JR, Tomaselli GF, Marban E (1999) Molecular dynamics of the sodium channel pore vary with gating: interactions between P-segment motions and inactivation. J Neurosci 19:1577–1585

    CAS  PubMed  Google Scholar 

  • Bezanilla F (2008) How membrane proteins sense voltage. Nat Rev Mol Cell Biol 9:323–332

    Article  CAS  PubMed  Google Scholar 

  • Brodie EDI, Brodie EDJ (1999) Costs of exploiting poisonous prey: evolutionary trade-offs in a predator-prey arms race. Evolution 53:626–631

    Article  Google Scholar 

  • Brodie EDJ, Hensel JLJ, Johnson JA (1974) Toxicity of the urodele amphibians Taricha, Notophthalmus, Cynops and Paramesotriton (Salamandridae). Copeia 1974:506–511

    Article  Google Scholar 

  • Catterall WA, Goldin AL, Waxman SG (2005) International union of pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol Rev 57:397–409

    Article  CAS  PubMed  Google Scholar 

  • Cha A, Ruben PC, George AL, Fujimoto E, Bezanilla F (1999) Voltage sensors in domains III and IV, but not I and II, are immobilized by Na channel fast inactivation. Neuron 22:73–87

    Article  CAS  PubMed  Google Scholar 

  • Chahine M, Bennett PB, George AL Jr, Horn R (1994) Functional expression and properties of the human skeletal muscle sodium channel. Pflügers Arch 427:136–142

    Article  CAS  PubMed  Google Scholar 

  • Chanda B, Bezanilla F (2002) Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation. J Gen Physiol 120:629–645

    Article  CAS  PubMed  Google Scholar 

  • Chanda B, Asamoah OK, Bezanilla F (2004) Coupling interactions between voltage sensors of the sodium channel as revealed by site-specific measurements. J Gen Physiol 123:217–230

    Article  CAS  PubMed  Google Scholar 

  • Chang NS, French RJ, Lipkind GM, Fozzard HA, Dudley S Jr (1998) Predominant interactions between [mu]-conotoxin Arg-13 and the skeletal muscle Na+ channel localized by mutant cycle analysis. Biochemistry 37:4407–4419

    Article  CAS  PubMed  Google Scholar 

  • Chiamvimonvat N, Perez-Garcia MT, Tomaselli GF, Marban E (1996) Control of ion flux and selectivity by negatively charged residues in the outer mouth of rat sodium channels. J Physiol 491:51–59

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Clare JJ, Tate SN, Nobbs M, Romanos MA (2000) Voltage-gated sodium channels as therapeutic targets. Drug Discov Today 5:506–520

    Article  CAS  PubMed  Google Scholar 

  • Cummins TR, Dib-Hajj SD, Black JA, Akopian AN, Wood JN, Waxman SG (1999) A novel persistent tetrodotoxin-resistant sodium current in SNS-null and wild-type small primary sensory neurons. J Neurosci 19(RC43):1–6

    Google Scholar 

  • Eaholtz G, Scheuer T, Catterall WA (1994) Restoration of inactivation and block of open sodium channels by an inactivation gate peptide. Neuron 12:1041

    Article  CAS  PubMed  Google Scholar 

  • Favre I, Moczydlowski E, Schild L (1996) On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel. Biophys J 71:3110–3125

    Article  CAS  PubMed  Google Scholar 

  • Featherstone DE, Richmond JE, Ruben PC (1996) Interaction between fast and slow inactivation in Skm1 sodium channels. Biophys J 71:3098–3109

    Article  CAS  PubMed  Google Scholar 

  • Featherstone DE, Fujimoto E, Ruben PC (1998) A defect in skeletal muscle sodium channel deactivation exacerbates hyperexcitability in human paramyotonia congenita. J Physiol 506:627–638

    Article  CAS  PubMed  Google Scholar 

  • Geffeney S, Brodie ED Jr, Ruben PC, Brodie ED 3rd (2002) Mechanisms of adaptation in a predator-prey arms race: TTX-resistant sodium channels. Science 297:1336–1339

    Article  CAS  PubMed  Google Scholar 

  • Geffeney SL, Fujimoto E, Brodie EDI, Brodle EDJ, Ruben PC (2005) Evolutionary diversification of TTX-resistant sodium channels in a predator-prey interaction. Nature 434:759–763

    Article  CAS  PubMed  Google Scholar 

  • Gellens ME, George AL Jr, Chen LQ, Chahine M, Horn R, Barchi RL, Kallen RG (1992) Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel. Proc Natl Acad Sci USA 89:554–558

    Article  CAS  PubMed  Google Scholar 

  • Hayward LJ, Brown RH, Cannon SC (1997) Slow inactivation differs among mutant Na channels associated with myotonia and periodic paralysis. Biophys J 72:1204–1219

    Article  CAS  PubMed  Google Scholar 

  • Hilber K, Sandtner W, Kudlacek O, Glaaser IW, Weisz E, Kyle JW, French RJ, Fozzard HA, Dudley SC, Todt H (2001) The selectivity filter of the voltage-gated sodium channel is involved in channel activation. J Biol Chem 276:27831–27839

    Article  CAS  PubMed  Google Scholar 

  • Hilber K, Sandtner W, Zarrabi T, Zebedin E, Kudlacek O, Fozzard HA, Todt H (2005) Selectivity filter residues contribute unequally to pore stabilization in voltage-gated sodium channels. Biochemistry 44:13874–13882

    Article  CAS  PubMed  Google Scholar 

  • Hille B (1971) The permeability of the sodium channel to organic cations in myelinated nerve. J Gen Physiol 58:599–619

    Article  CAS  PubMed  Google Scholar 

  • Hille B (1972) The permeability of the sodium channel to metal cations in myelinated nerve. J Gen Physiol 59:637–658

    Article  CAS  PubMed  Google Scholar 

  • Hille B (2001) Ion channels of excitable membranes, 3rd edn. Sinauer, Sunderland

    Google Scholar 

  • Horn R, Ding S, Gruber HJ (2000) Immobilizing the moving parts of voltage-gated ion channels. J Gen Physiol 116:461–476

    Article  CAS  PubMed  Google Scholar 

  • Jayne BC, Bennett AF (1990) Selection on locomotor performance capacity in a natural population of garter snakes. Evolution 44:1204–1229

    Article  Google Scholar 

  • Jost MC, Hillis DM, Lu Y, Kyle JW, Fozzard HA, Zakon HH (2008) Toxin-resistant sodium channels: parallel adaptive evolution across a complete gene family. Mol Biol Evol 25:1016–1024

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Lipkind GM, Fozzard HA (2000) KcsA crystal structure as framework for a molecular model of the Na(+) channel pore. Biochemistry 39:8161–8170

    Article  CAS  PubMed  Google Scholar 

  • Maruta S, Yamaoka K, Yotsu-Yamashita M (2007) Two critical residues in P-loop regions of puffer fish Na+ channels on TTX sensitivity. Toxicon 51(3):381–387

    Article  PubMed  Google Scholar 

  • Mitrovic N, George AL, Horn R (2000) Role of domain 4 in sodium channel slow inactivation. J Gen Physiol 115:707–718

    Article  CAS  PubMed  Google Scholar 

  • Noda M, Suzuki H, Numa S, Stuhmer W (1989) A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II. FEBS Lett 259:213–216

    Article  CAS  PubMed  Google Scholar 

  • Ong BH, Tomaselli GF, Balser JR (2000) A structural rearrangement in the sodium channel pore linked to slow inactivation and use dependence. J Gen Physiol 116:653–662

    Article  CAS  PubMed  Google Scholar 

  • Pathak M, Kurtz L, Tombola F, Isacoff E (2004) The cooperative voltage sensor motion that gates a potassium channel. J Gen Physiol 125:57–69

    Article  Google Scholar 

  • Pauling L (1960) The nature of the chemical bond and the structure of molecules and crystals: an introduction to modern structural chemistry. Cornell University Press, Ithaca

    Google Scholar 

  • Penzotti JL, Fozzard HA, Lipkind GM, Dudley SC Jr (1998) Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule. Biophys J 75:2647–2657

    Article  CAS  PubMed  Google Scholar 

  • Pérez-García MT, Chiamvimonvat N, Marban E, Tomaselli GF (1996) Structure of the sodium channel pore revealed by serial cysteine mutagenesis. Proc Natl Acad Sci USA 93:300–304

    Article  PubMed  Google Scholar 

  • Pérez-García MT, Chiamvimonvat N, Ranjan R, Balser JR, Tomaselli GF, Marban E (1997) Mechanisms of sodium/calcium selectivity in sodium channels probed by cysteine mutagenesis and sulfhydryl modification. Biophys J 72:989–996

    Article  PubMed  Google Scholar 

  • Richmond JE, VanDeCarr D, Featherstone DE, George AL, Ruben PC (1997) Defective fast inactivation recovery and deactivation account for sodium channel myotonia in the I1160V mutant. Biophys J 73:1896–1903

    Article  CAS  PubMed  Google Scholar 

  • Ruben PC, Starkus JG, Rayner MD (1990) Holding potential affects the apparent voltage-sensitivity of sodium channel activation in crayfish giant axons. Biophys J 58:1169–1181

    Article  CAS  PubMed  Google Scholar 

  • Ruben PC, Starkus JG, Rayner MD (1992) Steady-state availability of sodium channels. Interactions between activation and slow inactivation. Biophys J 61:941–955

    Article  CAS  PubMed  Google Scholar 

  • Santarelli VP, Eastwood AL, Dougherty DA, Horn R, Ahern CA (2007) A cation-pi interaction discriminates among sodium channels that are either sensitive or resistant to tetrodotoxin block. J Biol Chem 282:8044–8051

    Article  CAS  PubMed  Google Scholar 

  • Satin J, Kyle JW, Chen M, Bell P, Cribbs LL, Fozzard HA, Rogart RB (1992) A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. Science 256:1202–1205

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Strausberg RL, Feingold EA, Grouse LH, Derge JG, Klausner RD, Collins FS, Wagner L, Shenmen CM, Schuler GD, Altschul SF (2002) Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc Natl Acad Sci USA 99:16899–16903

    Article  PubMed  Google Scholar 

  • Terlau H, Heinemann SH, Stuhmer W, Pusch M, Conti F, Imoto K, Numa S (1991) Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. FEBS Lett 293:93–96

    Article  CAS  PubMed  Google Scholar 

  • Todt H, Dudley SC, Kyle JW, French RJ, Fozzard HA (1999) Ultra-slow inactivation in 1 Na+ channels is produced by a structural rearrangement of the outer vestibule. Biophys J 76:1335–1345

    Article  CAS  PubMed  Google Scholar 

  • Tomaselli GF, Chiamvimonvat N, Nuss HB, Balser JR, Perez-Garcia MT, Xu RH, Orias DW, Backx PH, Marban E (1995) A mutation in the pore of the sodium channel alters gating. Biophys J 68:1814–1827

    Article  CAS  PubMed  Google Scholar 

  • Tsushima RG, Li RA, Backx PH (1997) Altered ionic selectivity of the sodium channel revealed by cysteine mutations within the pore. J Gen Physiol 109:463–475

    Article  CAS  PubMed  Google Scholar 

  • Vedantham V, Cannon SC (2000) Rapid and slow voltage-dependent conformational changes in segment IVS6 of voltage-gated Na+ channels. Biophys J 78:2943–2958

    Article  CAS  PubMed  Google Scholar 

  • Venkatesh B, Lu SQ, Dandona N, See SL, Brenner S, Soong TW (2005) Genetic basis of tetrodotoxin resistance in pufferfishes. Curr Biol 15:2069–2072

    Article  CAS  PubMed  Google Scholar 

  • Vilin YY, Fujimoto E, Ruben PC (2001) A single residue differentiates between human cardiac and skeletal muscle Na+ channel slow inactivation. Biophys J 80:2221–2230

    Article  CAS  PubMed  Google Scholar 

  • Wang JZ, Rojas CV, Zhou JH, Schwartz LS, Nicholas H, Hoffman EP (1992) Sequence and genomic structure of the human adult skeletal muscle sodium channel alpha subunit gene on 17q. Biochem Biophys Res Commun 182:794–801

    Article  CAS  PubMed  Google Scholar 

  • Xiong W, Farukhi YZ, Tian Y, DiSilvestre D, Li RA, Tomaselli GF (2006) A conserved ring of charge in mammalian Na channels: a molecular regulator of the outer pore conformation during slow inactivation. J Physiol 576:739–754

    Article  CAS  PubMed  Google Scholar 

  • Yang YC, Hsieh JY, Kuo CC (2009) The external pore loop interacts with S6 and S3–S4 linker in domain 4 to assume an essential role in gating control and anticonvulsant action in the Na+ channel. J Gen Physiol 134:95–113

    Article  CAS  PubMed  Google Scholar 

  • Yotsu-Yamashita M, Nishimori K, Nitanai Y, Isemura M, Sugimoto A, Yasumoto T (2000) Binding properties of (3)H-PbTx-3 and (3)H-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

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This research was funded by a NSERC Discovery Grant to PCR. The authors thank Dr. Yuriy Vilin for invaluable assistance. Animals were housed and handled according to regulations established by the University Animal Care Committee and the Canadian Council on Animal Care.

Author information

Authors and Affiliations

  1. Department of Biomedical Physiology and Kinesiology, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada

    Chong Hyun Lee, David K. Jones & Peter C. Ruben

  2. Department of Anesthesiology, Pharmacology and Therapeutics, and Cellular and Physiological Sciences, University of British Columbia, 2329 West Mall, Vancouver, BC, Canada

    Christopher Ahern & Maen F. Sarhan

Authors
  1. Chong Hyun Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David K. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christopher Ahern
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maen F. Sarhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter C. Ruben
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter C. Ruben.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, C.H., Jones, D.K., Ahern, C. et al. Biophysical costs associated with tetrodotoxin resistance in the sodium channel pore of the garter snake, Thamnophis sirtalis . J Comp Physiol A 197, 33–43 (2011). https://doi.org/10.1007/s00359-010-0582-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00359-010-0582-9

Keywords

Search

Navigation