Letter | Published:

Black mamba venom peptides target acid-sensing ion channels to abolish pain

Nature volume 490, pages 552555 (25 October 2012) | Download Citation

Abstract

Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels1,2. In the field of pain, they led to important advances in basic research3,4,5,6 and even to clinical applications7,8. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway9, including in humans10. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain11. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia.

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Accessions

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

Mambalgin-1 cDNA and mambalgin-1 and -2 protein sequences have been deposited in GenBank and UniProt Knowledgebase under accession numbers JX428743, B3EWQ5 and B3EWQ4, respectively.

References

  1. 1.

    & Therapeutic potential of venom peptides. Nature Rev. Drug Discov. 2, 790–802 (2003)

  2. 2.

    & Conus venoms: a rich source of novel ion channel-targeted peptides. Physiol. Rev. 84, 41–68 (2004)

  3. 3.

    et al. Acid-sensing ion channels in postoperative pain. J. Neurosci. 31, 6059–6066 (2011)

  4. 4.

    et al. ASIC3, a sensor of acidic and primary inflammatory pain. EMBO J. 27, 3047–3055 (2008)

  5. 5.

    et al. A tarantula peptide against pain via ASIC1a channels and opioid mechanisms. Nature Neurosci. 10, 943–945 (2007)

  6. 6.

    & Effects of antagonists to high-threshold calcium channels upon spinal mechanisms of pain, hyperalgesia and allodynia. Pain 85, 9–18 (2000)

  7. 7.

    , , & Ziconotide for treatment of severe chronic pain. Lancet 375, 1569–1577 (2010)

  8. 8.

    & Effect of continuous intrathecal infusion of omega-conopeptides, N-type calcium-channel blockers, on behavior and antinociception in the formalin and hot-plate tests in rats. Pain 60, 83–90 (1995)

  9. 9.

    et al. Acid-sensing ion channels (ASICs): pharmacology and implication in pain. Pharmacol. Ther. 128, 549–558 (2010)

  10. 10.

    , , , & Acid-induced pain and its modulation in humans. J. Neurosci. 24, 10974–10979 (2004)

  11. 11.

    et al. A heteromeric Texas coral snake toxin targets acid-sensing ion channels to produce pain. Nature 479, 410–414 (2011)

  12. 12.

    & Structure, function and evolution of three-finger toxins: mini proteins with multiple targets. Toxicon 56, 855–867 (2010)

  13. 13.

    et al. A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons. EMBO J. 23, 1516–1525 (2004)

  14. 14.

    et al. Isolation of a tarantula toxin specific for a class of proton-gated Na+ channels. J. Biol. Chem. 275, 25116–25121 (2000)

  15. 15.

    , , , & Acid-sensing ion channel 2 (ASIC2) modulates ASIC1 H+-activated currents in hippocampal neurons. J. Biol. Chem. 279, 18296–18305 (2004)

  16. 16.

    , , & Acid sensing ion channels in dorsal spinal cord neurons. J. Neurosci. 28, 1498–1508 (2008)

  17. 17.

    et al. A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells. J. Biol. Chem. 272, 29778–29783 (1997)

  18. 18.

    , , , & A proton-gated cation channel involved in acid-sensing. Nature 386, 173–177 (1997)

  19. 19.

    , , & A sensory neuron-specific, proton-gated ion channel. Proc. Natl Acad. Sci. USA 95, 10240–10245 (1998)

  20. 20.

    Snake venom alpha-neurotoxins and other ‘three-finger’ proteins. Eur. J. Biochem. 264, 281–286 (1999)

  21. 21.

    , & ASIC-like, proton-activated currents in rat hippocampal neurons. J. Physiol. (Lond.) 539, 485–494 (2002)

  22. 22.

    , , & Heteromeric acid-sensing ion channels (ASICs) composed of ASIC2b and ASIC1a display novel channel properties and contribute to acidosis-induced neuronal death. J. Neurosci. 31, 9723–9734 (2011)

  23. 23.

    , , , & Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b. J. Biol. Chem. 276, 33782–33787 (2001)

  24. 24.

    , , , & Asic3 is a neuronal mechanosensor for pressure-induced vasodilation that protects against pressure ulcers. Nature Med. 18, 1205–1207 (2012)

  25. 25.

    , , & Cellular and molecular mechanisms of pain. Cell 139, 267–284 (2009)

  26. 26.

    Overcoming obstacles to developing new analgesics. Nature Med. 16, 1241–1247 (2010)

  27. 27.

    et al. The acid-activated ion channel ASIC contributes to synaptic plasticity, learning, and memory. Neuron 34, 463–477 (2002)

  28. 28.

    et al. Pain responses, anxiety and aggression in mice deficient in pre-proenkephalin. Nature 383, 535–538 (1996)

  29. 29.

    , , & Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. Nature 449, 316–323 (2007)

  30. 30.

    , & Purification and pharmacological characterization of peptide toxins from the black mamba (Dendroaspis polylepis) venom. Toxicon 28, 847–856 (1990)

  31. 31.

    et al. Unmasking venom gland transcriptomes in reptile venoms. Anal. Biochem. 311, 152–156 (2002)

  32. 32.

    et al. Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes. Proc. Natl Acad. Sci. USA 92, 5605–5609 (1995)

  33. 33.

    & Easier threading through web-based comparisons and cross-validations. Bioinformatics 17, 752–753 (2001)

  34. 34.

    Protein homology detection by HMM-HMM comparison. Bioinformatics 21, 951–960 (2005)

  35. 35.

    & SPARKS 2 and SP3 servers in CASP6. Proteins 61 (suppl. 7). 152–156 (2005)

  36. 36.

    et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997)

  37. 37.

    , & FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J. Mol. Biol. 310, 243–257 (2001)

  38. 38.

    MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004)

  39. 39.

    & Incremental threading optimization (TITO) to help alignment and modelling of remote homologues. Bioinformatics 14, 206–211 (1998)

  40. 40.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

  41. 41.

    , , , & Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

  42. 42.

    et al. An electrophysiological characterisation of long-term potentiation in cultured dissociated hippocampal neurones. Neuropharmacology 41, 693–699 (2001)

  43. 43.

    , , & Proinflammatory mediators, stimulators of sensory neuron excitability via the expression of acid-sensing ion channels. J. Neurosci. 22, 10662–10670 (2002)

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Acknowledgements

We are grateful to M. P. Price and M. J. Welsh for their gift of the ASIC1a–/– mice, to A. Zimmer for providing the Penk1–/– mice, to H. Schweitz and L. Beress for their gift of pre-purified peptidic fractions of black mamba venom, to J. Noël for cultures of hippocampal neurons and comments, to E. Deval, P. Inquimbert, A. Delaunay and M. Christin for discussions, to C. Heurteaux and N. Blondeau for help with stereotaxic injections, to A. Lazzari for support with plethysmography, to V. Thieffin, N. Leroudier, S. Boulakirba, T. Lemaire, C. Karoutchi and G. Marrane for technical assistance, and to C. Chevance for secretarial assistance. We thank E. Bourinet, F. Rassendren and M. B. Emerit for providing the Cav3.2, P2X2 and 5-HT3A cDNAs, respectively. This work was supported by the Fondation pour la Recherche Medicale, the Association Française contre les Myopathies and the Agence Nationale de la Recherche. Part of this work has been supported by EMMAservice under European Union contract Grant Agreement number 227490 of the EC FP7 Capacities Specific Programme.

Author information

Author notes

    • Sylvie Diochot
    •  & Anne Baron

    These authors contributed equally to this work.

    • Sabine Scarzello

    Present address: IRCAN, CNRS UMR 7284, Inserm U1081, UNS, 06107 Nice, France.

Affiliations

  1. CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, UMR 7275, 06560 Valbonne, France

    • Sylvie Diochot
    • , Anne Baron
    • , Miguel Salinas
    • , Dominique Douguet
    • , Sabine Scarzello
    • , Anne-Sophie Dabert-Gay
    • , Delphine Debayle
    • , Valérie Friend
    • , Michel Lazdunski
    •  & Eric Lingueglia
  2. Université de Nice-Sophia Antipolis, 06560 Valbonne, France

    • Sylvie Diochot
    • , Anne Baron
    • , Miguel Salinas
    • , Dominique Douguet
    • , Sabine Scarzello
    • , Anne-Sophie Dabert-Gay
    • , Delphine Debayle
    • , Valérie Friend
    • , Michel Lazdunski
    •  & Eric Lingueglia
  3. LabEx Ion Channel Science and Therapeutics, 06560 Valbonne, France

    • Sylvie Diochot
    • , Anne Baron
    • , Miguel Salinas
    • , Valérie Friend
    •  & Eric Lingueglia
  4. Clermont Université, Université d’Auvergne, NEURO-DOL, BP 10448, F-63000 Clermont-Ferrand, France

    • Abdelkrim Alloui
  5. Inserm U1107, F-63001 Clermont-Ferrand, France

    • Abdelkrim Alloui

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Contributions

S.D. and A.B. conducted a large part of the experiments including the screening and high-performance liquid chromatography purification of mambalgins (S.D.) and pain experiments, analysed the data and participated to the preparation of the manuscript. M.S. conducted the cloning of mambalgin cDNA and electrophysiological experiments. D. Douguet realized the three-dimensional modelling. S.S., A.-S.D.-G. and D. Debayle performed the mass spectrometry experiments and the amino-acid sequencing. V.F. performed validation of the siRNAs and provided technical support. A.A. was associated with pain behaviour experiments. M.L. contributed to initial aspects of the work and participated in the final preparation of the manuscript. E.L. supervised the project and participated in data analysis and manuscript preparation.

Competing interests

M. Lazdunski is a founder of Theralpha and the president of its scientific advisory board. The company has taken an option on the mambalgin patent. The other authors declare no competing financial interests.

Corresponding authors

Correspondence to Anne Baron or Eric Lingueglia.

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    Supplementary Information

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DOI

https://doi.org/10.1038/nature11494

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