The Effects of Botulinum Neurotoxin and Tetrodotoxin on Protein Phosphorylation in Pure Cholinergic Synaptosomes

  • Xavier Guitart
  • Jordi Marsal
  • Carles Solsona
Conference paper
Part of the NATO ASI Series book series (NSSA, volume 135)


Protein phosphorylation has been related to the molecular mechanisms of neurotransmitter release (1) and some phosphoproteins as Synapsin I are thought to be implicated in the exocytotic process (2). On the other hand, depolarization of isolated synaptic buttons (synaptosomes) results in the phosphorylation of specific proteins in rat brain synaptosomes (3) and in Torpedo electric organ synaptosomes (4). The electric organ of Torpedo is a useful model to study the acetylcholine release since it is innervated exclusively by cholinergic nerves (5), and presents an homologous structure with the neuromuscular junction. In contrast, in the electric organ of Torpedo the presynaptic nerve terminals represent as much as 2% to 3% of the total volume. A pure cholinergic synaptosomal fraction can be isolated from this organ (6). In these cholinergic synaptosomes the induced release of acetylcholine (7) and ATP (8) and the calcium fluxes (9) have been characterized. Acetylcholine release can be induced by several chemical depolarising agents as high external potassium concentration, or veratridine or the calcium ionophore A 23187. Botulinum toxin (BoNTx) blocks the acetylcholine release (see 10 and 11 for reviews) in several preparations. In cholinergic synaptosomes from the electric organ of Torpedo marmorata BoNTx inhibits the induced acetylcholine release whereas ATP release, calcium uptake and membrane potential are not affected by the toxin (12). We have used this pure cholinergic preparation to study the effect of botulinum neurotoxin type A under depolarising conditions on the rate of phosphorylation of synaptosomal proteins.


Botulinum Toxin Protein Phosphorylation Acetylcholine Release Electric Organ Botulinum Neurotoxin 
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  1. 1.
    E. J. Nestler and P. Greengard, Protein phosphorylation in the brain. Nature 305:583–588 (1983).PubMedCrossRefGoogle Scholar
  2. 2.
    F. Navone, P. Greengard and P. DeCamilli, Synapsin I in nerve terminals: selective association with small synaptic vesicles. Science 226:1209–1211 (1984).PubMedCrossRefGoogle Scholar
  3. 3.
    B. K. Krueger, J. Forn and P. Greengard, Depolarization-induced phosphorylation of specific proteins mediated by calcium ion influx in rat brain synaptosomes, J. Biol. Chem. 252:764–2773 (1977).Google Scholar
  4. 4.
    D. M. Michaelson and S. Avissar, Ca2+ –dependent protein phosphorylation of purely cholinergic Torpedo synaptosomes, J. Biol. Chem. 254:12542–12546 (1979)PubMedGoogle Scholar
  5. 5.
    W. Feldberg, A. Fessard and D. Nachmansohn, The cholinergic nature of the nervous supply of the electric organ of the Torpedo (Torpedo marmorata), J.Physiol. (Lond) 97:3P (1940).Google Scholar
  6. 6.
    M. Israel, R. Manaranche, P. Mastour-Frachon and N. Morel, Isolation of pure cholinergic nerve endings from the electric organ of Torpedo marmorata, Biochem. J. 160:113–115 (1976).PubMedGoogle Scholar
  7. 7.
    N. Morel, M. Israel, R. Manaranche and B. Lesbats, Stimulation of cholinergic synaptosomes isolated from Torpedo electric organ. Prog. Brain Res. 49:191–202 (1979).PubMedCrossRefGoogle Scholar
  8. 8.
    N. Morel and F.M. Meunier, Simultaneous release of acetylcholine and ATP from stimulated cholinergic synaptosomes, J. Neurochem. 36:1766–1773 (1981).PubMedCrossRefGoogle Scholar
  9. 9.
    J. Marsal, J.E. Esquerda, C. Fiol, C. Solsona and J. Tomas, Calcium fluxes in isolated pure cholinergic nerve endings from the electric organ of Torpedo marmorata, J. Physiol. (Paris) 76:443–457 (1980).Google Scholar
  10. 10.
    S. Thesleff and J. Molgó, A new type of transmitter release at the neuromuscular junction, Neurosci. 9:1–8 (1983).CrossRefGoogle Scholar
  11. 11.
    L. C. Sellin, The pharmacological mechanism of botulism. Trends Pharmacol. Sci.,Feb:80–82 (1985).CrossRefGoogle Scholar
  12. 12.
    J. Marsal, C. Solsona, X. Rabasseda, J. Blasi and A. Casanova, Depolarization-induced release of ATP from cholinergic synaptosomes is not blocked by botulinum toxin type A, Neurochem. Int. (in press) (1987).Google Scholar
  13. 13.
    H. Gower and R. Rodnight, Intrinsic protein phosphorylation in synaptic plasma membrane fragments from the rat. General characteristics and migration behaviour on Polyacrylamide gels of the main phosphate acceptors, Biochem. Bophys. Acta 716:45–52 (1982).CrossRefGoogle Scholar
  14. 14.
    Ü. K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T., Nature 227:680–685 (1970).PubMedCrossRefGoogle Scholar
  15. 15.
    K. Weber and M. Osborn, The reliability of molecular weight determination by dodecyl sulfate- polyacrylamide gel electrophoresis, J. Biol. Chem. 244:4406–4412 (1969).PubMedGoogle Scholar
  16. 16.
    M. Israel, B. Lesbats, N. Morel, R. Manaranche, T. Gulik-Krzywicki and J. C. Dedieu, Reconstitution of a functional synaptosomal membrane possessing the protein constituents involved in acetylcholine translocation, Proc. Natl. Acad. Sci. U.S.A. 81:277–281 (1984).PubMedCrossRefGoogle Scholar
  17. 17.
    R. L. Barchi, Protein components of the purified sodium channel from rat skeletal muscle sarcolemma, J Neurochem. 40:1377–1385 (1983).PubMedCrossRefGoogle Scholar
  18. 18.
    J. A. Miller, W. J. Agnew and S. R. Levinson, Principal glycopeptide of the tetrodotoxin/saxitoxin binding protein from Electrophorus electricus: isolation and partial chemical and physical characterization. Biochemistry 22:462–470 (1983).PubMedCrossRefGoogle Scholar
  19. 19.
    M. R. C. Costa and W. A. Catterall, Cyclic AMP-dependent phosphorylation of the K-subunit of the sodium channel in synaptic nerve endings particles, J. Biol. Chem. 259:8210–8218 (1984).PubMedGoogle Scholar
  20. 20.
    J. L. Brigant and A. Mallart, Presynaptic currents in mouse motor endings, J. Physiol. (Lond) 333:619–637 (1982).Google Scholar
  21. 21.
    J. H. Walker, C. M. Bousteaud, V. Witzemann, G. Shaw, K. Weber and M. Osborn, Cytoskeletal proteins at the cholinergic synapse: distribution of desmin, actin, fodrin, neurofilaments and tubulin in Torpedo electric organ, Eur. J. Cell Biol. 38:123–133 (1985).PubMedGoogle Scholar

Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • Xavier Guitart
    • 1
  • Jordi Marsal
    • 1
  • Carles Solsona
    • 1
  1. 1.Dpt. de Biologia Cel.lular i Anatomia Patològica Hosp. BellvitgeUniversitat de BarcelonaBarcelonaSpain

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