Mapping N-Type Calcium Channel Distributions with H-Conotoxins

  • Geula M. Bernstein
  • Owen T. Jones
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Natural toxins have revolutionized the study of ion channels (1). Owing to their specificity and potency, toxins form the basis of many of the most important ligands used by modern biologists to identify, discriminate, manipulate, or purify ion channels. An excellent example of the biological contribution of toxins is the use of analogs of H-conotoxin GVIA (H-CgTx) in visualizing the distribution of N-type voltage-dependent calcium channels (N-VDCCs) in neurons (Table 1). H-conotoxin GVIA was first isolated from the venom of the Pacific cone snail Conus geographus by Olivera and colleagues (2). This toxin drew attention because of its unusual pharmacological properties, which electrophysiological (3,(4) and biochemical studies (5,(6) now indicate are owing to blockade of voltage-dependent Ca2+ influx into neurons by its exclusive interaction with N-VDCCs (see Section 1.2.2.). Surprisingly, native H-CgTx confirmed both its structure and activity (7).


Live Imaging External Region Conjugation Reaction Ligand Internalization Modern Biologist 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Adams, M. E. and Olivera, B. M. (1994) Neurotoxins: overview of an emerging research technology. Trends Neurosci. 17, 151–155.PubMedCrossRefGoogle Scholar
  2. 2.
    Olivera, B. M., Rivier, J., Scott, J. K., Hillyard, D. R., and Cruz, L. J. (1991) Conotoxins. J. Biol. Chem. 266, 22,067–22,070.PubMedGoogle Scholar
  3. 3.
    Regan, L. J., Sah, D. W., and Bean B. P. (1991) Ca2+ channels in rat central and peripheral neurons: high-threshold current resistant to dihydropyridine blockers and omega-conotoxin. Neuron 6, 269–280.PubMedCrossRefGoogle Scholar
  4. 4.
    Ellinor, P. T., Zhang, J. F., Horne, W. A., and Tsien, R. W. (1994) Structural determinants of the blockade of N-type calcium channels by a peptide neurotoxin. Nature 372, 272–275.PubMedCrossRefGoogle Scholar
  5. 5.
    Witcher, D. R., De Waard, M., Sakamoto, J., Franzini-Armstrong, C., Pragnell, M., Kahl, S. D., and Campbell, K. P. (1993) Subunit identification and reconstitution of the N-type Ca2+ channel complex purified from brain. Science 261, 486–489.PubMedCrossRefGoogle Scholar
  6. 6.
    Jones, O. T., Bernstein, G. M., Jones, E. J., Jugloff, D. G., Law, M., Wong, W., and Mills, L. R. (1997) N-Type calcium channels in the developing rat hippocampus: subunit, complex, and regional expression. J. Neurosci. 17, 6152–6164.PubMedGoogle Scholar
  7. 7.
    Rivier, J., Galyean, R., Gray, W. R., Azimi-Zonooz, A., McIntosh, J. M., Cruz, L. J., and Olivera, B. M. (1987) Neuronal calcium channel inhibitors. Synthesis of omega-conotoxin GVIA and effects on 45Ca uptake by synaptosomes. J. Biol. Chem. 262, 1194–1198.PubMedGoogle Scholar
  8. 8.
    Davis, J. H., Bradley, E. K., Miljanich, G. P., Nadasdi, L., Ramachandran, J.,and Basus, V. J. (1993) Solution structure of omega-conotoxin GVIA using 2-D NMR spectroscopy and relaxation matrix analysis. Biochemistry 32, 7396–7405.PubMedCrossRefGoogle Scholar
  9. 9.
    Williams, M. E., Brust, P. F., Feldman, D. H., Patthi, S., Simerson, S., Maroufi, A., et al. (1992) Structure and functional expression of an H-conotoxin-sensitive human N-type calcium channel. Science 257, 389–395.PubMedCrossRefGoogle Scholar
  10. 10.
    Stocker, J. W., Nadasdi, L., Aldrich, R. W., and Tsien, R. W. (1997) Preferential interaction of H-Conotoxins with inactivated N-type Ca2+ channels. J. Neurosci. 17, 3002–3013.PubMedGoogle Scholar
  11. 11.
    Axelrod, D., Ravdin, P., Koppel, D. E., Schlessinger, J., Webb, W. W., Elson, E. L., and Podleski, T. R. (1976) Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc. Natl. Acad. Sci. USA 73, 4594–4598.PubMedCrossRefGoogle Scholar
  12. 12.
    Anderson, M. J., Cohen, M. W., and Zorychta, E. (1977) Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J. Physiol. (Lond.) 268, 731–756.Google Scholar
  13. 13.
    Jones, O. T., Kunze, D. L., and Angelides, K. J. (1989) Localization and mobility of H-conotoxin-sensitive Ca2+ channels in hippocampal CA1 neurons. Science 244, 1189–1193.PubMedCrossRefGoogle Scholar
  14. 14.
    Catterall, W. A. (1998) Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release. Cell Calcium. 24, 307–323.PubMedCrossRefGoogle Scholar
  15. 15.
    Cohen, M. W., Jones, O. T., and Angelides, K. J. (1991) Distribution of Ca2+ channels on frog motor nerve terminals revealed by fluorescent H-conotoxin. J. Neurosci. 11, 1032–1039.PubMedGoogle Scholar
  16. 16.
    Robitaille, R., Adler, E. M., and Charlton, M. P. (1990) Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses. Neuron 5, 773–779.PubMedCrossRefGoogle Scholar
  17. 17.
    Haydon, P. G., Henderson, E., and Stanley, E. F. (1994) Localization of individual calcium channels at the release face of a presynaptic nerve terminal. Neuron 13, 1275–1280.PubMedCrossRefGoogle Scholar
  18. 18.
    Farinas, I., Egea, G., Blasi, J., Cases, C., and Marsal, J. (1993) Calcium channel antagonist omega-conotoxin binds to intramembrane particles of isolated nerve terminals. Neuroscience 54, 745–752.PubMedCrossRefGoogle Scholar
  19. 19.
    Dunlap, K., Luebke, J. I., and Turner, T. J. (1995) Exocytotic Ca2+ channels in mammalian central neurons. Trends Neurosci. 18, 89–98.PubMedCrossRefGoogle Scholar
  20. 20.
    Mills, L. R., Niesen, C. E., So, A. P., Carlen, P. L., Spigelman, I., and Jones O. T. (1994) N-type Ca2+ channels are located on somata, dendrites, and a subpopulation of dendritic spines on live hippocampal pyramidal neurons. J. Neurosci. 14, 6815–6824.PubMedGoogle Scholar
  21. 21.
    Magee, J., Hoffman, D., Colbert, C., and Johnston, D. (1998) Electrical and calcium signaling in dendrites of hippocampal pyramidal neurons. Ann. Rev. Physiol. 60, 327–346.CrossRefGoogle Scholar
  22. 22.
    Westenbroek, R., Hell, J. W., Warner, C., Dubel, S. J., Snutch, T. P. and Catterall, W. A. (1992) Biochemical properties and subcellular distribution of an N-type calcium channel H1 subunit. Neuron 9, 1099–1115.PubMedCrossRefGoogle Scholar
  23. 23.
    Schiller, J., Schiller, Y., and Clapham, D. E. (1998) NMDA receptors amplify calcium influx into dendritic spines during associative pre-and postsynaptic activation. Nature Neurosci. 1, 114–118.PubMedCrossRefGoogle Scholar
  24. 24.
    Filloux, F., Schapper, A., Naisbitt, S. R., Olivera, B. M., McIntosh, J. M. (1994) Complex patterns of [125I]omega-conotoxin GVIA binding site expression during postnatal rat brain development. Brain Res. Dev. Brain Res. 78, 131–136.PubMedCrossRefGoogle Scholar
  25. 25.
    Bernstein, G. M., Mendonca, A., Wadia, J., Burnham, W. M., and Jones, O. T. (1999) Kindling enhances N-type calcium channels in the rat hippocampus. Neuroscience 94, 1083–1095.PubMedCrossRefGoogle Scholar
  26. 26.
    Bernstein, G. M., Mendonca, A., Wadia, J., Burnham, W. M., and Jones, O. T. (1999) Kindling induces an asymmetric enhancement of N-type calcium channels in the dendritic fields of the rat hippocampus. Neurosci. Lett. 268, 155–158.PubMedCrossRefGoogle Scholar
  27. 27.
    Albensi, B. C., Ryujin, K. T., McIntosh, J. M., Naisbitt, S. R., Olivera, B. M., and Filloux, F. (1993) Localization of [125I]omega-conotoxin GVIA binding in human hippocampus and cerebellum. Neuroreport 4, 1331–1334.PubMedCrossRefGoogle Scholar
  28. 28.
    Takemura, M., Kiyama, H., Fukui, H., Tohyama, M., and Wada, H. (1989) Distribution of the omega-conotoxin receptor in rat brain. An autoradiographic mapping. Neuroscience 32, 405–416.PubMedCrossRefGoogle Scholar
  29. 29.
    Maeda, N., Wada, K., Yuzaki, M., and Mikoshiba, K. (1989) Autoradiographic visualization of a calcium channel antagonist, [125I]omega-conotoxin GVIA, binding site in the brains of normal and cerebellar mutant mice (pcd and weaver). Brain Res. 489, 21–30.PubMedCrossRefGoogle Scholar
  30. 30.
    Kerr, L. M., Filloux, F., Olivera, B. M., Jackson, H., and Wamsley, J. K. (1988) Autoradiographic localization of calcium channels with [125I]omega-conotoxin in rat brain. Eur. J. Pharmacol. 146, 181–183.PubMedCrossRefGoogle Scholar
  31. 31.
    Takemura, M., Kiyama, H., Fukui, H., Tohyama, M., and Wada, H. (1988) Autoradiographic visualization in rat brain of receptors for omega-conotoxin GVIA, a newly discovered calcium antagonist. Brain Res. 451, 386–389.PubMedCrossRefGoogle Scholar
  32. 32.
    Quasthoff, S., Adelsberger, H., Grosskreutz, J., Arzberger, T., and Schroder, J. M. (1996) Immunohistochemical and electrophysiological evidence for omegaconotoxin-sensitive calcium channels in unmyelinated C-fibres of biopsied human sural nerve. Brain Res. 723, 29–36.PubMedCrossRefGoogle Scholar
  33. 33.
    Tharani, Y., Thurlow, G. A., and Turner, R. W. (1996) Distribution of omega-Conotoxin GVIA binding sites in teleost cerebellar and electrosensory neurons. J. Comp. Neurol. 364, 456–472.PubMedCrossRefGoogle Scholar
  34. 34.
    Fortier, L. P., Tremblay, J. P., Rafrafi, J., and Hawkes, R. (1991) A monoclonal antibody to conotoxin reveals the distribution of a subset of calcium channels in the rat cerebellar cortex. Brain Res. Mol. Brain Res. 9, 209–215.PubMedCrossRefGoogle Scholar
  35. 35.
    Grabner, M., Dirksen, R. T., and Beam, K. G. (1998) Tagging with green fluorescent protein reveals a distinct subcellular distribution of L-type and non-Ltype Ca2+ channels expressed in dysgenic myotubes. Proc. Natl. Acad. Sci. USA 95, 1903–1908.PubMedCrossRefGoogle Scholar
  36. 36.
    Whorlow, S. L., Loiacono, R. E., Angus, J. A., and Wright, C. E. (1996) Distribution of N-type Ca2+ channel binding sites in rabbit brain following central administration of omega-conotoxin GVIA. Eur. J. Pharmacol. 315, 11–18.PubMedCrossRefGoogle Scholar
  37. 37.
    Wilchek, M. and Bayer, E. A. (eds.) (1990) Methods in Enzymology, vol. 184. Academic, San Diego.Google Scholar
  38. 38.
    Jones, O. T. and So, A. P. (1993) Preparation and characterization of biotinylated analogs of the calcium channel blocker omega-conotoxin. Anal. Biochem. 214, 227–232.PubMedCrossRefGoogle Scholar
  39. 39.
    Komuro, H. and Rakic, P. (1992) Selective role of N-type calcium channels in neuronal migration. Science 257, 806–809.PubMedCrossRefGoogle Scholar
  40. 40.
    Jones, O. T., Koncz, E. J., and So, A. P. (1994) Imaging ion channels in live central neurons using fluorescent ligands. I. Ligand construction, in Three Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (Stevens, J. K., Mills, L. R., and Trogadis, J. E., eds.), Academic, Orlando, FL, pp. 183–213.Google Scholar
  41. 41.
    Baxes, G. A., ed. (1994) Digital Image Processing: Principles and Applications. Wiley and Sons, New York.Google Scholar
  42. 42.
    Jones, O. T., Koncz, E. J., and So, A. P. (1994) Imaging ion channels in live central neurons using fluorescent ligands. II. Labelling of cells and tissues, in Three Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (Stevens, J. K., Mills, L. R., and Trogadis, J. E., eds.), Academic, Orlando, FL, pp. 215–232.Google Scholar
  43. 43.
    Mukherjee, S., Ghosh, R. N., and Maxfield, F. R. (1997) Endocytosis. Physiol. Rev. 77, 759–803.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2001

Authors and Affiliations

  • Geula M. Bernstein
    • 1
  • Owen T. Jones
    • 1
  1. 1.Playfair Neuroscience UnitToronto Hospital Research InstituteTorontoCanada

Personalised recommendations