Production of Monoclonal Antibodies against Low-Abundance Ion-Channel-Associated Proteins in Neuronal Tissue

  • Masami Takahashi


The voltage-sensitive calcium channel is indispensable to the regulation of intracellular calcium concentration. Several types of calcium channels can be distinguished by electrophysiological and pharmacological properties (Bean, 1989; Hess, 1990). A dihydropyridine (DHP)-sensitive L-type calcium channel has been purified from transverse tubules of rabbit skeletal muscle and found to be made up of α 1, α 2,β, γ, and δ subunits (Catterall et al.,1988). The cDNAs of all of these subunits have been cloned and their amino acid sequences determined (Tanabe et al., 1987; Ellis et al., 1988; Ruth et al., 1989; Jay et al., 1990). The cDNAs of the skeletal α 1, subunit have been used as molecular probes, and four different α 1, subunit genes have been found to be expressed in the brain (Snuch et al., 1990). Further multiple subtypes are generated by alternative splicing, thus indicating various kinds of calcium channels to be present in the organ. The subunit structures and properties of these multiple calcium channels should be studied to provide some clarification of their roles in brain functions. It is quite difficult, however, to purify neuronal calcium channels, since they are present in very small quantities. The existence of many homologous structures among calcium channel subtypes makes purification even more difficult.


Calcium Channel Synaptic Membrane Chick Brain Brain Synaptic Membrane Calcium Channel Subtype 
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  1. Ahlijanian, M. K., Westenbroek, R. E., and Catterall, W. A. 1990. Subunit structure and localization of dihydropyridine-sensitive calcium channels in mammalian brain, spinal cord, and retina. Neuron, 4: 819–832.PubMedCrossRefGoogle Scholar
  2. Bean, B. P. 1989. Classes of calcium channels in vertebrate cells. Annu. Rev. Physiol., 51: 367–384.PubMedCrossRefGoogle Scholar
  3. Catterall, W. A., Seagar, M. J.. and Takahashi, M. 1988. Molecular properties of dihydropyridine-sensitive calcium channels in skeletal muscle. J. Biol. Chem., 263: 3535–3538.PubMedGoogle Scholar
  4. Ellis, S. B., Williams, M. E., Ways, N. R., Brenner, R., Sharp, A. H., Leung, A. T., Campbell, K. P., McKenna, E., Koch, W. J., Hui, A., Schwartz. A., and Harpold. M. M. 1988. Sequence and expression of mRNAs encoding a, and 02 subunits of a DHP-sensitive calcium channel. Science, 241: 1661–1664.Google Scholar
  5. Glossmann. H., and Striessnig, J. 1988. Calcium channels. Vitam. Horm., 44: 155–328.PubMedCrossRefGoogle Scholar
  6. Harlow, E., and Lane, D. 1988. Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory. Cold Spring Harbor, New York.Google Scholar
  7. Hess, P. 1990. Calcium channels in vertebrate cells. Annu. Rev. Neurosci., 13: 337–356.PubMedCrossRefGoogle Scholar
  8. Jay. S. D., Ellis, S. B., McCue, A. F.. Williams, M. E., Vedvick, T. S., Harpold, M. M., and Campbell, K. P. 1990. Primary structure of the y subunit of the DHP-sensitive calcium channel from skeletal muscle. Science, 248: 490–492.PubMedCrossRefGoogle Scholar
  9. Jones, D. H., and Matus. A. 1. 1974. Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation. Biochim. Biophvs. Acta, 356: 276–287.CrossRefGoogle Scholar
  10. Lai, Y., Seagar, M. J., Takahashi, M., and Catterall, W. A. 1990. Cyclic AMP-dependent phosphorylation of two size forms of a, subunits of L-type calcium channels in rat skeletal muscle cells. J. Biol. C’hein., 265: 20839–20848.Google Scholar
  11. Ruth, P., Röhrkasten, A., Biel, M., Bosse, E., Regulla, S., Meyer. H. E., Flockerzi, V.. and Hofmann, F. 1989. Primary structure of the ß subunit of the DHP-sensitive calcium channel from skeletal muscle. Science, 245: 1115–1118.Google Scholar
  12. Snutch, T. P., Leonard, J. P., Gilbert, M. M., Lester, H. A., and Davidson, N. 1990. Rat brain expresses a heterogeneous family of calcium channels. Proc. Natl. Acad. Sci. USA, 87: 3391–3395.PubMedCrossRefGoogle Scholar
  13. Takahashi, M., and Catterall, W. A. 1987a. Identification of ana subunit of dihydropyridine-sensitive brain calcium channels. Science, 236: 88–91.PubMedCrossRefGoogle Scholar
  14. Takahashi, M., and Catterall. W. A. 1987b. Dihydropyridine-sensitive calcium channel in cardiac and skeletal muscle membranes: Studies with antibodies against the a subunits. Biochemistry, 26: 5518–5526.PubMedCrossRefGoogle Scholar
  15. Takahashi, M., and Fujimoto, Y. 1989. Identification of a dihydropyridine-sensitive calcium channel in chick brain by a monoclonal antibody. Biochem. Biophvs. Res. Commun., 163: 1182–1188.CrossRefGoogle Scholar
  16. Takahashi, M., Seagar, M. J., Jones, J. F., Reber. B. F. X., and Catterall, W. A. 1987. Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle. Proc. Natl. Acad. Sci. USA, 84: 54785482.Google Scholar
  17. Takahashi, M., Arimatsu, Y., Fujita, S., Fujimoto, Y., Kondo, S., Hama. T., and Miyamoto, E. 1991. Protein kinase C and Ca2+ calmodulin-dependent protein kinase 11 phosphorylate a novel 58-kD protein in synaptic vesicles. Brain Res., 551: 279–292.Google Scholar
  18. Tanabe, T.. Takeshima, H., Mikami, A.. Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., and Numa, S. 1987. Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature, 328: 313–318.Google Scholar
  19. Vaitukaitis, J. L. 1981. Production of antisera with small doses of immunogen: Multiple intradermal injections. Meth. En_vmol., 73: 46–52.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1993

Authors and Affiliations

  • Masami Takahashi
    • 1
  1. 1.Mitsubishi Kasei Institute of Life SciencesMachida, TokyoJapan

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