Journal of Molecular Neuroscience

, Volume 7, Issue 4, pp 257–267 | Cite as

The molecular identity of Ca channel α1-subunits expressed in rat sympathetic neurons

  • Zhixin Lin
  • Charles Harris
  • Diane Lipscombe
Original Articles


Much of our understanding of the mechanisms of the gating, modulation, and function of neuronal Ca channels has its origins in investigations of sympathetic neurons. In this article, we use molecular analyses to identify the three Ca channel α1-subunits that are the likely counter-parts to the pharmacologically defined: ω-Conotoxin GVIA-sensitive N-type; dihydropyridine-sensitive L-type, and ω-Conotoxin GVIA-insensitive, dihydropyridine-insensitive Ca channel currents observed in sympathetic neurons. With a combination of degenerate and exact primers, small regions of Ca channel α1-subunit sequences were amplified by the polymerase chain reaction (PCR). Although all five Ca channel α1-subunit genes were expressed in rat sympathetic ganglia, α1B , α1D , and α1E-derived cDNAs were the dominant species. No novel Ca channel α1-sequences were identified in the regions selected for amplification, and we conclude that α1B, α1D, and α1E likely encode, respectively, N-type, L-type, and non-N/non-L-type channel currents of rat sympathetic neurons. In addition, we show that Ca channel β2 , β3 , and β4-subunit sequences are strongly represented in sympathetic ganglia. The results of this study also suggest that α1D, and not α1C, regulates Ca influx through dihydropyridine-sensitive Ca channel currents.

Index Entries

Calcium channels sympathetic neurons polymerase chain reaction calcium channel α1-subunits ω-conotoxin GVIA dihydropyridine nickel 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  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. Artalejo C. R., Adams M. E., and Fox A. P. (1994) Three types of Ca2+ channel trigger secretion with different efficacies in chromaffin cells.Nature 367, 72–76.PubMedCrossRefGoogle Scholar
  3. Birnbaumer L., Campbell K. P., Catterall W. A., Harpold M. M., Hofmann F., Horne W. A., et al. (1994) Matters arising: the naming of voltage-gated calcium channels.Neuron 13, 505,506.CrossRefGoogle Scholar
  4. Boland L. M., Morrill J. A., and Bean B. P. (1994) ω-Conotoxin block of N-type calcium channels in frog and rat sympathetic neurons.J. Neurosci. 14, 5011–5027.PubMedGoogle Scholar
  5. Castellano A., Wei X., Birnbaumer L., and Perez-Reyes E. (1993a) Cloning and expression of a neuronal calcium channel beta subunit.J. Biol. Chem. 268, 12,359–12,366.Google Scholar
  6. Castellano A., Wei X., Birnbaumer L., and Perez-Reyes E. (1993b) Cloning and expression of a third calcium channel beta subunit.J. Biol. Chem. 268, 3450–3455.PubMedGoogle Scholar
  7. De Koninck P. and Cooper E. (1995) Differential regulation of neuronal nicotinic ACh receptor subunit genes in cultured neonatal rat sympathetic neurons: specific induction of α2 by membrane depolarization through a Ca2+/calmodulin- dependent kinase pathway.J. Neurosci. 15, 7966–7978.PubMedGoogle Scholar
  8. DeWaard M., Pragnell M., and Campbell K. P. (1994) Ca2+ channel regulation by a conserved β subunit domain.Neuron 13, 495–503.CrossRefGoogle Scholar
  9. Dubel S. J., Starr T. V., Hell J. W., Ahlijanian M. K., Enyeart J. J., Catterall W. A., and Snutch T. P. (1992) Molecular cloning of the α1 subunit of an ω-conotoxin-sensitive calcium channel.Proc. Natl. Acad. Sci. USA 89, 5058–5062.PubMedCrossRefGoogle Scholar
  10. Dunlap K., Luebke J. I., and Turner T. J. (1995) Exocytotic Ca2+ channels in mammalian central neurons.Trends Neurosci. 18, 89–98.PubMedCrossRefGoogle Scholar
  11. Ellinor P. T., Zhang J. F., Randall A. D., Zhou M., Schwartz T. L., Tsien R. W., and Horne W. A. (1993) Functional expression of a rapidly inactivating neuronal calcium channel.Nature 363, 455–458.PubMedCrossRefGoogle Scholar
  12. Elmslie K. S., Kammermeier P. J., and Jones S. W. (1994) Reevaluation of Ca2+ channel types and their modulation in bullfrog sympathetic neurons.Neuron 13, 1–20.CrossRefGoogle Scholar
  13. Fujita Y., Mynlieff M., Dirksen R. T., Kim M.-S., Niidome T., Nakkai J., et al. (1993) Primary structure and functional expression of the (ω-conotoxin-sensitive N-type channel from rabbit brain.Neuron 10, 585–598.PubMedCrossRefGoogle Scholar
  14. Hamill O. P., Marty A., Neher E., Sakmann B., and Sigworth F. J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.Pflugers Arch. 391, 85–100.PubMedCrossRefGoogle Scholar
  15. Hawrot E. and Patterson P. H. (1979). Long-term culture of dissociated sympathetic neurons.Methods Enzymol. 58, 574–583.PubMedCrossRefGoogle Scholar
  16. Hell J. W., Westenbroek R. E., Warner C., Ahlijanian M. K., Prystay W., Gilbert M. M., Snutch T. P., and Catterall W. A. (1993) Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel α1 subunits.J. Cell Biol. 123, 949–962.PubMedCrossRefGoogle Scholar
  17. Hirning L. D., Fox A. P., McCleskey E. W., Miller R. J., Olivera B. M., Thayer S. A., and Tsien R. W. (1988) Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons.Science 239, 57–61.PubMedCrossRefGoogle Scholar
  18. Hui A., Ellinor P. T., Krizanova O., Wang J.-J., Diebold R. J. and Schwartz A. (1991) Molecular cloning of multiple subtypes of a novel rat brain isoform of the α1 subunit of the voltage-dependent calcium channel.Neuron 7, 35–44.PubMedCrossRefGoogle Scholar
  19. Ikeda S. R. (1991) Double-pulse calcium channel current facilitation in adult rat sympathetic neurones.J. Physiol. 439, 181–214.PubMedGoogle Scholar
  20. Isom L. L., DeJongh K. S., and Catterall W. A. (1994) Auxiliary subunits of voltage-gated ion channels.Neuron 12, 1183–1194.PubMedCrossRefGoogle Scholar
  21. Komuro H. and Rakic P. (1992) Selective role of N-type calcium channels in neuronal migration.Science 257, 806–809.PubMedCrossRefGoogle Scholar
  22. Lin Z., Haus S., Edgerton J., and Lipscombe D. (1997) Identification of functionally distinct isoforms of the N-type channel in rat sympathetic ganglia and brain.Neuron, in press.Google Scholar
  23. Mathie A., Bernheim L., and Hille B. (1992) Inhibition of N- and L-type calcium channels by muscarinic receptor activation in rat sympathetic neurons.Neuron 8, 907–914.PubMedCrossRefGoogle Scholar
  24. McCleskey E. W., Fox A. P., Feldman D. H., Cruz L. J., Olivera B. M., Tsien R. W., and Yoshikami D. (1987) ω-Conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle.Proc. Natl. Acad. Sci. USA 84, 4327–4331.PubMedCrossRefGoogle Scholar
  25. Miller R. J. (1987) Multiple calcium channels and neuronal function.Science 235, 46–52PubMedCrossRefGoogle Scholar
  26. Mintz I. M. and Bean B. P. (1993) Block of calcium channels in rat neurons by synthetic ω-Aga-IVA.Neuropharmacology 32, 1161–1169.PubMedCrossRefGoogle Scholar
  27. Mintz I. M., Adams M. E., and Bean B. P. (1992) P-type calcium channels in rat central and peripheral neurons.Neuron 9, 85–95.PubMedCrossRefGoogle Scholar
  28. Mori Y., Friedrich T., Kin M.-S., Mikami A., Nakai J., Ruth P., Bosse E., Hofmann F., Flockerzi V., Furuichi T., Mikoshiba K., Imoto K., Tanabe T., and Numa S. (1991) Primary structure and functional expression from complementary DNA of a brain calcium channel.Nature 350, 398–402.PubMedCrossRefGoogle Scholar
  29. Murphey T. H., Worley P. F., and Barban J. M. (1991) L-type voltage-sensitive calcium channels mediate synaptic activation of immediate early genes.Neuron 7, 625–635.CrossRefGoogle Scholar
  30. Neely A., Wei X., Olcese R., Birnbaumer L., and Stefani E. (1993) Potentiation of the β subunit of the ratio of the ionic current to the charge movement in the cardiac calcium channel.Science 262, 575–578.PubMedCrossRefGoogle Scholar
  31. Perez-Reyes E., Castellano A., Kim H. S., Bertrand P., Baggstrom E., Lacerda A. E., Wei X. Y., and Birnbaumer L. (1992) Cloning and expression of a cardiac/brain beta subunit of the L-type calcium channel.J. Biol. Chem. 267, 1792–1797.PubMedGoogle Scholar
  32. Plummer M. R., Logothetis D. E., and Hess P. (1989) Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons.Neuron 2, 1453–1463.PubMedCrossRefGoogle Scholar
  33. Pragnell M., Sakamoto J., Jay S. D., and Campbell K. P. (1991) Cloning and tissue-specific expression of the brain calcium channel beta-subunit.FEBS Lett. 291, 253–258.PubMedCrossRefGoogle Scholar
  34. Pragnell M., De Waard M., Mori Y., Tanabe T., Snutch T. P., and Campbell K. P. (1994) Calcium channel β-subunit binds to a conserved motif in the 1–11 cytoplasmic linker of the α1-subunit.Nature 368, 67–70.PubMedCrossRefGoogle Scholar
  35. Ruano D., Lambolez B., Rossier J., Paternain A. V., and Lerma J. (1995). Kainate receptor subunits expressed in single cultured hippocampal neurons: molecular and functional variants by RNA edging.Neuron 14, 1009–1017.PubMedCrossRefGoogle Scholar
  36. Sather W. A., Tanabe T., Zhang J.-F., Mori Y., Adams M. E., and Tsien R. W. (1993) Distinctive biophysical and pharmacological properties of class A (B1) calcium channel α1 subunits.Neuron 11, 291–303.PubMedCrossRefGoogle Scholar
  37. Scott V. E. S., DeWaard M., Liu M., Gurnett C. A., Venzke D. P., Lennon V. A., and Campbell K. P. (1996) β Subunit heterogeneity in N-type Ca2+ channels.J. Biol. Chem. 271, 3207–3212.PubMedCrossRefGoogle Scholar
  38. Snutch T. P. and Reiner P. B. (1992). Ca2+ channels: diversity of forms and function.Curr. Opinions Neurobiol. 2, 247–253.CrossRefGoogle Scholar
  39. Snutch T. P., Tomlinson W. J., Leonard J. P., and Gilbert M. M. (1991) Distinct calcium channels are generated by alternative splicing and are differentially expressed in the mammalian CNS.Neuron 7, 45–57.PubMedCrossRefGoogle Scholar
  40. Soong T. W., Stea A., Hodson C. D., Dubel S. J., Vincent S. R., and Snutch T. P. (1993) Structure and functional expression of a member of the low voltage-activated calcium channel family.Science 260, 1133–1136.PubMedCrossRefGoogle Scholar
  41. Starr T. V., Prystay W., and Snutch T. P. (1991) Primary structure of a calcium channel that is highly expressed in the rat cerebellum.Proc. Natl. Acad. Sci. USA 88, 5621–5625.PubMedCrossRefGoogle Scholar
  42. Stea A., Dubel S. J., Pragnell M., Leonard J. P., Campbell K. P., and Snutch T. P. (1993) A β-subunit normalizes the electrophysiological properties of a cloned N-type Ca2+ channel α1-subunit.Neuropharmacology 32, 1103–1116.PubMedCrossRefGoogle Scholar
  43. Stea A., Soong T. W., and Snutch T. P. (1995) Determinants of PKC-dependent modulation of a family of neuronal calcium channels.Neuron 15, 929–940.PubMedCrossRefGoogle Scholar
  44. 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.PubMedCrossRefGoogle Scholar
  45. Tanaka S. and Koike T. (1995) Up-regulation of L-type Ca2+ channel associated with the development of elevated K+-mediated survival of superior cervical ganglion cellsin vitro.Dev. Biol. 16, 166–178.CrossRefGoogle Scholar
  46. Tomlinson W. J., Stea A., Bourinet E., Charnet P., Nargeot J., and Snutch T. P. (1993) Functional properties of aneuronal class C L-type calcium channel.Neuropharmacology 32, 1117–1126.PubMedCrossRefGoogle Scholar
  47. Tsien R. W., Ellinor P. T., and Horne W. A. (1991) Molecular diversity of voltage-dependent Ca2+ channels.Trends Pharmacol. Sci. 12, 349–353.PubMedCrossRefGoogle Scholar
  48. Williams M. E., Brust P. F., Feldman D. H., Patthi S., Simerson S., Maroufi A., McCue A. F., Velicelebi G., Ellis S. B., and Harpold M. M. (1992a) Structure and functional expression of an ω-conotoxin-sensitive human N-type calcium channel.Science 257, 389–395.PubMedCrossRefGoogle Scholar
  49. Williams M. E., Feldman D. H., McCue A. F., Brenner R., Velicelebi G., Ellis S. B., and Harpold M. M. (1992b) Structure and functional expression of α1, α2, and b subunits of a novel human neuronal calcium channel subtype.Neuron 8, 71–84.PubMedCrossRefGoogle Scholar
  50. Wincher 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 rat brain.Science 261, 486–489.CrossRefGoogle Scholar
  51. Zhang J.-F., Randall A. D., Ellinor P. T., Horne W. A., Sather, W. A., Tanabe T., Schwartz T. L., and Tsien R. W. (1993) Distinctive pharmacology and kinetics of cloned neuronal Ca2+ channels and their possible counterparts in mammalian CNS neurons.Neuropharmacology 32, 1075–1088.PubMedCrossRefGoogle Scholar
  52. Zhu Y. and Ikeda S. R. (1993) Adenosine modulates voltage-gated Ca2+ channels in adult rat sympathetic neurons.J. Neurophysiol. 70, 610–620.PubMedGoogle Scholar

Copyright information

© Humana Press Inc 1996

Authors and Affiliations

  • Zhixin Lin
    • 2
  • Charles Harris
    • 1
  • Diane Lipscombe
    • 1
  1. 1.Department of NeuroscienceBrown UniversityProvidence
  2. 2.Department of Molecular Pharmacology and BiotechnologyBrown UniversityProvidence

Personalised recommendations