Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Jul 2;134(2):511–528. doi: 10.1083/jcb.134.2.511

Biochemical properties and subcellular distribution of the BI and rbA isoforms of alpha 1A subunits of brain calcium channels

PMCID: PMC2120867  PMID: 8707834

Abstract

Biochemical properties and subcellular distribution of the class A calcium channel alpha 1 subunits (alpha 1A) from rat and rabbit brain were examined using site-directed anti-peptide antibodies specific for rat rbA (anti-CNA3) and for rabbit BI (anti-NBI-1 and anti-NBI-2) isoforms of alpha 1A. In immunoblotting experiments, anti-CNA3 specifically identifies multiple alpha 1A polypeptides with apparent molecular masses of 210, 190, and 160 kD, and anti-NBI-1 and anti-NBI-2 specifically recognize 190-kD alpha 1A polypeptides in rat brain membrane. In rabbit brain, anti-NBI-1 or anti-NBI-2 specifically detect alpha 1A polypeptides with apparent molecular masses of 220, 200, and 190 kD, while anti-CNA3 specifically recognizes 190-kD alpha 1A polypeptides. These polypeptides evidently represent multiple isoforms of alpha 1A present in both rat and rabbit brain. Anti-CNA3 specifically immunoprecipitates high affinity receptor sites for omega- conotoxin MVIIC (Kd approximately 100 pM), whereas anti-NBI-2 immunoprecipitates two distinct affinity receptor sites for omega- conotoxin MVIIC (Kd approximately 100 pM and approximately 1 microM). Coimmunoprecipitation experiments indicate that alpha 1A subunits recognized by anti-CNA3 and anti-NBI-2 are associated with syntaxin in a stable, SDS-resistant complex and with synaptotagmin. Immunofluorescence studies reveal that calcium channels recognized by anti-NBI-2 are localized predominantly in dendrites and nerve terminals forming synapses on them, while calcium channels recognized by anti- CNA3 are localized more prominently in cell bodies and in nerve terminals. The mossy fiber terminals in hippocampus and the terminals of climbing and parallel fibers in cerebellum are differentially stained by these isoform-specific antibodies. These results indicate that both rbA and BI isoforms of alpha 1A are expressed in rat and rabbit brain and form calcium channels having alpha 1A subunits with distinct molecular mass, pharmacology, and subcellular localization.

Full Text

The Full Text of this article is available as a PDF (5.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baumert M., Maycox P. R., Navone F., De Camilli P., Jahn R. Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain. EMBO J. 1989 Feb;8(2):379–384. doi: 10.1002/j.1460-2075.1989.tb03388.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett M. K., Calakos N., Scheller R. H. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science. 1992 Jul 10;257(5067):255–259. doi: 10.1126/science.1321498. [DOI] [PubMed] [Google Scholar]
  3. Bezprozvanny I., Scheller R. H., Tsien R. W. Functional impact of syntaxin on gating of N-type and Q-type calcium channels. Nature. 1995 Dec 7;378(6557):623–626. doi: 10.1038/378623a0. [DOI] [PubMed] [Google Scholar]
  4. Bindokas V. P., Brorson J. R., Miller R. J. Characteristics of voltage sensitive calcium channels in dendrites of cultured rat cerebellar neurons. Neuropharmacology. 1993 Nov;32(11):1213–1220. doi: 10.1016/0028-3908(93)90015-u. [DOI] [PubMed] [Google Scholar]
  5. Birnbaumer L., Campbell K. P., Catterall W. A., Harpold M. M., Hofmann F., Horne W. A., Mori Y., Schwartz A., Snutch T. P., Tanabe T. The naming of voltage-gated calcium channels. Neuron. 1994 Sep;13(3):505–506. doi: 10.1016/0896-6273(94)90021-3. [DOI] [PubMed] [Google Scholar]
  6. Bowersox S. S., Miljanich G. P., Sugiura Y., Li C., Nadasdi L., Hoffman B. B., Ramachandran J., Ko C. P. Differential blockade of voltage-sensitive calcium channels at the mouse neuromuscular junction by novel omega-conopeptides and omega-agatoxin-IVA. J Pharmacol Exp Ther. 1995 Apr;273(1):248–256. [PubMed] [Google Scholar]
  7. Calakos N., Bennett M. K., Peterson K. E., Scheller R. H. Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking. Science. 1994 Feb 25;263(5150):1146–1149. doi: 10.1126/science.8108733. [DOI] [PubMed] [Google Scholar]
  8. Castillo P. E., Weisskopf M. G., Nicoll R. A. The role of Ca2+ channels in hippocampal mossy fiber synaptic transmission and long-term potentiation. Neuron. 1994 Feb;12(2):261–269. doi: 10.1016/0896-6273(94)90269-0. [DOI] [PubMed] [Google Scholar]
  9. Catterall W. A. Structure and function of voltage-sensitive ion channels. Science. 1988 Oct 7;242(4875):50–61. doi: 10.1126/science.2459775. [DOI] [PubMed] [Google Scholar]
  10. Chapman E. R., An S., Barton N., Jahn R. SNAP-25, a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils. J Biol Chem. 1994 Nov 4;269(44):27427–27432. [PubMed] [Google Scholar]
  11. De Waard M., Campbell K. P. Subunit regulation of the neuronal alpha 1A Ca2+ channel expressed in Xenopus oocytes. J Physiol. 1995 Jun 15;485(Pt 3):619–634. doi: 10.1113/jphysiol.1995.sp020757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dubel S. J., Starr T. V., Hell J., Ahlijanian M. K., Enyeart J. J., Catterall W. A., Snutch T. P. Molecular cloning of the alpha-1 subunit of an omega-conotoxin-sensitive calcium channel. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5058–5062. doi: 10.1073/pnas.89.11.5058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dunlap K., Luebke J. I., Turner T. J. Exocytotic Ca2+ channels in mammalian central neurons. Trends Neurosci. 1995 Feb;18(2):89–98. [PubMed] [Google Scholar]
  14. Fujita Y., Mynlieff M., Dirksen R. T., Kim M. S., Niidome T., Nakai J., Friedrich T., Iwabe N., Miyata T., Furuichi T. Primary structure and functional expression of the omega-conotoxin-sensitive N-type calcium channel from rabbit brain. Neuron. 1993 Apr;10(4):585–598. doi: 10.1016/0896-6273(93)90162-k. [DOI] [PubMed] [Google Scholar]
  15. Gordon D., Merrick D., Wollner D. A., Catterall W. A. Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies. Biochemistry. 1988 Sep 6;27(18):7032–7038. doi: 10.1021/bi00418a054. [DOI] [PubMed] [Google Scholar]
  16. Hayashi T., McMahon H., Yamasaki S., Binz T., Hata Y., Südhof T. C., Niemann H. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 1994 Nov 1;13(21):5051–5061. doi: 10.1002/j.1460-2075.1994.tb06834.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hell J. W., Westenbroek R. E., Warner C., Ahlijanian M. K., Prystay W., Gilbert M. M., Snutch T. P., Catterall W. A. Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits. J Cell Biol. 1993 Nov;123(4):949–962. doi: 10.1083/jcb.123.4.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hillyard D. R., Monje V. D., Mintz I. M., Bean B. P., Nadasdi L., Ramachandran J., Miljanich G., Azimi-Zoonooz A., McIntosh J. M., Cruz L. J. A new Conus peptide ligand for mammalian presynaptic Ca2+ channels. Neuron. 1992 Jul;9(1):69–77. doi: 10.1016/0896-6273(92)90221-x. [DOI] [PubMed] [Google Scholar]
  19. Horne A. L., Kemp J. A. The effect of omega-conotoxin GVIA on synaptic transmission within the nucleus accumbens and hippocampus of the rat in vitro. Br J Pharmacol. 1991 Jul;103(3):1733–1739. doi: 10.1111/j.1476-5381.1991.tb09855.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Inoue A., Obata K., Akagawa K. Cloning and sequence analysis of cDNA for a neuronal cell membrane antigen, HPC-1. J Biol Chem. 1992 May 25;267(15):10613–10619. [PubMed] [Google Scholar]
  21. Isom L. L., De Jongh K. S., Catterall W. A. Auxiliary subunits of voltage-gated ion channels. Neuron. 1994 Jun;12(6):1183–1194. doi: 10.1016/0896-6273(94)90436-7. [DOI] [PubMed] [Google Scholar]
  22. Jahn R., Südhof T. C. Synaptic vesicles and exocytosis. Annu Rev Neurosci. 1994;17:219–246. doi: 10.1146/annurev.ne.17.030194.001251. [DOI] [PubMed] [Google Scholar]
  23. Leveque C., Hoshino T., David P., Shoji-Kasai Y., Leys K., Omori A., Lang B., el Far O., Sato K., Martin-Moutot N. The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3625–3629. doi: 10.1073/pnas.89.8.3625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Llinás R., Sugimori M. Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices. J Physiol. 1980 Aug;305:197–213. doi: 10.1113/jphysiol.1980.sp013358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Llinás R., Sugimori M., Lin J. W., Cherksey B. Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1689–1693. doi: 10.1073/pnas.86.5.1689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Llinás R., Sugimori M., Silver R. B. Microdomains of high calcium concentration in a presynaptic terminal. Science. 1992 May 1;256(5057):677–679. doi: 10.1126/science.1350109. [DOI] [PubMed] [Google Scholar]
  27. Luebke J. I., Dunlap K., Turner T. J. Multiple calcium channel types control glutamatergic synaptic transmission in the hippocampus. Neuron. 1993 Nov;11(5):895–902. doi: 10.1016/0896-6273(93)90119-c. [DOI] [PubMed] [Google Scholar]
  28. Lévêque C., el Far O., Martin-Moutot N., Sato K., Kato R., Takahashi M., Seagar M. J. Purification of the N-type calcium channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis. J Biol Chem. 1994 Mar 4;269(9):6306–6312. [PubMed] [Google Scholar]
  29. Matthew W. D., Tsavaler L., Reichardt L. F. Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue. J Cell Biol. 1981 Oct;91(1):257–269. doi: 10.1083/jcb.91.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  31. McMahon H. T., Südhof T. C. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995 Feb 3;270(5):2213–2217. doi: 10.1074/jbc.270.5.2213. [DOI] [PubMed] [Google Scholar]
  32. Mori Y., Friedrich T., Kim M. S., Mikami A., Nakai J., Ruth P., Bosse E., Hofmann F., Flockerzi V., Furuichi T. Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature. 1991 Apr 4;350(6317):398–402. doi: 10.1038/350398a0. [DOI] [PubMed] [Google Scholar]
  33. O'Connor V. M., Shamotienko O., Grishin E., Betz H. On the structure of the 'synaptosecretosome'. Evidence for a neurexin/synaptotagmin/syntaxin/Ca2+ channel complex. FEBS Lett. 1993 Jul 12;326(1-3):255–260. doi: 10.1016/0014-5793(93)81802-7. [DOI] [PubMed] [Google Scholar]
  34. Olivera B. M., Miljanich G. P., Ramachandran J., Adams M. E. Calcium channel diversity and neurotransmitter release: the omega-conotoxins and omega-agatoxins. Annu Rev Biochem. 1994;63:823–867. doi: 10.1146/annurev.bi.63.070194.004135. [DOI] [PubMed] [Google Scholar]
  35. Ousley A. H., Froehner S. C. An anti-peptide antibody specific for the class A calcium channel alpha 1 subunit labels mammalian neuromuscular junction. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12263–12267. doi: 10.1073/pnas.91.25.12263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Oyler G. A., Higgins G. A., Hart R. A., Battenberg E., Billingsley M., Bloom F. E., Wilson M. C. The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations. J Cell Biol. 1989 Dec;109(6 Pt 1):3039–3052. doi: 10.1083/jcb.109.6.3039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Posnett D. N., McGrath H., Tam J. P. A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain. J Biol Chem. 1988 Feb 5;263(4):1719–1725. [PubMed] [Google Scholar]
  38. Randall A., Tsien R. W. Pharmacological dissection of multiple types of Ca2+ channel currents in rat cerebellar granule neurons. J Neurosci. 1995 Apr;15(4):2995–3012. doi: 10.1523/JNEUROSCI.15-04-02995.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Regehr W. G., Connor J. A., Tank D. W. Optical imaging of calcium accumulation in hippocampal pyramidal cells during synaptic activation. Nature. 1989 Oct 12;341(6242):533–536. doi: 10.1038/341533a0. [DOI] [PubMed] [Google Scholar]
  40. Regehr W. G., Mintz I. M. Participation of multiple calcium channel types in transmission at single climbing fiber to Purkinje cell synapses. Neuron. 1994 Mar;12(3):605–613. doi: 10.1016/0896-6273(94)90216-x. [DOI] [PubMed] [Google Scholar]
  41. Sakamoto J., Campbell K. P. A monoclonal antibody to the beta subunit of the skeletal muscle dihydropyridine receptor immunoprecipitates the brain omega-conotoxin GVIA receptor. J Biol Chem. 1991 Oct 5;266(28):18914–18919. [PubMed] [Google Scholar]
  42. Sakurai T., Hell J. W., Woppmann A., Miljanich G. P., Catterall W. A. Immunochemical identification and differential phosphorylation of alternatively spliced forms of the alpha 1A subunit of brain calcium channels. J Biol Chem. 1995 Sep 8;270(36):21234–21242. doi: 10.1074/jbc.270.36.21234. [DOI] [PubMed] [Google Scholar]
  43. Sather W. A., Tanabe T., Zhang J. F., Mori Y., Adams M. E., Tsien R. W. Distinctive biophysical and pharmacological properties of class A (BI) calcium channel alpha 1 subunits. Neuron. 1993 Aug;11(2):291–303. doi: 10.1016/0896-6273(93)90185-t. [DOI] [PubMed] [Google Scholar]
  44. Sheng Z. H., Rettig J., Cook T., Catterall W. A. Calcium-dependent interaction of N-type calcium channels with the synaptic core complex. Nature. 1996 Feb 1;379(6564):451–454. doi: 10.1038/379451a0. [DOI] [PubMed] [Google Scholar]
  45. Sheng Z. H., Rettig J., Takahashi M., Catterall W. A. Identification of a syntaxin-binding site on N-type calcium channels. Neuron. 1994 Dec;13(6):1303–1313. doi: 10.1016/0896-6273(94)90417-0. [DOI] [PubMed] [Google Scholar]
  46. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  47. Snutch T. P., Leonard J. P., Gilbert M. M., Lester H. A., Davidson N. Rat brain expresses a heterogeneous family of calcium channels. Proc Natl Acad Sci U S A. 1990 May;87(9):3391–3395. doi: 10.1073/pnas.87.9.3391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Snutch T. P., Reiner P. B. Ca2+ channels: diversity of form and function. Curr Opin Neurobiol. 1992 Jun;2(3):247–253. doi: 10.1016/0959-4388(92)90111-w. [DOI] [PubMed] [Google Scholar]
  49. Starr T. V., Prystay W., Snutch T. P. Primary structure of a calcium channel that is highly expressed in the rat cerebellum. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5621–5625. doi: 10.1073/pnas.88.13.5621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stea A., Tomlinson W. J., Soong T. W., Bourinet E., Dubel S. J., Vincent S. R., Snutch T. P. Localization and functional properties of a rat brain alpha 1A calcium channel reflect similarities to neuronal Q- and P-type channels. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10576–10580. doi: 10.1073/pnas.91.22.10576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Söllner T., Bennett M. K., Whiteheart S. W., Scheller R. H., Rothman J. E. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell. 1993 Nov 5;75(3):409–418. doi: 10.1016/0092-8674(93)90376-2. [DOI] [PubMed] [Google Scholar]
  52. Takahashi T., Momiyama A. Different types of calcium channels mediate central synaptic transmission. Nature. 1993 Nov 11;366(6451):156–158. doi: 10.1038/366156a0. [DOI] [PubMed] [Google Scholar]
  53. Tank D. W., Sugimori M., Connor J. A., Llinás R. R. Spatially resolved calcium dynamics of mammalian Purkinje cells in cerebellar slice. Science. 1988 Nov 4;242(4879):773–777. doi: 10.1126/science.2847315. [DOI] [PubMed] [Google Scholar]
  54. Trimble W. S., Gray T. S., Elferink L. A., Wilson M. C., Scheller R. H. Distinct patterns of expression of two VAMP genes within the rat brain. J Neurosci. 1990 Apr;10(4):1380–1387. doi: 10.1523/JNEUROSCI.10-04-01380.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Uchitel O. D., Protti D. A., Sanchez V., Cherksey B. D., Sugimori M., Llinás R. P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3330–3333. doi: 10.1073/pnas.89.8.3330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. West J. W., Numann R., Murphy B. J., Scheuer T., Catterall W. A. A phosphorylation site in the Na+ channel required for modulation by protein kinase C. Science. 1991 Nov 8;254(5033):866–868. doi: 10.1126/science.1658937. [DOI] [PubMed] [Google Scholar]
  57. Westenbroek R. E., Hell J. W., Warner C., Dubel S. J., Snutch T. P., Catterall W. A. Biochemical properties and subcellular distribution of an N-type calcium channel alpha 1 subunit. Neuron. 1992 Dec;9(6):1099–1115. doi: 10.1016/0896-6273(92)90069-p. [DOI] [PubMed] [Google Scholar]
  58. Westenbroek R. E., Sakurai T., Elliott E. M., Hell J. W., Starr T. V., Snutch T. P., Catterall W. A. Immunochemical identification and subcellular distribution of the alpha 1A subunits of brain calcium channels. J Neurosci. 1995 Oct;15(10):6403–6418. doi: 10.1523/JNEUROSCI.15-10-06403.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wheeler D. B., Randall A., Tsien R. W. Roles of N-type and Q-type Ca2+ channels in supporting hippocampal synaptic transmission. Science. 1994 Apr 1;264(5155):107–111. doi: 10.1126/science.7832825. [DOI] [PubMed] [Google Scholar]
  60. Williams M. E., Brust P. F., Feldman D. H., Patthi S., Simerson S., Maroufi A., McCue A. F., Veliçelebi G., Ellis S. B., Harpold M. M. Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel. Science. 1992 Jul 17;257(5068):389–395. doi: 10.1126/science.1321501. [DOI] [PubMed] [Google Scholar]
  61. Wu L. G., Saggau P. Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CA1 of hippocampus. Neuron. 1994 May;12(5):1139–1148. doi: 10.1016/0896-6273(94)90321-2. [DOI] [PubMed] [Google Scholar]
  62. Yoshida A., Oho C., Omori A., Kuwahara R., Ito T., Takahashi M. HPC-1 is associated with synaptotagmin and omega-conotoxin receptor. J Biol Chem. 1992 Dec 15;267(35):24925–24928. [PubMed] [Google Scholar]
  63. Zhang J. F., Randall A. D., Ellinor P. T., Horne W. A., Sather W. A., Tanabe T., Schwarz T. L., Tsien R. W. Distinctive pharmacology and kinetics of cloned neuronal Ca2+ channels and their possible counterparts in mammalian CNS neurons. Neuropharmacology. 1993 Nov;32(11):1075–1088. doi: 10.1016/0028-3908(93)90003-l. [DOI] [PubMed] [Google Scholar]
  64. el Far O., Charvin N., Leveque C., Martin-Moutot N., Takahashi M., Seagar M. J. Interaction of a synaptobrevin (VAMP)-syntaxin complex with presynaptic calcium channels. FEBS Lett. 1995 Mar 13;361(1):101–105. doi: 10.1016/0014-5793(95)00156-4. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES