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. 1993 Apr 1;291(Pt 1):199–204. doi: 10.1042/bj2910199

Intracellular Ca2+ stores of rat cerebellum: heterogeneity within and distinction from endoplasmic reticulum.

A Nori 1, A Villa 1, P Podini 1, D R Witcher 1, P Volpe 1
PMCID: PMC1132502  PMID: 8385931

Abstract

Rat cerebellum microsomes were subfractionated on isopycnic linear sucrose (20-42%)-density gradients. The distribution of endoplasmic reticulum (ER) markers (RNA, signal-sequence receptor alpha, calnexin, calreticulin, the immunoglobulin-binding protein Bip) and markers of intracellular rapidly exchanging Ca2+ stores [Ca2+ channels sensitive to either Ins(1,4,5)P3 or ryanodine) was investigated biochemically and immunologically. The comparison indicates that: (a) vesicles bearing the InsP3 receptor were separated from those bearing the ryanodine receptor; (b) ER markers, i.e. Bip, calnexin, signal-sequence receptor alpha, RNA, did not sediment as either InsP3 or ryanodine receptors did; (c) calreticulin, an intralumenal low-affinity high-capacity Ca(2+)-binding protein, had a widespread distribution, similar to that of Bip and calnexin, and was present in Purkinje, granule, Golgi and stellate neurons, as indicated by immunofluorescent labelling of cerebellum cortex cryosections. The present results show that the ER is not a homogeneous entity, and that Ca2+ stores are heterogeneous insofar as InsP3 receptors and ryanodine receptors are segregated, either to discrete intracellular organelles or to specialized ER subcompartments.

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Selected References

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  1. Alderson B. H., Volpe P. Distribution of endoplasmic reticulum and calciosome markers in membrane fractions isolated from different regions of the canine brain. Arch Biochem Biophys. 1989 Jul;272(1):162–174. doi: 10.1016/0003-9861(89)90207-5. [DOI] [PubMed] [Google Scholar]
  2. Brorson J. R., Bleakman D., Gibbons S. J., Miller R. J. The properties of intracellular calcium stores in cultured rat cerebellar neurons. J Neurosci. 1991 Dec;11(12):4024–4043. doi: 10.1523/JNEUROSCI.11-12-04024.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burgoyne R. D., Cheek T. R. Locating intracellular calcium stores. Trends Biochem Sci. 1991 Sep;16(9):319–320. doi: 10.1016/0968-0004(91)90131-e. [DOI] [PubMed] [Google Scholar]
  4. Cheek T. R., Barry V. A., Berridge M. J., Missiaen L. Bovine adrenal chromaffin cells contain an inositol 1,4,5-trisphosphate-insensitive but caffeine-sensitive Ca2+ store that can be regulated by intraluminal free Ca2+. Biochem J. 1991 May 1;275(Pt 3):697–701. doi: 10.1042/bj2750697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Danoff S. K., Ferris C. D., Donath C., Fischer G. A., Munemitsu S., Ullrich A., Snyder S. H., Ross C. A. Inositol 1,4,5-trisphosphate receptors: distinct neuronal and nonneuronal forms derived by alternative splicing differ in phosphorylation. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2951–2955. doi: 10.1073/pnas.88.7.2951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellisman M. H., Deerinck T. J., Ouyang Y., Beck C. F., Tanksley S. J., Walton P. D., Airey J. A., Sutko J. L. Identification and localization of ryanodine binding proteins in the avian central nervous system. Neuron. 1990 Aug;5(2):135–146. doi: 10.1016/0896-6273(90)90304-x. [DOI] [PubMed] [Google Scholar]
  7. Fasolato C., Zottini M., Clementi E., Zacchetti D., Meldolesi J., Pozzan T. Intracellular Ca2+ pools in PC12 cells. Three intracellular pools are distinguished by their turnover and mechanisms of Ca2+ accumulation, storage, and release. J Biol Chem. 1991 Oct 25;266(30):20159–20167. [PubMed] [Google Scholar]
  8. Fliegel L., Burns K., MacLennan D. H., Reithmeier R. A., Michalak M. Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. J Biol Chem. 1989 Dec 25;264(36):21522–21528. [PubMed] [Google Scholar]
  9. Fliegel L., Ohnishi M., Carpenter M. R., Khanna V. K., Reithmeier R. A., MacLennan D. H. Amino acid sequence of rabbit fast-twitch skeletal muscle calsequestrin deduced from cDNA and peptide sequencing. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1167–1171. doi: 10.1073/pnas.84.5.1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Furuichi T., Yoshikawa S., Miyawaki A., Wada K., Maeda N., Mikoshiba K. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature. 1989 Nov 2;342(6245):32–38. doi: 10.1038/342032a0. [DOI] [PubMed] [Google Scholar]
  11. Guillemette G., Balla T., Baukal A. J., Catt K. J. Characterization of inositol 1,4,5-trisphosphate receptors and calcium mobilization in a hepatic plasma membrane fraction. J Biol Chem. 1988 Apr 5;263(10):4541–4548. [PubMed] [Google Scholar]
  12. Hartmann E., Wiedmann M., Rapoport T. A. A membrane component of the endoplasmic reticulum that may be essential for protein translocation. EMBO J. 1989 Aug;8(8):2225–2229. doi: 10.1002/j.1460-2075.1989.tb08346.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaprielian Z., Campbell A. M., Fambrough D. M. Identification of a Ca2+-ATPase in cerebellar Purkinje cells. Brain Res Mol Brain Res. 1989 Jul;6(1):55–60. doi: 10.1016/0169-328x(89)90028-4. [DOI] [PubMed] [Google Scholar]
  14. Krause K. H., Lew P. D. Subcellular distribution of Ca2+ pumping sites in human neutrophils. J Clin Invest. 1987 Jul;80(1):107–116. doi: 10.1172/JCI113035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krause K. H., Simmerman H. K., Jones L. R., Campbell K. P. Sequence similarity of calreticulin with a Ca2(+)-binding protein that co-purifies with an Ins(1,4,5)P3-sensitive Ca2+ store in HL-60 cells. Biochem J. 1990 Sep 1;270(2):545–548. doi: 10.1042/bj2700545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. Lieu T. S., Newkirk M. M., Capra J. D., Sontheimer R. D. Molecular characterization of human Ro/SS-A antigen. Amino terminal sequence of the protein moiety of human Ro/SS-A antigen and immunological activity of a corresponding synthetic peptide. J Clin Invest. 1988 Jul;82(1):96–101. doi: 10.1172/JCI113607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lytton J., Zarain-Herzberg A., Periasamy M., MacLennan D. H. Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase. J Biol Chem. 1989 Apr 25;264(12):7059–7065. [PubMed] [Google Scholar]
  20. MacLennan D. H., Wong P. T. Isolation of a calcium-sequestering protein from sarcoplasmic reticulum. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1231–1235. doi: 10.1073/pnas.68.6.1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Malgaroli A., Fesce R., Meldolesi J. Spontaneous [Ca2+]i fluctuations in rat chromaffin cells do not require inositol 1,4,5-trisphosphate elevations but are generated by a caffeine- and ryanodine-sensitive intracellular Ca2+ store. J Biol Chem. 1990 Feb 25;265(6):3005–3008. [PubMed] [Google Scholar]
  22. McPherson P. S., Campbell K. P. Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes. J Biol Chem. 1990 Oct 25;265(30):18454–18460. [PubMed] [Google Scholar]
  23. Meldolesi J., Villa A., Volpe P., Pozzan T. Cellular sites of IP3 action. Adv Second Messenger Phosphoprotein Res. 1992;26:187–208. [PubMed] [Google Scholar]
  24. Michalak M., Baksh S., Opas M. Identification and immunolocalization of calreticulin in pancreatic cells: no evidence for "calciosomes". Exp Cell Res. 1991 Nov;197(1):91–99. doi: 10.1016/0014-4827(91)90484-c. [DOI] [PubMed] [Google Scholar]
  25. Michalak M., Milner R. E., Burns K., Opas M. Calreticulin. Biochem J. 1992 Aug 1;285(Pt 3):681–692. doi: 10.1042/bj2850681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mignery G. A., Newton C. L., Archer B. T., 3rd, Südhof T. C. Structure and expression of the rat inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1990 Jul 25;265(21):12679–12685. [PubMed] [Google Scholar]
  27. Milner R. E., Baksh S., Shemanko C., Carpenter M. R., Smillie L., Vance J. E., Opas M., Michalak M. Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum. J Biol Chem. 1991 Apr 15;266(11):7155–7165. [PubMed] [Google Scholar]
  28. Mészáros L. G., Volpe P. Caffeine- and ryanodine-sensitive Ca2+ stores of canine cerebrum and cerebellum neurons. Am J Physiol. 1991 Dec;261(6 Pt 1):C1048–C1054. doi: 10.1152/ajpcell.1991.261.6.C1048. [DOI] [PubMed] [Google Scholar]
  29. Nakagawa T., Shiota C., Okano H., Mikoshiba K. Differential localization of alternative spliced transcripts encoding inositol 1,4,5-trisphosphate receptors in mouse cerebellum and hippocampus: in situ hybridization study. J Neurochem. 1991 Nov;57(5):1807–1810. doi: 10.1111/j.1471-4159.1991.tb06385.x. [DOI] [PubMed] [Google Scholar]
  30. Peter F., Nguyen Van P., Söling H. D. Different sorting of Lys-Asp-Glu-Leu proteins in rat liver. J Biol Chem. 1992 May 25;267(15):10631–10637. [PubMed] [Google Scholar]
  31. Plessers L., Eggermont J. A., Wuytack F., Casteels R. A study of the organellar Ca2(+)-transport ATPase isozymes in pig cerebellar Purkinje neurons. J Neurosci. 1991 Mar;11(3):650–656. doi: 10.1523/JNEUROSCI.11-03-00650.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Robinson I. M., Burgoyne R. D. Characterisation of distinct inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive calcium stores in digitonin-permeabilised adrenal chromaffin cells. J Neurochem. 1991 May;56(5):1587–1593. doi: 10.1111/j.1471-4159.1991.tb02055.x. [DOI] [PubMed] [Google Scholar]
  33. Ross C. A., Danoff S. K., Schell M. J., Snyder S. H., Ullrich A. Three additional inositol 1,4,5-trisphosphate receptors: molecular cloning and differential localization in brain and peripheral tissues. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4265–4269. doi: 10.1073/pnas.89.10.4265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rossier M. F., Bird G. S., Putney J. W., Jr Subcellular distribution of the calcium-storing inositol 1,4,5-trisphosphate-sensitive organelle in rat liver. Possible linkage to the plasma membrane through the actin microfilaments. Biochem J. 1991 Mar 15;274(Pt 3):643–650. doi: 10.1042/bj2740643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Satoh T., Ross C. A., Villa A., Supattapone S., Pozzan T., Snyder S. H., Meldolesi J. The inositol 1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: quantitative immunogold labeling reveals concentration in an ER subcompartment. J Cell Biol. 1990 Aug;111(2):615–624. doi: 10.1083/jcb.111.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Scott B. T., Simmerman H. K., Collins J. H., Nadal-Ginard B., Jones L. R. Complete amino acid sequence of canine cardiac calsequestrin deduced by cDNA cloning. J Biol Chem. 1988 Jun 25;263(18):8958–8964. [PubMed] [Google Scholar]
  37. Stauderman K. A., McKinney R. A., Murawsky M. M. The role of caffeine-sensitive Ca2+ stores in agonist- and inositol 1,4,5-trisphosphate-induced Ca2+ release from bovine adrenal chromaffin cells. Biochem J. 1991 Sep 15;278(Pt 3):643–650. doi: 10.1042/bj2780643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Supattapone S., Worley P. F., Baraban J. M., Snyder S. H. Solubilization, purification, and characterization of an inositol trisphosphate receptor. J Biol Chem. 1988 Jan 25;263(3):1530–1534. [PubMed] [Google Scholar]
  39. Südhof T. C., Newton C. L., Archer B. T., 3rd, Ushkaryov Y. A., Mignery G. A. Structure of a novel InsP3 receptor. EMBO J. 1991 Nov;10(11):3199–3206. doi: 10.1002/j.1460-2075.1991.tb04882.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takei K., Stukenbrok H., Metcalf A., Mignery G. A., Südhof T. C., Volpe P., De Camilli P. Ca2+ stores in Purkinje neurons: endoplasmic reticulum subcompartments demonstrated by the heterogeneous distribution of the InsP3 receptor, Ca(2+)-ATPase, and calsequestrin. J Neurosci. 1992 Feb;12(2):489–505. doi: 10.1523/JNEUROSCI.12-02-00489.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thayer S. A., Perney T. M., Miller R. J. Regulation of calcium homeostasis in sensory neurons by bradykinin. J Neurosci. 1988 Nov;8(11):4089–4097. doi: 10.1523/JNEUROSCI.08-11-04089.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Treves S., De Mattei M., Landfredi M., Villa A., Green N. M., MacLennan D. H., Meldolesi J., Pozzan T. Calreticulin is a candidate for a calsequestrin-like function in Ca2(+)-storage compartments (calciosomes) of liver and brain. Biochem J. 1990 Oct 15;271(2):473–480. doi: 10.1042/bj2710473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tsien R. W., Tsien R. Y. Calcium channels, stores, and oscillations. Annu Rev Cell Biol. 1990;6:715–760. doi: 10.1146/annurev.cb.06.110190.003435. [DOI] [PubMed] [Google Scholar]
  44. Van Delden C., Favre C., Spät A., Cerny E., Krause K. H., Lew D. P. Purification of an inositol 1,4,5-trisphosphate-binding calreticulin-containing intracellular compartment of HL-60 cells. Biochem J. 1992 Feb 1;281(Pt 3):651–656. doi: 10.1042/bj2810651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Verma A., Ross C. A., Verma D., Supattapone S., Snyder S. H. Rat brain endoplasmic reticulum calcium pools are anatomically and functionally segregated. Cell Regul. 1990 Sep;1(10):781–790. doi: 10.1091/mbc.1.10.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Villa A., Podini P., Clegg D. O., Pozzan T., Meldolesi J. Intracellular Ca2+ stores in chicken Purkinje neurons: differential distribution of the low affinity-high capacity Ca2+ binding protein, calsequestrin, of Ca2+ ATPase and of the ER lumenal protein, Bip. J Cell Biol. 1991 May;113(4):779–791. doi: 10.1083/jcb.113.4.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Villa A., Sharp A. H., Racchetti G., Podini P., Bole D. G., Dunn W. A., Pozzan T., Snyder S. H., Meldolesi J. The endoplasmic reticulum of Purkinje neuron body and dendrites: molecular identity and specializations for Ca2+ transport. Neuroscience. 1992 Jul;49(2):467–477. doi: 10.1016/0306-4522(92)90111-e. [DOI] [PubMed] [Google Scholar]
  48. Volpe P., Alderson-Lang B. H., Madeddu L., Damiani E., Collins J. H., Margreth A. Calsequestrin, a component of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store of chicken cerebellum. Neuron. 1990 Nov;5(5):713–721. doi: 10.1016/0896-6273(90)90225-5. [DOI] [PubMed] [Google Scholar]
  49. Volpe P., Furlan S., Damiani E. Purification and characterization of calsequestrin from chicken cerebellum. Biochem Biophys Res Commun. 1991 Nov 27;181(1):28–35. doi: 10.1016/s0006-291x(05)81377-4. [DOI] [PubMed] [Google Scholar]
  50. Volpe P., Pozzan T., Meldolesi J. Rapidly exchanging Ca2+ stores of non-muscle cells. Semin Cell Biol. 1990 Aug;1(4):297–304. [PubMed] [Google Scholar]
  51. Volpe P., Villa A., Damiani E., Sharp A. H., Podini P., Snyder S. H., Meldolesi J. Heterogeneity of microsomal Ca2+ stores in chicken Purkinje neurons. EMBO J. 1991 Nov;10(11):3183–3189. doi: 10.1002/j.1460-2075.1991.tb04880.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wada I., Rindress D., Cameron P. H., Ou W. J., Doherty J. J., 2nd, Louvard D., Bell A. W., Dignard D., Thomas D. Y., Bergeron J. J. SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J Biol Chem. 1991 Oct 15;266(29):19599–19610. [PubMed] [Google Scholar]
  53. Walton P. D., Airey J. A., Sutko J. L., Beck C. F., Mignery G. A., Südhof T. C., Deerinck T. J., Ellisman M. H. Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons. J Cell Biol. 1991 Jun;113(5):1145–1157. doi: 10.1083/jcb.113.5.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Watras J., Bezprozvanny I., Ehrlich B. E. Inositol 1,4,5-trisphosphate-gated channels in cerebellum: presence of multiple conductance states. J Neurosci. 1991 Oct;11(10):3239–3245. doi: 10.1523/JNEUROSCI.11-10-03239.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Witcher D. R., Strifler B. A., Jones L. R. Cardiac-specific phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase is conserved in the brain ryanodine receptor. J Biol Chem. 1992 Mar 5;267(7):4963–4967. [PubMed] [Google Scholar]
  56. Zimanyi I., Pessah I. N. Pharmacological characterization of the specific binding of [3H]ryanodine to rat brain microsomal membranes. Brain Res. 1991 Oct 11;561(2):181–191. doi: 10.1016/0006-8993(91)91594-q. [DOI] [PubMed] [Google Scholar]

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