Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Dec 1;123(5):1161–1174. doi: 10.1083/jcb.123.5.1161

Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro

PMCID: PMC2119885  PMID: 8245124

Abstract

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95- kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential- induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.

Full Text

The Full Text of this article is available as a PDF (5.0 MB).

Selected References

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

  1. Adams B. A., Beam K. G. A novel calcium current in dysgenic skeletal muscle. J Gen Physiol. 1989 Sep;94(3):429–444. doi: 10.1085/jgp.94.3.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams B. A., Beam K. G. Muscular dysgenesis in mice: a model system for studying excitation-contraction coupling. FASEB J. 1990 Jul;4(10):2809–2816. doi: 10.1096/fasebj.4.10.2165014. [DOI] [PubMed] [Google Scholar]
  3. Adams B. A., Tanabe T., Mikami A., Numa S., Beam K. G. Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs. Nature. 1990 Aug 9;346(6284):569–572. doi: 10.1038/346569a0. [DOI] [PubMed] [Google Scholar]
  4. Allbritton N. L., Meyer T., Stryer L. Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science. 1992 Dec 11;258(5089):1812–1815. doi: 10.1126/science.1465619. [DOI] [PubMed] [Google Scholar]
  5. Banker B. Q. Muscular dysgenesis in the mouse (mdg/mdg). I. Ultrastructural study of skeletal and cardiac muscle. J Neuropathol Exp Neurol. 1977 Jan;36(1):100–127. doi: 10.1097/00005072-197701000-00010. [DOI] [PubMed] [Google Scholar]
  6. Beam K. G., Knudson C. M., Powell J. A. A lethal mutation in mice eliminates the slow calcium current in skeletal muscle cells. Nature. 1986 Mar 13;320(6058):168–170. doi: 10.1038/320168a0. [DOI] [PubMed] [Google Scholar]
  7. Block B. A., Imagawa T., Campbell K. P., Franzini-Armstrong C. Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle. J Cell Biol. 1988 Dec;107(6 Pt 2):2587–2600. doi: 10.1083/jcb.107.6.2587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Borsotto M., Barhanin J., Fosset M., Lazdunski M. The 1,4-dihydropyridine receptor associated with the skeletal muscle voltage-dependent Ca2+ channel. Purification and subunit composition. J Biol Chem. 1985 Nov 15;260(26):14255–14263. [PubMed] [Google Scholar]
  9. Bowden-Essien F. An in vitro study of normal and mutant myogenesis in the mouse. Dev Biol. 1972 Mar;27(3):351–364. doi: 10.1016/0012-1606(72)90174-1. [DOI] [PubMed] [Google Scholar]
  10. Brandt N. R., Caswell A. H., Wen S. R., Talvenheimo J. A. Molecular interactions of the junctional foot protein and dihydropyridine receptor in skeletal muscle triads. J Membr Biol. 1990 Feb;113(3):237–251. doi: 10.1007/BF01870075. [DOI] [PubMed] [Google Scholar]
  11. Caswell A. H., Brandt N. R., Brunschwig J. P., Purkerson S. Localization and partial characterization of the oligomeric disulfide-linked molecular weight 95,000 protein (triadin) which binds the ryanodine and dihydropyridine receptors in skeletal muscle triadic vesicles. Biochemistry. 1991 Jul 30;30(30):7507–7513. doi: 10.1021/bi00244a020. [DOI] [PubMed] [Google Scholar]
  12. Chaudhari N. A single nucleotide deletion in the skeletal muscle-specific calcium channel transcript of muscular dysgenesis (mdg) mice. J Biol Chem. 1992 Dec 25;267(36):25636–25639. [PubMed] [Google Scholar]
  13. Chaudhari N., Beam K. G. The muscular dysgenesis mutation in mice leads to arrest of the genetic program for muscle differentiation. Dev Biol. 1989 Jun;133(2):456–467. doi: 10.1016/0012-1606(89)90049-3. [DOI] [PubMed] [Google Scholar]
  14. Chaudhari N., Beam K. G. mRNA for cardiac calcium channel is expressed during development of skeletal muscle. Dev Biol. 1993 Feb;155(2):507–515. doi: 10.1006/dbio.1993.1048. [DOI] [PubMed] [Google Scholar]
  15. Courbin P., Do Thi A., Ressouches A., Dussartre C., Powell J. A., Koenig J. Restoration of dysgenic murine (mdg) myotube contraction after addition of Schwann cells from normal mice in vitro. Biol Cell. 1989;67(3):355–358. [PubMed] [Google Scholar]
  16. Courbin P., Koenig J., Ressouches A., Beam K. G., Powell J. A. Rescue of excitation-contraction coupling in dysgenic muscle by addition of fibroblasts in vitro. Neuron. 1989 Apr;2(4):1341–1350. doi: 10.1016/0896-6273(89)90072-x. [DOI] [PubMed] [Google Scholar]
  17. Fleischer S., Inui M. Biochemistry and biophysics of excitation-contraction coupling. Annu Rev Biophys Biophys Chem. 1989;18:333–364. doi: 10.1146/annurev.bb.18.060189.002001. [DOI] [PubMed] [Google Scholar]
  18. Flockerzi V., Oeken H. J., Hofmann F., Pelzer D., Cavalié A., Trautwein W. Purified dihydropyridine-binding site from skeletal muscle t-tubules is a functional calcium channel. Nature. 1986 Sep 4;323(6083):66–68. doi: 10.1038/323066a0. [DOI] [PubMed] [Google Scholar]
  19. Flucher B. E., Andrews S. B. Characterization of spontaneous and action potential-induced calcium transients in developing myotubes in vitro. Cell Motil Cytoskeleton. 1993;25(2):143–157. doi: 10.1002/cm.970250204. [DOI] [PubMed] [Google Scholar]
  20. Flucher B. E., Morton M. E., Froehner S. C., Daniels M. P. Localization of the alpha 1 and alpha 2 subunits of the dihydropyridine receptor and ankyrin in skeletal muscle triads. Neuron. 1990 Sep;5(3):339–351. doi: 10.1016/0896-6273(90)90170-k. [DOI] [PubMed] [Google Scholar]
  21. Flucher B. E., Phillips J. L., Powell J. A., Andrews S. B., Daniels M. P. Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro. Dev Biol. 1992 Apr;150(2):266–280. doi: 10.1016/0012-1606(92)90241-8. [DOI] [PubMed] [Google Scholar]
  22. Flucher B. E., Phillips J. L., Powell J. A. Dihydropyridine receptor alpha subunits in normal and dysgenic muscle in vitro: expression of alpha 1 is required for proper targeting and distribution of alpha 2. J Cell Biol. 1991 Dec;115(5):1345–1356. doi: 10.1083/jcb.115.5.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Flucher B. E. Structural analysis of muscle development: transverse tubules, sarcoplasmic reticulum, and the triad. Dev Biol. 1992 Dec;154(2):245–260. doi: 10.1016/0012-1606(92)90065-o. [DOI] [PubMed] [Google Scholar]
  24. Flucher B. E., Terasaki M., Chin H. M., Beeler T. J., Daniels M. P. Biogenesis of transverse tubules in skeletal muscle in vitro. Dev Biol. 1991 May;145(1):77–90. doi: 10.1016/0012-1606(91)90214-n. [DOI] [PubMed] [Google Scholar]
  25. Fosset M., Jaimovich E., Delpont E., Lazdunski M. [3H]nitrendipine receptors in skeletal muscle. J Biol Chem. 1983 May 25;258(10):6086–6092. [PubMed] [Google Scholar]
  26. Franzini-Armstrong C., Pincon-Raymond M., Rieger F. Muscle fibers from dysgenic mouse in vivo lack a surface component of peripheral couplings. Dev Biol. 1991 Aug;146(2):364–376. doi: 10.1016/0012-1606(91)90238-x. [DOI] [PubMed] [Google Scholar]
  27. Franzini-Armstrong C. Simultaneous maturation of transverse tubules and sarcoplasmic reticulum during muscle differentiation in the mouse. Dev Biol. 1991 Aug;146(2):353–363. doi: 10.1016/0012-1606(91)90237-w. [DOI] [PubMed] [Google Scholar]
  28. Gonatas J. O., Gonatas N. K., Stieber A., Louvard D. Polypeptides of the Golgi apparatus of neurons from rat brain. J Neurochem. 1987 Nov;49(5):1498–1506. doi: 10.1111/j.1471-4159.1987.tb01020.x. [DOI] [PubMed] [Google Scholar]
  29. Inui M., Saito A., Fleischer S. Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle. J Biol Chem. 1987 Feb 5;262(4):1740–1747. [PubMed] [Google Scholar]
  30. Jaffe L. F. The path of calcium in cytosolic calcium oscillations: a unifying hypothesis. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9883–9887. doi: 10.1073/pnas.88.21.9883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Jorgensen A. O., Kalnins V. I., Zubrzycka E., MacLennan D. H. Assembly of the sarcoplasmic reticulum. Localization by immunofluorescence of sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures. J Cell Biol. 1977 Jul;74(1):287–298. doi: 10.1083/jcb.74.1.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jorgensen A. O., Shen A. C., Arnold W., Leung A. T., Campbell K. P. Subcellular distribution of the 1,4-dihydropyridine receptor in rabbit skeletal muscle in situ: an immunofluorescence and immunocolloidal gold-labeling study. J Cell Biol. 1989 Jul;109(1):135–147. doi: 10.1083/jcb.109.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Jorgensen A. O., Shen A. C., Campbell K. P., MacLennan D. H. Ultrastructural localization of calsequestrin in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections. J Cell Biol. 1983 Nov;97(5 Pt 1):1573–1581. doi: 10.1083/jcb.97.5.1573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kaprielian Z., Fambrough D. M. Expression of fast and slow isoforms of the Ca2+-ATPase in developing chick skeletal muscle. Dev Biol. 1987 Dec;124(2):490–503. doi: 10.1016/0012-1606(87)90502-1. [DOI] [PubMed] [Google Scholar]
  35. Kim K. C., Caswell A. H., Talvenheimo J. A., Brandt N. R. Isolation of a terminal cisterna protein which may link the dihydropyridine receptor to the junctional foot protein in skeletal muscle. Biochemistry. 1990 Oct 2;29(39):9281–9289. doi: 10.1021/bi00491a025. [DOI] [PubMed] [Google Scholar]
  36. Klaus M. M., Scordilis S. P., Rapalus J. M., Briggs R. T., Powell J. A. Evidence for dysfunction in the regulation of cytosolic Ca2+ in excitation-contraction uncoupled dysgenic muscle. Dev Biol. 1983 Sep;99(1):152–165. doi: 10.1016/0012-1606(83)90262-2. [DOI] [PubMed] [Google Scholar]
  37. Knudson C. M., Chaudhari N., Sharp A. H., Powell J. A., Beam K. G., Campbell K. P. Specific absence of the alpha 1 subunit of the dihydropyridine receptor in mice with muscular dysgenesis. J Biol Chem. 1989 Jan 25;264(3):1345–1348. [PubMed] [Google Scholar]
  38. Lai F. A., Erickson H. P., Rousseau E., Liu Q. Y., Meissner G. Purification and reconstitution of the calcium release channel from skeletal muscle. Nature. 1988 Jan 28;331(6154):315–319. doi: 10.1038/331315a0. [DOI] [PubMed] [Google Scholar]
  39. Lechleiter J. D., Clapham D. E. Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes. Cell. 1992 Apr 17;69(2):283–294. doi: 10.1016/0092-8674(92)90409-6. [DOI] [PubMed] [Google Scholar]
  40. McCray J., Werner G. Production and properties of site-specific antibodies to synthetic peptide antigens related to potential cell surface receptor sites for rhinovirus. Methods Enzymol. 1989;178:676–692. doi: 10.1016/0076-6879(89)78045-9. [DOI] [PubMed] [Google Scholar]
  41. Meissner G., Conner G. E., Fleischer S. Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca 2+ -pump and Ca 2+ -binding proteins. Biochim Biophys Acta. 1973 Mar 16;298(2):246–269. doi: 10.1016/0005-2736(73)90355-6. [DOI] [PubMed] [Google Scholar]
  42. Morton M. E., Froehner S. C. Monoclonal antibody identifies a 200-kDa subunit of the dihydropyridine-sensitive calcium channel. J Biol Chem. 1987 Sep 5;262(25):11904–11907. [PubMed] [Google Scholar]
  43. PAI A. C. DEVELOPMENTAL GENETICS OF A LETHAL MUTATION, MUSCULAR DYSGENESIS (MDG), IN THE MOUSE. I. GENETIC ANALYSIS AND GROSS MORPHOLOGY. Dev Biol. 1965 Feb;11:82–92. doi: 10.1016/0012-1606(65)90038-2. [DOI] [PubMed] [Google Scholar]
  44. Pinçon-Raymond M., García L., Romey G., Houenou L., Lazdunski M., Rieger F. A genetic model for the study of abnormal nerve-muscle interactions at the level of excitation-contraction coupling: the mutation muscular dysgenesis. J Physiol (Paris) 1990;84(1):82–87. [PubMed] [Google Scholar]
  45. Pinçon-Raymond M., Rieger F., Fosset M., Lazdunski M. Abnormal transverse tubule system and abnormal amount of receptors for Ca2+ channel inhibitors of the dihydropyridine family in skeletal muscle from mice with embryonic muscular dysgenesis. Dev Biol. 1985 Dec;112(2):458–466. doi: 10.1016/0012-1606(85)90418-x. [DOI] [PubMed] [Google Scholar]
  46. Powell J. A., Fambrough D. M. Electrical properties of normal and dysgenic mouse skeletal muscle in culture. J Cell Physiol. 1973 Aug;82(1):21–38. doi: 10.1002/jcp.1040820104. [DOI] [PubMed] [Google Scholar]
  47. Rieger F., Pinçon-Raymond M., Tassin A. M., Garcia L., Romey G., Fosset M., Lazdunski M. Excitation-contraction uncoupling in the developing skeletal muscle of the muscular dysgenesis mouse embryo. Biochimie. 1987 Apr;69(4):411–417. doi: 10.1016/0300-9084(87)90033-2. [DOI] [PubMed] [Google Scholar]
  48. Rios E., Brum G. Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle. Nature. 1987 Feb 19;325(6106):717–720. doi: 10.1038/325717a0. [DOI] [PubMed] [Google Scholar]
  49. Ríos E., Ma J. J., González A. The mechanical hypothesis of excitation-contraction (EC) coupling in skeletal muscle. J Muscle Res Cell Motil. 1991 Apr;12(2):127–135. doi: 10.1007/BF01774031. [DOI] [PubMed] [Google Scholar]
  50. Saito A., Seiler S., Chu A., Fleischer S. Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle. J Cell Biol. 1984 Sep;99(3):875–885. doi: 10.1083/jcb.99.3.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schneider M. F., Chandler W. K. Voltage dependent charge movement of skeletal muscle: a possible step in excitation-contraction coupling. Nature. 1973 Mar 23;242(5395):244–246. doi: 10.1038/242244a0. [DOI] [PubMed] [Google Scholar]
  52. Striessnig J., Goll A., Moosburger K., Glossmann H. Purified calcium channels have three allosterically coupled drug receptors. FEBS Lett. 1986 Mar 3;197(1-2):204–210. doi: 10.1016/0014-5793(86)80327-1. [DOI] [PubMed] [Google Scholar]
  53. Takahashi M., Seagar M. J., Jones J. F., Reber B. F., Catterall W. A. Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5478–5482. doi: 10.1073/pnas.84.15.5478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Takeshima H., Nishimura S., Matsumoto T., Ishida H., Kangawa K., Minamino N., Matsuo H., Ueda M., Hanaoka M., Hirose T. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature. 1989 Jun 8;339(6224):439–445. doi: 10.1038/339439a0. [DOI] [PubMed] [Google Scholar]
  55. Tanabe T., Beam K. G., Powell J. A., Numa S. Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature. 1988 Nov 10;336(6195):134–139. doi: 10.1038/336134a0. [DOI] [PubMed] [Google Scholar]
  56. Wagenknecht T., Grassucci R., Frank J., Saito A., Inui M., Fleischer S. Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum. Nature. 1989 Mar 9;338(6211):167–170. doi: 10.1038/338167a0. [DOI] [PubMed] [Google Scholar]
  57. Yuan S. H., Arnold W., Jorgensen A. O. Biogenesis of transverse tubules and triads: immunolocalization of the 1,4-dihydropyridine receptor, TS28, and the ryanodine receptor in rabbit skeletal muscle developing in situ. J Cell Biol. 1991 Jan;112(2):289–301. doi: 10.1083/jcb.112.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES