The Journal of Membrane Biology

, Volume 147, Issue 1, pp 7–22 | Cite as

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle

  • D. R. Laver
  • L. D. Roden
  • G. P. Ahern
  • K. R. Eager
  • P. R. Junankar
  • A. F. Dulhunty


Ca2+-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca2+ inhibited cardiac RyRs with an average K m = 15 mm, skeletal RyRs with K m = 0.7 mm and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca2+ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [3H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca2+-inhibition in cardiac RyRs (Chu, Fill, Stefani & Entman (1993) J. Membrane Biol. 135, 49–59). Ryanodine prevented Ca2+ from inhibiting either cardiac or skeletal RyRs. Ca2+-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mm Cs+, but not by 500 mm K+, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca2+-activation. Ca2+-inhibition in skeletal RyRs was retained in the presence of 500 mm Cs+. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mm Cs+ in the solutions fail to demonstrate Ca2+-inhibition, while Ca2+-inhibition of Ca2+ release is observed in vesicle studies where K+ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca2+-inhibition in skeletal RyRs and cardiac RyRs, with different Ca2+ affinities for inhibition. We conclude that differences in the Ca2+-inhibition in cardiac and skeletal channels depends on their Ca2+ binding properties.

Key words

Ca2+-inhibition Sarcoplasmic reticulum Cardiac muscle Ryanodine receptor Artificial BLM 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ahern, G.P., Junankar, P.R., Dulhunty, A.F. (1994). Single channel activity of the ryanodine receptor calcium release channel is modulated by FK-506. FEBS Lett. 352:194–197Google Scholar
  2. Airey, J.A., Beck, C.F., Murakami, K., Tanksley, S.J., Deerinck, T.J., Ellisman, M.H., Sutko, J.L. (1990). Identification and localization of two triad junctional foot protein isoforms in mature avian fast twitch skeletal muscle. J. Biol. Chem. 265:14187–14194Google Scholar
  3. Ashley, C.C., Mulligan, I.P., Lea, T.J. (1991). Ca2+ and activation mechanisms in skeletal muscle. Q. Rev. Biophys. 24:1–73Google Scholar
  4. Ashley, R.H., Williams, A.J. (1990). Divalent cation activation and inhibition of single calcium release channels from sheep cardiac sarcoplasmic reticulum. J. Gen. Physiol. 95:981–1005Google Scholar
  5. Brandt, N.R., Caswell, A.H., Wen, S.R., Talvenheimo, J.A. (1990). Molecular interactions of the junctional foot protein and dihydropyridine receptor in skeletal muscle triads. J. Membrane Biol. 113:237–251Google Scholar
  6. Caswell, A.H., Brandt, N.R., Brunschwig, J.P., Purkerson, S. (1991). 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 30:7507–7513Google Scholar
  7. Chamberlain, B.K., Volpe, P., Fleischer, S. (1984). Calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles. General characteristics. J. Biol.Chem. 259:7540–7546Google Scholar
  8. Chen, S.R., Zhang, L., MacLennan, D.H. (1992). Characterization of a Ca2+ binding and regulatory site in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 267:23318–23326Google Scholar
  9. Chu, A., Dixon, M.C., Saito, A., Seiler, S., Fleischer, S. (1988). Isolation of sarcoplasmic reticulum fractions referable to longitudinal tabules and junctional terminal cisternae from rabbit skeletal muscle. Methods Enzymol. 157:36–46Google Scholar
  10. Chu, A., Fill, M., Stefani, E., Entman, M.L. (1993). Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel, although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles. J. Membrane Biol. 135:49–59Google Scholar
  11. Chung, S.H., Moore, J.B., Xia, L.G., Premkumar, L.S., Gage, P.W. (1990). Characterization of single channel currents using digital signal processing techniques based on Hidden Markov Models. Philos. Trans. R. Soc. Lond. Biol. 329:265–285Google Scholar
  12. Collins, K.D., Washabaugh, M.W. (1985). The Hofmeister effect and the behavior of water at interfaces. Q. Rev. Biophys. 18:323–422Google Scholar
  13. Fabiato, A. (1985). Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J. Gen. Physiol. 85:247–289Google Scholar
  14. Fettiplace, R., Gordon, L.G.M., Hladky, S.B., Requena, J., Zingsheim, H.P., Haydon, D.A., Methods of Membrane Biol. In: Methods of Membrane Biol, E.D. Korn, editor, pp. 1–75. Plenum Press, New York 1975Google Scholar
  15. Fruen, B.R., Mickelson, J.R., Shomer, N.H., Roghair, T.J., Louis, C.F. (1994). Regulation of the sarcoplasmic reticulum ryanodine receptor by inorganic phosphate. J. Biol. Chem. 269:192–198Google Scholar
  16. Herrmann-Frank, A., Varsanyi, M. (1993). Enhancement of Ca2+ release channel activity by phosphorylation of the skeletal muscle ryanodine receptor. FEBS Lett. 332:237–242Google Scholar
  17. Ikemoto, N., Ronjat, M., Meszaros, L.G., Koshita, M. (1989). Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum. Biochemistry 28:6764–6771Google Scholar
  18. Imagawa, T., Smith, J.S., Coronado, R., Campbell, K.P. (1987). Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel. J. Biol. Chem. 262:16636–16643Google Scholar
  19. Jayaraman, T., Brillantes, A.M., Timerman, A.P., Fleischer, S., Erdjuent-Bromage, H. Tempst, P., Marks, A.R. (1992). FK-506 binding protein associated with the calcium release channel (ryanodine receptor). J. Biol. Chem. 267:9474–9477Google Scholar
  20. Lai, F.A., Anderson, K., Rousseau, E., Lui, Q.Y., Meissner, G. (1988). Evidence for a Ca2+ channel within the ryanodine receptor complex from cardiac sarcoplasmic reticulum. Biochem. Biophys. Res. Commun. 151:441–449Google Scholar
  21. Laver, D.R., Walker, N.A. (1991). Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops from Chara australis. J. Membrane Biol. 120:131–139Google Scholar
  22. Lindsay, A.R., Williams, A.J. (1991). Functional characterisation of the ryanodine receptor purified from sheep cardiac muscle sarcoplasmic reticulum. Biochim. Biophys. Acta 1064:89–102Google Scholar
  23. Lindsay, A.R., Manning, S.D., Williams, A.J. (1991). Monovalent cation conductance in the ryanodine receptor-channel of sheep cardiac muscle sarcoplasmic reticulum J. Physiol. Lond. 439:463–480Google Scholar
  24. Ma., J. (1995). Desensitization of the skeletal muscle ryanodine receptor: Evidence of hetergeneity of calcium release channels. Biophys. J. 68:893–899Google Scholar
  25. Marty, I., Robert, M., Villaz, M., De Jongh, K.S., Lai, Y., Catterall, W.A., Ronjat, M. (1994). Biochemical evidence for a complex in-volving dihydropyridine receptor and ryanodine receptor in triad junctions of skeletal muscle. Proc. Natl. Acad. Sci. USA 91:2270–2274Google Scholar
  26. Meissner, G. (1986). Ryanodine activation and inhibition of the Ca2+ release channel of sarcoplasmic reticulum. J. Biol. Chem. 261:6300–6306Google Scholar
  27. Menegazzi, P., Larini, F., Treves, S., Guerrini, R., Quadroni, M., Zorzato, F. (1994). Identification and characterization of three calmodulin binding sites of the skeletal muscle ryanodine receptor. Biochemistry 33:9078–9084Google Scholar
  28. Miller, C., Racker, E. (1976). Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers. Cell 9:283–300Google Scholar
  29. Moczydlowski, E., Alvarez, O., Vergara, C., Latorre, R. (1985). Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers. J. Membrane Biol. 83:273–282Google Scholar
  30. Moczydlowski, E., Latorre, R. (1983). Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage-dependent Ca2+ binding reactions. J. Gen. Physiol. 82:511–542Google Scholar
  31. Mueller, P., Rudin, D.O., Tien, H.T., Westcott, W.C. (1962). Reconstitution of cell membrane structure in vitro and its transformation into an excitable system. Nature 194:979–981Google Scholar
  32. Murayama, T., Ogawa, Y. (1992). Purification and characterization of two ryanodine-binding protein isoforms from sarcoplasmic reticulum of bullfrog skeletal muscle. J. Biochem. Tokyo. 112:514–522Google Scholar
  33. Nabauer, M., Callewaert, G., Cleemann, L., Morad, M. (1989). Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes. Science 244:800–803Google Scholar
  34. Otsu, K., Willard, H.F., Khanna, V.J., Zorzato, F., Green, N.M., MacLennan, D.H. (1990). Molecular cloning of cDNA encoding the Ca++ release channel (Ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. J. Biol. Chem. 265:13472–13483Google Scholar
  35. Peng, M., Fan, H., Kirley, T.L., Caswell, A.M., Schwartz, A. (1994). Structural diversity of triadin in skeletal muscle and evidence of its existence in heart. FEBS Lett. 348:17–20Google Scholar
  36. Perrin, D.D., Sayce, I.G. (1967). Computer calculation of equilibrium concentrations in mixtures of metal ions and complexing species. Talanta 14:833–842Google Scholar
  37. Pessah, I.N., Waterhouse, A.L., Casida, J.E. (1985). The calciumryanodine receptor complex of skeletal and cardiac muscle. Biochem. Biophys. Res. Commun. 128:449–456Google Scholar
  38. Rardon, D.P., Cefali, D.C., Mitchell, R.D., Seiler, S.M., Hathaway, D.R., Jones, L.R. (1990). Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II. Effects on the Ca2+ release channel. Circ. Res. 67:84–96ISGoogle Scholar
  39. Rousseau, E. (1989). Single chloride-selective channel from cardiac sarcoplasmic reticulum studied in planar lipid bilayers. J. Membrane Biol. 110:39–47Google Scholar
  40. Rousseau, E., Meissner, G. (1989). Single cardiac sarcoplasmic reticulum Ca2+-release channel: activation by caffeine. Am. J. Physiol. 256:H328-H333Google Scholar
  41. Rousseau, E., Smith, J.S., Henderson, J.S., Meissner, G. (1986). Single channel and 45Ca2+ flux measurements of the cardiac sarcoplasmic reticulum calcium channel. Biophys. J. 50:1009–1014Google Scholar
  42. Rousseau, E., Smith, J.S., Meissner, G. (1987). Ryanodine modifies conductance and gating behavior of single Ca2+ release channel. Am. J. Physiol. 253:C364-C368Google Scholar
  43. Shomer, N.H.; Louis, C.F.; Fill, M.; Litterer, L.A.; Mickelson, J.R. (1993). Reconstitution of abnormalities in the malignant hyperthermiasusceptible pig ryanodine receptor Am. J. Physiol. 264:C125-C135Google Scholar
  44. Sigworth, F.J., Sine, S.M. (1987). Data transformations for improved display and fitting of single-channel dwell time histograms. Biophys. J. 52:1047–1054Google Scholar
  45. Sitsapesan, R., Montgomery, R.A., MacLeod, K.T., Williams, A.J. (1991). Sheep cardiac sarcoplasmic reticulum calcium-release channels: modification of conductance and gating by temperature. J. Physiol. Lond. 434:469–488Google Scholar
  46. Sitsapesan, R., Williams, A.J. (1990). Mechanisms of caffeine activation of single calcium-release channels of sheep cardiac sarcoplasmic reticulum. J. Physiol. Lond. 423:425–439Google Scholar
  47. Sitsapesan, R., Williams, A.J. (1994). Gating of the native and purified cardiac SR Ca2+-release channel with monovalent cations as permeant species. Biophys. J. 67:1484–1494Google Scholar
  48. Smith, J.S., Coronado, R., Meissner, G. (1986). Single channel measurements of the calcium release channel from skeletal muscle sarcoplasmic reticulum. Activation by Ca2+ and ATP and modulation by Mg2+. J. Gen. Physiol. 88:573–588Google Scholar
  49. Timerman, A.P., Jayaraman, T., Wiederrecht, G., Onoue, H., Marks, A.R., Fleischer, S. (1994). The ryanodine receptor from canine heart sarcoplasmic reticulum is associated with a novel FK-506 binding protein. Biochem. Biophys. Res. Commun. 198:701–706Google Scholar
  50. Tinker, A., Lindsay, A.R., Williams, A.J. (1992). A model for ionic conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum. J. Gen. Physiol. 100:495–517Google Scholar
  51. Tsien, R.Y. (1980). New calcium indicators and buffers with high selectivity against magnesium and protons; Design, synthesis, and properties of prototype structures. Biochemistry 19:2396–2404Google Scholar
  52. White, S.H., Thompson, T.E. (1973). Capacitance, area, and thickness variations in thin lipid films. Biochim. Biophys. Acta. 323:7–22Google Scholar
  53. Witcher, D.R., Kovacs, R.J., Schulman, H., Cefali, D.C., Jones, L.R. (1991). Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity. J. Biol. Chem. 266:11144–11152Google Scholar
  54. Woodhull, A.M. (1973). Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61:687–708CrossRefPubMedGoogle Scholar
  55. Zahradnikova, A., Palade, P. (1993). Procaine effects on single sarcoplasmic reticulum Ca2+ release channels. Biophys. J. 64:991–1003Google Scholar
  56. Zimanyi, I., Pessah, I.N. (1991). Comparison of [3H]ryanodine receptors and Ca++ release from rat cardiac and rabbit skeletal muscle sarcoplasmic reticulum. J. Pharmacol. Exp. Ther. 256:938–946Google Scholar

Copyright information

© Springer-Verlag New York Inc 1995

Authors and Affiliations

  • D. R. Laver
    • 1
  • L. D. Roden
    • 1
  • G. P. Ahern
    • 1
  • K. R. Eager
    • 1
  • P. R. Junankar
    • 1
  • A. F. Dulhunty
    • 1
  1. 1.Muscle Research Group, Division of NeuroscienceJohn Curtin School of Medical Research, Australian National UniversityCanberraAustralia

Personalised recommendations