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. 2002 May;82(5):2436–2447. doi: 10.1016/s0006-3495(02)75587-2

Role of the proposed pore-forming segment of the Ca2+ release channel (ryanodine receptor) in ryanodine interaction.

S R Wayne Chen 1, Pin Li 1, Mingcai Zhao 1, Xiaoli Li 1, Lin Zhang 1
PMCID: PMC1302034  PMID: 11964232

Abstract

In earlier studies we showed that point mutations introduced into the proposed pore-forming segment, GVRAGGGIGD (amino acids 4820-4829), of the mouse cardiac ryanodine receptor reduced or abolished high affinity [3H]ryanodine binding. Here we investigate the effects of these mutations on the affinity and dissociation properties of [3H]ryanodine binding and on ryanodine modification of the ryanodine receptor channel at the single channel and whole cell levels. Scatchard analysis and dissociation studies reveal that mutation G4824A decreases the equilibrium dissociation constant (K(d)) and the dissociation rate constant (k(off)), whereas mutations G4828A and D4829A increase the K(d) and k(off) values. The effect of ryanodine on single G4828A and D4829A mutant channels is reversible on the time scale of single channel experiments, in contrast to the irreversible effect of ryanodine on single wild-type channels. Ryanodine alone is able to induce a large and sustained Ca2+ release in HEK293 cells transfected with the R4822A or G4825A mutant cDNA at the resting cytoplasmic Ca2+ but causes little or no Ca2+ release in cells transfected with the wild-type cDNA. Mutation G4826C diminishes the functional effect of ryanodine on Ca2+ release but spares caffeine-induced Ca2+ release in HEK293 cells. Co-expression of the wild-type and G4826C mutant proteins produces single channels that interact with ryanodine reversibly and display altered conductance and ryanodine response. These results are consistent with the view that the proposed pore-forming segment is a critical determinant of ryanodine interaction. A putative model of ryanodine-ryanodine receptor interaction is proposed.

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Selected References

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  1. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  2. Bhat M. B., Zhao J., Takeshima H., Ma J. Functional calcium release channel formed by the carboxyl-terminal portion of ryanodine receptor. Biophys J. 1997 Sep;73(3):1329–1336. doi: 10.1016/S0006-3495(97)78166-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Callaway C., Seryshev A., Wang J. P., Slavik K. J., Needleman D. H., Cantu C., 3rd, Wu Y., Jayaraman T., Marks A. R., Hamilton S. L. Localization of the high and low affinity [3H]ryanodine binding sites on the skeletal muscle Ca2+ release channel. J Biol Chem. 1994 Jun 3;269(22):15876–15884. [PubMed] [Google Scholar]
  4. Chen S. R., Ebisawa K., Li X., Zhang L. Molecular identification of the ryanodine receptor Ca2+ sensor. J Biol Chem. 1998 Jun 12;273(24):14675–14678. doi: 10.1074/jbc.273.24.14675. [DOI] [PubMed] [Google Scholar]
  5. Clapham D. E. Calcium signaling. Cell. 1995 Jan 27;80(2):259–268. doi: 10.1016/0092-8674(95)90408-5. [DOI] [PubMed] [Google Scholar]
  6. Coronado R., Morrissette J., Sukhareva M., Vaughan D. M. Structure and function of ryanodine receptors. Am J Physiol. 1994 Jun;266(6 Pt 1):C1485–C1504. doi: 10.1152/ajpcell.1994.266.6.C1485. [DOI] [PubMed] [Google Scholar]
  7. Doyle D. A., Morais Cabral J., Pfuetzner R. A., Kuo A., Gulbis J. M., Cohen S. L., Chait B. T., MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998 Apr 3;280(5360):69–77. doi: 10.1126/science.280.5360.69. [DOI] [PubMed] [Google Scholar]
  8. Du G. G., Guo X., Khanna V. K., MacLennan D. H. Functional characterization of mutants in the predicted pore region of the rabbit cardiac muscle Ca(2+) release channel (ryanodine receptor isoform 2). J Biol Chem. 2001 Jun 26;276(34):31760–31771. doi: 10.1074/jbc.M102751200. [DOI] [PubMed] [Google Scholar]
  9. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  10. Franzini-Armstrong C., Protasi F. Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions. Physiol Rev. 1997 Jul;77(3):699–729. doi: 10.1152/physrev.1997.77.3.699. [DOI] [PubMed] [Google Scholar]
  11. Gao L., Balshaw D., Xu L., Tripathy A., Xin C., Meissner G. Evidence for a role of the lumenal M3-M4 loop in skeletal muscle Ca(2+) release channel (ryanodine receptor) activity and conductance. Biophys J. 2000 Aug;79(2):828–840. doi: 10.1016/S0006-3495(00)76339-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hawkes M. J., Nelson T. E., Hamilton S. L. [3H]ryanodine as a probe of changes in the functional state of the Ca(2+)-release channel in malignant hyperthermia. J Biol Chem. 1992 Apr 5;267(10):6702–6709. [PubMed] [Google Scholar]
  13. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  14. Lai F. A., Misra M., Xu L., Smith H. A., Meissner G. The ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum. Evidence for a cooperatively coupled, negatively charged homotetramer. J Biol Chem. 1989 Oct 5;264(28):16776–16785. [PubMed] [Google Scholar]
  15. Li P., Chen S. R. Molecular basis of Ca(2)+ activation of the mouse cardiac Ca(2)+ release channel (ryanodine receptor). J Gen Physiol. 2001 Jul;118(1):33–44. doi: 10.1085/jgp.118.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lindsay A. R., Tinker A., Williams A. J. How does ryanodine modify ion handling in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel? J Gen Physiol. 1994 Sep;104(3):425–447. doi: 10.1085/jgp.104.3.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McGrew S. G., Wolleben C., Siegl P., Inui M., Fleischer S. Positive cooperativity of ryanodine binding to the calcium release channel of sarcoplasmic reticulum from heart and skeletal muscle. Biochemistry. 1989 Feb 21;28(4):1686–1691. doi: 10.1021/bi00430a039. [DOI] [PubMed] [Google Scholar]
  18. Mead F. C., Sullivan D., Williams A. J. Evidence for negative charge in the conduction pathway of the cardiac ryanodine receptor channel provided by the interaction of K+ channel N-type inactivation peptides. J Membr Biol. 1998 Jun 1;163(3):225–234. doi: 10.1007/s002329900386. [DOI] [PubMed] [Google Scholar]
  19. Meissner G. Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors. Annu Rev Physiol. 1994;56:485–508. doi: 10.1146/annurev.ph.56.030194.002413. [DOI] [PubMed] [Google Scholar]
  20. Muschol M., Dasgupta B. R., Salzberg B. M. Caffeine interaction with fluorescent calcium indicator dyes. Biophys J. 1999 Jul;77(1):577–586. doi: 10.1016/S0006-3495(99)76914-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ogawa Y. Role of ryanodine receptors. Crit Rev Biochem Mol Biol. 1994;29(4):229–274. doi: 10.3109/10409239409083482. [DOI] [PubMed] [Google Scholar]
  22. Orlova E. V., Serysheva I. I., van Heel M., Hamilton S. L., Chiu W. Two structural configurations of the skeletal muscle calcium release channel. Nat Struct Biol. 1996 Jun;3(6):547–552. doi: 10.1038/nsb0696-547. [DOI] [PubMed] [Google Scholar]
  23. Pessah I. N., Zimanyi I. Characterization of multiple [3H]ryanodine binding sites on the Ca2+ release channel of sarcoplasmic reticulum from skeletal and cardiac muscle: evidence for a sequential mechanism in ryanodine action. Mol Pharmacol. 1991 May;39(5):679–689. [PubMed] [Google Scholar]
  24. Sharma M. R., Penczek P., Grassucci R., Xin H. B., Fleischer S., Wagenknecht T. Cryoelectron microscopy and image analysis of the cardiac ryanodine receptor. J Biol Chem. 1998 Jul 17;273(29):18429–18434. doi: 10.1074/jbc.273.29.18429. [DOI] [PubMed] [Google Scholar]
  25. Sutko J. L., Airey J. A., Welch W., Ruest L. The pharmacology of ryanodine and related compounds. Pharmacol Rev. 1997 Mar;49(1):53–98. [PubMed] [Google Scholar]
  26. Tanna B., Welch W., Ruest L., Sutko J. L., Williams A. J. Interactions of a reversible ryanoid (21-amino-9alpha-hydroxy-ryanodine) with single sheep cardiac ryanodine receptor channels. J Gen Physiol. 1998 Jul;112(1):55–69. doi: 10.1085/jgp.112.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tanna B., Welch W., Ruest L., Sutko J. L., Williams A. J. The interaction of a neutral ryanoid with the ryanodine receptor channel provides insights into the mechanisms by which ryanoid binding is modulated by voltage. J Gen Physiol. 2000 Jul 1;116(1):1–9. doi: 10.1085/jgp.116.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tinker A., Lindsay A. R., Williams A. J. Large tetraalkyl ammonium cations produce a reduced conductance state in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel. Biophys J. 1992 May;61(5):1122–1132. doi: 10.1016/S0006-3495(92)81922-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tinker A., Sutko J. L., Ruest L., Deslongchamps P., Welch W., Airey J. A., Gerzon K., Bidasee K. R., Besch H. R., Jr, Williams A. J. Electrophysiological effects of ryanodine derivatives on the sheep cardiac sarcoplasmic reticulum calcium-release channel. Biophys J. 1996 May;70(5):2110–2119. doi: 10.1016/S0006-3495(96)79777-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weiland G. A., Molinoff P. B. Quantitative analysis of drug-receptor interactions: I. Determination of kinetic and equilibrium properties. Life Sci. 1981 Jul 27;29(4):313–330. doi: 10.1016/0024-3205(81)90324-6. [DOI] [PubMed] [Google Scholar]
  31. Welch W., Ahmad S., Airey J. A., Gerzon K., Humerickhouse R. A., Besch H. R., Jr, Ruest L., Deslongchamps P., Sutko J. L. Structural determinants of high-affinity binding of ryanoids to the vertebrate skeletal muscle ryanodine receptor: a comparative molecular field analysis. Biochemistry. 1994 May 24;33(20):6074–6085. doi: 10.1021/bi00186a006. [DOI] [PubMed] [Google Scholar]
  32. Welch W., Sutko J. L., Mitchell K. E., Airey J., Ruest L. The pyrrole locus is the major orienting factor in ryanodine binding. Biochemistry. 1996 Jun 4;35(22):7165–7173. doi: 10.1021/bi9527294. [DOI] [PubMed] [Google Scholar]
  33. Welch W., Williams A. J., Tinker A., Mitchell K. E., Deslongchamps P., Lamothe J., Gerzon K., Bidasee K. R., Besch H. R., Jr, Airey J. A. Structural components of ryanodine responsible for modulation of sarcoplasmic reticulum calcium channel function. Biochemistry. 1997 Mar 11;36(10):2939–2950. doi: 10.1021/bi9623901. [DOI] [PubMed] [Google Scholar]
  34. Williams A. J., West D. J., Sitsapesan R. Light at the end of the Ca(2+)-release channel tunnel: structures and mechanisms involved in ion translocation in ryanodine receptor channels. Q Rev Biophys. 2001 Feb;34(1):61–104. doi: 10.1017/s0033583501003675. [DOI] [PubMed] [Google Scholar]
  35. Witcher D. R., McPherson P. S., Kahl S. D., Lewis T., Bentley P., Mullinnix M. J., Windass J. D., Campbell K. P. Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine. J Biol Chem. 1994 May 6;269(18):13076–13079. [PubMed] [Google Scholar]
  36. Zhao M., Li P., Li X., Zhang L., Winkfein R. J., Chen S. R. Molecular identification of the ryanodine receptor pore-forming segment. J Biol Chem. 1999 Sep 10;274(37):25971–25974. doi: 10.1074/jbc.274.37.25971. [DOI] [PubMed] [Google Scholar]
  37. Zhou M., Morais-Cabral J. H., Mann S., MacKinnon R. Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors. Nature. 2001 Jun 7;411(6838):657–661. doi: 10.1038/35079500. [DOI] [PubMed] [Google Scholar]
  38. Zorzato F., Fujii J., Otsu K., Phillips M., Green N. M., Lai F. A., Meissner G., MacLennan D. H. Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J Biol Chem. 1990 Feb 5;265(4):2244–2256. [PubMed] [Google Scholar]

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