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. 2000 Mar;78(3):1270–1281. doi: 10.1016/S0006-3495(00)76683-5

Molecular cloning of cDNA encoding a drosophila ryanodine receptor and functional studies of the carboxyl-terminal calcium release channel.

X Xu 1, M B Bhat 1, M Nishi 1, H Takeshima 1, J Ma 1
PMCID: PMC1300728  PMID: 10692315

Abstract

Ryanodine is a plant alkaloid that was originally used as an insecticide. To study the function and regulation of the ryanodine receptor (RyR) from insect cells, we have cloned the entire cDNA sequence of RyR from the fruit fly Drosophila melanogaster. The primary sequence of the Drosophila RyR contains 5134 amino acids, which shares approximately 45% identity with RyRs from mammalian cells, with a large cytoplasmic domain at the amino-terminal end and a small transmembrane domain at the carboxyl-terminal end. To characterize the Ca(2+) release channel activity of the cloned Drosophila RyR, we expressed both full-length and a deletion mutant of Drosophila RyR lacking amino acids 277-3650 (Drosophila RyR-C) in Chinese hamster ovary cells. For subcellular localization of the expressed Drosophila RyR and Drosophila RyR-C proteins, green fluorescent protein (GFP)-Drosophila RyR and GFP-Drosophila RyR-C fusion constructs were generated. Confocal microscopic imaging identified GFP-Drosophila RyR and GFP-Drosophila RyR-C on the endoplasmic reticulum membranes of transfected cells. Upon reconstitution into the lipid bilayer membrane, Drosophila RyR-C formed a large conductance cation-selective channel, which was sensitive to modulation by ryanodine. Opening of the Drosophila RyR-C channel required the presence of microM concentration of Ca(2+) in the cytosolic solution, but the channel was insensitive to inhibition by Ca(2+) at concentrations as high as 20 mM. Our data are consistent with our previous observation with the mammalian RyR that the conduction pore of the calcium release channel resides within the carboxyl-terminal end of the protein and further demonstrate that structural and functional features are essentially shared by mammalian and insect RyRs.

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Selected References

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  1. Bhat M. B., Hayek S. M., Zhao J., Zang W., Takeshima H., Wier W. G., Ma J. Expression and functional characterization of the cardiac muscle ryanodine receptor Ca(2+) release channel in Chinese hamster ovary cells. Biophys J. 1999 Aug;77(2):808–816. doi: 10.1016/S0006-3495(99)76933-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhat M. B., Zhao J., Hayek S., Freeman E. C., Takeshima H., Ma J. Deletion of amino acids 1641-2437 from the foot region of skeletal muscle ryanodine receptor alters the conduction properties of the Ca release channel. Biophys J. 1997 Sep;73(3):1320–1328. doi: 10.1016/S0006-3495(97)78165-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bhat M. B., Zhao J., Zang W., Balke C. W., Takeshima H., Wier W. G., Ma J. Caffeine-induced release of intracellular Ca2+ from Chinese hamster ovary cells expressing skeletal muscle ryanodine receptor. Effects on full-length and carboxyl-terminal portion of Ca2+ release channels. J Gen Physiol. 1997 Dec;110(6):749–762. doi: 10.1085/jgp.110.6.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bidasee K. R., Besch H. R., Jr Structure-function relationships among ryanodine derivatives. Pyridyl ryanodine definitively separates activation potency from high affinity. J Biol Chem. 1998 May 15;273(20):12176–12186. doi: 10.1074/jbc.273.20.12176. [DOI] [PubMed] [Google Scholar]
  6. Brillantes A. B., Ondrias K., Scott A., Kobrinsky E., Ondriasová E., Moschella M. C., Jayaraman T., Landers M., Ehrlich B. E., Marks A. R. Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell. 1994 May 20;77(4):513–523. doi: 10.1016/0092-8674(94)90214-3. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chen S. R., Leong P., Imredy J. P., Bartlett C., Zhang L., MacLennan D. H. Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor). Biophys J. 1997 Oct;73(4):1904–1912. doi: 10.1016/S0006-3495(97)78221-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Du G. G., Imredy J. P., MacLennan D. H. Characterization of recombinant rabbit cardiac and skeletal muscle Ca2+ release channels (ryanodine receptors) with a novel [3H]ryanodine binding assay. J Biol Chem. 1998 Dec 11;273(50):33259–33266. doi: 10.1074/jbc.273.50.33259. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Gao L., Tripathy A., Lu X., Meissner G. Evidence for a role of C-terminal amino acid residues in skeletal muscle Ca2+ release channel (ryanodine receptor) function. FEBS Lett. 1997 Jul 21;412(1):223–226. doi: 10.1016/s0014-5793(97)00781-3. [DOI] [PubMed] [Google Scholar]
  12. Hakamata Y., Nakai J., Takeshima H., Imoto K. Primary structure and distribution of a novel ryanodine receptor/calcium release channel from rabbit brain. FEBS Lett. 1992 Nov 9;312(2-3):229–235. doi: 10.1016/0014-5793(92)80941-9. [DOI] [PubMed] [Google Scholar]
  13. Hasan G., Rosbash M. Drosophila homologs of two mammalian intracellular Ca(2+)-release channels: identification and expression patterns of the inositol 1,4,5-triphosphate and the ryanodine receptor genes. Development. 1992 Dec;116(4):967–975. doi: 10.1242/dev.116.4.967. [DOI] [PubMed] [Google Scholar]
  14. Jenden D. J., Fairhurst A. S. The pharmacology of ryanodine. Pharmacol Rev. 1969 Mar;21(1):1–25. [PubMed] [Google Scholar]
  15. Ma J. Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle. J Gen Physiol. 1993 Dec;102(6):1031–1056. doi: 10.1085/jgp.102.6.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ma J., Zhao J. Highly cooperative and hysteretic response of the skeletal muscle ryanodine receptor to changes in proton concentrations. Biophys J. 1994 Aug;67(2):626–633. doi: 10.1016/S0006-3495(94)80522-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McPherson P. S., Campbell K. P. The ryanodine receptor/Ca2+ release channel. J Biol Chem. 1993 Jul 5;268(19):13765–13768. [PubMed] [Google Scholar]
  18. Meissner G. Adenine nucleotide stimulation of Ca2+-induced Ca2+ release in sarcoplasmic reticulum. J Biol Chem. 1984 Feb 25;259(4):2365–2374. [PubMed] [Google Scholar]
  19. Otsu K., Willard H. F., Khanna V. K., Zorzato F., Green N. M., MacLennan D. H. Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. J Biol Chem. 1990 Aug 15;265(23):13472–13483. [PubMed] [Google Scholar]
  20. Qi Y., Ogunbunmi E. M., Freund E. A., Timerman A. P., Fleischer S. FK-binding protein is associated with the ryanodine receptor of skeletal muscle in vertebrate animals. J Biol Chem. 1998 Dec 25;273(52):34813–34819. doi: 10.1074/jbc.273.52.34813. [DOI] [PubMed] [Google Scholar]
  21. Schmitt M., Turberg A., Londershausen M. Characterization of a ryanodine receptor in Periplaneta americana. J Recept Signal Transduct Res. 1997 Jan-May;17(1-3):185–197. doi: 10.3109/10799899709036603. [DOI] [PubMed] [Google Scholar]
  22. Sutko J. L., Airey J. A. Ryanodine receptor Ca2+ release channels: does diversity in form equal diversity in function? Physiol Rev. 1996 Oct;76(4):1027–1071. doi: 10.1152/physrev.1996.76.4.1027. [DOI] [PubMed] [Google Scholar]
  23. Takeshima H., Nishi M., Iwabe N., Miyata T., Hosoya T., Masai I., Hotta Y. Isolation and characterization of a gene for a ryanodine receptor/calcium release channel in Drosophila melanogaster. FEBS Lett. 1994 Jan 3;337(1):81–87. doi: 10.1016/0014-5793(94)80634-9. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Takeshima H. Primary structure and expression from cDNAs of the ryanodine receptor. Ann N Y Acad Sci. 1993 Dec 20;707:165–177. doi: 10.1111/j.1749-6632.1993.tb38051.x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Usherwood P. N., Vais H. Towards the development of ryanoid insecticides with low mammalian toxicity. Toxicol Lett. 1995 Dec;82-83:247–254. doi: 10.1016/0378-4274(95)03558-3. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Waterhouse A. L., Pessah I. N., Francini A. O., Casida J. E. Structural aspects of ryanodine action and selectivity. J Med Chem. 1987 Apr;30(4):710–716. doi: 10.1021/jm00387a022. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]

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