Molecular cloning of cDNA encoding a drosophila ryanodine receptor and functional studies of the carboxyl-terminal calcium release channel.
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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Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Bhat
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Nishi
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Takeshima
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Ma
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D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D004331:Drosophila melanogaster / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D008322:Mammals; D008564:Membrane Potentials / Q000502:physiology; D008861:Microsomes / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017384:Sequence Deletion; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D049452:Green Fluorescent Proteins; D012433:Ryanodine