Molecular Cloning of cDNA Encoding a Drosophila Ryanodine Receptor and Functional Studies of the Carboxyl-Terminal Calcium Release Channel

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.

Molecular characteristics, mRNA expression, and alternative splicing of a ryanodine receptor gene in the Oriental fruit fly,Bactrocera dorsalis(Hendel)

“…The five types of conserved domain (Figure 3) in the N-terminal region of BdRyR included: a mannosyltransferase, IP3R and RyR domain (MIR, pfam02815) at position 211-392; two RyR and IP3R homology domains (RIH, pfam01365) at positions 439-648 and 2227-2456; three spore lysis The C-terminal region of RyR protein can form functionally important domains shared by mammalian and insect RyRs [9,20]. Thus, the C-terminal region of BdRyR was compared with other reported insect RyRs, including DmRyR from D. melanogaster, sRyR from B. mori, PxRyR from P. xylostella, and NlRyR from N. lugens (Figure 4).…”

“…In birds, amphibians and fish, two RyRs, RyRA and RyRB are homologous to mammalian RyR1 and RyR3, respectively [17][18][19]. In contrast to mammals, only one gene encoding a RyR is present in Drosophila melanogaster, and it shares a 44 to 46% amino acid identity with the RyR mammalian isoforms [20]. Interestingly, mammalian and insect RyRs are more different in sequence than their corresponding inositol trisphosphate receptors (InsP3Rs), which may make RyRs better targets for insecticidal molecules with low mammalian toxicity.…”

Co-Induction of LTP and LTD and Its Regulation by Protein Kinases and Phosphatases

“…Third, the role of ryanodine receptor (RyR)-mediated release from intracellular Ca 2ϩ stores was also examined. Ryanodine, at micromolar concentrations (Sutko et al 1997), is a selective inhibitor for RyRs and has been shown to be effective in a variety of invertebrates, including Aplysia, Hirudo, and Drosophila (Geiger and Magoski 2008; Trueta et al 2004;Xu et al 2000). Application of ryanodine (50 M) in conjunction with the pairing protocol blocked LTP [ryanodine ϩ pairing, 102 Ϯ 9%, n ϭ 10; ryanodine control, 93 Ϯ 9%, n ϭ 11; P Ͻ 0.05; F (8,68) ϭ 10.898, P Ͻ 0.001; Fig.…”

Verticilide, a new ryanodine-binding inhibitor, produced by Verticillium sp. FKI-1033

“…5 Therefore, insect RyR is a potential target for new insecticides. 6 The molecular target of some new synthetic insecticides, flubendiamide and anthranilic diamide, was shown to be RyR.…”