Identification of a critical region in the Drosophila ryanodine receptor that confers sensitivity to diamide insecticides

Anthranilic diamides, which include the new commercial insecticide, chlorantraniliprole, are an exciting new class of chemistry that target insect ryanodine receptors. These receptors regulate release of stored intracellular calcium and play a critical role in muscle contraction. As with insects, nematodes express ryanodine receptors and are sensitive to the plant alkaloid, ryanodine. However the plant parasitic nematode, Meloidogyne incognita, is insensitive to anthranilic diamides. Expression of a full-length Drosophila melanogaster ryanodine receptor in an insect cell line confers sensitivity to the receptor agents, caffeine and ryanodine along with nanomolar sensitivity to anthranilic diamides. Replacement of a 46 amino acid segment in a highly divergent region of the Drosophila C-terminus with that from Meloidogyne results in a functional RyR which lack sensitivity to diamide insecticides. These findings indicate that this region is critical to diamide sensitivity in insect ryanodine receptors. Furthermore, this region may contribute to our understanding of the differential selectivity diamides exhibit for insect over mammalian ryanodine receptors.     

Molecular characterization of a ryanodine receptor gene in the rice leaffolder, Cnaphalocrocis medinalis (Guenée)

Ryanodine receptors (RyRs) are the targets of two novel classes of synthetic insecticidal chemicals, phthalic acid diamides and anthranilic diamides. Isolation of full-length RyR cDNAs is a critical step towards the structural and functional characterization of insect RyRs and an understanding of the molecular mechanisms underlying the species selective toxicity of diamide insecticides. However, there has been little research on the insect RyR genes due to the high molecular weight of the RyR proteins. In this study, we isolated a full-length RyR cDNA (named as CmRyR) from Cnaphalocrocis medinalis, an important rice pest throughout Southeast Asia. The composite CmRyR gene contains an ORF of 15264 bp encoding a protein of 5087 amino acid residues, which shares 79% overall identity with its Drosophila melanogaster homologue. All hallmarks of the RyR proteins are conserved in the CmRyR protein, suggesting that CmRyR is a structural and functional analogue of known RyRs. A multiple sequence alignment illustrates that the insect RyRs share high levels of amino acid sequence identity at the the COOH-terminal region. However, the amino acid residues analogous to the CmRyR residues N(4922), N(4924), N(4935), L(4950), L(4981), N(5013) and T(5064) are unique to lepidopteran RyRs compared with non-lepidopteran insect RyRs. This finding suggests that these residues may be involved in the differences in channel properties between lepidopteran and non-lepidopteran insect RyRs and in the species selective toxicity of diamide insecticides. Furthermore, two alternative splicing sites were identified in the CmRyR gene, one of which was located in the central part of the predicted second SPRY domain. Diagnostic PCR showed that the inclusion frequencies of two mutually exclusive exons (a/b) and one optional exon (c) differed between developmental stages or adult anatomical regions. Our results imply that alternative splicing may be a major means of generating functional diversity in C. medinalis RyR channel.     

Synthesis,insecticidal evaluation and mode of action of novel anthranilic diamide derivatives containing sulfur moiety as potential ryanodine receptor activators

Anthranilic diamide insecticide could control lepidopteran pests by selectively binding and activating insect ryanodine receptors (RyRs), and the unique mode of action is different from other conventional insecticides. In order to discover new anthranilic diamide insecticide as ryanodine receptors activators, a series of 11 novel anthranilic diamides derivatives (Ia-k) were synthesized and confirmed by melting point, ¹H NMR, ¹³C NMR and elemental analyses. The preliminary bioactivity revealed that most title compounds showed moderate to remarkable activities against oriental armyworm (Mythimna separata) and diamondback moth (Plutella xylostella). Especially, compounds Ia and If, which exhibited 100% larvicidal activity against oriental armyworm at 1.0?mg?L^?1, and comparable to that of chlorantraniliprole (100% at 1?mg?L^?1). If displayed 60% insecticidal activity against diamondback moth at 0.01?mg?L^?1, better than chlorantraniliprole (45% at 0.01?mg?L^?1). The preliminary structure activity relationships were discussed. In addition, the calcium imaging experiment indicated that the insect ryanodine receptor is the potential target of If.     

新型二酰胺类杀虫剂对鱼尼丁受体作用的分子机理

最近发现了一类新型二酰胺类杀虫剂——氟虫酰胺和氯虫酰胺,其作用靶标是鱼尼丁受体 (ryanodine receptors, RyRs)。本文对RyR的结构与功能、电压门控钙离子通道和鱼尼丁受体钙离子释放通道对细胞质钙离子内环境稳定的调节以及二酰胺类杀虫剂对RyRs作用的分子机理进行综述。二酰胺类杀虫剂使昆虫RyR通道处于持续的开放状态,引发钙离子从肌质网腔内大量释放,破坏了细胞质钙离子内环境的稳定,从而产生不同的药物学特性。这些变化都是由一个不同于鱼尼丁在RyR上的结合部位介导的。该类杀虫剂的作用对昆虫RyR s是高度专一的,结果产生选择毒性。由于二酰胺类杀虫剂的结构独特,作用方式新颖,对鳞翅目害虫效果好、杀虫谱广,对各种益虫和天敌安全,并对现用的杀虫剂无交互抗性,故它们非常适合于抗性治理和IPM。     

Modulation of the expression of ryanodine receptor mRNA from Plutella xylostella as a result of diamide insecticide application

Ryanodine receptors (RyRs), members of the largest family of calcium channel proteins, have been studied because of their key roles in calcium signalling within cells. With the development of diamide insecticides that exhibit a novel mode of action on the RyRs from Lepidoptera, research on insect RyRs has become more attractive in the field of plant protection. To enhance our understanding of the effects of diamides on RyRs, we cloned the Plutella xylostella RyR gene (Px-RyR), which is the most serious pest of Brassicaceae plants throughout the world. Furthermore, we investigated the modulation of the expression of Px-RyR as a result of the application of diamide insecticides. The full-length cDNAs of Px-RyR contain an open reading frame (ORF) of 15,372 bp with a predicted protein consisting of 5123 amino acids. Px-RyR possesses a high level of overall amino acid homology with other isoforms (77–92% identity with insect isoforms and 45–47% identity with vertebrate isoforms). The weight of Px. gradually decreased as the concentration of the diamides increased. However, the relative expression levels of the RyRs from larvae were dependent on the insecticide concentration and gradually increased with increasing insecticide concentrations.     

Phthalic acid diamides activate ryanodine-sensitive Ca2+ release channels in insects

Flubendiamide represents a novel chemical family of substituted phthalic acid diamides with potent insecticidal activity. So far, the molecular target and the mechanism of action were not known. Here we present for the first time evidence that phthalic acid diamides activate ryanodine-sensitive intracellular calcium release channels (ryanodine receptors, RyR) in insects. With Ca(2+) measurements, we showed that flubendiamide and related compounds induced ryanodine-sensitive cytosolic calcium transients that were independent of the extracellular calcium concentration in isolated neurons from the pest insect Heliothis virescens as well as in transfected CHO cells expressing the ryanodine receptor from Drosophila melanogaster. Binding studies on microsomal membranes from Heliothis flight muscles revealed that flubendiamide and related compounds interacted with a site distinct from the ryanodine binding site and disrupted the calcium regulation of ryanodine binding by an allosteric mechanism. This novel insecticide mode of action seems to be restricted to specific RyR subtypes because the phthalic acid diamides reported here had almost no effect on mammalian type 1 ryanodine receptors.     

Discovery of cyantraniliprole,a potent and selective anthranilic diamide ryanodine receptor activator with cross-spectrum insecticidal activity

Anthranilic diamides are an exceptionally active class of insect control chemistry that selectively activates insect ryanodine receptors causing mortality from uncontrolled release of calcium ion stores in muscle cells. Work in this area led to the successful commercialization of chlorantraniliprole for control of Lepidoptera and other insect pests at very low application rates. In search of lower log P analogs with improved plant systemic properties, exploration of cyano-substituted anthranilic diamides culminated in the discovery of a second product candidate, cyantraniliprole, having excellent activity against a wide range of pests from multiple insect orders. Here we report on the chemistry, biology and structure–activity trends for a series of cyanoanthranilic diamides from which cyantraniliprole was selected for commercial development.     

New and selective ryanodine receptor activators for insect control

Diamide insecticides have emerged as one of the most promising new classes of insecticide chemistry owing to their excellent insecticidal efficacy and high margins of mammalian safety. Chlorantraniliprole and flubendiamide, the first two insecticides from this class, demonstrate exceptional activity across a broad range of pests in the order Lepidoptera. This chemistry has been confirmed to control insects via activation of ryanodine receptors which leads to uncontrolled calcium release in muscle. The high levels of mammalian safety are attributed to a strong selectivity for insect over mammalian receptors.     

CRISPR/Cas9 mediated G4946E substitution in the ryanodine receptor of Spodoptera exigua confers high levels of resistance to diamide insecticides

Diamide insecticides selectively activate insect ryanodine receptors (RyRs), inducing uncontrolled release of calcium ions, and causing muscle contraction, paralysis and eventually death. The RyR^G4946E substitution associated with diamide resistance has been identified in three lepidopteran pests, Plutella xylostella, Tuta absoluta and Chilo suppressalis. Recently, the T. absoluta RyR^G4946V mutation was knocked into the model insect Drosophila melanogaster by CRISPR/Cas9 mediated genome editing and provided in vivo functional confirmation for its role in diamide resistance. In the present study, we successfully introduced the RyR^G4946E mutation with CRISPR/Cas9 technology into a lepidopteran pest of global importance, Spodoptera exigua. The genome-edited strain (named 4946E) homozygous for the SeRyR^G4946E mutation exhibited 223-, 336- and >1000-fold resistance to chlorantraniliprole, cyantraniliprole and flubendiamide, respectively when compared to the wild type strain (WHS) of S. exigua. Reciprocal crossing experiments revealed that the target-site resistance in strain 4946E underlies an autosomal and almost recessive mode of inheritance for anthranilic diamides, whereas it was completely recessive for flubendiamide. Our results not only provided in vivo functional validation of the RyR^G4946E mutation in conferring high levels of resistance to diamide insecticides for the first time in a controlled genetic background of a lepidopteran pest, but also revealed slight differences on the level of resistance between anthranilic diamides (chlorantraniliprole and cyantraniliprole) and flubendiamide conferred by the SeRyR^G4946E mutation.     

Nicotinic acetylcholine receptors: targets for commercially important insecticides

Nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in both vertebrates and invertebrates. In insects, nAChRs are the target site for several naturally occurring and synthetic compounds that exhibit potent insecticidal activity. Several compounds isolated from plants are potent agonists or antagonists of nAChRs, suggesting that these may have evolved as a defence mechanism against insects and other herbivores. Nicotine, isolated from the tobacco plant, has insecticidal activity and has been used extensively as a commercial insecticide. Spinosad, a naturally occurring mixture of two macrocyclic lactones isolated from the microorganism Saccharopolyspora spinosa, acts upon nAChRs and has been developed as a commercial insecticide. Since the early 1990s, one of the most widely used and rapidly growing classes of insecticides has been the neonicotinoids. Neonicotinoid insecticides are potent selective agonists of insect nAChRs and are used extensively in both crop protection and animal health applications. As with other classes of insecticides, there is growing evidence for the evolution of resistance to insecticides that act on nAChRs.     

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.     

Bicyclic heterocyclic anthranilic diamides as ryanodine receptor modulators with insecticidal activity

The diamide insecticides act on the ryanodine receptor (RyR). The synthesis of various bicyclic anthranilic derivatives is reported. Their activity against the insect ryanodine receptor (RyR) and their insecticidal activity in the greenhouse is presented, as well as structure activity relationship considerations.     

A molecular model and Monte Carlo simulation of flavivirus envelope building block

Ryanodine receptors (RyRs) are mainly located on the endoplasmic reticulum (ER) and play an important role in regulating glucose-induced cytosolic Ca(2+) oscillation in pancreatic β-cells. However, subcellular locations and functions of RyRs on other cell organelles such as nuclear envelope are not well understood. In order to investigate the role of RyRs in nuclear Ca(2+) oscillation we designed and conducted experiments in intact primary pancreatic β-cells. Immunocytochemistry was used to examine the expression of RYRs on the nuclear envelope. Confocal microscopy was used to evaluate the function of RYRs on the nuclear envelope. We found that RyRs are expressed on the nuclear envelope in single primary pancreatic β-cells and isolated nuclei. Laser scanning confocal microscopy studies indicated that application of glucose to the cells co-incubated with Ca(2+) indicator Fluo-4 AM and cell-permeable nuclear indicator Hoechst 33342 resulted in nuclear Ca(2+) oscillation. The pattern of glucose-induced Ca(2+) oscillation in the nucleus and cytosol was similar. The reduction of Ca(2+) oscillation amplitude by ryanodine was much greater in the nucleus though both the cytosol and the nucleus Ca(2+) amplitude decreased by ryanodine. Our results suggest that functional ryanodine receptors not only exist in endoplasmic reticulum but are also expressed in nuclear envelope of pancreatic β-cells.     

The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues

Ryanodine receptors (RyRs) are intracellular calcium release channels that participate in controlling cytosolic calcium levels. At variance with the probably ubiquitous inositol 1,4,5-trisphosphate-operated calcium channels (1,4,5-trisphosphate receptors), RyRs have been mainly regarded as the calcium release channels controlling skeletal and cardiac muscle contraction. Increasing evidence has recently suggested that RyRs may be more widely expressed, but this has never been extensively examined. Therefore, we cloned three cDNAs corresponding to murine RyR homologues to carry a comprehensive analysis of their expression in murine tissues. Here, we report that the three genes are expressed in almost all tissues analyzed, where tissue-specific patterns of expression were observed. In the uterus and vas deferens, expression of RyR3 was localized to the smooth muscle component of these organs. In the testis, expression of RyR1 and RyR3 was detected in germ cells. RyR mRNAs were also detected in in vitro-cultured cell lines. RyR1, RyR2, and RyR3 mRNA were detected in the cerebrum and in the cerebellum. In situ analysis revealed a cell type-specific pattern of expression in the different regions of the central nervous system. The differential expression of the three ryanodine receptor genes in the central nervous system was also confirmed using specific antibodies against the respective proteins. This widespread pattern of expression suggests that RyRs may participate in the regulation of intracellular calcium homeostasis in a range of cells wider than previously recognized.     

Insecticidal anthranilic diamides: a new class of potent ryanodine receptor activators

A novel class of anthranilic diamides has been discovered with exceptional insecticidal activity on a range of Lepidoptera. These compounds have been found to exhibit their action by release of intracellular Ca2+ stores mediated by the ryanodine receptor. The discovery, synthesis, structure-activity, and biological results are presented.     

Ryanodine receptors in liver

The ryanodine receptor has been mainly regarded as the Ca2+ release channel from sarcoplasmic reticulum controlling skeletal and cardiac muscle contraction. However, many studies have shown that it is widely expressed, with functions not restricted to muscular contraction. This study examined whether ryanodine receptor plays a role in calcium signaling in the liver. RT-PCR analysis of isolated hepatocytes showed expression of a truncated type 1 ryanodine receptor, but no type 2 or type 3 message was detected. We also detected binding sites for [3H]ryanodine in the microsomal cellular fraction and in permeabilized hepatocytes. This binding was displaced by caffeine and dantrolene, but not by ruthenium red, heparin or cyclic ADP-Ribose. Ryanodine, by itself, did not trigger Ca2+ oscillations in either primary cultured hepatocytes or hepatocytes within the intact perfused rat liver. In both preparations, however, ryanodine significantly increased the frequency of the cytosolic free [Ca2+] oscillations evoked by an alpha1 adrenergic receptor agonist. Experiments in permeabilized hepatocytes showed that both ryanodine and cyclic ADP-ribose evoked a slow Ca2+ leak from intracellular stores and were able to increase the Ca2+-released response to a subthreshold dose of inositol 1,4,5-trisphosphate. Our findings suggest the presence of a novel truncated form of the type 1 ryanodine receptor in rat hepatocytes. Ryanodine modulates the pattern of cytosolic free [Ca2+] oscillations by increasing oscillation frequency. We propose that the Ca2+ released from ryanodine receptors on the endoplasmic reticulum provides an increased pool of Ca2+ for positive feedback on inositol 1,4,5-trisphosphate receptors.     

Local control models of cardiac excitation-contraction coupling. A possible role for allosteric interactions between ryanodine receptors

In cardiac muscle, release of activator calcium from the sarcoplasmic reticulum occurs by calcium- induced calcium release through ryanodine receptors (RyRs), which are clustered in a dense, regular, two-dimensional lattice array at the diad junction. We simulated numerically the stochastic dynamics of RyRs and L-type sarcolemmal calcium channels interacting via calcium nano-domains in the junctional cleft. Four putative RyR gating schemes based on single-channel measurements in lipid bilayers all failed to give stable excitation-contraction coupling, due either to insufficiently strong inactivation to terminate locally regenerative calcium-induced calcium release or insufficient cooperativity to discriminate against RyR activation by background calcium. If the ryanodine receptor was represented, instead, by a phenomenological four-state gating scheme, with channel opening resulting from simultaneous binding of two Ca2+ ions, and either calcium-dependent or activation-linked inactivation, the simulations gave a good semiquantitative accounting for the macroscopic features of excitation-contraction coupling. It was possible to restore stability to a model based on a bilayer-derived gating scheme, by introducing allosteric interactions between nearest-neighbor RyRs so as to stabilize the inactivated state and produce cooperativity among calcium binding sites on different RyRs. Such allosteric coupling between RyRs may be a function of the foot process and lattice array, explaining their conservation during evolution.     

Molecular Characteristics,mRNA Expression,and Alternative Splicing of a Ryanodine Receptor Gene in the Oriental Fruit Fly,Bactrocera dorsalis (Hendel)

Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis.     

Functional and genetic characteristics of Chlorantraniliprole resistance in the diamondback moth,Plutella xylostella (Lepidoptera: Plutellidae)

The diamondback moth (Plutella xylostella) is a globally distributed and important economic pest, and it has developed resistance to all conventional insecticide classes used in the field. Chlorantraniliprole is a new chemical class of insecticide that acts as a conformation‐sensitive activator of the insect ryanodine receptor (RyR). In the present study, a field strain (16.3‐fold resistance to chlorantraniliprole) was collected in Korea and lab‐selected with chlorantraniliprole for more than one year. The resulting strain presented 2,157‐fold resistance to chlorantraniliprole. A point mutation (G4946E) in the RyR gene was observed at a high frequency in the resistant strain. Enzyme assays indicated that glutathione S‐transferase (GST) and P450 activity in the resistant strain were 2.4‐ and 1.96‐times higher than that of the susceptible strain, respectively. The expression of the RyR, GST (sigma, omega, and zeta) and CYP321E1 gene was higher in the resistant strain than in the susceptible strain. The F₁ progeny resulting from reciprocal crosses did not reveal maternal effects or a diamide‐susceptible phenotype, which suggests an autosomal nearly recessive mode of inheritance. In addition, we surveyed the susceptibility to 13 insecticides (3 diamides, 2 synthetic pyrethroids, 2 spinosyns, 1 organophosphate, 1 oxadiazine, 1 avermectin, and 3 others) in the chlorantraniliprole‐resistant strain. The resistant strain exhibited high cross‐resistance to flubendiamide (5,910 fold) and showed no cross‐resistance to spinetoram, spinosad, indoxacarb, and metaflumizone. These results can serve as an important basis for guiding the use of insecticides in the field.     

Ryanodine receptors, voltage-gated calcium channels and their relationship with protein kinase A in the myocardium

We present a review about the relationship between ryanodine receptors and voltage-gated calcium channels in myocardium, and also how both of them are related to protein kinase A. Ryanodine receptors, which have three subtypes (RyR1-3), are located on the membrane of sarcoplasmic reticulum. Different subtypes of voltage-gated calcium channels interact with ryanodine receptors in skeletal and cardiac muscle tissue. The mechanism of excitation-contraction coupling is therefore different in the skeletal and cardiac muscle. However, in both tissues ryanodine receptors and voltage-gated calcium channels seem to be physically connected. FK-506 binding proteins (FKBPs) are bound to ryanodine receptors, thus allowing their concerted activity, called coupled gating. The activity of both ryanodine receptors and voltage-gated calcium channels is positively regulated by protein kinase A. These effects are, therefore, components of the mechanism of sympathetic stimulation of myocytes. The specificity of this enzyme's targeting is achieved by using different A kinase adapting proteins. Different diseases are related to inborn or acquired changes in ryanodine receptor activity in cardiac myocytes. Mutations in the cardiac ryanodine receptor gene can cause catecholamine-provoked ventricular tachycardia. Changes in phosphorylation state of ryanodine receptors can provide a credible explanation for the development of heart failure. The restoration of their normal level of phosphorylation could explain the positive effect of beta-blockers in the treatment of this disease. In conclusion, molecular interactions of ryanodine receptors and voltage-gated calcium channels with PKA have a significant physiological role. However, their defects and alterations can result in serious disturbances.