Invertebrate Neuroscience

, 8:107

Insect ryanodine receptors: molecular targets for novel pest control chemicals

Authors

  • David B. Sattelle
    • MRC Functional Genomics Unit, Department of Physiology, Anatomy and GeneticsUniversity of Oxford
    • Dupont Crop ProtectionStine-Haskell Research Center
  • Timothy R. Cheek
    • Institute for Cell and Molecular Biosciences, The Medical SchoolUniversity of Newcastle upon Tyne
Review

DOI: 10.1007/s10158-008-0076-4

Cite this article as:
Sattelle, D.B., Cordova, D. & Cheek, T.R. Invert Neurosci (2008) 8: 107. doi:10.1007/s10158-008-0076-4

Abstract

Ryanodine receptors (RyRs) are a distinct class of ligand-gated calcium channels controlling the release of calcium from intracellular stores. They are located on the sarcoplasmic reticulum of muscle and the endoplasmic reticulum of neurons and many other cell types. Ryanodine, a plant alkaloid and an important ligand used to characterize and purify the receptor, has served as a natural botanical insecticide, but attempts to generate synthetic commercial analogues of ryanodine have proved unsuccessful. Recently two classes of synthetic chemicals have emerged resulting in commercial insecticides that target insect RyRs. The phthalic acid diamide class has yielded flubendiamide, the first synthetic ryanodine receptor insecticide to be commercialized. Shortly after the discovery of the phthalic diamides, the anthranilic diamides were discovered. This class has produced the insecticides Rynaxypyr® and Cyazypyr™. Here we review the structure and functions of insect RyRs and address the modes of action of phthalic acid diamides and anthranilic diamides on insect ryanodine receptors. Particularly intersting is the inherent selectivity both chemical classes exhibit for insect RyRs over their mammalian counterparts. The future prospects for RyRs as a commercially-validated target site for insect control chemicals are also considered.

Introduction

Ryanodine is a toxic natural alkaloid (Fig. 1) present in the tropical South American and Caribbean plant Ryania speciosa Vahl (Flacourtiaceae). Ryanodine is best known for its defining role in the characterization and purification of an important class of ion channels (Fill and Copello 2002; Jenden and Fairhurst 1969) and for its use as a natural insect control chemical (Nauen 2006). For example, ryanodine shows high affinity (KD ~ 5–15 nM) for a distinct class of ligand-gated calcium channels which control the release of calcium (Ca2+) from intracellular stores and are referred to as ryanodine receptors (RyRs). These transmembrane receptors are located in the sarcoplasmic reticulum of muscle and the endoplasmic reticulum of neurons, epithelial cells and many other cell types. Radiolabeled ryanodine ([3H]ryanodine) has been used to purify RyRs from a variety of tissues. Ryanodine itself has long been utilized as an insecticide (Pepper and Carruth 1945), but its mammalian toxicity has precluded its continued use. Synthetic derivatives of the Ryania alkaloids have been explored and extensive structure-activity studies pursued (Lehmberg and Casida 1994; Waterhouse et al. 1987). This has resulted in compounds of considerable interest but no commercial products targeting RyRs have emerged via this approach.
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Fig. 1

The chemical structure of ryanodine, a neutral alkaloid with insecticidal properties originally isolated from the tropical plant Ryania speciosa. Ryanodine is a high affinity ligand for ryanodine receptor molecules, calcium channels which regulate release of Ca2+ from intracellular stores and play a key signalling role in cells. A radiolabeled form of the molecule, [3H]ryanodine, has been used to characterize and purify ryanodine receptors

Recently, there has been renewed interest in this field based on two new areas of synthetic chemistry, both of which have yielded important commercial products targeting RyRs. The first chemical class is the phthalic acid diamides, which include the compound flubendiamide, discovered by Nihon Nohyaku and jointly developed with Bayer (Ebbinghaus-Kintscher et al. 2006; Masaki et al. 2006; Nauen 2006; Tohnishi et al. 2005). The second chemical class is the anthranilic diamides which include chlorantraniliprole (known by its trade name, Rynaxypyr®), discovered and developed into a commercial product by Dupont (Lahm et al. 2007; Cordova et al. 2006, 2007a; Lahm et al. 2005). Both phthalic acid diamides and anthranilic diamides are potent activators of insect RyRs. The successful introduction of these new classes of insecticidal RyR modulators has led to the designation of an important new mode of action group (Group 28) by the Insecticide Resistance Action Committee (IRAC) (www.irac-online.org). Here we review current knowledge on insect ryanodine receptors and the modes of action of these two classes of important new synthetic insecticides, both of which show intrinsic target-site selectivity for insects over mammals. Future prospects for insect control chemicals targeting ryanodine receptors are also examined.

Ryanodine receptors: structure and function

Inositol 1,4,5-trisphosphate receptors (InsP3Rs) (Berridge 1993) and RyRs (Hamilton 2005; Meissner 1994) form two major classes of calcium channels mediating release of Ca2+ from intracellular stores. The RyRs are homomeric tetramers (Fig. 2) (Hamilton 2005; Meissner 1994) and have been studied intensively because of their key roles in calcium signaling in cells. RyR molecules are among the largest of all ion channels (~2–2.5 MDa). Their overall structure in situ has been determined by cryoelectron microscopy (Radermacher et al. 1994; Serysheva et al. 1995), revealing a complex tetrameric molecule with the four N-terminal domains forming a large quatrefoil structure extending into the cytoplasm. In mammals there are three types of ryanodine receptor (RyR1, RyR2 and RyR3), each the product of a separate gene (Table 1). They show ~65% homology at the amino acid level. In contrast to mammals, birds, amphibians and fish possess only two RyRs (RyRA and RyRB) (Ogawa et al. 1999; Ottini et al. 1996). The RyRA receptor shows homology with mammalian RyR1, whereas RyRB most closely resemble the RyR3 isoform (Oyamada et al. 1994).
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Fig. 2

Schematic representation of the ryanodine receptor and important associated proteins. The receptor is a tetramer of identical subunits; for simplicity just two of the four subunits are shown. Each subunit has 4 transmembrane regions in the endoplasmic reticulum membrane (sarcoplasmic reticulum in muscle cells) close to the C-terminus and a very long N-terminal region, which extends into the cytoplasm. Some of the important associated proteins that interact directly with ryanodine receptors are shown. CAM calmodulin, FKBP FK506-binding protein, CSQ calsequestrin, Ca2+ calcium ions

Table 1

Characteristics of human and Drosophila ryanodine receptors

Type

Ensembl ID & link

Amino acids

Tissue expression

Reference

RyR1

ENSG00000196218

http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000196218

5037

skeletal muscle (SM)

Takeshima et al. 1989

RyR2

ENSG00000198626

http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000198626

4968

cardiac muscle, CNS

Nakai et al. 1990

RyR3

ENSG00000198838

http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000198838

4872

epithelia, CNS, SM

Sorrentino et al. 1993

DmRyR

FBgn0011286

http://www.ensembl.org/Drosophila_melanogaster/geneview?gene=FBgn0011286

5216

muscle, CNS, adult, embryo

Takeshima et al. 1994

The table describes the gene annotation, number of amino acids present and the tissues and developmental stages in which RyRs play an important role. RyR1-3 refer to human ryanodine receptors. DmRyR refers to the Drosophila ryanodine receptor

Localization of the three mammalian isoforms is tissue-dependent. RyR1 is present mainly on skeletal muscle (Marks et al. 1989) although lower levels of RyR1 expression are also found in aortic smooth muscle and non-muscle tissues such as adrenal gland, cerebellum (Purkinje cells), ovary and testis (Berridge et al. 1996; Fill and Copello 2002; MacKenzie et al. 1990; Otsu et al. 1990; Sorrentino et al. 1993). RyR2 is the major isoform in cardiac muscle. It is also present in aortic smooth muscle (Fill and Copello 2002), neurons, notably cerebellar Purkinje and granule cells (Kuwajima et al. 1992), adrenal gland cells (Ledbetter et al. 1994) and non-excitable cells such as lung (Giannini et al. 1995) and HeLa cells (Bennett et al. 1996). The RyR3 protein is found in striated (skeletal and cardiac) muscle (Bennett et al. 1996), but at relatively low levels, amounting to <5% of the overall RyR population in the diaphragm (Fill and Copello 2002). RyR3 is also expressed in brain and smooth muscle as well as non-excitable cells such as kidney (Bennett et al. 1996; Fill and Copello 2002; Giannini et al. 1995).

The transmembrane regions of the three human isoforms contain three sites of diversity (D1-3) and differences within these sites account for several of the functional differences between receptor subtypes. D2 is implicated in the coupling of RyR1 to Cav1.1, something not seen for RyR2 (Perez et al. 2003), and D1 of RyR1 modulates receptor sensitivity to Ca2+ and caffeine (Du et al. 2000). It is nevertheless interesting to note that functional diversity of RyRs is by no means restricted to regions D1-D3. For example, the extensive N-terminal, cytoplasmic domain (75% of the receptor) contains the majority of human disease-causing mutations (Fig. 3). Mutations in RyR1 can lead to muscle wasting and weakness as seen in malignant hyperthermia syndrome (MHS) and central core disease (CCD) (Quane et al. 1993). MHS is a rare but life-threatening condition triggered by exposure to anesthetics, particularly volatile and gaseous anesthetics, as well as the neuromuscular blocking agent succinylcholine. Susceptibility can be inherited in an autosomal dominant manner. Susceptibility of this kind is phenotypically and genetically related to central core disease (CCD), another autosomal dominant disorder characterized by MHS symptoms as well as myopathy. The RyR2 channel region mutations affect cardiac function resulting in diseases such as arrythmogenic right ventricular dysplasia type 2 (ARVD2) (Tiso et al. 2001; Thiene et al. 1997). Mutations which underlie these disorders are in homologous regions of the protein, affecting amino acid sequences in the N-terminus (RyR1, 35–614 and RyR2, 176–420) the central cytoplasmic region (RyR1,2,162–2,458 and RyR2, 2,246–2,504) and in the C-terminal transmembrane region (RyR1, 4,647–4,914 and RyR2, 3,778–4,950). In the case of RyR2 mutations, the results are relevant to human diseases as alteration of Ca2+ signaling and the disruption of Ca2+ homeostasis in cardiac muscle can lead to an inability of the heart to fill with and eject blood effectively, resulting in heart failure and secondary pathologies such as diabetes.
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Fig. 3

Amino acid structures of RyR isoforms. Regions of diversity between RyR isoforms are contained in three major gene regions, as are clusters of disease-causing mutations. Regions of gene diversity are designated D1 (*), D2 (**) and D3 (***) and represent the main contributory regions to the overall 35% amino acid variation seen between RyR isoforms. Three further regions are thought to contribute toward disease mutations in RyR1 and RyR2. These clusters are found in the N-terminal region (aa 35–614 RyR1, aa 176–420 RyR2), central cytoplasmic region (aa 2,162–2,458 RyR1, aa 2,246–2,504 RyR2) and the transmembrane region (aa 4,647–4,914, aa 3,778–4,950 RyR2) and are designated regions R1, R2 and R3 respectively. Some functional properties associated with the N-terminal, central cytoplasmic and transmembrane (C-terminal) regions of RyRs are shown. FKBP binding ?—location of the FKBP12.6 binding site(s) on RyR2 are controversial. Three different locations have been reported, two in the central region and one in the C-terminal region (Yano et al. 2005)

Destabilization of the RyR complex has been implicated in the development of heart failure independent of mutations in the receptor itself. Chronic hyperactivity of the β-adrenergic signaling pathway leads to PKA-hyperphosphorylation of RyR2 (Marx et al. 2000). The resulting dissociation of the stabilizing protein FKBP12.6 from the RyR2 complex then results in diastolic leak of Ca2+ from the sarcoplasmic reticulum (Lehnart et al. 2004; Marx et al. 2000; Wehrens and Marks 2004; Yano et al. 2005) into the heart muscle cytoplasm. This activates inward currents, thereby introducing cardiac arrhythmias (George et al. 2005; Matsuda et al. 1982).

Ryanodine receptors in Ca2+ signaling

Cells draw on both internal and external sources of calcium (Ca2+) to generate intracellular Ca2+ signals. The internal Ca2+ stores are maintained within the endoplasmic reticulum, or in the case of muscle cells, the sarcoplasmic reticulum, and Ca2+ release from these stores is mediated by InsP3Rs (Berridge 1993) and RyRs (Meissner 1994). InsP3Rs rely on the diffusion of the second messenger, inositol 1,4,5-trisphosphate, which binds to InsP3Rs and activates Ca2+ release from the endoplasmic reticulum. In muscle sarcoplasmic reticulum, Ca2+ release through RyRs is dependent on an interaction of the receptor with a voltage-operated Ca2+ channel (the dihydropyridine receptor, DHPR) in the T-tubule membrane. In the case of RyR1 in skeletal muscle, the α1S subunit of the DHPR interacts directly with the cytoplasmic head of RyR1. A conformational change in the DHPR following T-tubule membrane depolarization is transmitted directly to RyR1 resulting in Ca2+ release from the sarcoplasmic reticulum. For RyR2 in cardiac cells, the α1C subunit of the DHPR (an L-type Ca2+ channel) gates a small amount of Ca2+ (‘trigger Ca2+’) which diffuses across the plasma membrane and activates the RyR2 channel by calcium-induced calcium release (Bers 2002). Ca2+ release through the RyR3 protein is also mediated by calcium-induced calcium release following a pulse of trigger Ca2+ (Sonnleitner et al. 1998). In the case of non-muscle cells, ‘trigger Ca2+’ leading to RyR2 and RyR3 activation can be supplied either by the influx of external Ca2+, or by Ca2+ which has been supplied via InsP3Rs (Berridge 1998; Fill and Copello 2002). By these means, stimulus-induced Ca2+ waves are able to propagate throughout a variety of cell types (Berridge 1998; Riddoch et al. 2007; Riddoch et al. 2005; Verkhratsky 2005). Ca2+ release through RyRs can be mediated or modulated by cyclic ADP-ribose (cADPR) (Clapper et al. 1987) and nicotinic acid adenine dinucleotide phosphate (NAADP), both metabolites of NADP, but the effects of these messengers in striated muscle remains controversial (Bai et al. 2005; Copello et al. 2002, 2001). Studies using skeletal muscle have shown that RyR1 proteins not coupled to DHPRs (so-called ‘uncoupled’ RyRs) can be activated by calcium-induced calcium release (Fill and Copello 2002).

Ca2+ signals mediated by RyRs play key roles in muscle and neuronal function, where the RyR-bearing Ca2+ store acts as both a Ca2+ sink and a Ca2+ source, by sequestering Ca2+ that enters the cell from outside and releasing Ca2+ in response to stimulation with InsP3-mobilizing hormone (Berridge 1998; Riddoch et al. 2005; Verkhratsky 2005). The capacity to switch between “sink” or “source” is dynamic and depends on, for example, the repletion state of the endoplasmic reticulum, the amount of Ca2+ entering the cell and the stimulus (Riddoch et al. 2007). The transient nature of amplification by RyR-mediated by calcium-induced calcium release is important because it provides neurons with a coincidence detection mechanism and a short-term memory of neuronal activity (Berridge 1998; Verkhratsky 2005). For example, by sequestering and retaining Ca2+ associated with successive depolarizations the store can function as an integrator, keeping track of neuronal activity. If a second stimulus (e.g. Ca2+ discharge via InsP3Rs) occurs during the period when the store is primed, RyRs act as coincidence detectors and release stored Ca2+ by calcium-induced calcium release (Berridge 1998; Riddoch et al. 2007; Verkhratsky 2005).

Therefore, since Ca2+ is such an ubiquitous cellular signaling molecule, RyR-mediated Ca2+ signaling is the basis for a wide range of cellular processes including ion channel activation, neurite outgrowth, synaptic plasticity, neurotransmitter release, gene transcription and neurodegeneration, in addition to E-C coupling in skeletal and cardiac muscles.

Chemical modulators of ryanodine receptor function

As chemical modulators of RyRs have been extensively reviewed, we will briefly touch upon this subject here. Table 2 summarizes the important modulators of ryanodine receptors, with particular emphasis on those active against invertebrate RyRs. The endogenous activators are cytosolic Ca2+ and adenine nucleotides (Coronado et al. 1994). Ca2+ acts on the RyR channel by increasing open channel probability under conditions of low (micromolar) cytosolic concentrations. As cytosolic Ca2+ concentrations reach millimolar concentrations, RyR channel activation becomes inhibited (Meissner 1986, 1994; Schmitt et al. 1996; Scott-Ward et al. 2001; Ebbinghaus-Kintscher et al. 2006). Like cytosolic Ca2+, adenine nucleotides also have a biphasic effect on [3H]ryanodine binding (Zimanyi and Pessah 1991). ATP is more effective at activating RyR1 than RyR2 and RyR3 (Fill and Copello 2002). The biphasic response for these endogenous activators is consistent with two or more binding sites having differing affinities, though that remains to be proven (Hadad et al. 1999). In addition to cytosolic Ca2+ and adenine nucleotides, RyR activity is also modulated by depolarization (via the dihydropyridine receptor for RyR1), luminal Ca2+, calmodulin, CAM kinase and PKA.
Table 2

Examples of invertebrate and vertebrate ryanodine receptor modulators

Effector

Action

Tissue source

Activity

Binding site

Reference

Ryanodine (nM-μM)

Modulator

P. americana, H. virescens

Channels locked in sub-conductance state

Ryanodine site

Cordova et al. 2006; Scott-Ward et al. 2001

Ryanodine (>100 μM)

Blocker

lobster

Channels locked in closed state

Ryanodine site

Seok et al. 1992

Ruthenium red

Blocker

D. melanogaster, M. domestica, H. virescens, P. americana

Decrease in [3H]ryanodine binding; decrease in open channel probability

Ryanodine site

Vazquez-Martinez et al. 2003; Lehmberg and Casida 1994; Scott-Ward et al. 2001

Dantrolene

Blocker

D. melanogaster

Decrease in [3H]ryanodine binding

Ryanodine site

Vazquez-Martinez et al. 2003

Procaine

Blocker

rabbit

Decrease in [3H]ryanodine binding (10 mM)

Ryanodine site

Murayama and Ogawa 1997; Xu et al. 1993

Cytosolic Mg2+

Antagonist

D. melanogaster, H. virescens

Decrease in [3H]ryanodine binding

Ca2+ site

Vazquez-Martinez et al. 2003; Scott-Ward et al. 2001

3,5-Di-t-butylcatechol

Activator

rat

[3H]ryanodine displacement IC50 = 310 μM

Ryanodine site

Fusi et al. 2005

4-Chloro-m-cresol

Activator

rabbit

Increase in ryanodine binding affinity (75 μM)

 

Herrmann-Frank et al. 1996

Cytosolic Ca2+ (μM)

Activator

P. americana, H. virescens

Increase in open channel probability; increase in [3H]ryanodine binding

Ca2+ site

Schmitt et al. 1996; Ebbinghaus-Kintscher et al. 2006; Scott-Ward et al. 2001

Cytosolic Ca2+ (>1 mM)

Antagonist

P. americana, H. virescens

Decrease inopen channel probability; decrease in [3H]ryanodine binding

Ca2+ site

Schmitt et al. 1996; Ebbinghaus-Kintscher et al. 2006; Scott-Ward et al. 2001

ATP

Activator

P. americana

Enhancement in ryanodine binding (5 mM)

 

Lehmberg and Casida 1994

  

lobster

Ca2+-dependent enhancement in ryanodine binding (1 mM)

 

Zhang et al. 1999

Caffeine

Activator

P. americana, H. virescens, D. melanogaster (native & recomb)

Calcium release at mM concentrations. Enhances ryanodine binding under low Ca2+ conditions

 

Cordova et al. 2006; Lehmberg and Casida 1994; Ebbinghaus-Kintscher et al. 2006

Suramin

Activator

rabbit, sheep

1 mM (binding enhancement); binds to CAM site

CAM site

Suko et al. 2001; Hohenegger et al. 1996; Sitsapesan and Williams 1996

Phthalic diamides

Activator

H. virescens, D. melanogaster (partial recomb)

Ca2+ release EC50 = 0.7 μM (flubendiamide); Ca2+-dependent increase in [3H]ryanodine binding

Novel site

Ebbinghaus-Kintscher et al. 2006; Luemmen et al. 2007

Anthranilic diamides

Activator

P. americana, D. melanogaster (recomb), H. virescens (recomb)

Ca2+ release EC50 = 0.04–0.05 μM (Rynaxypyr®)

Novel site

Lahm et al. 2007; Cordova et al. 2007a, b; Cordova et al. 2006; Gutteridge et al. 2003

Numerous exogenous modulators of RyRs are known that provide useful tools for pharmacological characterization. All three mammalian RyR isoforms are modulated by ryanodine and caffeine, whereas the polysulfonated naphthylurea, suramin, activates only RyR1 and RyR2. Caffeine binds to a site distinct from ryanodine and stimulates channel activation by decreasing the threshold for Ca2+ activation (Pessah et al. 1987 ). Dantrolene, the primary therapeutic agent used for the prevention and treatment of MH, blocks RyR1 and RyR3, but not RyR2 (Zhao et al. 2001). The basis for this selectivity may be dantrolene’s poor accessibility to the binding site in RyR2 (Paul-Pletzer et al. 2005). Ruthenium red is a channel blocker of all RyR. This molecule inhibits RyRs by at least two mechanisms, noncompetitive interaction with the Ca2+ regulatory site and formation of RyR substates via a voltage-dependent mechanism (Xu et al. 1999). Procaine, and related local anesthetics, in addition to blocking voltage-gated Na+ channels, have also been shown to block RyRs (Murayama and Ogawa 1997; Xu et al. 1993). Single channel studies have shown that procaine stabilizes a long-lived closed state of the channel.

Ryanodine receptors as molecular scaffolds for interacting proteins

The RyR molecule, in particular its extensive cytoplasmic region, can serve as a scaffold for proteins that regulate channel function. Among such proteins are those which bind directly to RyR molecules such as phosphatases, kinases, CAM, FKBP12 and FKBP12.6 (also known as calstabin1 and 2) and sorcin (MacKrill 1999). Triadin and junctin are associated proteins that appear to anchor calsequestrin, the high-capacity, sarcoplasmic reticulum luminal Ca2+-binding protein to the RyR (Oyamada et al. 1994).

Accessory proteins bind at the cytoplasmic region of the receptor and regulate function in response to signals generated extracellularly by secondary messengers. For example, kinases, such as PKA, bind to RyR2 through the accessory binding protein mAKAP (Marx et al. 2000) and modulation of the RyR2 channel via this mechanism has been linked to cardiac arrhythmias (Marx et al. 2000). Phosphatases such as PP1 & PP2A also bind to RyR2 but through their own binding intermediates, spinophilin (Allen et al. 1997) and PR130 (Marx et al. 2001). The mAKAP, spinophilin and PR130 adapter proteins all contain leucine-isoleucine zipper (LIZ) motifs which bind to three highly conserved similar LIZs in RyR2 (Marx et al. 2001), thereby anchoring the enzyme in the macromolecular complex. There is evidence that CaMKII may also bind to RyR2 (Currie et al. 2004) but the binding site is still unknown. The effect of CaMKII-dependent phosphorylation on RyR2 channel activity remains to be resolved (Currie et al. 2004; MacKrill 1999).

The calcium binding protein, CaM, binds to RyR1, RyR2 and RyR3, at three highly conserved binding sites in the vicinity of the transmembrane region of the channel (MacKrill 1999). Application of CaM can either activate (at nanomolar Ca2+ concentrations) or inhibit (at micromolar Ca2+ concentrations) RyR1 and RyR3 channels (Fill and Copello 2002). However, for RyR2, only inhibitory effects have been reported (Balshaw et al. 2001). Thus, apoCaM (the Ca2+-free form) activates channel opening in RyR1, but not in RyR2, whereas the Ca2+-CaM form inhibits channel opening in both RyR1 and RyR2 (Yano et al. 2005). Sorcin is considered to act in a similar manner to FKBP12.6 as a controller of the release of Ca2+ from the SR. At nanomolar concentrations, sorcin inhibits the RyR2 channel in a Ca2+-independent manner. However, PKA-dependent phosphorylation of sorcin stops it from binding to RyR2 and therefore prevents it from affecting the opening probability of the RyR2 channel itself (MacKrill 1999).

In functional terms, the FK506-binding proteins (FKBPs, calstabins), in particular FKBP12.6 (calstabin2), are the best understood of the many molecules known to make up the RyR macromolecular complex. These proteins are Ca2+ channel stabilizing proteins which associate with a single RyR monomer. RyR1 and RyR3 bind both FKBP12 and FKBP12.6, though the receptor affinity for FKBP12 is greater than for FKBP12.6 (Timerman et al. 1996). FKBP12.6 has a higher affinity for RyR2 channels and is the predominant form bound to that receptor (Timerman et al. 1996). Both proteins stabilize the open and closed channel states of the receptor to which they are bound. One model proposed to account for RyR2 dysfunction in heart failure is dissociation of FKBP12.6 from RyR2 by either PKA-dependent phosphorylation of the receptor, or by the receptor mutation Ser2809Asp. FKBP12.6 dissociation results in an increase in diastolic Ca2+ leak from the SR, which in turn results in aberrant increased cardiac contractions (George et al. 2005; Marx et al. 2000; Wehrens and Marks 2004). Thus, understanding the modulation of RyRs by cellular components that form part of the macromolecular complex, inorganic ions (Ca2+, Mg2+), pharmacological substances (caffeine) or by protein modification (phosphorylation) is vitally important as discrepancies in these regulatory mechanisms can result in a number of human diseases. While extensive structural and functional studies of mammalian RyRs has advanced our understanding of their roles in calcium signaling and human disease, much less is known about insect RyR function. However, recent progress on sequencing and genome analysis of the genetic model organism, Drosophila melanogaster, and other insect species is accelerating this effort.

Insect ryanodine receptors

Ryanodine receptors have been studied in several insect species. We first discuss the ryanodine receptors described in the genetic model organism Drosophila melanogaster as well as those reported from several other insect species. As shown in Table 3, insect RyRs show a strong similarity to one another in amino acid sequence but are strikingly different from those of their mammalian counterparts.
Table 3

Comparison of amino acid sequence identity among ryanodine receptors from selected insect and mammalian species

 

Green peach aphid (M. persicae)

Cotton melon aphid A. gossypii)

Corn planthopper (P. maidis)

Fruitfly (D. melanogaster)

Mosquito (A. gambiae)

Mouse RyR2

Rabbit RyR2

Human RyR2

Tobacco budworm (H. virescens)

76.8

77.5

79.6

78.2

79.0

47.1

47.2

47.2

Green peach aphid (M. persicae)

***

97.9

81.9

75.4

76.5

47.0

46.9

46.9

Cotton melon aphid (A. gossypii)

 

***

82.2

75.8

76.8

47.1

47.3

47.4

Corn planthopper (P. maidis)

  

***

78.4

80.2

47.2

47.3

47.2

Fruitfly (D. melanogaster)

   

***

82.3

46.6

46.8

46.7

Mosquito (A. gambiae)

    

***

46.7

46.7

46.7

Mouse RyR2

     

***

97.1

97.2

Rabbit RyR2

      

***

98.6

Values correspond to the percentage of identical amino acids between paired species. Insects are Heliothis virescens, Myzus persicae, Aphis gossypi, Peregrinus maidis, Drosophila melanogaster and Anopheles gambiae. Table is modified from Gutteridge et al. (2003)

Ryanodine receptors of a genetic model organism: the fruitfly, Drosophila melanogaster

Studies on the ryanodine receptor of the genetic model organism Drosophila melanogaster have been instructive. In 1994, Takeshima and colleagues described a 25.7 kb genomic DNA fragment containing a gene for a ryanodine receptor homologue (Takeshima et al. 1994). In all, 26 exons were found to make up the protein coding sequence and a predicted protein of 5216 or 5112 amino acids was reported. This receptor showed 45–47% identity with the three mammalian RyRs. In contrast to mammals, only one gene (Rya-r44F) encoding a ryanodine receptor is present in the fly. Interestingly, mammalian and insect RyRs are strikingly more different in sequence than the corresponding InsP3Rs, which may make RyRs better candidate targets in the search for insecticidal molecules with low mammalian toxicity.

In 2000, the Ma lab (Xu et al. 2000) demonstrated functional expression of Drosophila RyRs in CHO cells. However due to the transient and low levels of protein expression, biophysical characterization was limited to a 20% fragment of the C-terminal encoding the pore-forming region (RyR-C). Comparative studies with a homologous C-terminal fragment from rabbit skeletal muscle demonstrated that the Drosophila channels exhibited a 35% larger full conductance than their rabbit counterpart. Furthermore, the Drosophila RyR-C channels exhibited frequent sub-conductance state transitions. As expected, ryanodine modified gating of the Drosophila RyR-C channel; however conductance was reduced to 30% of the fully-open state rather than 50% as observed with full-length RyR or RyR-C channels from rabbit. The basis of such difference remains unclear but could reflect structural differences between mammalian and Drosophila RyRs.

Robust functional expression of the full-length Drosophila RyR was achieved in a Spodoptera frugiperda cell line (Sf9) by Tao and colleagues (Cordova et al. 2006, 2007a; Gutteridge et al. 2003). Fura-2 imaging studies conducted on Sf9 cells transiently expressing Drosophila RyRs showed that low micromolar concentrations of ryanodine induced calcium store depletion upon channel activation, consistent with these channels having become locked in a partially open state. Sf9 cells stably expressing this receptor (Sf9-DRyR-15b) were activated by caffeine with an EC50 value (4.4 mM) comparable to that reported in literature for mammalian cells. Detailed single-channel characterization remains for this full-length RyR. Such work may enable further elucidation of structure-function differences between insect and mammalian RyRs.

Ryanodine receptors of housefly, cockroach and the agricultural pest, Heliothis virescens

John Casida, who has pioneered so many innovations in insect toxicology and advanced our knowledge of insecticide mode of action, was the first to generate a specific radioligand ([3H]ryanodine) for RyRs. This probe has been utilized to characterize ryanodine’s membrane binding properties and its correlation with murine and housefly (Musca domestica) toxicity (Pessah et al. 1985; Waterhouse et al. 1987). Numerous ryanoid derivatives were generated and their potencies compared. The optimal potency of ryanodine and didehydroryanodine was reduced 3–14 fold by hydroxylation at an isopropyl methyl substituent (Waterhouse et al. 1987). Despite strong correlation between mammalian and insect toxicity for most ryanoids investigated, the hydrolysis products of ryanodine and didehydroryanodine (ryanodol and 9,21-didehydroryanodol, respectively) were found to exhibit low murine toxicity yet potent insect activity. In a later study these hydrolysis products were found to be poor displacers of [3H]ryanodine in rabbit muscle preparations yet potent against housefly (Jeffries et al. 1997). These findings suggested a possible difference in the target sites of mammals and insects (Waterhouse et al. 1987). Nevertheless, ryanoid-based insecticides have not been commercialized beyond the limited exception of ryanodine itself. Such work did however highlight a very important principle concerning selective toxicity that would prove to be an invaluable attribute of future commercial insecticides.

In addition to Drosophila, [3H]ryanodine binding has also been studied in preparations from the American cockroach, Periplaneta americana (Schmitt et al. 1996; Lehmberg and Casida 1994). A single high-affinity binding site was observed in muscle with a KD and Bmax value of 4.4 nM and 2,520 fmol/mg, respectively (Lehmberg and Casida, 1994). Interestingly, caffeine enhanced [3H]ryanodine binding but only in Ca2+-free incubation medium. Relatively low sensitivity was observed to the pharmacological differences exist RyR blocker, ruthenium red (IC50 = 20.2 and 6.3 μM for cockroach and mouse, respectively), again suggesting significant between insect and mammalian RyRs. A RyR gene has been identified in the Heliothis virescens lepidopteran pest, (Puente et al. 2000). This work was advanced further by (Scott-Ward et al. 2001), who initiated a functional characterization of RyR from Heliothis thoracic muscle. They demonstrated that binding was dependent on ryanodine concentration, and stimulated by ATP. In this preparation, millimolar concentrations of caffeine failed to stimulate the binding in the presence of either nanomolar or micromolar Ca2+. A specific anti-Heliothis RyR antibody identified a 400 kDa membrane protein. Microsomal membranes from Heliothis muscle were reconstituted into artificial lipid bilayers to form functional RyR channels. These channels yielded a Ca2+ conductance of ~110 pS, responded to 100 nM ryanodine, and were sensitive to ATP and ruthenium red (as is the case for mammalian RyRs). However, single channel studies were limited by difficulties in obtaining stable lipid bilayers with channels incorporated. Despite the limitations, these studies were important in that they were the first to directly explore [3H]ryanodine binding and single channel characteristics of a native pest insect RyR. Tao and colleagues recently reported success in achieving stable functional expression of a full-length RyR from H. virescens (Gutteridge et al. 2003). As was the case with the recombinant Drosophila RyR, H. virescens receptors expressed in Sf9 cells stimulate release of internal calcium stores in the presence of millimolar concentrations of caffeine and are highly sensitive to insecticidally-active chemistry.

Ryanodine and ryanoids—insecticides based on a natural product

The ground stem wood of the shrub Ryania speciosa, a native plant of tropical America has been used as an insecticide (Heal 1949; Pepper and Carruth 1945). The main alkaloid in such extracts, ryanodine (Fig. 1; Rogers et al. 1948), makes up 0.16–0.2% of the insecticide. Ryanodine and 9,21-dehydroryanodine, another active component extracted from the plant, are very toxic to insects and mammals. Other, less abundant ryanoids are also present in R. speciosa. The natural botanical insecticide ‘Ryania’, based on ryanodine, is effective against the fruit moth, coddling moth and corn earworm, European corn borer, and citrus thrips, but it is ineffective against the cabbage maggot, cauliflower worms or the boll weevil. Ryanodol, the hydrolysis product of ryanodine is also a botanical insecticide (Gonzalez-Coloma et al. 1999).

New synthetic insecticides targeting ryanodine receptors

Phthalic diamides (flubendiamide)

A novel family of substituted phthalic diamides, recently discovered by Nihon Nohyaku, has yielded an important new commercial insecticide, flubendiamide (Fig. 4a; Masaki et al. 2006; Seo et al. 2007). Interestingly, its discovery originated out of a pyrazinedicarboxamide herbicide program (Tsubata et al. 2007). Flubendiamide shows strong insecticidal activity on lepidopteran pests, including Plutella xylostella (EC50 = 0.004 ppm), Spodoptera litura (EC50 = 0.19 ppm) and Homona magnanima (EC50 = 0.58 ppm). Additionally, P. xylostella that were resistant to pyrethroids, benzoylphenylureas, organophosphates and carbamates exhibited no signs of cross-resistance to flubendiamide (Tohnishi et al. 2005). Ecotoxicological studies found flubendiamide to have no acute toxicity among rats and birds (>2,000 mg/kg). Furthermore, no acute toxicity was observed among freshwater fish when tested at the limit of aqueous solubility (29.8 ppb) (Hall 2007).
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Fig. 4

Chemical structures of the insecticides flubendiamide (A) and Rynaxypyr® (chloratraniliprole) (B), both of which selectively target insect ryanodine receptors

Ebbinghaus-Kintscher and colleagues have detailed flubendiamide’s mode of action (Ebbinghaus-Kintscher et al. 2007; Ebbinghaus-Kintscher et al. 2006; Luemmen et al. 2007). Studies on isolated H. virescens neurons showed that phthalic diamides activate ryanodine-sensitive release of internal Ca2+ stores. Similar findings were reported for CHO cells transfected with the Drosophila melanogaster RyR-C-terminal fragment (RyR-C) cloned in the Ma lab. Binding studies performed on microsomal membranes from H.virescens flight muscle showed that flubendiamide acts at a site on the receptor distinct from that of ryanodine (Ebbinghaus-Kintscher et al. 2006). Flubendiamide allosterically enhanced [3H]ryanodine binding under conditions of low Ca2+. In binding studies on H. virescens microsomal membranes [3H]flubendiamide was found to have a KD value of 4.7 nM and a Bmax ranging between 6–7 pmol/mg. No change in [3H]flubendiamide binding was observed with ryanodine, caffeine, cADP-ribose or dantrolene, indicating that the phthalic diamides bind to a new site on the RyR complex. In a separate study Masaki and colleagues showed that flubendiamide stimulates SERCA Ca2+ pump activity (Masaki et al. 2006, 2007). Such stimulation was attributed to a decrease in luminal calcium as a result of tight coupling between RyRs and the SERCA pump. To test activity on mammalian RyRs, flubendiamide and the somewhat more water-soluble phthalic diamide, flubendiamide sulfoxide, were tested against mammalian cell lines that naturally express the three RyR isoforms (Ebbinghaus-Kintscher et al. 2006; Ebbinghaus-Kintscher et al. 2007). These compounds were without effect at micromolar concentrations. Given the extremely low aqueous solubility for this chemical class (29.8 ppb for flubendiamide), the true concentration that these mammalian receptors were exposed to is unclear. Nevertheless, the ability of flubendiamide and related phthalic diamides to modulate insect but not mammalian RyRs clearly indicates that this chemistry exhibits target-based insect selectivity.

Anthranilic diamides (Rynaxypyr®, Cyazypyr™)

Lahm and colleagues at Dupont described another new class of insecticide chemistry, the anthranilic diamides (Fig. 4b; Lahm et al. 2007; Cordova et al. 2007a, b; Lahm et al. 2005; Cordova et al. 2006). They have exceptional insecticidal activity on a range of Lepidopteran pests. For Rynaxypyr®, LC50 values of 0.02 ppm were found for P. xylostella and S. frugiperda with an LC50 value of 0.04 ppm for H. virescens (Lahm et al. 2007). Comparison of insecticidal activity against these pests and Ca2+ mobilization in neurons from the American cockroach, P. americana, illustrates the strong relationship between insect toxicity and Ca2+ mobilization for numerous molecules of this class.

Activity on insect RyRs for this chemistry was first disclosed in a patent application published in 2004 (Gutteridge et al. 2003). Here the inventors demonstrated that Sf9 cells expressing recombinant insect RyRs released intracellular calcium stores in the presence of nanomolar concentrations of various anthranilic diamides. In contrast, non-transfected cells exhibited no response in the presence of micromolar concentrations. Rynaxypyr®, the first commercialized insecticide from this chemical class, was shown to have EC50 values of 40–50 nM when tested on P. americana neurons as well as on Sf9 cells expressing Drosophila or H. virescens RyRs (Lahm et al. 2007; Cordova et al. 2007b). As was the case with the phthalic diamides, anthranilic diamides bind to a site distinct from ryanodine. This chemistry failed to displace or enhance [3H]ryanodine binding in membranes prepared from P. americana leg muscle (Cordova et al. 2006, 2007a). Using a radiolabeled anthranilic diamide, [3H]DP010, a high-affinity saturable binding site was described. Interestingly, the presence of micromolar concentrations of ryanodine significantly enhanced binding of this radiolabeled anthranilic diamide. In the presence of micromolar ryanodine, [3H]DP010 was found to have KD and Bmax values of 28 nM and 8,410 fmol/mg, respectively. No displacement or enhancement in binding was observed in the presence of caffeine. To date, no published studies have appeared addressing whether the anthranilic and phthalic diamides bind to the same site on the RyR.

A key feature of Rynaxypyr® is its superb toxicological profile. It possesses low mammalian toxicity with an acute oral LD50 value of >5,000 mg/kg in rats (Lahm et al. 2007). Exceptional safety was also reported on avian and aquatic organisms (Rynaxypyr® technical bulletin). A major factor contributing to Rynaxypyr®’s mammalian safety is the intrinsic selectivity at the RyR (Lahm et al. 2007; Cordova et al. 2006, 2007a). The molecule was found to be ~300-fold less potent on mouse cells that naturally express RyR1 than on insect cells (Fig. 5). Even greater selectivity (>2,000-fold) was shown for a rat cell line that natively expresses the cardiac isoform, RyR2.
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Fig. 5

Comparison of the action of the anthranilic diamide, Rynaxypyr®, on mammalian and insect cells reveals an intrinsic selectivity at the ryanodine receptor target site. Insect receptors were tested from native (Periplaneta) or recombinant (Drosophila and Heliothis) cells. C2C12 and PC12 cell lines predominantly express RyR1 and RyR2, respectively. The specific isoforms expressed in the human cell line, IMR32, have not been characterized

Conclusions and prospects

Insect RyRs differ quite strikingly in their sequences from those of their mammalian counterparts, nevertheless they appear to share overall similarities in topology and share some common modulators. Ryanodine has proven to be a highly potent insecticide and effective probe for characterizing receptor structure and function of invertebrate and mammalian RyRs. Analogues of ryanodine, although explored extensively, have not yielded a commercial insecticide though early work demonstrated that ryanoids more active than ryanodine can be generated with some exhibiting improved selectivity for insect receptors. Most exciting for insect pest management, is the recent discovery and development of synthetic insecticides such as flubendiamide and Rynaxypyr® (chlorantraniliprole). These RyR modulators exhibit excellent insect potency, bind to a new site on the receptor and show exceptional selectivity for insect compared to mammalian RyRs.

Future insect control prospects look exciting. In addition to Rynaxypyr®, Dupont has just recently announced that a second anthranilic diamide, Cyazypyr™ is in commercial development (June 2008 press release: http://vocuspr.vocus.com/VocusPR30/Newsroom/Query.aspx?SiteName=DupontNew&Entity=PRAsset&SF_PRAsset_PRAssetID_EQ=110201&XSL=PressRelease&Cache=). Cyazypyr™ targets sucking pests as well as Lepidoptera. With these new commercial products showing such promise, one can only expect a renewed search for additional chemotypes. Phthalic and anthranilic diamides also offer novel probes for characterizing structure-function differences between insect and mammalian RyRs. Furthermore, such chemical probes may enhance our understanding of Ca2+ signaling mechanisms in invertebrate and vertebrate systems.

Acknowledgments

The authors are indebted to their colleagues, Dr. S.D. Buckingham and Dr. A.K. Jones (Oxford), Prof. K. Matsuda (Kinki University, Nara), Dr. Y. Tao, Dr. J.J. Rauh, Dr. G.P. Lahm and E.A. Benner (Dupont) for helpful discussions during the preparation of the manuscript.

Copyright information

© Springer-Verlag 2008