The insecticide chlorantraniliprole is a weak activator of mammalian skeletal ryanodine receptor/Ca²⁺ release channel

Highlights

• Chlorantraniliprobe is a weak activator of mammalian skeletal RyR1 channel.

• Chlorantraniliprobe activates mammalian skeletal RyR1 at high concentrations.

• Chlorantraniliprobe has much lower affinity to mammalian RyR1 than insect RyR.

• Chlorantraniliprobe has a binding site distinct from all known RyR channel modulators.

Abstract

Chlorantraniliprobe (Chlo), a potent insecticide, demolishes intracellular Ca²⁺ homeostasis of insects by inducing uncontrolled Ca²⁺ release through ryanodine receptors (RyRs). Chlo is lethal to insects but has low toxicity to mammals. In this study, we investigated the effects of Chlo on RyR1 from mammalian skeletal muscle. Ca²⁺ release assay indicated that Chlo at high concentrations promoted Ca²⁺ release from sarcoplasmic reticulum through RyR1 channels. Single channel recording of purified RyR1 showed that Chlo activated RyR1 channel, increased channel open probability P_o, reduced channel mean close time T_c, but did not change the channel mean open time T_o, suggesting that Chlo destabilized the closed RyR1 channel, rendered the channel easy to open. The dissociation constant K_d values of Chlo for RyR1 were of micromolar level, approximately 100-fold larger than that for insect RyR. The K_d values were smaller for open states than for closed/blocked states of the RyR1 channel. The maximal binding capacity B_max did not change in the presence of either channel activators or inhibitors/blockers. Our results demonstrate that the insecticide Chlo is a weak activator of mammalian RyR1. It can interact with mammalian RyR1 and activate RyR1 channel but with much lower affinity compared with insect RyR; Chlo has a binding site distinct from all known RyR channel modulators and represents a novel type of RyR channel modulator. Our data provide biochemical and pharmacological insights into its high specificity to insect RyR and high selectivity of poisoning to insects over mammals.

Introduction

Diamide insecticides have becoming one of the emerging classes of molecules for insect control [1,2]. Chlorantraniliprole (hereafter referred as Chlo) developed by DuPont, one of the first two insecticides from this class, is most widely used [3]. Diamide insecticides have outstanding insecticidal potency and high selectivity for insects over other species, particularly mammals [[3], [4], [5]]. The target of Chlo is identified as the ryanodine receptor (RyR)/Ca²⁺ release channels and the lethal dysfunctions of muscle were related to the uncontrolled Ca²⁺ release through RyR/Ca²⁺ release channels [6,7]. Further studies conclude that Chlo can bind to insect RyRs and lock the channels in an open state, leading to uncontrolled Ca²⁺ release, which impairs Ca²⁺ homeostasis within cells [3,8].

RyRs are Ca²⁺ release channels located in the sarcoplasmic/endoplasmic reticulum membranes [9], responsible for releasing Ca²⁺ from intracellular stores during excitation-contraction coupling in muscle [[10], [11], [12]]. As the largest known ion channel (∼2300 kDa), RyR is a homo-tetramer consisting of four identical subunits, which form an ion-conducting pore in the center of RyR [[13], [14], [15], [16], [17], [18], [19]]. Generally, the pore-forming region merely corresponds to a small portion of the C-terminal of each monomer; in contrast, most of the mass of RyR localizes in the cytoplasmic region. Various substances modulate RyR [20,21], including a number of physiological agents (e.g. Ca²⁺, adenine nucleotides), and pharmacological chemicals (e.g. ryanodine, ruthenium red, caffeine). Among them, Ca²⁺ is the most important physiological modulator. Low concentrations of Ca²⁺ (100 nM–100 μM) activate RyR, while high concentrations (>1 mM) inhibit it. Adenine nucleotides, especially ATP, serve as another endogenous activator. Ryanodine, a natural plant metabolite from Ryania speciosa, has been found to modify channel activity with a complex mode— locking the channel in the open or sub-conductance state at nanomolar level and inhibiting it at high concentrations (>100 μM). Ruthenium red, a polycationic dye, has been demonstrated to interact with RyR and block its ion channel [17].

In mammals, at least three isoforms of RyRs (named RyR1∼3) are expressed, each is encoded by a distinct gene (ryr1, ryr2, ryr3) [20]. Among them, RyR1 is the most thoroughly studied isoform because of its high expression in skeletal muscle and relatively convenience of purification. In contrast to mammals, there is only one isoform in insects, named insect RyR [4], but alternative splicing and expressing of the insect RyR exist [22,23]. RyRs from insects show high (75%–80%) sequence homology with one another, but is strikingly different from their mammalian counterparts (only 45%–47% identity). All RyRs appear to share the same topological structure and several common modulators, such as Ca²⁺, ATP, and ryanodine, in spite of some pharmaco-dynamic differences.

Previous studies have been focused on the interaction of Chlo with insect RyR [6,24,25]. Comparatively, little information has been available on the interaction of Chlo with mammalian RyR. To date, only one research reported the effects of Chlo on Ca²⁺ release from sarcoplasmic reticulum (SR) vesicles and ryanodine binding to rabbit wild type and malignant hyperthermia-susceptible ryanodine receptors [26]. However, no information exists about binding parameters (disassociation constant K_d, maximal binding capacity B_max) of Chlo to mammalian RyR and effect of Chlo on single channel characteristics of mammalian RyR. In the present study, we investigated the effects of Chlo on Ca²⁺ release from rabbit skeletal sarcoplasmic reticulum (SR) and single channel characteristics of purified rabbit skeletal RyR1, and determined the binding parameters K_d and B_max of Chlo to rabbit skeletal RyR1. We demonstrate that Chlo can interact with mammalian RyR1 and modulate its channel activity but is a weak activator of mammalian RyR1. Our studies on mammalian RyR1 together with previous studies on insect RyR provide the biochemical and pharmacological insights into high specificity of Chlo to insect RyR and high selectivity of poisoning to insects over mammals.