A Fungal Metabolite Asperparaline A Strongly and Selectively Blocks Insect Nicotinic Acetylcholine Receptors: The First Report on the Mode of Action

Asperparalines produced by Aspergillus japonicus JV-23 induce paralysis in silkworm (Bombyx mori) larvae, but the target underlying insect toxicity remains unknown. In the present study, we have investigated the actions of asperparaline A on ligand-gated ion channels expressed in cultured larval brain neurons of the silkworm using patch-clamp electrophysiology. Bath-application of asperparaline A (10 µM) had no effect on the membrane current, but when delivered for 1 min prior to co-application with 10 µM acetylcholine (ACh), it blocked completely the ACh-induced current that was sensitive to mecamylamine, a nicotinic acetylcholine receptor (nAChR)-selective antaogonist. In contrast, 10 µM asperparaline A was ineffective on the γ-aminobutyric acid- and L-glutamate-induced responses of the Bombyx larval neurons. The fungal alkaloid showed no-use dependency in blocking the ACh-induced response with distinct affinity for the peak and slowly-desensitizing current amplitudes of the response to 10 µM ACh in terms of IC50 values of 20.2 and 39.6 nM, respectively. Asperparaline A (100 nM) reduced the maximum neuron response to ACh with a minimal shift in EC50, suggesting that the alkaloid is non-competitive with ACh. In contrast to showing marked blocking action on the insect nAChRs, it exhibited only a weak blocking action on chicken α3β4, α4β2 and α7 nAChRs expressed in Xenopus laevis oocytes, suggesting a high selectivity for insect over certain vertebrate nAChRs

The fungal alkaloid Okaramine-B activates an L-glutamate-gated chloride channel from Ixodes scapularis, a tick vector of Lyme disease

This work was supported by Merial Ltd., The Japan Society for the Promotion of Sciences (KAKENHI, Grant number: 17H01472) and The UK Medical Research Council.A novel L-glutamate-gated anion channel (IscaGluCl1) has been cloned from the black-legged tick, Ixodes scapularis, which transmits multiple pathogens including the agents of Lyme disease and human granulocytic anaplasmosis. When mRNA encoding IscaGluCl1 was expressed in Xenopus laevis oocytes, we detected robust 50–400 nA currents in response to 100 μM L-glutamate. Responses to L-glutamate were concentration-dependent (pEC50 3.64 ± 0.11). Ibotenate was a partial agonist on IscaGluCl1. We detected no response to 100 μM aspartate, quisqualate, kainate, AMPA or NMDA. Ivermectin at 1 μM activated IscaGluCl1, whereas picrotoxinin (pIC50 6.20 ± 0.04) and the phenylpyrazole fipronil (pIC50 6.90 ± 0.04) showed concentration-dependent block of the L-glutamate response. The indole alkaloid okaramine B, isolated from fermentation products of Penicillium simplicissimum (strain AK40) grown on okara pulp, activated IscaGluCl1 in a concentration-dependent manner (pEC50 5.43 ± 0.43) and may serve as a candidate lead compound for the development of new acaricides.Publisher PDFPeer reviewe

Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels.

Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to 10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs

Acetylcholine (ACh)-induced currents (A), the effects of blockers (mecamylamine and fipronil) on the ACh- (B), γ-aminobutyric acid (GABA) (C)- and L-glutamate (D)-induced currents and the actions of asperparaline A on the resting-state (E) and neurotransmitter-evoked currents (F–H) in the silkworm (<i>Bombyx mori</i>) larval neurons.

<p>The holding potential was −60 mV. ACh (10 µM), L-glutamate (30 µM) and GABA (30 µM) was applied for 2 s using the U-tube, whereas mecamylamine and fipronil were bath-applied for 1 min prior to co-application with the agonists. In (E), asperparaline A was applied alone at 1 µM for 2 s using the U-tube, whereas in (F–H), it was bath-applied for 1 min prior to co-application with neurotransmitters ACh (F), GABA (G) and L-glutamate (H). Note that both peak and slowly desensitizing current amplitudes of the ACh-evoked response were blocked reversibly, selectively and almost completely by 1 µM asperparaline A (F).</p

The effects of repeated application of ACh on the blocking action of asperparaline A.

<p>After recording the control response to ACh at 10 µM, asperparaline A was continuously bath-applied at 30 nM, during which ACh was also applied at 10 µM for 2 s every minute using the U-tube. (A) Traces of the ACh-induced current responses in the presence of 30 nM asperparaline A. (B) Normalized peak current amplitude of the ACh responses recorded during the continuous application of asperparaline A. The peak current amplitude of each response was normalized by that of the response recorded before the application of asperparaline A. Each plot represents the mean ± standard error of the mean of 4 separate experiments.</p

Concentration-inhibition curves for asperparaline A in terms of attenuation of the responses to ACh of the silkworm larval neurons.

<p>(A) The ACh-induced responses recorded before and after bath-application of asperparaline A for 1 min prior to co-application with 10 µM ACh. The peak and slowly desensitizing currents are indicated by “a” and “b”, respectively. (B) Concentration-inhibition curves for asperparaline A. Data were normalized to the maximum response to ACh (10 µM). Each plot represents the mean ± the standard error of the mean of 4 experiments. The concentration-inhibition curves were obtained by fitting the data to Eq. (1) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018354#s2" target="_blank">Materials and Methods</a>). The pIC₅₀ ( = log(1/IC₅₀) values for the peak and slowly desensitizing currents were 7.69±0.02 (n = 4, IC₅₀ = 20.2 nM) and 7.40±0.04 (n = 4, IC₅₀ = 39.6 nM), respectively. These two values are significantly different (<i>p</i><0.05, <i>t</i>-test).</p

Effects of asperparaline A on the ACh-induced responses of chicken α3β4 (A), α4β2 (B) and α7 (C) nAChRs expressed in <i>Xenopus laevis</i> oocytes.

<p>After three successive control applications of ACh, 10 µM asperparaline A was continuously bath-applied and then co-applied with 100 µM ACh. Asperparaline A blocked the ACh-response of α3β4 nAChR by 33.4±3.3% (n = 3), whereas it scarcely influenced the response of α4β2 (n = 4) and α7 (n = 3) nAChRs.</p

Effects of asperparaline A on the concentration-response curve for ACh in the silkworm larval neurons.

<p>The ACh-induced responses were measured at various concentrations in the presence and absence of 100 nM asperparaline A. The concentration-response curves were obtained by fitting the data to Eq. (2) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018354#s2" target="_blank">Materials and Methods</a>). The pEC₅₀ ( = log(1/EC₅₀)) values determined in the presence and absence of asperparaline A were 4.98±0.10 (n = 4, EC₅₀ = 10.5 µM) and 4.94±0.04 (n = 7, EC₅₀ = 11.4 µM), respectively. No significant shift in EC₅₀ was observed by the application of asperparaline A.</p

Effects of pre-application on the antagonist action of asperparaline A.

<p>(A) Asperparaline A was co-applied at 30 nM with 10 µM ACh for 2 s without pre-application, or applied for 1, 2 and 5 min prior to co-application with 10 µM ACh. (B) The antagonist action of asperparaline A with and without pre-application for 1, 2 and 5 min. Each bar graph represents the mean ± standard error of the mean (n = 4) of the peak current amplitude of the ACh-induced response normalized by that taken before the application of asperparaline A. The pre-application of asperparaline A significantly enhanced the antagonist action (<i>p</i><0.05, One-way ANOVA, Tukey's test), but there were no significant differences in the blocking action between 1, 2, and 5 min pre-applications.</p

Chemical structure of asperparaline A.

<p>Chemical structure of asperparaline A.</p