Hirata K et al. (2011), A fungal metabolite asperparaline a strongly an...

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PLoS One 2011 Apr 01;64:e18354. doi: 10.1371/journal.pone.0018354.

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A fungal metabolite asperparaline a strongly and selectively blocks insect nicotinic acetylcholine receptors: the first report on the mode of action.

Hirata K , Kataoka S , Furutani S , Hayashi H , Matsuda K .

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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 IC(50) 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 EC(50), 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.

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Species referenced: Xenopus laevis
Genes referenced: chrna4

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	Figure 1. Chemical structure of asperparaline A.
	Figure 2. 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 (Bombyx mori) larval neurons.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).
	Figure 3. The effects of repeated application of ACh on the blocking action of asperparaline A.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.
	Figure 4. Effects of pre-application on the antagonist action of asperparaline A.(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 (p<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.
	Figure 5. Concentration-inhibition curves for asperparaline A in terms of attenuation of the responses to ACh of the silkworm larval neurons.(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 Materials and Methods). 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 (p<0.05, t-test).
	Figure 6. Effects of asperparaline A on the concentration-response curve for ACh in the silkworm larval neurons.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 Materials and Methods). 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.
	Figure 7. Effects of asperparaline A on the ACh-induced responses of chicken α3β4 (A), α4β2 (B) and α7 (C) nAChRs expressed in Xenopus laevis oocytes.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.

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