Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, Nakamachi, Nara, Japan.
PLoS One. 2011 Apr 1;6(4):e18354. doi: 10.1371/journal.pone.0018354.
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.
由日本曲霉（Aspergillus japonicus） JV-23 产生的asperparaline A 会导致家蚕（Bombyx mori）幼虫瘫痪，但昆虫毒性的靶标尚不清楚。在本研究中，我们使用膜片钳电生理学技术研究了 asperparaline A 在培养的家蚕幼虫脑神经元表达的配体门控离子通道上的作用。10µM 的 asperparaline A 对膜电流没有影响，但在与 10µM 乙酰胆碱（ACh）共同应用前给予 1 分钟时，它完全阻断了对烟碱型乙酰胆碱受体（nAChR）选择性拮抗剂美加明敏感的 ACh 诱导电流。相比之下，10µM 的 asperparaline A 对γ-氨基丁酸和 L-谷氨酸诱导的家蚕幼虫神经元反应没有作用。真菌生物碱在阻断 ACh 诱导反应时没有使用依赖性，对 10µM ACh 反应的峰值和缓慢脱敏电流幅度具有明显的亲和力，其 IC50 值分别为 20.2 和 39.6 nM。asperparaline A（100 nM）降低了 ACh 对神经元的最大反应，EC50 仅有最小的偏移，表明该生物碱是非竞争性的。与在家蚕 nAChRs 上表现出明显的阻断作用相反，它在家蚕卵母细胞中表达的鸡α3β4、α4β2 和α7 nAChRs 上仅表现出较弱的阻断作用，表明其对昆虫 nAChRs 的选择性高于某些脊椎动物 nAChRs。
