戊吡虫胍对棉铃虫中枢神经细胞电压门控钙通道和钾通道的影响 *

doi:10.7679/j.issn.2095-1353.2020.086

应用昆虫学报 ›› 2020, Vol. 57 ›› Issue (4): 841-849.doi: 10.7679/j.issn.2095-1353.2020.086

• 昆虫抗药性专栏 • 上一篇

戊吡虫胍对棉铃虫中枢神经细胞电压门控钙通道和钾通道的影响 ^*

关丹阳¹(), 姜笑维¹, 李清亚¹, 刘晓¹, 马永强², 陈强¹, Li-Byarlay Hongmei³, 贺秉军¹^,^***()

1. 南开大学生命科学学院,生物活性材料教育部重点实验室,天津 300071
2. 中国农业大学理学院应用化学系,北京 100193
3. 中央州立大学农业与生命科学系农业研究与开发计划,Wilberforce OH 45384

收稿日期:2020-01-10 接受日期:2020-04-09 出版日期:2020-07-27 发布日期:2020-09-02
通讯作者: 贺秉军 E-mail:dy13512892573@163.com;hebj@nankai.edu.cn
基金资助:
*国家自然科学基金(31871992,31371974)

Effects of Guadipyr on voltage-gated calcium and potassium channels in central neurons of Helicoverpa armigera

Dan-Yang GUAN¹(), Xiao-Wei JIANG¹, Qing-Ya LI¹, Xiao LIU¹, Yong-Qiang MA², Qiang CHEN¹, Hongmei LI-Byarlay³, Bing-Jun HE¹^,^***()

1. The Key Laboratory of Bioactive Materials of Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China
2. Department of Applied Chemistry, China Agricultural University, Beijing 100093, China
3. Agricultural Research and Develop Program, Department of Agriculture and Life Science, Central State University, Wilberforce OH 45384, U.S.A

Received:2020-01-10 Accepted:2020-04-09 Online:2020-07-27 Published:2020-09-02
Contact: Bing-Jun HE E-mail:dy13512892573@163.com;hebj@nankai.edu.cn

摘要/Abstract

摘要：

【目的】 戊吡虫胍是将新烟碱类和缩氨脲类杀虫剂杀虫活性部分重新组合的新型杀虫剂。但对于该类杀虫剂究竟如何影响离子通道,通道门控特性和功能是如何变化目前尚未见报道。本实验旨在明确该杀虫剂是否影响电压门控钙通道和钾通道的门控过程,探究其是否为该杀虫剂的潜在作用靶标。 【方法】 应用全细胞膜片钳技术检测戊吡虫胍对棉铃虫Helicoverpa armigera Hübner中枢神经细胞电压门控Ca ²⁺通道和K ⁺通道的影响。【结果】 戊吡虫胍作用后Ⅰ-Ⅴ曲线和激活曲线均向超极化方向移动10-15 mV,具有显著性统计学差异（P<0.05）。稳态失活曲线向超极化方向移动约5 mV,不具有统计学差异（P>0.05）。电压门控Ca ²⁺通道峰值电流（I_peak）有不同程度的降低。随着浓度增大I_peak降低有减小的趋势。此外,1 μmol?L ^–1戊吡虫胍作用后钙离子的窗口电流（I_w）面积增加幅度较10 μmol?L ^–1和100 μmol?L ^–1大,为93.20%。提示在一定的测试电压下,该药物作用后处于激活状态的Ca ²⁺通道数目增多。另外,其作用后电压门控钾通道I_peak降低。随着浓度增大I_peak降低有减小的趋势。同时Ⅰ-Ⅴ曲线下移,激活曲线向去极化方向移动约8 mV,不具有统计学差异（P>0.05）。这表明戊吡虫胍作用后K ⁺通道在较高电位下才能激活。【结论】 戊吡虫胍能够有效抑制Ca ²⁺通道和K ⁺通道I_peak,并使通道的激活曲线和失活曲线发生移动,影响Ca ²⁺通道和K ⁺通道的门控特性。表明棉铃虫中枢神经细胞上的电压门控Ca ²⁺通道和K ⁺通道是戊吡虫胍的潜在作用靶标之一。

关键词: 戊吡虫胍, 棉铃虫, 电压门控Ca ²⁺通道, 电压门控K ⁺通道, 全细胞膜片钳

Abstract:

[Objectives] Guadipyr is a novel insecticide that has both neonicotinoid and semicarbazone insecticidal activity. How it affects ion channels and channel gating characteristics and functions is currently unknown. We examined the effects of Guadipyr on voltage-gated calcium and potassium channels, and explored whether these channels are potential targets for this pesticide. [Methods] The effects of three concentrations (1, 10, and 100 μmol?L ^–1) of Guadipyr on Ca ²⁺and K ⁺ channels were studied using the patch clamp technique. [Results] All three concentrations of Guadipyr significantly (P<0.05) shifted Ca ²⁺channel Ⅰ-Ⅴ and activation curves about 10-15 mV in the negative direction. The inactivation curves of Ca ²⁺channels were also shifted 5 mV towards the hyperpolarizing direction, which is a not significant shift (P>0.05). The Ca ²⁺peak current was significantly decreased by Guadipyr treatment relative to the control. The I_peak decreased more slowly as the concentration of Guadipyr increased. The area of window current increased the most (93.20%) following treatment with 1 μmol?L ^–1 Guadipyr than by treatment with 10 μmol?L ^–1 and 100 μmol?L ^–1. The number of Ca ²⁺ channels activated increased after Guadipyr treatment at a test voltage of ﹣60 mV to +10 mV. The K ⁺peak current decreased gradually when Guadipyr was added to the external solution; the I_peak decreased more slowly as the Guadipyr concentration increased from 10 μmol?L ^–1 to 100 μmol?L ^–1. Ⅰ-Ⅴ curves declined and the activation of K ⁺channels was shifted 8 mV towards the depolarizing direction, which was not significant (P>0.05). This shows that K ⁺ channels can be activated at higher potentials by Guadipyr. [Conclusion] Guadipyr can effectively inhibit the I_peak of the Ca ²⁺and K ⁺channels, and shift the activation and inactivation curves of these channels, thereby affecting the gating of Ca ²⁺ channels and K ⁺ channels. Therefore, Ca ²⁺ and K ⁺ channels in the central neurons of H. armigera are potential Guadipyr target sites.

Key words: Guadipyr, Helicoverpa armigera, voltage-gated calcium channels, voltage-gated potassium channels, Whole-cell Patch Clamp Technique

关丹阳, 姜笑维, 李清亚, 刘晓, 马永强, 陈强, Li-Byarlay Hongmei, 贺秉军. 戊吡虫胍对棉铃虫中枢神经细胞电压门控钙通道和钾通道的影响 ^*[J]. 应用昆虫学报, 2020, 57(4): 841-849.

Dan-Yang GUAN, Xiao-Wei JIANG, Qing-Ya LI, Xiao LIU, Yong-Qiang MA, Qiang CHEN, Hongmei LI-Byarlay, Bing-Jun HE. Effects of Guadipyr on voltage-gated calcium and potassium channels in central neurons of Helicoverpa armigera[J]. Chinese Journal of Applied Entomology, 2020, 57(4): 841-849.

图/表 11

图1 戊吡虫胍作用前后棉铃虫中枢神经细胞Ca2+通道电流 A. 对照;B. 戊吡虫胍（10 μmol?L–1）作用后的ICa。

Fig. 1 ICa of the Helicoverpa armigera before and after the application of Gua A. Control; B. ICa after Gua (10 μmol?L–1).

图2 不同浓度戊吡虫胍作用下Ca2+通道的Ⅰ-Ⅴ曲线及Ipeak A. 戊吡虫胍对棉铃虫中枢神经细胞Ca2+通道Ⅰ-Ⅴ曲线的影响: 电流幅值标准化;B. 戊吡虫胍作用后Ca2+通道Ipeak变化。* 表示对照组相比具有显著性差异（P<0.05,单因素方差分析）。图7同。

Fig. 2 TheⅠ-Ⅴcurves and Ipeak of Ca2+ channels before and after the application of Gua A. Gua's effects on the Ⅰ-Ⅴ curve of Ca2+ channels: Standardized current amplitude; B. The Ipeak of Ca2+ channels after Gua. * indicats significant difference compared with the control (P<0.05, One-way analysis of variance). The same as Fig.7.

图3 不同浓度戊吡虫胍对Ca2+通道稳定性激活的影响

Fig. 3 Effects of Gua on the steady-state activation of ICa

表1 戊吡虫胍对棉铃虫中枢神经细胞Ca2+通道稳定性激活的影响

Table 1 Effects of Gua on the steady-state activation of ICa

条件 Condition	激活 Activation
条件 Condition	V_0.5^a	k^b	n
对照 Control	–9.83±0.73	4.55±0.38	10
1 μmol?L^–1戊吡虫胍 1 μmol?L^–1Gua	–22.85±5.02*	5.27±0.26	5
10 μmol?L^–1戊吡虫胍 10 μmol?L^–1Gua	–23.45±3.36*	5.48±0.21	8
100 μmol?L^–1戊吡虫胍 100 μmol?L^–1Gua	–24.21±3.74*	5.46±0.26	5

图4 不同浓度戊吡虫胍对Ca2+通道失活的影响

Fig. 4 Effects of Gua on the steady-state inactivation of ICa

表2 戊吡虫胍对棉铃虫中枢神经细胞Ca2+通道失活的影响

Table 2 Effects of Gua on the steady-state inactivation of ICa

条件 Condition	失活 Inactivation
条件 Condition	V_0.5^a	k^b	n
对照 Control	–22.30±2.46	18.45±3.60	7
1 μmol?L^–1戊吡虫胍 1 μmol?L^–1Gua	–26.26±2.86	13.58±3.36	5
10 μmol?L^–1戊吡虫胍 10 μmol?L^–1Gua	–24.76±2.44	12.28±2.95	6
100 μmol?L^–1戊吡虫胍 100 μmol?L^–1Gua	–28.69±3.12	13.41±3.13	5

图5 戊吡虫胍对棉铃虫中枢神经细胞Ca2+通道Window current的影响对比 A. 1 μmol?L–1戊吡虫胍;B. 10 μmol?L–1戊吡虫胍;C. 100 μmol?L–1戊吡虫胍。

Fig. 5 Comparative analysis about the effect of Gua on the Window current of ICa A. 1 μmol?L–1 Gua; B. 10 μmol?L–1 Gua; C. 100 μmol?L–1 Gua.

图6 戊吡虫胍作用前后棉铃虫中枢神经细胞K+通道电流 A. 对照;B. 戊吡虫胍（100 μmol?L–1）作用后的IK。

Fig. 6 IK of the Helicoverpa armigera before and after the application of Gua A. Control; B. IK after Gua (100 μmol?L–1).

图7 不同浓度戊吡虫胍作用下IK的Ⅰ-Ⅴ曲线及Ipeak A. 不同浓度戊吡虫胍对棉铃虫中枢神经细胞K+通道I-V曲线的影响：电流幅值未标准化;B. 戊吡虫胍对棉铃虫中枢神经细胞K+通道电流的峰值影响。

Fig. 7 TheⅠ-Ⅴ curves and Ipeak of IK before and after the application of Gua A. Gua’s effects on the I-V curve of K+ channels: Current amplitude is not standardized; B. The Ipeak of K+ channels after Gua.

图8 不同浓度戊吡虫胍对K+通道稳定性激活的影响

Fig. 8 Effects of Gua on the steady-state activation of IK

表3 戊吡虫胍对棉铃虫中枢神经细胞K+通道激活的影响

Table 3 Effects of Gua on the steady-state activation of IK

条件 Condition	激活 Activation
条件 Condition	V_0.5^a	k^b	n
对照Control	22.15±1.72	21.10±0.74	13
10 μmol?L^–1戊吡虫胍 10 μmol?L^–1Gua	31.72±1.07*	22.31±0.78	7
100 μmol?L^–1戊吡虫胍 100 μmol?L^–1Gua	29.35±1.55	24.16±1.93	4

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戊吡虫胍对棉铃虫中枢神经细胞电压门控钙通道和钾通道的影响 ^*

Effects of Guadipyr on voltage-gated calcium and potassium channels in central neurons of Helicoverpa armigera

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