The effects of knock-down resistance mutations and alternative splicing on voltage-gated sodium channels in Musca domestica and Drosophila melanogaster.
The effects of knock-down resistance mutations and alternative splicing on voltage-gated sodium channels in Musca domestica and Drosophila melanogaster.
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DOI：
10.1016/j.ibmb.2020.103388
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发表时间：
2020-05
影响因子：
3.8
通讯作者：
A. J. Thompson;Paul S Verdin;M. Burton;T. Davies;M. Williamson;L. Field;R. Baines;I. Mellor;I. Duce
中科院分区：
文献类型：
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作者：
A. J. Thompson;Paul S Verdin;M. Burton;T. Davies;M. Williamson;L. Field;R. Baines;I. Mellor;I. Duce
电压门控钠通道（VGSC）是拟除虫菊酯类杀虫剂作用的主要靶点，对拟除虫菊酯类杀虫剂的抗性被归因于VGSC基因的突变。昆虫的VGSCs仅由一个基因编码，其结构和功能的多样性源于转录后修饰，特别是选择性剪接。通过对拟除虫菊酯易感（野生型）家蝇和抗性家蝇（s-kdr）的神经元进行全细胞片箝，我们发现与野生型相比，钠电流（INa+）激活和稳态失活的v50在kdr神经元中显著去极化，而10 nM溴氰菊酯在易感而非kdr家蝇的神经元中显著超极化。同样，尾电流对溴氰菊酯野生型神经元（EC1514.5 nM）比-kdr（EC15133 nM）更敏感。我们还发现，在这两个菌株中，INa+有两种类型：一种是强灭活电流（峰值的6.8%），另一种是更持久的电流（峰值的17.1%）。尾电流分析表明，两种菌株的持续电流（野生型ec155.84 nM）对溴氰菊酯的敏感性高于灭活型菌株（野生型ec1535.1 nM）。先前已有研究表明，与含有外显子k的剪接变体相比，黑腹果蝇VGSC中外显子1的存在会产生更持久的INa+，我们使用家蝇头cDNA PCR来证实剪接变体k和1的家蝇同源物的存在。通过检测表达果蝇旁avgsc k或l变体的异种卵母细胞中的INa+来确定它们对溴氰菊酯敏感性的影响。在不同浓度溴氰菊酯存在下，尾电流分析表明，l剪接变异（EC5042 nM）比k剪接变异（EC50866 nM）更敏感。我们得出结论，除了点突变的存在，靶位点对拟除虫菊酯的抗性可能与剪接变体的差异表达有关。
Voltage-gated sodium channels (VGSCs) are a major target site for the action of pyrethroid insecticides and resistance to pyrethroids has been ascribed to mutations in the VGSC gene. VGSCs in insects are encoded by only one gene and their structural and functional diversity results from posttranscriptional modification, particularly, alternative splicing. Using whole cell patch clamping of neurons from pyrethroid susceptible (wild-type) and resistant strains (s-kdr) of housefly,Musca domestica, we have shown that the V50for activation and steady state inactivation of sodium currents (INa+) is significantly depolarised ins-kdrneurons compared withwild-typeand that 10 nM deltamethrin significantly hyperpolarised both of these parameters in the neurons from susceptible but nots-kdrhouseflies. Similarly, tail currents were more sensitive to deltamethrin inwild-typeneurons (EC1514.5 nM) thans-kdr(EC15133 nM). We also found that in both strains, INa+are of two types: a strongly inactivating (to 6.8% of peak) current, and a more persistent (to 17.1% of peak) current. Analysis of tail currents showed that the persistent current in both strains (wild-typeEC155.84 nM) was more sensitive to deltamethrin than was the inactivating type (wild-typeEC1535.1 nM). It has been shown previously, that the presence of exon l in the Drosophila melanogaster VGSC gives rise to a more persistent INa+than does the alternative splice variant containing exon k and we used PCR with housefly head cDNA to confirm the presence of the housefly orthologues of splice variants k and l. Their effect on deltamethrin sensitivity was determined by examining INa+inXenopusoocytes expressing either the k or l variants of theDrosophila paraVGSC. Analysis of tail currents, in the presence of various concentrations of deltamethrin, showed that the l splice variant was significantly more sensitive (EC5042 nM) than the k splice variant (EC50866 nM). We conclude that in addition to the presence of point mutations, target site resistance to pyrethroids may involve the differential expression of splice variants.