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A unique primary structure of RDL (resistant to dieldrin) confers resistance to GABA-gated chloride channel blockers in the two-spotted spider mite Tetranychus urticae Koch.
Journal of Neurochemistry ( IF 4.7 ) Pub Date : 2020-09-07 , DOI: 10.1111/jnc.15179
Takeru Kobayashi 1 , Susumu Hiragaki 1 , Takeshi Suzuki 1 , Noriaki Ochiai 1 , Liza J Canlas 1 , Muhammad Tufail 1 , Naotaka Hayashi 1 , Ahmed A M Mohamed 1 , Mark A Dekeyser 2 , Kazuhiko Matsuda 3, 4 , Makio Takeda 1
Affiliation  

The primary structure of the second transmembrane (M2) segment of resistant to dieldrin (RDL), an ionotropic γ‐aminobutyric acid receptor (GABAR) subunit, and the structure–function relationships in RDL are well conserved among insect species. An amino acid substitution at the 2′ position in the M2 segment (Ala to Ser or Gly) confers resistance to non‐competitive antagonists (NCAs) of GABARs. Here, a cDNA encoding RDL was cloned from the two‐spotted spider mite Tetranychus urticae Koch. Unlike insect homologs, native TuRDL has His at the 2′ position (H305) and Ile at 6′ (I309) in the M2 segment and is insensitive to NCAs. Single and multiple mutations were introduced in the M2 segment of TuRDL, and the mutant proteins were expressed in Xenopus oocytes and examined for the restoration of sensitivity to NCAs. The sensitivity of a double mutant (H305A and I309T in the M2 segment) was greatly increased but was still considerably lower than that of insect RDLs. We therefore constructed chimeric RDLs consisting of TuRDL and Drosophila melanogaster RDL and examined their sensitivities to NCAs. The results show that the N‐terminal region containing the Cys‐loop as well as the M2 segment confers functional specificity; thus, our current understanding of the mechanism underlying NCA binding to GABARs requires reappraisal.

中文翻译:

RDL的独特一级结构(对狄氏剂具有抗性)可在两斑叶螨Tetranychus urticae Koch中赋予对GABA控氯通道阻滞剂的抗性。

抵抗狄氏剂(RDL),离子型γ-氨基丁酸受体(GABAR)亚基的第二跨膜(M2)区段的一级结构以及RDL中的结构-功能关系在昆虫物种之间都非常保守。M2区段(Ala到Ser或Gly)2'位置的氨基酸取代赋予对GABARs非竞争性拮抗剂(NCA)的抗性。在这里,从二斑叶螨Tetranychus urticae Koch克隆了编码RDL的cDNA 。与昆虫同源物不同,天然Tu RDL在M2段的2'位置(H305)具有His,在6'(I309)处具有Ile,并且对NCA不敏感。在Tu RDL的M2片段中引入了单个和多个突变,突变蛋白在非洲爪蟾中表达。卵母细胞并检查其对NCA敏感性的恢复。双重突变体(M2段中的H305A和I309T)的敏感性大大提高,但仍远低于昆虫RDL的敏感性。因此,我们构建了由Tu RDL和Drosophila melanogaster RDL组成的嵌合RDL,并检查了它们对NCA的敏感性。结果表明,含有Cys环和M2节段的N末端区域赋予了功能特异性;因此,我们目前对NCA与GABAR结合的潜在机制的了解需要重新评估。
更新日期:2020-09-07
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