High-resolution insights into insecticide interactions with voltage-gated sodium channels

项目摘要 电压门控钠通道（Navs）是多种天然神经毒素和杀虫剂的靶标，包括 拟除虫菊酯和钠通道阻断剂杀虫剂（SCBI）。拟除虫菊酯是目前 这是消灭蚊子和其他传播人类疾病的节肢动物媒介的重要和有效的武器库。 SCBI属于一类新的选择性杀虫剂。有效控制病媒的一个主要挑战是全球性的 杀虫剂抗药性的出现。特别是，Nav基因的突变赋予了一种主要形式的抗性， 称为击倒抗性（KDR）。在真核细胞Navs的3-D结构不存在时， 由于拟除虫菊酯或SCBI的存在，在了解其相互作用方面仍存在重大不确定性（包括 各自的受体位点）和KDR机制。在这份拨款提案中，我们利用最近的 co-PI实验室在确定几种真核Navs的cryo-EM结构方面取得了突破。 这些Nav结构的可用性现在承诺对Nav的理解达到前所未有的新水平 与杀虫剂和其他神经毒素的相互作用。我们建议确定Navs的冷冻电镜结构， 与拟除虫菊酯，SCBI和其他三种神经毒素复合，并使用这些结构作为指导， 全面的一系列突变，电生理和计算建模分析， Nav-ligand相互作用。该更新建议的两个具体目标是：1）确定 真核Nav与各种拟除虫菊酯形成复合物，以深入了解拟除虫菊酯的原子机制 2）研究Nav与SCBI（DCJW和氰氟虫腙）、甾体 激动剂（BTX和藜芦啶）和烷基酰胺杀虫剂BTG 502。这项研究的结果将推动我们的 将拟除虫菊酯、SCBI和其他神经毒素的分子作用理解到一个新的水平， 促进在全世界范围内发展对杀虫剂抗药性的精确监测和管理， 发现新的安全的Nav-targeting化学品，以应对节肢动物疾病传播的全球威胁 向量。

Project Summary Voltage-gated sodium channels (Navs) are targets of a variety of natural neurotoxins and insecticides, including pyrethroids and sodium-channel-blocker insecticides (SCBIs). Pyrethroids are currently one of the most important and effective arsenals against mosquitoes and other arthropod vectors that transmit human diseases. SCBIs belong to a newer class of selective insecticides. A major challenge for effective vector control is global emergence of insecticide resistance. In particular, mutations in Nav genes confer a major form of resistance known as knockdown resistance (kdr). In the absence of 3-D structures of eukaryotic Navs in complex with pyrethroids or SCBIs, major uncertainties remain in understanding their interactions (including the nature of respective receptor sites) and kdr mechanisms. In this grant proposal, we take advantage of recent breakthroughs in the co-PI’s laboratory in the determination of the cryo-EM structures of several eukaryotic Navs. The availability of these Nav structures now promises an unprecedented new level of understanding of Nav interactions with insecticides and other neurotoxins. We propose to determine cryo-EM structures of Navs in complex with pyrethroids, SCBIs and three other neurotoxins and use these structures as a guide to conduct a comprehensive series of mutational, electrophysiological and computational modeling analyses of respective Nav-ligand interactions. Two specific aims of this renewal proposal are: 1) determine cryo-EM structures of eukaryotic Nav complexes with various pyrethroids to gain insights into the atomic mechanisms of pyrethroids and kdr, and 2) Investigate the mechanisms of Nav interactions with SCBIs (DCJW and metaflumizone), steroidal agonists (BTX and veratridine) and the alkylamide insecticide BTG 502. Results from this research will push our understanding of the molecular actions of pyrethroids, SCBIs and other neurotoxins to a new level, thereby facilitating the development of precision monitoring and management of insecticide resistance worldwide and discovery of new and safe Nav-targeting chemicals to combat global threats from arthropod disease-transmitting vectors.