Molecular Determinants of Pyrethroid Neurotoxicity

拟除虫菊酯神经毒性的分子决定因素

• 批准号: 7761693
• 负责人: DAVID M SODERLUND
• 金额: $ 25.4万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7761693
• 关键词: Action Potentials; Acute; Affect; Agriculture; Amino Acid Sequence; Animals; Binding; Cells; Development; Drug or chemical Tissue Distribution; Electrodes; Exhibits; Family; Funding; Human; Insect Vectors; Insecta; Insecticides; Intoxication; Kinetics; Mammalian Cell; Maps; Mediating; Modeling; Modification; Molecular; Molecular Structure; Multigene Family; Mutate; Myocardium; Nature; Nerve; Nervous system structure; Neurons; Oocytes; Permeability; Pharmacology; Property; Protein Isoforms; Public Health; Rana; Rattus; Regulation; Research; Research Personnel; Resistance; Role; Severities; Site; Site-Directed Mutagenesis; Sodium; Sodium Channel; Testing; Tissues; Toxic effect; Xenopus laevis; Xenopus oocyte; base; human disease; insight; member; neurotoxic; neurotoxicity; patch clamp; programs; pyrethroid; receptor; skeletal; voltage; voltage clamp

DESCRIPTION (provided by applicant): Synthetic pyrethroids, an important class of neurotoxic insecticides used worldwide in agriculture and public health, cause toxic effects in animals by modifying the normal function of voltage-sensitive sodium channels. Sodium channels in mammalian tissues exist as multiple isoforms that exhibit differential tissue distribution and developmental regulation and are encoded by members of a multigene family. Previous studies of pyrethroid action have not considered the differential sensitivity of sodium channel isoforms as a determining factor in the toxicity of these compounds. The proposed research will test the hypothesis that differences in the pyrethroid sensitivity of mammalian sodium channel isoforms are important determinants of the nature and severity of pyrethroid intoxication. Specific experimental aims are: (1) to define the actions of pyrethroid insecticides on cloned rat sodium channel isoforms expressed in frog (Xenopus laevis) oocytes or transfected mammalian cells; (2) to compare the actions of pyrethroids on orthologous rat and human sodium channel isoforms expressed in Xenopus oocytes or trnasfected mammalian cells; and (3) to map the the structural determinants of pyrethroid action by the construction, functional expression and pharmacological characterization of specifically mutated sodium channels. Cloned sodium channel isoforms will be expressed in Xenopus oocytes or transiently transfected mammalian cells (e.g., HEK-293) and the actions of pyrethroid insecticides on expressed channels will be assessed using electrophysiological recordings of macroscopic sodium currents under two-electrode voltage-clamp or whole cell patch-clamp conditions. Structural determinants of pyrethroid sensitivity will be identified by site-directed mutagenesis, expression, and pharmacological comparison of native a mutated sodium channel isoforms. Results of these studies will define the role of different sodium channel isoforms in pyrethroid toxicity, establish whether rat sodium channels are valid toxicological models for the sensitivity of human sodium channels to pyrethroids, and identify sodium channel domains that determine pyrethroid sensitivity.

描述（由申请人提供）：合成拟除虫菊酯是世界范围内用于农业和公共卫生的一类重要的神经毒性杀虫剂，通过改变电压敏感钠通道的正常功能对动物产生毒性作用。哺乳动物组织中的钠通道以多种亚型存在，表现出不同的组织分布和发育调节，并由多基因家族的成员编码。先前对拟除虫菊酯作用的研究并未考虑钠通道亚型的不同敏感性作为这些化合物毒性的决定因素。拟议的研究将检验以下假设：哺乳动物钠通道亚型对拟除虫菊酯敏感性的差异是拟除虫菊酯中毒的性质和严重程度的重要决定因素。具体的实验目的是：（1）确定拟除虫菊酯杀虫剂对青蛙（非洲爪蟾）卵母细胞或转染的哺乳动物细胞中表达的克隆大鼠钠通道亚型的作用； (2) 比较拟除虫菊酯对非洲爪蟾卵母细胞或转染哺乳动物细胞中表达的直系同源大鼠和人钠通道亚型的作用； (3) 通过特异性突变的钠通道的构建、功能表达和药理学表征来绘制拟除虫菊酯作用的结构决定因素。克隆的钠通道亚型将在非洲爪蟾卵母细胞或瞬时转染的哺乳动物细胞（例如 HEK-293）中表达，并且将使用两电极电压钳或整体电压钳下宏观钠电流的电生理学记录来评估拟除虫菊酯杀虫剂对表达通道的作用细胞膜片钳条件。拟除虫菊酯敏感性的结构决定因素将通过定点诱变、表达和天然突变钠通道亚型的药理学比较来鉴定。这些研究的结果将定义不同钠通道亚型在拟除虫菊酯毒性中的作用，确定大鼠钠通道是否是人类钠通道对拟除虫菊酯敏感性的有效毒理学模型，并确定决定拟除虫菊酯敏感性的钠通道结构域。