PHOTOLABELING OF ACETYLCHOLINE BINDING PROTEIN BY NICOTINOIDS & NEONICOTINOIDS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 乙酰胆碱结合蛋白（Acetylcholine binding protein，AChBP）是烟碱型乙酰胆碱受体/离子通道复合物（nAChR）胞外结构域的模型/替代物。 与nAChR的细胞外结构域一样，AChBP具有五个ACh或药物结合口袋。 乙酰胆碱结合蛋白由5个同源亚基组成，在亚基间的界面区域存在乙酰胆碱结合位点。 哺乳动物nAChR选择性激动剂epibatidine（EPI）和昆虫nAChR选择性激动剂imidacloprid（IMI）具有一些共同的结构部分，但它们的理化性质明显不同。 EPI具有质子化的氮原子（阳离子），但IMI具有不可质子化的电负性硝基（NO2）药效团。 这些结构差异决定了哺乳动物和昆虫之间的选择性。 作为证据，如果IMI失去硝基（desnitro-IMI），它现在是哺乳动物选择性化合物。 因此，与EPI的阳离子和IMI的电负性硝基氧相互作用的氨基酸残基必须是完全不同的，即使两种化合物都埋在相同的孔中，即，结合方向不同。 为了探讨选择性相互作用的分子基础，我们设计并制备了两种光亲和配体。 AChBP结合阳离子EPI激动剂和负电性IMI杀虫剂。 因此，本研究涉及LC/MS/MS分析的实验目标是鉴定由这两种类型的光亲和配体标记的特定氨基酸残基。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Acetylcholine (ACh) binding protein (AChBP) is the model/surrogate of the extra cellular domain for the nicotinic ACh receptor/ion channel complex (nAChR). As with the extra cellular domain of nAChR, the AChBP has five binding pockets for ACh or drug. The AChBP is assembled with five homologous subunits (homo-pentamer) and the ACh binding site exists on the interface region between subunits. Mammalian selective nAChR agonist epibatidine (EPI) and insect nAChR selective agonist imidacloprid (IMI, the representation neonicotinoid insecticide) share some common structural moieties, but they are distinctly different in their physicochemical properties. EPI has a protonated nitrogen atom (cation), but IMI has a non-protonatable and electronegative nitro (NO2) pharmacophore. These structural differences determine the selectivity between mammals and insects. As a proof, if IMI loses the nitro group (desnitro-IMI), it is now a mammalian selective compound. Therefore, amino acid residue(s) interacting with the cation of EPI and the electronegative nitro oxygen(s) of IMI must be quite different even if both compounds are buried in the identical hole, i.e., the binding orientations are different. To approach the molecular basis for the selective interaction, we designed and prepared two types of photoaffinity ligands. The Aplysia AChBP binds both the cationic EPI agonist and the electronegative IMI insecticide. Therefore, the experimental goals of this study involving the LC/MS/MS analyses are to identify specific amino acid residue(s) labeled by these two types of photoaffinity ligands.