Physiology of Insect Amino Acid Transport

描述（由申请人提供）：苔藓虫幼虫已经进化出有效的系统来吸收中肠上皮细胞中的氨基酸。这种摄取似乎是由二级活性氨基酸转运蛋白（AAT）介导的，并由初级H+ V-ATP酶与二级无机离子交换剂协同作用提供能量;整个蛋白质系综构成了“氨基酸摄取代谢子”。冈比亚按蚊基因组的完成为研究代谢子提供了一个工具。基因组的网络筛选证实，V-ATP酶亚基都存在，并揭示了3个阴离子交换剂，5个阳离子转运蛋白和92个氨基酸转运蛋白的推定基因。虽然不是所有这些基因将在幼虫中是活跃的，这种相对简单的氨基酸摄取代谢提供了一个很好的主题遗传和分子评价。幼虫中肠上皮由几千个大（50 - 100微米）细胞组成，简单易行，为分析体内氨基酸转运代谢子的生理学/电化学提供了独特的机会。 该提案的目标是通过鉴定、克隆和表征An中推定的营养AAT的整个群体来探索蚊子幼虫中的氨基酸转运代谢子。冈比亚幼虫和定量的生理相互作用与ATP酶和无机离子转运蛋白在体内。目的1：通过筛选中肠组织特异性cDNA文库，鉴定营养型AAT基因并克隆其转录本。冈比亚基因组。目的2是通过在非洲爪蟾卵母细胞或细胞系中异源表达ATTs，然后通过标记氨基酸摄取测定和摄取性质的电化学分析来表征ATTs。目的3：利用原位杂交、真实的时间PCR和免疫细胞化学技术，研究这些营养型AAT在冈比亚按蚊幼虫中肠的分布。目的4是利用高分辨率毛细管电泳以及常规和自参比离子选择性微电极来表征营养氨基酸在体内摄取的电化学性质。特征的一个。冈比亚氨基酸摄取代谢物将作为其他动物氨基酸摄取的模型，并将为开发蚊子控制剂提供特定目标。

DESCRIPTION (provided by applicant): Mosquito larvae have evolved efficient systems to take up amino acids across the midgut epithelium. This uptake appears to be mediated by secondary active amino acid transporters (AATs) and to be energized by primary H+ V-ATPases in synergy with secondary inorganic ion exchangers; the entire protein ensemble constitutes an "amino acid uptake metabolon". The completed Anopheles gambiae genome provides a tool with which to study the metabolon. Cyber-screening of the genome confirmed that the V-ATPase subunits are all present, and revealed putative genes for 3 anion exchangers, 5 cation transporters, and 92 amino acid transporters. Although not all of these genes will be active in larvae, this relatively simple amino acid uptake metabolon provides an excellent subject for genetic and molecular evaluation. The larval midgut epithelium consists of a few thousand large (50 - 100 micron) cells, is simple and accessible, and provides a unique opportunity to analyze the physiology/electrochemistry of the amino acid transport metabolon in vivo. The goal of this proposal is to explore the amino acid transport metabolon in mosquito larvae by identifying, cloning, and characterizing the entire population of putative nutrient AATs in An. gambiae larvae and quantifying their physiological interplay with the ATPase and inorganic ion transporters in vivo. Aim 1 is to identify genes and clone transcripts encoding nutrient AATs by screening tissue specific midgut cDNA libraries with exact primers for putative AAT genes in the An. gambiae genome. Aim 2 is to characterize the ATTs by heterologous expression in Xenopus oocytes or cell lines, followed by labeled amino acid uptake assays and electrochemical analysis of the uptake properties. Aim 3 is to define the distribution of these nutrient AATs in larval An gambiae midgut using in situ hybridization, real time PCR, and immunocytochemistry. Aim 4 is to characterize the electrochemical properties of nutrient amino acid uptake in vivo using high-resolution capillary electrophoresis as well as conventional and self-referencing ion selective microelectrodes. The characterized An. gambiae amino acid uptake metabolon will serve as a model for amino acid uptake in other animals and will provide specific targets for developing mosquito control agents.