Physiology of Insect Amino Acid Transport

描述(申请人提供)：蚊子幼虫已经进化出有效的系统，通过中肠上皮吸收氨基酸。这种摄取似乎是由次级活性氨基酸转运体(AATS)介导的，并由初级H+V-ATPase与次级无机离子交换器协同作用而提供能量；整个蛋白质组合构成了一个“氨基酸摄取代谢”。冈比亚按蚊的完整基因组为研究新陈代谢提供了工具。基因组的计算机筛选证实V-ATPase亚基全部存在，并揭示了3个阴离子交换器，5个阳离子转运体和92个氨基酸转运体的推测基因。虽然不是所有这些基因在幼虫中都是活跃的，但这种相对简单的氨基酸吸收代谢为遗传和分子评估提供了一个很好的主题。幼虫中肠上皮由几千个大的(50-100微米)细胞组成，简单易得，为分析体内氨基酸运输代谢的生理/电化学提供了独特的机会。 这项建议的目的是通过鉴定、克隆和鉴定蚊子幼虫中可能的营养物质AATS的整个种群来探索蚊子幼虫中的氨基酸运输代谢。冈比亚幼虫并量化它们与体内ATPase和无机离子转运体的生理相互作用。目的1通过筛选组织特异性AAT基因文库，筛选AN中可能存在的AAT基因，鉴定并克隆营养AATS基因的转录本。冈比亚亚纲基因组。目的2是通过在非洲爪哇卵母细胞或细胞系中异源表达、标记氨基酸摄取试验和电化学分析摄取特性来表征ATTS。目的3利用原位杂交、实时荧光定量聚合酶链式反应和免疫细胞化学的方法，确定这些营养物质在冈比亚幼虫中肠中的分布。目的4利用高分辨率毛细管电泳法以及常规和自参比离子选择微电极对体内营养氨基酸摄取的电化学性质进行表征。特色化的An。冈比亚的氨基酸摄取代谢系统将作为其他动物摄取氨基酸的模型，并将为开发蚊子控制剂提供特定的靶标。

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.