Physiology of Insect Amino Acid Transport

昆虫氨基酸运输的生理学

• 批准号: 6783635
• 负责人: DMITRI Y. BOUDKO
• 金额: $ 28.38万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-12-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6783635
• 关键词: Insecta; Xenopus; adenosinetriphosphatase; aminoacid transport; arthropod genetics; capillary electrophoresis; cell line; complementary DNA; genetic screening; immunocytochemistry; in situ hybridization; ion transport; larva; metabolomics; microelectrodes; polymerase chain reaction

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.

描述（由申请人提供）：蚊子幼虫已经进化出有效的系统来跨中肠上皮吸收氨基酸。这种吸收似乎是由次级活性氨基酸转运蛋白 (AAT) 介导的，并由初级 H+ V-ATP 酶与次级无机离子交换剂协同作用提供能量；整个蛋白质整体构成了“氨基酸摄取代谢”。完整的冈比亚按蚊基因组提供了研究代谢的工具。基因组的网络筛选证实 V-ATP 酶亚基全部存在，并揭示了 3 个阴离子交换蛋白、5 个阳离子转运蛋白和 92 个氨基酸转运蛋白的推定基因。尽管并非所有这些基因都会在幼虫中活跃，但这种相对简单的氨基酸摄取代谢为遗传和分子评估提供了一个极好的对象。幼虫中肠上皮由数千个大（50 - 100 微米）细胞组成，简单易行，为分析体内氨基酸转运代谢的生理学/电化学提供了独特的机会。 该提案的目标是通过识别、克隆和表征蚊子幼虫中假定的营养 AAT 的整个种群，探索蚊子幼虫中的氨基酸转运代谢。冈比亚幼虫并量化它们与体内 ATP 酶和无机离子转运蛋白的生理相互作用。目标 1 是通过使用 An 中假定的 AAT 基因的精确引物筛选组织特异性中肠 cDNA 文库来鉴定编码营养 AAT 的基因和克隆转录本。冈比亚基因组。目标 2 是通过非洲爪蟾卵母细胞或细胞系中的异源表达来表征 ATT，然后进行标记氨基酸摄取测定和摄取特性的电化学分析。目标 3 是利用原位杂交、实时 PCR 和免疫细胞化学来确定这些营养 AAT 在冈比亚幼虫中肠中的分布。目标 4 是使用高分辨率毛细管电泳以及传统和自参考离子选择性微电极来表征体内营养氨基酸吸收的电化学特性。所表征的 An.冈比亚氨基酸摄取代谢将作为其他动物氨基酸摄取的模型，并将为开发蚊虫控制剂提供具体目标。