Physiology of Insect Amino Acid Transport

描述（由申请人提供）：蚊子幼虫已经进化出有效的系统来吸收氨基酸穿过中肠上皮。这种摄取似乎是由二级活性氨基酸转运蛋白（AATs）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激活；整个蛋白质集合构成一个“氨基酸摄取代谢物”。完整的冈比亚按蚊基因组为研究其代谢提供了工具。基因组的网络筛选证实了v - atp酶亚基全部存在，并揭示了3个阴离子交换体、5个阳离子转运体和92个氨基酸转运体的假定基因。虽然不是所有这些基因在幼虫中都有活性，但这种相对简单的氨基酸摄取代谢为遗传和分子评价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，简单易接近，为体内氨基酸转运代谢的生理/电化学分析提供了独特的机会。

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.