PHYSIOLOGY OF INSECT AMINO ACID TRANSPORT

描述（改编自申请人的摘要）：长期 目的克隆氨基酸：碱金属离子同向转运体， 来自天蛾和埃及伊蚊的碱性中肠的蛋白质， 分析化学渗透偶联氨基酸中共转运蛋白的作用 摄取，并开发这些独特转运蛋白的抑制剂， 对环境安全的灭蚊剂。 假设是， 毛虫和蚊子幼虫中肠的碱性含量反映了 一种独特的吸收营养的方法， 通过一个H+易位的液泡型ATP酶和一个 K +/2H+反向转运蛋白。 电压通过通电的膜驱动氨基 酸：K+同向运输进入细胞。 独特的K+：氨基酸转运蛋白， KAAT 1是最近从M. sexta中肠和其他毛虫 据推测，转运蛋白介导蚊子幼虫的氨基酸摄取 也 目的1分析KAAT 1及其亚型在M. Sexta和Ae.在松弛动力学和定点 阳离子和氨基酸结合位点的诱变。 目标二：克隆 并表征编码来自M的系统B、Pro-Gly和R+的cDNA。Sexta和 AE.埃及人。 目的3是定位M. sexta 和Ae。原位杂交和免疫细胞化学方法检测细胞凋亡。 目的 4是在平面脂质双层中重建KAAT 1和其他共转运蛋白， 研究特定的膜脂对其动力学的影响 特色 目标5，与一家商业公司（罗门和哈斯）合作， 鉴定KAAT 1和其它昆虫转运蛋白特异性抑制剂， 将其开发为环境安全的杀幼虫剂。 该项目具有科学 因为像KAAT 1一样，其他转运蛋白可能是独一无二的， 是K +-而不是Na +-耦合的，由电压驱动， 在pH9.5 - 11.5之间运行。 很可能， KAAT 1和这些推定的转运蛋白可能被开发为安全的蚊子 杀幼虫剂，可以笼和选择性激活，只有在高肠道pH值。

DESCRIPTION (Adapted from the Applicant's Abstract): The long term objective is to clone and characterize amino acid:alkali metal ion symporter proteins from alkaline midguts of Manduca sexta and Aedes aegypti, to analyze the role of the symporters in chemiosmotically coupled amino acid uptake and to develop inhibitors of these unique transporters as environmentally safe mosquitocides. The hypothesis is that the uniquely alkaline midgut contents in caterpillars and larval mosquitoes reflect a unique method of nutrient uptake in which the plasma membranes are energized, aerobically, by an H+ translocating, vacuolar-type ATPase and a K+/2H+ antiporter. The voltage across the energized membrane drives amino acid:K+ symport into the cells. The unique K+:Amino Acid Transporter, KAAT1, recently cloned from M. sexta midgut, and other caterpillar transporters are postulated to mediate amino acid uptake in mosquito larvae as well. Aim 1 is to analyze the mechanism of action of KAAT1 and its isoforms in M. sexta and Ae. aegypti in terms of relaxation kinetics and site-directed mutagenesis of both cation and amino acid binding sites. Aim 2 is to clone and characterize cDNAs encoding Systems B, Pro-Gly, and R+ from M. sexta and Ae. aegypti. Aim 3 is to localize KAAT1 and other transporters in M. sexta and Ae. aegypti cells by in situ hybridization and immunocytochemistry. Aim 4 is to reconstitute KAAT1 and other symporters in planar lipid bilayers and to study the influence of specific membrane lipids on their kinetic characteristics. Aim 5, with a commercial firm (Rohm and Haas) is to identify specific inhibitors of KAAT1 and other insect transporters and to develop them as environmentally safe larvacides. The project has scientific merit because, like KAAT1, the other transporters are likely to be unique, to be K+ -rather than Na+ -coupled, to be driven by the voltage and to operate between pH 9.5 and 11.5 in vivo. It is likely that inhibitors of KAAT1 and these putative transporters may be developed as safe mosquito larvacides that can be caged and selectively activated only at high gut pH.