PHYSIOLOGY OF INSECT AMINO ACID TRANSPORT

描述(改编自申请人的摘要)：长期 目的克隆和鉴定氨基酸：碱金属离子转运体 六分白纹伊蚊和埃及伊蚊碱性中肠蛋白 分析共转运体在化学渗透偶联氨基酸中的作用 摄取并开发这些独特转运蛋白的抑制剂，如 环境安全的灭蚊剂。假设是唯一的 毛虫和蚊子幼虫体内的碱性中肠内容物反映了 独特的营养吸收方法，其中质膜是 由H+转运的液泡型ATPase和 K+/2H+逆向转运蛋白。通电的膜上的电压驱动氨基 酸：K+进入细胞内。独特的K+：氨基酸转运蛋白， KAAT1，最近从中肠毛虫和其他毛虫中克隆出来的 转运蛋白被认为参与蚊子幼虫对氨基酸的摄取 也是。 目的1分析KAAT1及其异构体在M。 Sexta和Ae.松弛动力学和定点定向的埃及伊蚊 阳离子和氨基酸结合位点的诱变。目标2是克隆 和编码系统B、Pro-Gly和R+的cDNA的特征 天哪。埃及伊蚊。目标3是在六分枝杆菌中定位KAAT1和其他转运蛋白 和Ae.埃及伊蚊细胞的原位杂交和免疫细胞化学。目标 4是重组KAAT1和其他平面脂质双层中的共转运体，以及 研究特定膜脂对其动力学的影响 特点。与一家商业公司(Rohm And Haas)合作的目标5是 确定KAAT1和其他昆虫转运蛋白的特定抑制剂，并 将它们开发为环境安全的杀幼虫剂。该项目具有科学性 优点是，像KAAT1一样，其他转运体可能是独一无二的， K+偶联而不是Na+偶联，由电压驱动， 在体内可在pH 9.5至11.5范围内工作。很可能是药物的抑制剂 KAAT1和这些假定的转运蛋白可能被开发为安全蚊子 只有在肠道pH值较高的情况下才能被关在笼子里并有选择地激活的杀幼虫。

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.