PHYSIOLOGY OF INSECT AMINO ACID TRANSPORT

描述（改编自申请人摘要）：长期

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.