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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI030464-08
Application #
2628753
Study Section
Special Emphasis Panel (ZRG5-TMP (01))
Project Start
1990-12-01
Project End
2003-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
8
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
University of Florida
Department
Type
Organized Research Units
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611

  Publications

Metzler, Ryan; Meleshkevitch, Ella A; Fox, Jeffrey et al. (2013) An SLC6 transporter of the novel B(0,)- system aids in absorption and detection of nutrient amino acids in Caenorhabditis elegans. J Exp Biol 216:2843-57
Meleshkevitch, Ella A; Voronov, Dmitri A; Miller, Melissa M et al. (2013) A novel eukaryotic Na+ methionine selective symporter is essential for mosquito development. Insect Biochem Mol Biol 43:755-67
Sterling, Kenneth M; Okech, Bernard A; Xiang, Minghui A et al. (2012) High affinity (3)H-phenylalanine uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AaBBMVw). J Insect Physiol 58:580-9
Boudko, Dmitri Y (2012) Molecular basis of essential amino acid transport from studies of insect nutrient amino acid transporters of the SLC6 family (NAT-SLC6). J Insect Physiol 58:433-49
Xiang, Minghui A; Linser, Paul J; Price, David A et al. (2012) Localization of two Na+- or K+-H+ antiporters, AgNHA1 and AgNHA2, in Anopheles gambiae larval Malpighian tubules and the functional expression of AgNHA2 in yeast. J Insect Physiol 58:570-9
Harvey, William R; Xiang, Minghui A (2012) K+ pump: from caterpillar midgut to human cochlea. J Insect Physiol 58:590-8
Hansen, Immo A; Boudko, Dmitri Y; Shiao, Shin-Hong et al. (2011) AaCAT1 of the yellow fever mosquito, Aedes aegypti: a novel histidine-specific amino acid transporter from the SLC7 family. J Biol Chem 286:10803-13
Harvey, William R; Okech, Bernard A; Linser, Paul J et al. (2010) H(+) V-ATPase-energized transporters in brush border membrane vesicles from whole larvae of Aedes aegypti. J Insect Physiol 56:1377-89
Harvey, William R; Boudko, Dmitri Y; Rheault, Mark R et al. (2009) NHE(VNAT): an H+ V-ATPase electrically coupled to a Na+:nutrient amino acid transporter (NAT) forms an Na+/H+ exchanger (NHE). J Exp Biol 212:347-57
Meleshkevitch, Ella A; Robinson, Marvin; Popova, Lyudmila B et al. (2009) Cloning and functional expression of the first eukaryotic Na+-tryptophan symporter, AgNAT6. J Exp Biol 212:1559-67

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