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.
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