PHYSIOLOGY OF INSECT AMINO ACID TRANSPORT

长期目标是比较初级结构和

The LONG TERM OBJECTIVE is to compare the primary structure and functional characteristics of amino acid/alkali metal ion symporter proteins from alkaline midguts of Manduca sexta and Aedes aegypti, to analyses the symporters's roles in chemiosmotically coupled amino acid uptake and to exploit this unusual symport to develop new, environmentally safe mosquitocides. The HYPOTHESIS is that the high alkalinity of caterpillar and mosquito larval midgut is continuously generated by a novel V-ATPase that uses ATP to energize the apical cell membrane by imposing a transmembrane proton electrochemical gradient, delta/mu/H+, lumen positive. The delta/mu/H+ is transformed by a K+/nH+ antiporter to a K+ electrochemical gradient, delta/mu/K+, raising the carbonate-rich lumen pH to >10.5. delta/mu/K+, expressed mainly as delta/psi, chemiosmotically drives amino acid (AA) uptake by alkali adapted AA/K+ symporters (see sketch below). AIM 1 is to characterize glycine/K+ and proline/K+ symporters by rapid filtration studies in brush border membrane vesicles. AIM 2 is to sequence M. sexta symporter cDNA clones, screened by injection of transcribed, sized mRNA fractions from feeding larvae into Xenopus oocytes. AIM 3 is to isolate and sequence symporter cDNA clones from M. sexta and Ae. aegypti midgut cDNA libraries by hybridization with xenic cDNAs encoding known amino acid symporters from vertebrate kidney, brain and other tissues. AIM 4 is to reconstitute purified, recombinant symporters into functional liposomes and characterize them in terms of ionic specificities, kinetic parameters and substrate selectivity, using rapid filtration and fluorescence quenching measurements of amino acid uptake. AIM 5 is to localize amino acid/K+ symporters in midguts from M. sexta and Ae. aegypti by immunocytochemistry and in situ hybridization. SCIENTIFIC MERIT: The use of ATP hydrolysis, chemiosmotically coupled by delta/mu/H+ and delta/mu/K+, to drive amino acid uptake is the first example of such coupling in animal cell plasma membranes. HEALTH RELEVANCE: If larval Ae. aegypti midguts are also energized chemiosmotically by transapical membrane delta/mu/H+ transformed to delta/mu/K+, and if unusual AA/K+ symporters have evolved in the resulting alkaline milieu of disease vector dipteran midguts then agents which interfere with the unusual symporters can be developed as safe mosquitocides.