The long term objective is to analyse the structure, function regulation of the plasma membrane H+ V-ATPase in Manduca sexta, to isolate the k+/2H+ antiporter and to understand how activity of the ATPase-antiporter couple alkalinizes the larval midgut lumen. A concomitant objective is to provide a new basis for vector mosquito control by using cDNA probes and antibodies from the transport model, M. sexta to study midgut alkalinization in a disease vector model, Aedes aegypti. The hypothesis is that the high pH of both caterpillar and mosquito midgut is generated by an H+ translocating, plasma membrane V-ATPase. The voltage component of the protonmotive force energized the apical membrane and drives electrophoretic K+/H+ exchange by a novel K+/2H+ antiporter. A structure/function analysis of the ATPase- antiporter couple will be worthwhile in itself and will provide molecular details of the alkalinization mechanism. Selective inhibition of the antiporter should prevent mosquito midgut alkalinization, eventually killing larvae with little environmental impact.
Aim 1 is to analyse the structure and function of the V1 complex; ATP binding and hydrolysis as well as subunit assembly will be studied using recombinant subunits produced by in vitro transcription/translation or over-expression; eventually the catalytic site will be systematically modified by site directed mutagenesis.
Aim 2 is to analyse the regulation of subunit gene expression using an existing genomic DNA library; promoter elements will be isolated and their activity will be monitored in transfected cells using reporter genes.
Aim 3 is to isolate the K+/2H+ antiporter by protein biochemistry as well as to clone and sequence its encoding cDNA by hybridization cloning with cDNA probes from vertebrate Na+/H+ antiporter, by complementation cloning in Na+/H+ antiport-deficient yeast or by expression cloning in Xenopus oocytes.
Aim 4 is to analyze the alkalinization mechanism by comparing ATPase-antiporter structure/function in the structurally complex caterpillar midgut with that in the structurally simple mosquito midgut using in situ hybridization and immunocytochemistry and to evaluate alkalinization blocking with microencapsulated amiloride derivatives. The proposal has SCIENTIFIC MERIT because energization of animal cell plasma membranes by a protonmotive force and reversal of acidification direction by secondary active cation/proton antiport are both novel concepts. It has
because the new basic science is immediately applied to a disease vector model.
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