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

Public Health Relevance

because the new basic science is immediately applied to a disease vector model.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022444-13
Application #
2633449
Study Section
Special Emphasis Panel (ZRG5-TMP (01))
Project Start
1985-09-01
Project End
1999-12-31
Budget Start
1998-01-01
Budget End
1998-12-31
Support Year
13
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Florida
Department
Type
Organized Research Units
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Boudko, D Y; Moroz, L L; Harvey, W R et al. (2001) Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut. Proc Natl Acad Sci U S A 98:15354-9
Gruber, G; Wieczorek, H; Harvey, W R et al. (2001) Structure-function relationships of A-, F- and V-ATPases. J Exp Biol 204:2597-605
Boudko, D Y; Moroz, L L; Linser, P J et al. (2001) In situ analysis of pH gradients in mosquito larvae using non-invasive, self-referencing, pH-sensitive microelectrodes. J Exp Biol 204:691-9
Wieczorek, H; Grber, G; Harvey, W R et al. (2000) Structure and regulation of insect plasma membrane H(+)V-ATPase. J Exp Biol 203:127-35
Merzendorfer, H; Reineke, S; Zhao, X F et al. (2000) The multigene family of the tobacco hornworm V-ATPase: novel subunits a, C, D, H, and putative isoforms. Biochim Biophys Acta 1467:369-79
Gruber, G; Svergun, D I; Godovac-Zimmermann, J et al. (2000) Evidence for major structural changes in the Manduca sexta midgut V1 ATPase due to redox modulation. A small angle X-ray scattering study. J Biol Chem 275:30082-7
Svergun, D I; Becirevic, A; Schrempf, H et al. (2000) Solution structure and conformational changes of the Streptomyces chitin-binding protein (CHB1). Biochemistry 39:10677-83
Wieczorek, H; Gruber, G; Harvey, W R et al. (1999) The plasma membrane H+-V-ATPase from tobacco hornworm midgut. J Bioenerg Biomembr 31:67-74
Wieczorek, H; Brown, D; Grinstein, S et al. (1999) Animal plasma membrane energization by proton-motive V-ATPases. Bioessays 21:637-48
Zhuang, Z; Linser, P J; Harvey, W R (1999) Antibody to H(+) V-ATPase subunit E colocalizes with portasomes in alkaline larval midgut of a freshwater mosquito (Aedes aegypti). J Exp Biol 202:2449-60

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