The overall goal is to characterize lepidopteran midgut K(+) homeostasis; according to the midgut model, K(+) enters midgut cells via channels in the basal membranes and is propelled across goblet cell apical membrane, GCAM, to goblet cavity via an electrogenic H(+) pump in parallel with an nH(+)/K(+) antiporter; the apical membrane is energized to a PD >180 mV. The PD supports a lumen pH >10 compared to the cell pH of 7.0; it drives amino acid/K(+) symport from lumen to columnar cells, resulting in amino acid absorption and completing the K(+) homeostatic cycle. This renewal application deals with GCAM. The hypothesis is that a proton pumping electrogenic V-ATPase and an electrogenic nH(+)/K(+) antiporter inserted into a cation impermeable lipid bilayer are key components of the homeostatic pathway.
Aim 1 is to complete sequencing cDNAs encoding GCAM V-ATPase subunits of 67 and 56 kDa and to clone and sequence cDNAs for 43, 28, and 16 kDa subunits.
Aim 2 is to solubilize and purify the antiporter protein(s).
Aim 3 is to clone and sequence the cDNA encoding the antiporter(s).
Aim 4 is to localize the V-ATPase and nH(+) /K(+) antiporter in K(+) transporting epithelia and describe the ontogeny of GCAM from vacuolar membranes. GCAM will be isolated by sonication and gradient centrifugation. Antiporter will be solubilized with nonionic detergents and purified by density gradient centrifugation and/or FPLC using reconstitution into liposomes as an assay. Midgut larval cDNA library screening will use either antibodies to midgut proteins or oligonucleotide probes to highly homologous components from other sources. ATPase and antiporter localization will use fluorescent and gold labelled antibodies to appropriate subunit proteins in both light and EM immunocytochemistry. The midgut model may provide insight into V-ATPase energization of plasma membranes of other insects and into energization of vertebrate urinary and bone membranes. Because the midgut model deals with an unusual cation impermeable apical membrane protecting cells from a highly alkaline lumen, it has provided insight into the action of the Bacillus thuringiensis endotoxin; it may lead to other environmentally safe caterpillar and mosquito larval controls.
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