The midgut model explains the constancy of lumen, cell and blood (K+) in terms of a pump-leak steady state. K+ moves from goblet cells to lumen via and electrogenic K+ pump in globet cell apical membrane (GCAM) rendering the lumen alkaline. K+ leaks back, down its electrochemical gradient, into columnar cells via amino acid-K+ symports in columnar cell apical membrane (CCAM). K+ diffuses between columnar and goblet cells via lateral junctions and from blood to cells via K+ channels. A similar K+ pump-leak steady state drives secretion by insect salivary glands and Malpighian tubules, develops receptor potentials in insect sensory epithelia, and may dive K+ flow during mammalian hearing. Our goal is to characterize this insect cellular K+ homeostasis and to exploit its disruption for environmentally safe insect control. During the prior period we have solubilized and characterized the K+ ATPase activity of GCAM and identified subunit proteins. We have shown that Ba2+ blocks the K+ channels induced in CCAM by Bacillus thuringiensis var kurstaki (Btk) toxin and reverses the intoxication process.
Our first aim i s to purify the K+ ATPase and to show that it is localized in portasomes where it is a key component of the K+ pump. Using biochemistry and molecular biology we will sequence the gene coding for the ATPase and deduce the primary structure of the enzyme. Using immunohistochemistry we will study the distribution of K+ portasomes in midgut and other K+ transporting epithelia.
Our second aim i s to characterize the midgut K+ homeostasis. We will use the short circuit current, radiolabelled ion fluxes. and vesicle transport techniques to determine specific cation permeabilities of CCAM in its active state and when it is intoxicated with Btk endotoxin. Midgut K+ homeostasis is important scientifically as a model for insect gastrointestinal, and sensory function. Disruption of midgut K+ homeostasis by Btk and other agents which selectively form ion channels in CCAM provides the basis for enviromentally safe insect control.
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