The mechanism of insecticidal action of Bacillus thuringiensis delta-endotoxin on lepidopteran larvae involves specific binding between the toxin and one or more proteins of the brush border membrane of midgut columnar cells. Interaction between this well characterized bacterial protein toxin and the unidentified insect membrane proteins leads to a large increase in the ion permeability of the cell membrane. Formation of this new ion channel in the cell membrane appears to be the primary lesion in the cytolytic action of Bacillus thuringiensis toxin. Our objective is to determine the structure of the insect proteins involved in formation of this primary lesion, the nature of the lesion, and the mechanism of its formation. We will begin by using the purified delta-endotoxin as an affinity reagent in the isolation of the integral insect membrane proteins which combine with the bacterial endotoxin. We will show that the isolated bacterial and insect proteins are sufficient to form a functional ion channel in a phospholipid bilayer and characterize the ionic conductance of the channel. Using techniques of modern molecular biology we will isolate the genes that encode the insect membrane proteins of the channel, determine their nucleotide sequences, and deduce the primary structure of the channel proteins. We will determine the stochiometery of these proteins in the functional channel and propose a three dimensional structure for the channel. New knowledge gained from these studies will increase our understanding of basic cell membrane biology and mechanisms involved in ionic homeostasis in insect gastrointestinal cells as well as the mode of action of lepidopteran-active Bacillus thuringiensis delta-endotoxins. All of this information will be useful in guiding future efforts in biorational insect control.
