The role played by ␣-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-D-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that ␣-helix 4 plays a crucial role in the mechanism of pore formation.Bacillus thuringiensis is the most extensively used commercial biopesticide worldwide and is presently the sole source of toxin genes for the development of insect-resistant transgenic plants (13,15,42). The insecticidal activity of B. thuringiensis is primarily associated with its ability to synthesize a crystalline parasporal inclusion body containing highly specific insecticidal proteins (22,36). The mode of action of these insecticides involves solubilization of the crystal in the highly alkaline lepidopteran midgut lumen, activation of the toxins by intestinal proteases, recognition of one or more binding sites on the midgut brush border membrane surface followed by pore formation, and cell lysis leading ultimately to insect death (36).The elucidation by X-ray diffraction analysis of the threedimensional structures of the activated Cry1Aa (21), Cry2Aa (31), Cry3Aa (28), and Cry3Bb (16) toxins has revealed a common three-domain folding pattern. Domain I is made of seven ␣-helices, and domains II and III are composed mostly of ␤-sheets. While domain I is considered to be responsible for pore formation (37, 43, 44), domains II and III are involved in receptor binding and host specificity (11,12,24,47). Domain III is also thought to play a role in protein stability (28). The domains of the activated toxins were shown to interact with each other to yield their overall toxic effect (33, 34). Exchanging domain I from different toxins can affect crystal formation, stability, pore formation, and membrane permeability as well as the size of the pores and toxicity.The toxin is thought to form pores in the cell membrane by first inserting a hairpin composed of the hydrophobic ␣5 and the amphipathic ␣4 h...