SINGLE-SITE MUTATIONS IN THE CONSERVED ALTERNATING-ARGININE REGION AFFECT IONIC CHANNELS FORMED BY CRYIAA, A BACILLUS-THURINGIENSIS TOXIN

The role of the third domain of CryIAa, a Bacillus thuringiensis insec ticidal toxin, in toxin-induced membrane permeabilization in a recepto r-free environment was investigated, Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by s ite-directed mutagenesis in block 4, an arginine-rich, highly conserve d region of the protein, Four mutants were constructed by replacing th e first arginine in position 21 by a lysine (R521K), a glutamine (R521 Q), a histidine (R521H), or a glutamic acid (R521E), A fifth mutant wa s obtained by replacing the fourth arginine by a lysine (R527K), Like CryIAa, the mutants formed cation-selective channels, A limited but si gnificant reduction in channel conductance was observed for all mutant s except R521H. The effect was more dramatic for the voltage dependenc e of the channels formed by R521K and R521Q, which was reversed compar ed to that of the parental toxin, This study provides the first direct evidence of a functional role for domain III in membrane permeabiliza tion, Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.