Bacillus thuringiensis CrylAa δ-Endotoxin Affects the K⁺/Amino Acid Symport in Bombyx mori Larval Midgut

Abstract.

We have examined the type of inhibition exerted by an activated preparation of the Bacillus thuringiensisδ-endotoxin CrylAa on K⁺-dependent leucine transport into midgut brush border membrane vesicles or epithelial cells of the isolated midgut from Bombyx mori to study its possible interaction with the amino acid symporter.

K⁺ permeability and the cation-dependent amino acid translocation into brush border membrane vesicles were evaluated by monitoring the fluorescence of the voltage-sensitive cyanine dye 3,3′-dipropylthiadicarbocyanine iodide. The symporter ability to accept Na⁺ instead of K⁺ was exploited and the dissipation of an imposed inside-negative potential (K⁺ gradient in>out and valinomycin) was registered in the presence of a Na⁺ gradient (out>in) and of the amino acid. The fluorescence quenching dissipated more rapidly when the amino acid was present. Preincubation of brush border membrane vesicles with CrylAa caused a significant decrease of the amino acid-dependent recovery of fluorescence, whereas K⁺ permeability was sparely affected.

In the isolated midgut, CrylAa inhibits leucine uptake as well as the transepithelial electrical potential difference. The strong inhibition exerted by the δ-endotoxin was observed also in the absence of potassium and the transepithelial electrical potential difference. The results obtained strongly suggest a direct interaction of CrylAa δ-endotoxin with the K⁺/amino acid symporter.