Potency of agarose gel-supported lipid bilayers for electrophysiologic analysis of channel pores formed by Bacillus thuringiensis insecticidal proteins.

Abstract

Electrophysiologic analysis using artificial lipid bilayers is useful for studying the formation of pores by insecticidal proteins, especially the ion permeability of toxin pores. However, such studies are time-consuming and require special skills, particularly regarding the construction of lipid bilayers and promoting toxin pore formation. To facilitate the analysis of toxin pore formation in the present study, we evaluated the usefulness of agarose gel-supported lipid bilayers for electrophysiologic measurements using two structurally different mosquito-larvicidal proteins, Mpp46Ab and Cry4Aa. The agarose gel-supported lipid bilayers enabled the measurement of channel currents through pores made by both toxins and, notably, the lipid bilayers could be easily reconstructed even after disruption of the lipid bilayer. Using this system, measurements could be repeated at least five times using the same apparatus and toxins. We also investigated the effect of the lipid bilayer component on toxin pore formation and found that the incorporation of both cholesterol and sphingomyelin into the lipid bilayer facilitates the formation of pores by both Mpp46Ab and Cry4Aa. Both cholesterol and sphingomyelin are major components of lipid raft microdomains, suggesting that, in addition to recruiting toxin receptors, raft microdomains play a key role in membrane insertion and pore formation by insecticidal proteins.