Molecular Action of Insecticides on Ion Channels - ACS Publications

Chapter 19

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Permeability of Bacillus thuringiensis CryI Toxin Channels Michael G. Wolfersberger Department of Biology, Temple University, Philadelphia, PA 19122

During sporulation Bacillus thuringiensis produces parasporal inclusions with insecticidal activity. These parasporal inclusions consist of one or more Cry proteins. Type-I Cry proteins are active only against the larvae of lepidopteran insects. Their mode of action includes binding to specific components of the brush border membrane of larval midgut columnar cells and culminates with formation of a pore in the cell membrane. The resulting increase in cell membrane permeability leads eventually to cell lysis, disruption of gut integrity, and finally death of the insect. Recently we have been able to reconstitute both CryIA(c) toxin proteins and toxin binding proteins from the brush border membrane of insect midgut cells into planar lipid bilayers and determine the conductance of the resulting pores. The results of these determinations are compared with previous estimates of pore size obtained by less direct methods.

The species Bacillus thuringiensis consists of numerous strains of gram-positive rodshaped bacteria which during sporulation produce crystalline proteinaceous parasporal bodies. The parasporal body proteins produced by many strains of B. thuringiensis are toxic to certain insect larvae. Therefore, they are frequently called either delta-endotoxins (older literature) or insecticidal crystal proteins (more recent literature). For many years B. thuringiensis strains have been classified primarily on the basis offlagellarantigens (7). This classification system brought some order to the huge collection of strains but proved to be of very limited utility in predicting the spectrum of larvicidal activity of the various strains. The application of modern molecular genetic methods to B. thuringiensis revealed that genetic information for most insecticidal parasporal body proteins was encoded on transmissible plasmids rather than on the bacterial chromosomes. Furthermore, it was determined that a single plasmid could carry the information for more than one insecticidal parasporal body protein and that a single bacterium could contain more than one plasmid (2). 0097-6156/95A)591-O294$12.00y0 © 199S American Chemical Society Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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These findings explained why seemingly closely related strains could have rather different spectra of larvicidal activity. When a critical mass of information, including primary structure, about the genes encoding B. thuringiensis insecticidal parasporal body proteins had accumulated a classification system was proposed (2). This system grouped the proteins on the basis of their similarities in both primary structure and insecticidal spectra. The rapid adoption of this classification system by the B. thuringiensis research community has made it possible to compare and combine the more recent results from various laboratories and in some cases even make sense of the older literature. Relatedness of R thuringiensis Crystal Proteins Briefly, the B. thuringiensis insecticidal crystal protein (ICP) classification system divided all parasporal body proteins into five groups. Four of these groups contain Cry proteins that are encoded by cry genes. The fifth group contains Cyt proteins that are encoded by cyt genes. Unlike Cry proteins, Cyt proteins are cytolytic to a variety of non-insect cells. Cyt proteins seem to be genetically unrelated to Cry proteins. A l l Cry proteins seem to be genetically related. Cry proteins in the first and by far the largest group (Cryl) have greater than 50% amino acid sequence identity and are active against lepidopteran larvae. CryEI proteins show activity against lepidopteran and/or dipteran larvae. Although they are often found together in the same bacterium, Cryll proteins are rather distant relatives of Cryl proteins. Cryin proteins are more closely related genetically to Cryl proteins than are Cryll proteins. However, CrylH proteins are toxic only to certain coleopteran larvae. A major breakthrough in B. thuringiensis research occurred when the crystal structure of a Crym toxin was determined at 2.5 A resolution (3). Members of the fourth class of Cry proteins (CrylV) often occur together with one another as well as Cyt proteins in the parasporal bodies of B. thuringiensis strains that show larvicidal activity against certain dipteran insects. The CrylV class of ICPs is a comparatively diverse group. CrylVA and CrylVB are about as closely related to one another as are the proteins in the Cryl class. CrylVA and CrylVB proteins are also related, about equally, to Cryl and CrylH ICPs. However, on the basis of amino acid sequence, CrylVC proteins are only slightly more closely related to CrylVA and CrylVB proteins than to Cryl or Crylll proteins. Finally, CrylVD proteins are related most closely to Cryll ICPs. A somewhat controversial update of the original classification system introduced two new classes of Cry proteins (4). ICPs in one of these new proposed classes were said to be completely unrelated to any other Cry proteins. These authors also proposed that CrylVC proteins are much more closely related to CrylVA and CrylVB proteins than to any other ICPs. Hopefully, a concensus update of the 1989 classification will be fourthcomming following the Second International Conference on Bacillus thuringiensis during the summer of 1994. Processing of Cry Proteins Most Cry ICPs are regarded to be protoxins because full larvicidal activity is contained within a portion of the ICP. Ingested IPCs are partially digested in the midguts of susceptible insect larvae. In the case of CrylVA and CrylVB as well as all

Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Cryl ICPs the "toxin" portion of the ICP resides in the N-terminal half of each molecule, the entire C-terminal half being dispensible for toxicity if not stability of the molecules (5). Cryll, Cryffl and CrylVC ICPs occur naturally C-terminally truncated. Removal of only a few amino acids from the C-termini of these ICPs results in loss of larvicidal activity. However, at least the Cryin ICPs, like Cryl ICPs, undergo limited N-terminal digestion in the midguts of susceptible insect larvae (6). Crystal Structure of CrymA Toxin The sequence of about a dozen amino acids near the C-termini of Cryl, CrylQ, CrylVA, CrylVB, and CrylVC toxins is extremely similar. This is one of five "blocks" of amino acid sequences shared by all of these toxins (2). As mentioned previously, the crystal structure of CrylHA toxin has been determined. It was found to consist of three domains. Domain I consists of a bundle of seven a-helixes connected by short loops. Amino acids in the conserved sequence block closest to the N-terminus of all Cry toxins, including Cryll and CrylVD, are involved in forming hydrophobic helix 5 in the center of the seven helix bundle. Amino acids in conserved sequence block 2 participate in forming the sixth and seventh helixes of the bundle. Domain II consists of three antiparallel 6 sheets stacked roughly parallel to the helix bundle of domain I. The only conserved sequence block found in domain II consists of several amino acids at the C-terminal end of block 2 which connect the end of helix 7 of domain I with the first sheet of domain II. Amino acids of conserved sequence block 2 that are involved in forming helix 7 of domain I are also in contact with one of the sheets in domain II. Domain III again consists of a group of antiparallel 6 sheets laying atop domain n at its junction with domain I. Three of the five conserved amino acid sequence blocks are located in domain III. As mentioned above, block 5 amino acids are found at the C-terminus of the toxin. Amino acids of block 3 are found in loops connecting domain II with domain HI. Amino acids of block 4 constitute a 13 strand adjacent to the strand containing block 5 amino acids. Both of these strands are buried within the molecule at the junction of all three of its structural domains. Since nearly all conserved amino acid sequences are found at sites within the CrylllA toxin molecule involved in stabilizing its three dimensional structure, it is thought likely that other molecules containing these sequence blocks would adopt a similar structure (5). Mode of Action of Cryl Toxins Studies of the mode of action of B. thuringiensis delta-endotoxins have been in progress since the 1950s. Nearly all of the early studies were conducted on whole larvae and by far the most widely used experimental method was microscopic examination of tissues removed from the larvae and fixed at various times after they had ingested a delta-endotoxin preparation. These studies were invariably conducted with lepidopteran larvae and Cryl delta-endotoxin preparations. The first CrylV and Cyt ICP producing strain of B. thuringiensis was discovered in 1977 (7) and the first Cryin ICP producing strain of B. thuringiensis was discovered in 1983 (8). We now know that the delta-endotoxin preparations used in these early studies often contained more than one Cryl ICP and were sometimes contaminated with Cryll proteins.


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Nonetheless, as seen in the summary by Luethy and Ebersold (9), a surprisingly complete picture of critical steps in the mode of action of Cryl ICPs emerged from these studies. The primary site of toxin action was clearly the larval midgut. The importance of high lumen pH for solubilization of parasporal bodies as well as the role of lumen proteinases in cleaving ICPs to toxins were recognized. The potential consequences of disrupting the large pH gradient between midgut lumen and midgut cell cytoplasm were discussed. The primary effect of the toxins seemed to be on cell membrane permeability and evidence favored the primary site of toxin action being the brush border membrane of midgut columnar cells. A requirement for specific toxin binding proteins (receptors) in the brush border membrane was not mentioned explicitly but was implied strongly in the Luethy and Ebersold (9) review. The authors of a recent article on the mode of action of B. thuringiensis Cry proteins (10) endorse the idea, based on the presence of conserved blocks of amino acid sequences in most Cry toxins and strengthened by the locations of these blocks in the structure of the CrylUA toxin, that the mode of action of all Cry toxins is basically similar. They summarize the mode of action of Cry toxins in four steps: 1) Ingestion, 2) Solubilisation and proteolytic activation, 3) Receptor binding, 4) Formation of toxic lesion. Studies with Insect Cell Lines. It may seem that advances in understanding the mode of action of Cryl toxins between 1981 and 1993 were limited. If this is the case, it might be due in part to the introduction of insect cell lines into B. thuringiensis research (77). The prospects of eliminating or at least reducing insect rearing plus convenient access to cells in vitro lured many investigators into working with insect cell lines. These cell lines were neither derived from nor resembled larval midgut columnar cells. Furthermore, they were 50 to 100 times less sensitive to Cry toxins. Nonetheless, working with these cell lines was not without some rewards. Some of the first evidence for the existence and concerning the nature of Cryl toxin binding cell membrane components (12) as well as the concept of colloid-osmotic lysis as a general mechanism for the larvicidal action of B. thuringiensis deltaendotoxins (73) came from studies using insect cell lines. However, quantitative characterization of binding between Cry toxins and membranes of cultured insect cells has never been achieved and the prospects for isolation of toxin binding proteins from insect cell lines continue to appear slim. The use of cultured insect cell lines in B. thuringiensis research seemed to decrease considerably after publication of a study showing a lack of correlation between the toxicity of several Cryl toxins to spruce budworm larvae and a cell line derived from spruce budworm larvae (14). Following the introduction of larval midgut brush border membrane vesicles (BBMV) to Cryl mode of action studies (75) and especially after the quantitative demonstration of specific high affinity binding between B B M V and Cryl toxins (16; 17), the amount of effort expended by all but a few laboratories on studies of the effects of B. thuringiensis toxins on established insect cell lines seems to have decreased even further. Molecular Mode of Action. Although the overall course of a successful intoxication of a lepidopteran larva by a Cryl B. thuringiensis toxin seems to be well established at the level of the major steps involved, many questions remain unanswered at a


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molecular level. The concentration and affinity of toxin binding sites on its midgut brush border membranes often but not always correlates positively with the susceptibility of a larva to a toxin (75). The irreversible step that follows binding of toxin to the brush border membrane is thought to be associated with insertion of all or part of the toxin into the membrane. This insertion of toxin molecules into the membrane is believed to be necessary for formation of a pore which mediates the potentially lethal increase in membrane permeability (10). However, the size and composition of this membrane pore is unknown. Membrane pores formed by Cryl toxin proteins in the presence of insect receptor proteins could consist of toxin molecules alone or they could consist of some combination of toxin and receptor molecules (10). Pores consisting of toxin molecules alone might be expected to exhibit the same permeability properties whether or not receptor molecules are also present. However, pores composed of both receptor molecules and toxin molecules seem likely to differ significantly in their permeability properties from pores formed by toxin alone. The permeability properties of pores formed by CryIA(c) and CrylC toxins in planar phospholipid bilayers have been determined quantitatively (79; 20). Several studies of the effects of Cryl toxins on the permeability of lepidopteran insect tissue culture cells (75; 27) and brush border membrane vesicles prepared from midguts of lepidopteran larvae (75; 22-25) have been published. For reasons discussed above the results of studies with tissue culture cells must be interpreted with caution. Midgut B B M V have the advantage of being the true target of the toxins and contain authentic toxin binding proteins. Although Hendrickx and associates (22) attempted to measure the effect of toxin on alanine permeability, all but the most recent of the studies with B B M V cited above used indirect methods which limited them to detecting changes in membrane permeability only for certain inorganic ions. With their light-scattering assay, Carroll and Ellar (25) were able to study the effects of CryIA(c) toxin on the permeability of larval Manduca sexta midgut B B M V to a variety of solutes. However, they were able to draw only qualitative conclusions about relative changes in permeability for the different solutes. By incorporating larval M. sexta midgut B B M V into planar phospholipid bilayers we have been able to measure directly and quantitatively the current flow through pores formed by CryIA(c) toxin in the presence of insect midgut proteins that interact specifically with this bacterial protein (Martin, F. G.; Wolfersberger, M . G. J Esq). Biol., in press). Toxin Pores in Hybrid Bilayers. Fusions of B B M V with planar bilayer lipid membranes (BLMs) were detected as small step increases in membrane current. After evidence of one or more B L M - B B M V fusions had been observed, addition of a small amount (final [toxin] < 2 nM) of CryIA(c) toxin to the chamber supporting the B L M bathed by pH 9.6 KC1 solution resulted in one or more large step increases in membrane current. The smallest step increase recorded corresponded to an increase in membrane conductance of 13 nS. However, current increases corresponding to changes in membrane conductance of approximately 26 nS or 39 nS were most common. Membrane current never decreased following one of these step increases; any subsequent increases simply added to the total membrane current. Similar increases in membrane current were never recorded from B L M s exposed to only


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B B M V . CryIA(c) toxin at the concentrations used in these experiments had no affect on B L M s that had not been exposed to B B M V . The quantal nature of changes in membrane conductance following addition of toxin to the solutions bathing BLMs that showed evidence of having fused with B B M V (all changes in membrane conductance were within experimental error some multiple of 13 nS) lead us to propose that 13 nS was most likely to be the conductance of a single pore. A cylindrical pore spaning the insulating portion of the bilayer filled with the buffer used in these studies with a conductance of 13 nS would have a diameter of 22 A. This is approximately twice the diameter of the pore required to allow passage of the largest solutes shown by light scattering measurements to enter CrylA(c) toxin treated B B M V prepared from larval M sexta midguts (25). However, it is within the range of diameters of the larger of the two most likely six toxin molecule model pores constructed by Hodgman and Ellar (2d). p H Sensitivity of Pores. All light scattering studies of the effects of Cryl toxins on the permeability of B B M V have been conducted at pH 7.5. We have not attempted to measure the effects of CryIA(c) toxin on B B M V containing B L M s at this pH. However, a few experiments conducted at pH 8.8 gave results that were qualitatively similar to but quantitatively different from those obtained at pH 9.6. At pH 8.8 the seemingly irreversible stepwise increases in membrane current that occured after addition of toxin were never more than 25% as large as the smallest increases observed at pH 9.6. Pores with conductances in this range would be expected to have diameters of about 9 A and solute permeabilies very similar to those seen in the light scattering studies at pH 7.5. The pore diameter of the smaller of the two most likely six toxin molecule pore models constructed by Hodgman and Ellar (26) brackets this caluclated pore diameter. The model pores constructed by Hodgman & Ellar (26) consisted only of conserved amino acid sequences found in Cry toxins that fulfilled certain criteria for potentially forming membrane spanning helixes. pH was not considered explicitly in model construction. Slatin and associates (79) were the first to demonstrate that Cry toxins alone actually could form pores in phospholipid bilayers. Neither CryIA(c) nor CryllLA toxin pores were observed at pH 7 but both toxins formed pores at pH > 9.5. The pores formed by CryIA(c) toxin at pH 9.7 were highly cation-selective and showed two major conductance states. The higher and more frequently observed "single channel" conductance was 600 pS. The other major state had a conductance of 200 pS. Both states showed the same 25:1 P K C I * selectivity. Both high and low conductance channels alternated, in what appeared to be a completely independent manner, between open and closed on a time scale of seconds. :P

o n

Pore Composition. We have independently confirmed the conductance and gating characteristics reported by Slatin and associates (79) for pores formed by CryIA(c) toxin in planar phospholipid bilayers (Martin F. G.; Wolfersberger, M . G., unpublished data). However, as mentioned above, when B B M V prepared from the midguts of M sexta larvae were introduced into an otherwise identical system, very different results were obtained. The toxin concentration required for pore formation was at least two orders of magnitude lower, the pores formed were much larger, and after forming or opening they have never been observed to close. The great


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differences between the properties of CryIA(c) toxin pores formed in phospholipid bilayers in the presence and absence of specific toxin-binding components of target insect cell membranes favors insect cell membrane components playing an active role in pore formation. Knowles and Dow (70) discuss two ways in which insect membrane receptors might interact with Cry toxins to form pores different from those formed in their absence. In one case the receptor molecules, which are presumed to be integral membrane proteins, combine with the toxin to form the walls of the pore. The resulting hybrid pores would differ in structure and almost certainly also in functional propertiesfrompores composed of toxin molecules alone. In their second model, the pore is lined only with toxin molecules. However, binding between toxin and receptor triggers a change in the conformation of the toxin so that the portions of the toxin that insert into the membrane and line the pore are ones that are buried within the structure of the toxin molecules in aqueous solution. In this case, although the pore is lined only by portions of toxin molecules, the portions of toxin molecules lining the pore could be different from those lining pores formed in the absence of receptors. Knowles and Dow (JO) seem to favor their second model. It is certainly not without appeal and fits well with the results of molecular modeling (26). However, they suggest that high pH and/or proximity to a hydrophobic membrane might have similar effects on toxin conformation as receptor binding. Proximity to a hydrophobic membrane is necessarily part of all studies of the effects of toxins on natural or artificial membranes. pH clearly affects toxin pore formation as well as the properties of the resulting pores (20) but not nearly as much as the presence or absence of toxin binding proteins in the membrane under study. Acknowledgments My research on the mode of action of Bacillus thuringiensis toxins has been supported by grantsfromthe United States Department of Agriculture and the National Institutes of Health. Literature Cited 1. deBarjac, H.; Bonnefoi, A. Entomophaga 1973, 18, 5-17. 2. Hoefte, H.; Whiteley, H. R. Microbiol. Rev. 1989, 53, 242-255. 3. Li, J.; Carroll, J.; Ellar, D.J. Nature 1991, 353, 815-821. 4. Feitelson, J.S.; Payne, J.; Kim, L. Bio/Technol. 1992, 10, 271-275. 5. Aronson, A. I.; Beckman, W.; Dunn, P. Microbiol. Rev. 1986, 50, 1-12. 6. Gill, S.S.; Cowles, E.A.; Pietrantonio, P.V. Ann. Rev. Entomol. 1992, 37, 615-636. 7. Goldberg, L.; Margalit, J. Mosquito News 1977, 37, 355-358. 8. Krieg, A.; Huger, A.M.; Langenbruch, G.A.; Schnetter, W. Z. Angew. Entomol. 1983, 96, 500-508. 9. Luethy, P.; Ebersold, H.R. In Pathogenesis of Invertebrate Microbial Diseases; Davidson, E.W. Ed.; Allanheld Osmun.Totowa, NJ, 1981; pp 235-267. 10. Knowles B.H.; Dow, J.AT. BioEssays 1993, 15, 469-476. 11. Murphy, D.W.; Sohi, S.S.; Fast, P.G. Science, 1976 194, 954-956. 12. Knowles, B.H.; Thomas, W.E.; Ellar, D.J. FEBS Lett. 1984, 168, 197-202.


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13. Knowles, B.H.; Ellar, D.J. Biochim. Biophys. Acta 1987, 924, 509-518. 14. Witt, D.P.; Carson, H.; Hodgdon, J.C. In Fundamental and Applied Aspects of Invertebrate Pathology; Samson, R.A.; Vlak, J.M.; Peters, D. Eds.; Foundation of the Fourth International Colloquium of Invertebrate Pathology: Wageningen, 1986; pp. 3-6. 15. Sacchi, V.F.; Parenti, P.; Hanozet, G.M.; Giordana, B.; Luethy, P.; Wolfersberger, M.G. FEBS Lett. 1986, 204, 213-218. 16. Hofmann, C.; Luethy, P.; Huetter, R.; Pliska, V. Eur. J. Biochem. 1988, 173, 85-91. 17. Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; VanRie, J.; Jansens, S.; VanMellaert, H. Proc. Natl.Acad.Sci. USA 1988, 85, 7844-7848. 18. Wolfersberger, M.G. Experientia 1990, 46, 475-477. 19. Slatin, S.L.; Abrams, C.K.; English, L. Biochem. Biophys. Res. Commun. 1990, 169, 765-772. 20. Schwartz, J-L.; Garneau, L.; Savaria, D.; Masson, L.; Brousseau, R. J. Membrane Biol. 1993, 132, 53-62. 21. Schwartz, J-L.; Garneau, L.; Masson, L.; Brousseau, R. Biochim. Biophys. Acta 1991, 1065, 250-260. 22. Hendrickx, K.; deLoof, A.; VanMellaert, H. Comp. Biochem. Physiol. 1989, 95C, 241-245. 23. Wolfersberger, M.G. Arch. Insect Biochem. Physiol. 1989, 12, 267-277. 24. Uemura, T.; Ihara, H.; Wadano, A.; Himeno, M. Biosci. Biotech. Biochem. 1992, 56, 1976-1979. 25. Carroll, J.; Ellar, D.J. Eur. J. Biochem. 1993, 214, 771-778. 26. Hodgman, T.C.; Ellar, D.J. DNA Sequence 1990, 1, 97-106. RECEIVED January 12,

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Molecular Action of Insecticides on Ion Channels - ACS Publications

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