Vol.
169,
No.
June
15,
1990
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 765-772
DELTA-XNDOTOXINS
FORM
Slatin#,
CATION-SELBCTIVX LIPID BILAYXRS Charles
K. Abrams#,
CXAmXLS
and Leigh
IN
PLAWAB
English*
Stephen
L.
#Albert
Einstein College of Medicine, Dept. of Physiology and Biophysics, Bronx, NY 10461
*Ecogen Received
April
Inc., 30,
2005
Cabot
Blvd.
W.,
Langhorne,
PA 19047
1990
Delta-endotoxins CryIA(c) and CryIIIA, two members of a large family of toxic proteins from Bacillus thuringiensis, were each allowed to interact with planar lipid bilayers and were analyzed for their ability to form ion-conducting channels. Both of these toxins made clearly resolved channels in the membranes and exhibited several conductance states, which ranged from 200 pS to about 4000 pS (in 300 mM KCl). The channels formed by both toxins were highly cation-selective, but not ideally so. The permeability ratio of K+ to Cl- was about 25 for both channels. The ability of these proteins to form such channels may account for their toxic action on sensitive cells, and suggests that this family of toxins may act by a common mechanism. 01990 Academic Press, Inc.
Delta-endotoxins kDa-27 kDa) produced
are a large family of protein toxins (135 as crystalline inclusions by Bacillus thuringiensis (1). These proteins are of enormous agricultural importance because of their acute insecticidal properties. When ingested by a susceptible organism, the toxins are solubilized, and in some cases proteolytically activated; and in the samples of the toxin that have been evaluated, they bind to specific receptors localized on brush border membrane (2,3). In the toxin induces leakage of association with the brush border, cations in the epithelium (4), likely a direct effect of the toxin on the membrane, as demonstrated by the ability of the toxin to induce ion leakage in phospholipid vesicles in the absence of brush border epithelial membrane containing specific Other researchers demonstrated the ability of receptors (5). some Bacillus thurfngiensis toxins to disrupt the integrity of It was suggested that this the phospholipid vesicles (6,7,8). large class of toxins behave as ion channels in association with the epithelium (9,lO). CytA, a small hemolytic protein (27 kDa)
165
0006-291x190 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 169, No. 2, 1990
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
from Bacillus thuringiensis var. israelensis with no sequence similarity to Bacillus thuringiensis crystalline Cry-toxin proteins, was reported to have cation channel-like properties in Unlike the CytA protein, the Cry-type planar bilayers (11). toxin proteins have not been reported to have hemolytic activity. We report here that delta-endotoxins CryIA(c) (55 kDa) and CryIIIA (67 kDa), representative of the Cry-toxins, form cationselective channels. Materials
and
Methods
Bacterial
Cultures
Bacillus thuringiensis var. kurstaki strain EG2244, thuringiensis producing a single CryIA(c) toxin, and Bacillus EG2158 producing a single CryIIIA toxin, were grown to sporulation in chemically defined C2 medium (12). Cultures were concentrated by removing excess water, salts, and soluble macromolecules by filtration through a 500,000 Mr cut-off Romicon filter (Woburn, MA) and stored at 4'C until used. Fractions of this concentrate were washed with water equal to 5,000 times the fraction volume, and the solids separated by centrifugation (7000 xg for 20 minutes).
Toxin
Purification
Trypsin-activated 55 kDa toxin was prepared from EG2244 according to the method of English et al. (13). This method yields a highly purified 55 kDa toxin by trypsin activation of the protoxin and subsequent pIi-dependent precipitation of the activated toxin at pH 7.5. Activated CryIA(c) toxin was stored as a lyophilized powder. CryIIIA was purified by solubilizing the toxin in 4 M NaBr, removing the insoluble cellular debris, spores, and other insoluble fermentation ingredients by centrifugation 7000 xg, and dialyzing the supernatant to remove NaBr and precipitate the toxin. The precipitate was washed in 10x volume of water and stored at -20 C. Planar
Lioid
Bilavers
Planar lipid bilayers were formed by either the brush technique of Meuller et al. (14) or the wsolvent-free" technique of Montal (15). Black lipid membranes (BLMs) were formed by painting a 3% solution of lipid in decane across a 0.6 mm hole in a Teflon partition which separated buffered salt solutions. The initially thick film thins to a bilayer in a process that can be observed optically. The lipid was either asolectin (Sigma, St. Louis, MO) from which neutral lipids were extracted in acetone (16), diphytanoyl phosphotidyl choline (Avanti Polar Lipids, Pelham, AL), or mixtures of asolectin and cholesterol. The aqueous phase was typically 0.3 M KCl, 5 mM 3-[cyclohexyl-amino]l-propane sulfonic acid (CAPS), pH 9.7, 5 mr4 Cac12, 0.5 IIIM EDTA. Solvent-free films were made by raising two lipid monolayers on opposite sides of a Teflon partion containing a 100 micron hole which had been pretreated with squalene. Monolayers were formed by spreading a 1% solution of lipid in hexane on the surfaces of the buffers, and allowing the hexane to evaporate. For both 766
Vol.
169, No. 2, 1990
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
electrical measurements were made under of membrane, clamp conditions using a homemade I-to-V converter based on a Burr Brown OPA104 operational amplifier. The inverting imput of the op amp, the virtual ground side, was connected to the tram (as opposed to the cis, or protein-containing) compartment with a Ag/AgCl electrode which contacted the solution via a RCl/agar bridge. The cis compartment was similarly source. connected to a voltage Current was monitored on an oscilloscope and recorded on a strip chart recorder. Samples of delta endotoxin, prepared either as an insoluble slurry or dry, were vortexed with 12 mM KOH for several minutes before adding 10 or 20 ul volumes to the cis compartment, while stirring with magnetic fleas. types
voltage
Results Preparations CryIIIA) were activated toxin, sample of that polyacrylamide The molecular regression of
of purified delta-endotoxins (CryIA(c) and used in all experiments. 55 kDa trypsin-WA(c), was precipitated from solution at pH 7.4. A toxin was resolved by continuous SDSgel electrophoresis illustrated in Fig. 1, Lane 1. weight of this toxin was estimated by linear log molecular weight standards (STD) versus
1 CrylA(c)
2
3
STD
CrylIlA
4 STD
67
Activated and purified CryIA(c) and CryIIIA resolved by cant nuous SDS polyacrylamide -+ (5-20%) gel electrophoresis. CryIA(c) (Lane 1) migrated at 55 kDa when compared with molecular weight standards: myosin (200 kDa), 6-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa),ovalbumin (43 kDa) and carbonic anhydrase (31 kDa) (Lane 2 - STD). CryIIIA (bane 3) migrated at 67 kDa when compared with the same molecular weight standards (Lane 4 -. STD). Lanes I,2 and Lanes 3,4 are from two different gels. 767
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records of delta-endotoxin. Fiq. 2. Single channel current A. CryIA(c) (32 bg/ml) in asolectin BIJ4 separating symmetric solutions of 0.3 M KCl, 5 mM CaC12, 0.5 mM EDTA, 5 mM CABS, pH 9.7. Holding voltage was +40 mV. Channels open up. B. CryIIIA (23 /.rg/ml) in asolectin: cholesterol, 2:1, BLM separating symmetric solutions of 0.3 M KCl, 5 mM CaCl2, 0.5 ml4 EDTA, 8 mM CAPS, pxi 9.7. Holding voltage was -10 mV. Channels open down.
A sample of recrystallized migration distance (Fig. 1, Lane 2). CryIIIA was resolved by continuous SDS-polyacrylamide gel electrophoresis illustrated in Fig. 1, Lane 3. The molecular weight of this toxin was 67 kDa estimated by linear regression of the log molecular weight standards (STD) versus migration distance (Fig. 1, Lane 4). The electrophoretic mobility of CryIIIA was greater than bovine serum albumin (66 kDa), but when compared with other standards in this and other gels, the best size estimate for CryIIIA was 67 kDa. Figure 2 shows current records from two black lipid membranes in symmetric 300 mM KCl, pH 9.7 buffered solutions treated with CryIA(c) (Fig. 2A) and CryIIIA (Fig. 2B). Both toxins made channels that gate on a time scale of seconds and lack a strong voltage dependence. Histograms of the conductance jumps (not shown) indicated an overlapping range of sizes for the two toxins. Under the conditions examined, the most common single channel conductance of CryIA(c) was 600 pS, and of CryIIIA was 4000 pS. Channels were observed in membranes made from pure diphytanoyl phosphatidyl choline, asolectin, and mixtures of asolectin and cholesterol. Most experiments were done with BLMs, but channels were also observed using @Wontal/Meullerw membranes, which contain much less hydrocarbon than BLMs and are slightly thinner. Both toxins induced at least as much conductance in 768
Vol. 169, No. 2, 1990
BIOCHEMICAL
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AND BIOPHYSICAL
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Grodieoi
Fig.,3. Reversal potentials of delta-endotoxins in KC1 gradients. Channels were incorporated in BLMs in symmetric solutions (volume = 3 ml) of 0.3 M KCl, 5 mM CaCl3, 0.5 mM EDTA, 5 mM CAPS, pIi 9.7. After good channel activity was verified, a 100 ul aliquot of 4 M XC1 was stirred into the cis compartment (followed immediately by removal of an equal volume of the mixed solution to maintain the total volume). The potential required to zero the current through the channel was determined, and the process was repeated. Cation-selectivity gives negative reversal
potentials
with
our sign
convention,
but is shown here
as
positive for convenience. Results shown here have been corrected for electrode offset but not for nonideal salt activity.
these "solvent-free I' films as in the decane films, which were about 30 times larger in surface area, suggesting that the toxins are actually much more active on the thinner membranes. No channels were observed at pH 7, and most experiments were therefore carried out at pH 9.5 or above. Reversal potentials were determined for channels of both toxins in BLMs separating asymmetric solutions of KCl. Despite the range of channel conductances encountered in various experiments, the reversal potentials all fell on a straight line when plotted against the log of the concentration gradient (Fig. 3). Thus, the various channel states formed by the toxin The channels were highly seem to have the same selectivity. cation-selective. Channels formed by both CryIA(c) and CryIIIA reversed at the same potential within experimental error. Extrapolating the data lead to a predicted reversal potential of 48 mV for a 1O:l KC1 gradient. Correcting for the nonideality of the solutions (17), and using the Goldman-Hodgkin-Katz equation (18), this corresponds to a permeability ratio, Pk:pCl, of about When a large excess of the potassium ionophore valinomycin 25:l. was added to the chamber, the membrane became a few mV more 769
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deviation of cation-selective (Fig. 3), showing that the small the channel's reversal potential from ideal cation selectivity is both channels have a nonzero chloride conductance real; i.e., under these conditions. only a few channels were induced in a given Usually, Greater conductances membrane by tens of pg of toxin protein. were often seen when the membrane was broken and reformed in the or when the nonionic detergent octylglucoside presence of toxin, This low activity may be due to poor was added to the protein. binding of the toxin to the membrane in the absence of any endogenous receptor. Discussion The various delta-endotoxins display a range of specificities for particular targets, but in all instances where susceptible cells invariably develop toxicity has been examined, an abnormal leakiness to cations, which is evidently crucial to the toxic action (4,19). This permeability could come about through a direct effect of the toxin on the cell membrane, or could be the result of cellular processes induced by the toxin. Studies with phospholipid vesicles suggested that the toxin could act directly on the lipid membrane, and it was proposed that the toxin might form an ion channel in target membranes which would lead directly to colloid osmotic lysis (6,7,8). In this paper, we show that two different delta-endotoxins, CryIA(c) and CryIIIA, do indeed form highly cation-selective ion channels in planar lipid bilayers. Channel formation was not limited to a particular lipid or solvent system used to form the membrane, but was observed only at high pH. p&dependent channel formation is a fairly common feature of protein toxins, such as diphtheria toxin, botulinum toxin, and some of the colicins (20,21). The biological action of the delta-endotoxins results in increased cation permeability of the brush border membrane of the insect midgut, so it is plausible to conclude that the channels observed in these artificial systems are central to the toxicity of the proteins, and may represent a general mechanism of delta-endotoxin action. The channels formed by the Cry-type toxin proteins appear to have more than one "single channel" conductance level. This may mean that the channel has several different substates; i.e., different conformations in the membrane, perhaps due to different 770
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orders of aggregation, such as is seen with alamethecin (22). Alternately, seemingly different conductance levels could be due to cooperative gating of more than one identical channel; i.e., the opening of one channel could induce another nearby channel to faster than can be resolved by the also open, on a time scale recording system (about 30 ms with our bandwidth). This mechanism has been invoked to explain the anomolously large channels formed by diphtheria toxin under some conditions (23). Our data indicate that the different delta-endotoxin conductance states have a similar ability to select K+ over Cl-, which suggests that the substates are structurally rather similar, lending support to the cooperative gating hypothesis. The toxin preparations used in these experiments were not highly efficient at forming channels. In most experiments, far fewer channels were seen than one would expect from a comparable quantity of many of the known channel-forming toxins (such as diphtheria or anthrax) under ideal conditions for the particular toxin. This low efficiency may mean that we have not used the best conditions for promoting channel formation (e.g., we have not systematically examined the effect of divalent cations on channel formation). In addition, the delta-endotoxin proteins are not very soluble, even at high pH, and we cannot be certain that all of the protein added to the chamber is available to interact with the membrane. And, of course, in these membranes which presumably play a crucial there are no protein receptors, role in promoting toxin/membrane interaction biologically. Since both preparations used in these experiments were electrophoretically pure, and both induced channels which had the expected ion-selectivity and pH dependence, the channel-forming activity is probably a fundamental property of Cry-toxin proteins. These data support the suggestion that the deltaendotoxins, in general, behave as ion channels in association independent of other membrane-bound with lipid membranes, proteins (9,lO). The data also suggest that measurements of specific receptor binding in cell membranes is likely to include components from toxin bound both to endogenous membrane-bound protein and to membrane lipid. We are continuing to elucidate the structure and function of the channels formed by deltaendotoxins, as well as to evaluate the role of channel formation in toxicity. 771
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Aaknowledgments
We thank A. Slaney and A. Bastian for and S. Mapes for reviewing the manuscript.
technical
assistance
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
HiSfte, H. and Whiteley, H.R. (1989) Microb. Rev. 53, 242-255. Hofmann, C., Luthy, P., Hutter, R., and Pliska, V. (1988a) Eur. J. Biochem. 173, 85-91. Hofmann, C., Vanderbruggen, H., Hafte, H., Van Rie, J., Jansens, S., and Van Mellaert, H. (1988b) Proc. Natl. Acad. Sci. USA. 85, 7844-7848. Sacchi, V.F., Parenti, P., Hanozet, G.M., Giordana, B., Luthy, P. and Wolfersberger, M.G. (1986) FEBS Letts. 240, 213 -218. English, L. H. and T. R. Readdy. (1990) Submitted to Insect Biochemistry, December, 1989. Yunovitz, H. and Yawetz, A. (1988) FEBS Letts. 230, 106-108. Drobniewski, F.A., Knowles, B.H., and Ellar, D.J. (1987) Current Microbial. 15, 295-299. Haider, M.Z. and Ellar, D.J. (1989) Biochem. Biophys. Acta 978, 216-222. Convents, D., Houssier, C., Lasters, I. and Lauwereys, M. (1990) J. Biol. Chem. 265, 1369-1375. Price, R.C. (1990) Trends Biochem. Sci. 15, 2-3. Knowles, B.H., Blatt, M.R., Tester, M., Horsnell, J.M., Carroll, J., Menestrina, G., and Ellar, D. (1989) FEBS Letts. 244, 259-262. Donovan, W.P., Dankocsik, C.C., Gilbert, M.P. Gawron-Burke, C Groat, R.G., and Carlton, B.C. (1988) J. Biol. Chem. 263, 5&567. English, L-H., Basian, A.E., and Readdy, T-L. (1990) Submitted to Appl. Env. Toxicol. Mueller, P., Rudin, D.O., Tein, H.T., and Wescott, W.C. (1963) J. Phys. Chem. 67, 534-535. Montal, M.C. (1974) Methods Enzymol. 32, 545-554. Kagawa, Y. and Racker, E. (1971) J. Biol. Chem. 246, 5477-5487. Robinson, R.A. and Stokes, R.H. (1959) Electrolyte Solutions, Butterworths, London. Hodgkin, A.L. and Katz, B. (1949) J. Physiol. (London) 108, 37-77. Crawford, D.N. and Harvey, W. (1988) J. Exp. Biol. 137, 277-286. Hoch, D.H., Romero-Mira, M., Ehrlich, B.E., Finkelstein, A., DasGupta, B.R., and Simpson, L.L. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1692-1696. Slatin, S.L. (1988) Int. J. Biochem. 20, 737-744. Baumann, G. and Mueller, P. (1974) J. Supramolecular Structure. 2, 538-557. Romero, M. (1988) The relationships between small and large conductance channels formed by diphtheria toxin on planar lipid bilayers, Ph.D. Thesis, Albert Einstein College of Medicine.
772
169,
No.
June
15,
1990
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 765-772
DELTA-XNDOTOXINS
FORM
Slatin#,
CATION-SELBCTIVX LIPID BILAYXRS Charles
K. Abrams#,
CXAmXLS
and Leigh
IN
PLAWAB
English*
Stephen
L.
#Albert
Einstein College of Medicine, Dept. of Physiology and Biophysics, Bronx, NY 10461
*Ecogen Received
April
Inc., 30,
2005
Cabot
Blvd.
W.,
Langhorne,
PA 19047
1990
Delta-endotoxins CryIA(c) and CryIIIA, two members of a large family of toxic proteins from Bacillus thuringiensis, were each allowed to interact with planar lipid bilayers and were analyzed for their ability to form ion-conducting channels. Both of these toxins made clearly resolved channels in the membranes and exhibited several conductance states, which ranged from 200 pS to about 4000 pS (in 300 mM KCl). The channels formed by both toxins were highly cation-selective, but not ideally so. The permeability ratio of K+ to Cl- was about 25 for both channels. The ability of these proteins to form such channels may account for their toxic action on sensitive cells, and suggests that this family of toxins may act by a common mechanism. 01990 Academic Press, Inc.
Delta-endotoxins kDa-27 kDa) produced
are a large family of protein toxins (135 as crystalline inclusions by Bacillus thuringiensis (1). These proteins are of enormous agricultural importance because of their acute insecticidal properties. When ingested by a susceptible organism, the toxins are solubilized, and in some cases proteolytically activated; and in the samples of the toxin that have been evaluated, they bind to specific receptors localized on brush border membrane (2,3). In the toxin induces leakage of association with the brush border, cations in the epithelium (4), likely a direct effect of the toxin on the membrane, as demonstrated by the ability of the toxin to induce ion leakage in phospholipid vesicles in the absence of brush border epithelial membrane containing specific Other researchers demonstrated the ability of receptors (5). some Bacillus thurfngiensis toxins to disrupt the integrity of It was suggested that this the phospholipid vesicles (6,7,8). large class of toxins behave as ion channels in association with the epithelium (9,lO). CytA, a small hemolytic protein (27 kDa)
165
0006-291x190 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
from Bacillus thuringiensis var. israelensis with no sequence similarity to Bacillus thuringiensis crystalline Cry-toxin proteins, was reported to have cation channel-like properties in Unlike the CytA protein, the Cry-type planar bilayers (11). toxin proteins have not been reported to have hemolytic activity. We report here that delta-endotoxins CryIA(c) (55 kDa) and CryIIIA (67 kDa), representative of the Cry-toxins, form cationselective channels. Materials
and
Methods
Bacterial
Cultures
Bacillus thuringiensis var. kurstaki strain EG2244, thuringiensis producing a single CryIA(c) toxin, and Bacillus EG2158 producing a single CryIIIA toxin, were grown to sporulation in chemically defined C2 medium (12). Cultures were concentrated by removing excess water, salts, and soluble macromolecules by filtration through a 500,000 Mr cut-off Romicon filter (Woburn, MA) and stored at 4'C until used. Fractions of this concentrate were washed with water equal to 5,000 times the fraction volume, and the solids separated by centrifugation (7000 xg for 20 minutes).
Toxin
Purification
Trypsin-activated 55 kDa toxin was prepared from EG2244 according to the method of English et al. (13). This method yields a highly purified 55 kDa toxin by trypsin activation of the protoxin and subsequent pIi-dependent precipitation of the activated toxin at pH 7.5. Activated CryIA(c) toxin was stored as a lyophilized powder. CryIIIA was purified by solubilizing the toxin in 4 M NaBr, removing the insoluble cellular debris, spores, and other insoluble fermentation ingredients by centrifugation 7000 xg, and dialyzing the supernatant to remove NaBr and precipitate the toxin. The precipitate was washed in 10x volume of water and stored at -20 C. Planar
Lioid
Bilavers
Planar lipid bilayers were formed by either the brush technique of Meuller et al. (14) or the wsolvent-free" technique of Montal (15). Black lipid membranes (BLMs) were formed by painting a 3% solution of lipid in decane across a 0.6 mm hole in a Teflon partition which separated buffered salt solutions. The initially thick film thins to a bilayer in a process that can be observed optically. The lipid was either asolectin (Sigma, St. Louis, MO) from which neutral lipids were extracted in acetone (16), diphytanoyl phosphotidyl choline (Avanti Polar Lipids, Pelham, AL), or mixtures of asolectin and cholesterol. The aqueous phase was typically 0.3 M KCl, 5 mM 3-[cyclohexyl-amino]l-propane sulfonic acid (CAPS), pH 9.7, 5 mr4 Cac12, 0.5 IIIM EDTA. Solvent-free films were made by raising two lipid monolayers on opposite sides of a Teflon partion containing a 100 micron hole which had been pretreated with squalene. Monolayers were formed by spreading a 1% solution of lipid in hexane on the surfaces of the buffers, and allowing the hexane to evaporate. For both 766
Vol.
169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
electrical measurements were made under of membrane, clamp conditions using a homemade I-to-V converter based on a Burr Brown OPA104 operational amplifier. The inverting imput of the op amp, the virtual ground side, was connected to the tram (as opposed to the cis, or protein-containing) compartment with a Ag/AgCl electrode which contacted the solution via a RCl/agar bridge. The cis compartment was similarly source. connected to a voltage Current was monitored on an oscilloscope and recorded on a strip chart recorder. Samples of delta endotoxin, prepared either as an insoluble slurry or dry, were vortexed with 12 mM KOH for several minutes before adding 10 or 20 ul volumes to the cis compartment, while stirring with magnetic fleas. types
voltage
Results Preparations CryIIIA) were activated toxin, sample of that polyacrylamide The molecular regression of
of purified delta-endotoxins (CryIA(c) and used in all experiments. 55 kDa trypsin-WA(c), was precipitated from solution at pH 7.4. A toxin was resolved by continuous SDSgel electrophoresis illustrated in Fig. 1, Lane 1. weight of this toxin was estimated by linear log molecular weight standards (STD) versus
1 CrylA(c)
2
3
STD
CrylIlA
4 STD
67
Activated and purified CryIA(c) and CryIIIA resolved by cant nuous SDS polyacrylamide -+ (5-20%) gel electrophoresis. CryIA(c) (Lane 1) migrated at 55 kDa when compared with molecular weight standards: myosin (200 kDa), 6-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa),ovalbumin (43 kDa) and carbonic anhydrase (31 kDa) (Lane 2 - STD). CryIIIA (bane 3) migrated at 67 kDa when compared with the same molecular weight standards (Lane 4 -. STD). Lanes I,2 and Lanes 3,4 are from two different gels. 767
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records of delta-endotoxin. Fiq. 2. Single channel current A. CryIA(c) (32 bg/ml) in asolectin BIJ4 separating symmetric solutions of 0.3 M KCl, 5 mM CaC12, 0.5 mM EDTA, 5 mM CABS, pH 9.7. Holding voltage was +40 mV. Channels open up. B. CryIIIA (23 /.rg/ml) in asolectin: cholesterol, 2:1, BLM separating symmetric solutions of 0.3 M KCl, 5 mM CaCl2, 0.5 ml4 EDTA, 8 mM CAPS, pxi 9.7. Holding voltage was -10 mV. Channels open down.
A sample of recrystallized migration distance (Fig. 1, Lane 2). CryIIIA was resolved by continuous SDS-polyacrylamide gel electrophoresis illustrated in Fig. 1, Lane 3. The molecular weight of this toxin was 67 kDa estimated by linear regression of the log molecular weight standards (STD) versus migration distance (Fig. 1, Lane 4). The electrophoretic mobility of CryIIIA was greater than bovine serum albumin (66 kDa), but when compared with other standards in this and other gels, the best size estimate for CryIIIA was 67 kDa. Figure 2 shows current records from two black lipid membranes in symmetric 300 mM KCl, pH 9.7 buffered solutions treated with CryIA(c) (Fig. 2A) and CryIIIA (Fig. 2B). Both toxins made channels that gate on a time scale of seconds and lack a strong voltage dependence. Histograms of the conductance jumps (not shown) indicated an overlapping range of sizes for the two toxins. Under the conditions examined, the most common single channel conductance of CryIA(c) was 600 pS, and of CryIIIA was 4000 pS. Channels were observed in membranes made from pure diphytanoyl phosphatidyl choline, asolectin, and mixtures of asolectin and cholesterol. Most experiments were done with BLMs, but channels were also observed using @Wontal/Meullerw membranes, which contain much less hydrocarbon than BLMs and are slightly thinner. Both toxins induced at least as much conductance in 768
Vol. 169, No. 2, 1990
BIOCHEMICAL
KCI
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Grodieoi
Fig.,3. Reversal potentials of delta-endotoxins in KC1 gradients. Channels were incorporated in BLMs in symmetric solutions (volume = 3 ml) of 0.3 M KCl, 5 mM CaCl3, 0.5 mM EDTA, 5 mM CAPS, pIi 9.7. After good channel activity was verified, a 100 ul aliquot of 4 M XC1 was stirred into the cis compartment (followed immediately by removal of an equal volume of the mixed solution to maintain the total volume). The potential required to zero the current through the channel was determined, and the process was repeated. Cation-selectivity gives negative reversal
potentials
with
our sign
convention,
but is shown here
as
positive for convenience. Results shown here have been corrected for electrode offset but not for nonideal salt activity.
these "solvent-free I' films as in the decane films, which were about 30 times larger in surface area, suggesting that the toxins are actually much more active on the thinner membranes. No channels were observed at pH 7, and most experiments were therefore carried out at pH 9.5 or above. Reversal potentials were determined for channels of both toxins in BLMs separating asymmetric solutions of KCl. Despite the range of channel conductances encountered in various experiments, the reversal potentials all fell on a straight line when plotted against the log of the concentration gradient (Fig. 3). Thus, the various channel states formed by the toxin The channels were highly seem to have the same selectivity. cation-selective. Channels formed by both CryIA(c) and CryIIIA reversed at the same potential within experimental error. Extrapolating the data lead to a predicted reversal potential of 48 mV for a 1O:l KC1 gradient. Correcting for the nonideality of the solutions (17), and using the Goldman-Hodgkin-Katz equation (18), this corresponds to a permeability ratio, Pk:pCl, of about When a large excess of the potassium ionophore valinomycin 25:l. was added to the chamber, the membrane became a few mV more 769
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169, No. 2, 1990
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deviation of cation-selective (Fig. 3), showing that the small the channel's reversal potential from ideal cation selectivity is both channels have a nonzero chloride conductance real; i.e., under these conditions. only a few channels were induced in a given Usually, Greater conductances membrane by tens of pg of toxin protein. were often seen when the membrane was broken and reformed in the or when the nonionic detergent octylglucoside presence of toxin, This low activity may be due to poor was added to the protein. binding of the toxin to the membrane in the absence of any endogenous receptor. Discussion The various delta-endotoxins display a range of specificities for particular targets, but in all instances where susceptible cells invariably develop toxicity has been examined, an abnormal leakiness to cations, which is evidently crucial to the toxic action (4,19). This permeability could come about through a direct effect of the toxin on the cell membrane, or could be the result of cellular processes induced by the toxin. Studies with phospholipid vesicles suggested that the toxin could act directly on the lipid membrane, and it was proposed that the toxin might form an ion channel in target membranes which would lead directly to colloid osmotic lysis (6,7,8). In this paper, we show that two different delta-endotoxins, CryIA(c) and CryIIIA, do indeed form highly cation-selective ion channels in planar lipid bilayers. Channel formation was not limited to a particular lipid or solvent system used to form the membrane, but was observed only at high pH. p&dependent channel formation is a fairly common feature of protein toxins, such as diphtheria toxin, botulinum toxin, and some of the colicins (20,21). The biological action of the delta-endotoxins results in increased cation permeability of the brush border membrane of the insect midgut, so it is plausible to conclude that the channels observed in these artificial systems are central to the toxicity of the proteins, and may represent a general mechanism of delta-endotoxin action. The channels formed by the Cry-type toxin proteins appear to have more than one "single channel" conductance level. This may mean that the channel has several different substates; i.e., different conformations in the membrane, perhaps due to different 770
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orders of aggregation, such as is seen with alamethecin (22). Alternately, seemingly different conductance levels could be due to cooperative gating of more than one identical channel; i.e., the opening of one channel could induce another nearby channel to faster than can be resolved by the also open, on a time scale recording system (about 30 ms with our bandwidth). This mechanism has been invoked to explain the anomolously large channels formed by diphtheria toxin under some conditions (23). Our data indicate that the different delta-endotoxin conductance states have a similar ability to select K+ over Cl-, which suggests that the substates are structurally rather similar, lending support to the cooperative gating hypothesis. The toxin preparations used in these experiments were not highly efficient at forming channels. In most experiments, far fewer channels were seen than one would expect from a comparable quantity of many of the known channel-forming toxins (such as diphtheria or anthrax) under ideal conditions for the particular toxin. This low efficiency may mean that we have not used the best conditions for promoting channel formation (e.g., we have not systematically examined the effect of divalent cations on channel formation). In addition, the delta-endotoxin proteins are not very soluble, even at high pH, and we cannot be certain that all of the protein added to the chamber is available to interact with the membrane. And, of course, in these membranes which presumably play a crucial there are no protein receptors, role in promoting toxin/membrane interaction biologically. Since both preparations used in these experiments were electrophoretically pure, and both induced channels which had the expected ion-selectivity and pH dependence, the channel-forming activity is probably a fundamental property of Cry-toxin proteins. These data support the suggestion that the deltaendotoxins, in general, behave as ion channels in association independent of other membrane-bound with lipid membranes, proteins (9,lO). The data also suggest that measurements of specific receptor binding in cell membranes is likely to include components from toxin bound both to endogenous membrane-bound protein and to membrane lipid. We are continuing to elucidate the structure and function of the channels formed by deltaendotoxins, as well as to evaluate the role of channel formation in toxicity. 771
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Aaknowledgments
We thank A. Slaney and A. Bastian for and S. Mapes for reviewing the manuscript.
technical
assistance
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
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