Channel Formation by the Glycosylphosphatidylinositol-anchored Protein Binding Toxin Aerolysin Is Not Promoted by Lipid Rafts

Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-β-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin. Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-β-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin. glycosylphosphatidylinositol placental alkaline phosphatase phosphatidylcholine phosphatidylethanolamine cholesterol sphingomyelin Dulbecco's modified Eagle's high glucose medium phosphate-buffered saline The possibility that lateral phase separations of specific bilayer components might lead to the occurrence of microdomains in cell membranes has received a great deal of recent attention (1.Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2551) Google Scholar, 2.Brown D.A. London E. J. Memb. Biol. 1998; 164: 103-114Crossref PubMed Scopus (837) Google Scholar, 3.Jacobson K. Dietrich C. Trends Cell Biol. 1999; 9: 87-91Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 4.Kurzchalia T.V. Parton R.G. Curr. Opin. Cell Biol. 1999; 11: 424-431Crossref PubMed Scopus (513) Google Scholar). These “lipid rafts” are largely defined by their resistance to extraction with nonionic detergents (5.Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar, 6.Ostermeyer A. Beckrich B. Ivarson K. Grove K. Brown D. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 7.Schroeder R. London E. Brown D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar). They are enriched in sphingolipids and cholesterol as well as in a number of membrane proteins, including several signal-transducing molecules and glycosylphosphatidylinositol (GPI)1-anchored proteins (7.Schroeder R. London E. Brown D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar, 8.Benting J. Rietveld A. Ansorge I. Simons K. FEBS. 1999; 462: 47-50Crossref PubMed Scopus (77) Google Scholar, 9.Friedrichson T. Kurzchalia T.V. Nature. 1998; 394: 802-805Crossref PubMed Scopus (479) Google Scholar, 10.Harder T. Simons K. Eur. J. Immunol. 1999; 29: 556-562Crossref PubMed Scopus (305) Google Scholar, 11.Varma R. Mayor S. Nature. 1998; 394: 798-801Crossref PubMed Scopus (1027) Google Scholar). Some members of this latter group of proteins are themselves involved in cell signaling, while some are enzymes and others have less well defined functions (12.Brown D. Curr. Opin. Immun. 1993; 5: 349-354Crossref PubMed Scopus (189) Google Scholar, 13.Janes P.W. Ley S.C. Magee A.I. J. Cell Biol. 1999; 147: 447-461Crossref PubMed Scopus (696) Google Scholar, 14.McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (803) Google Scholar, 15.Redman C.A. Thomas-Oates J.E. Ogata S. Ikehara Y. Ferguson M.A. Biochem. J. 1994; 302: 861-865Crossref PubMed Scopus (54) Google Scholar, 16.Roberts W.L. Kim B.H. Rosenberry T.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7817-7821Crossref PubMed Scopus (99) Google Scholar). The channel-forming protein toxin aerolysin and its inactive precursor proaerolysin have the unique ability to bind specifically and with high affinity to GPI-anchored proteins on the surfaces of target cells (17.Cowell S. Wolfgang A. Gruber H.J. Nelson K.L. Buckley J.T. Molec. Microbiol. 1997; 25: 343-350Crossref PubMed Scopus (52) Google Scholar, 18.Nelson K.L. Raja S.M. Buckley J.T. J. Biol. Chem. 1997; 272: 12170-12174Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 19.Diep D.B. Nelson K.L. Raja S.M. Pleshak E.N. Buckley J.T. J. Biol. Chem. 1998; 273: 2355-2360Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 20.MacKenzie C.R. Hirama T. Buckley J.T. J. Biol. Chem. 1999; 274: 22604-22609Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Once bound, proaerolysin may be converted to aerolysin by surface proteases (21.Abrami L. Fivaz M. Decroly E. Seidah N.G. Jean F. Thomas G. Leppla S.H. Buckley J.T. van der Goot F.G. J. Biol. Chem. 1998; 273: 32656-32661Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Bound aerolysin then forms heptameric oligomers that can insert into the plasma membrane, producing discrete channels (22.van der Goot F.G. Pattus F. Wong K.R. Buckley J.T. Biochemistry. 1993; 32: 2636-2642Crossref PubMed Scopus (84) Google Scholar). Because binding effectively concentrates the toxin on the cell surface, promoting oligomerization, cells that display GPI-anchored proteins are far more sensitive to aerolysin than those that do not (23.Nelson K.L. Brodsky R.A. Buckley J.T. Cell. Micro. 1999; 1: 69-74Crossref PubMed Scopus (65) Google Scholar, 24.Brodsky R.A. Mukhina G. Nelson K.L. Lawrence T.S. Jones R.J. Buckley J.T. Blood. 1999; 93: 1749-1756Crossref PubMed Google Scholar). Thus, normal T lymphocytes, which contain several GPI-anchored proteins that bind aerolysin, including Thy-1, are killed by 1-h exposure to 10−10maerolysin or proaerolysin, whereas T lymphocytes that lack GPI-anchored proteins because they are unable to synthesize the anchor are approximately 104-fold less sensitive. Similarly, we have shown that channel formation in artificial lipid bilayers occurs at far lower aerolysin concentrations if the bilayers contain incorporated GPI-anchored proteins, such as Thy-1 from brain or lymphocytes, or the erythrocyte aerolysin receptor, a novel aerolysin-binding GPI-anchored protein purified from erythrocytes (17.Cowell S. Wolfgang A. Gruber H.J. Nelson K.L. Buckley J.T. Molec. Microbiol. 1997; 25: 343-350Crossref PubMed Scopus (52) Google Scholar, 18.Nelson K.L. Raja S.M. Buckley J.T. J. Biol. Chem. 1997; 272: 12170-12174Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Recently, it has been proposed that lipid rafts promote channel formation by aerolysin because the increased density of GPI-anchored proteins therein leads to higher toxin concentrations than elsewhere on the cell surface, thereby, it was argued, increasing the rate of oligomerization (25.Abrami L. van der Goot F.G. J. Cell Biol. 1999; 147: 175-184Crossref PubMed Scopus (138) Google Scholar). However, only circumstantial evidence was presented to support the proposal. It was shown that aerolysin comigrates with the Triton X-100-insoluble fraction upon density gradient centrifugation, which is consistent with the fact that GPI-anchored proteins also tend to migrate there, and with our observation that aerolysin binds these proteins with high affinity (17.Cowell S. Wolfgang A. Gruber H.J. Nelson K.L. Buckley J.T. Molec. Microbiol. 1997; 25: 343-350Crossref PubMed Scopus (52) Google Scholar, 18.Nelson K.L. Raja S.M. Buckley J.T. J. Biol. Chem. 1997; 272: 12170-12174Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 19.Diep D.B. Nelson K.L. Raja S.M. Pleshak E.N. Buckley J.T. J. Biol. Chem. 1998; 273: 2355-2360Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 20.MacKenzie C.R. Hirama T. Buckley J.T. J. Biol. Chem. 1999; 274: 22604-22609Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). It was also shown that treating cells with a cholesterol-lowering agent, which is known to lower the amount of GPI-anchored protein that is detergent-insoluble, also lowered the amount of detergent-insoluble aerolysin (25.Abrami L. van der Goot F.G. J. Cell Biol. 1999; 147: 175-184Crossref PubMed Scopus (138) Google Scholar). Surprisingly, however, no comparison was made of the rate of channel formation by the toxin in the normal and treated cells. Although it is possible that the concentration of GPI-anchored proteins in rafts might promote oligomerization of aerolysin, rafts could conceivably have the opposite effect. These regions are thought to be enriched in saturated lipids, so that the lateral mobility of GPI-anchored proteins may actually be lower when they are in rafts than when they are in the bulk of the membrane (2.Brown D.A. London E. J. Memb. Biol. 1998; 164: 103-114Crossref PubMed Scopus (837) Google Scholar, 3.Jacobson K. Dietrich C. Trends Cell Biol. 1999; 9: 87-91Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Restricted motion of bound aerolysin would tend to lower oligomerization rates. In any case, whether or not aerolysin binding to raft-associated GPI-anchored proteins does affect the kinetics of oligomerization of the toxin, it seems unlikely that there would be a significant change in the overall rate of channel formation. This is because binding rather than oligomerization is the rate-limiting step in channel formation, especially at low toxin concentrations (26.Garland W.J. Buckley J.T. Infect. Immun. 1988; 56: 1249-1253Crossref PubMed Google Scholar), so that any influence of lipid rafts on oligomerization would probably be masked. In the present study, we looked for direct evidence of an effect of lipid rafts on channel formation by aerolysin. Cell sensitivity to the toxin was compared before and after treatment with methyl-β-cyclodextrin, which abolishes lipid rafts by reducing plasma membrane cholesterol levels (27.Ilangumaran S. Hoessli D.C. Biochem. J. 1998; 335: 433-440Crossref PubMed Scopus (401) Google Scholar). We found that the sensitivity of a T cell line was unaffected by cholesterol extraction; nor was the sensitivity of erythrocytes decreased by cholesterol removal. We also studied liposomes containing incorporated GPI-anchored placental alkaline phosphatase (PLAP), which we show acts as an aerolysin receptor. Liposomes containing PLAP associated with rafts were no more sensitive to aerolysin than liposomes that were raft-free. Liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and brain sphingomyelin (SM) were obtained from Avanti Polar Lipids. Cholesterol and a crude preparation of human placental alkaline phosphatase were purchased from Sigma. Proaerolysin and the inactive variant Y221G were purified as described previously. The purified variant was labeled with the fluorescent probe Alexa 488 (Molecular Probes, Inc., Eugene, OR), using a procedure provided by the manufacturer. The murine lymphocyte cell line EL4 was generously provided by Dr. R. Hyman (Salk Institute). Cells were grown in Dulbecco's modified Eagle's high glucose medium (DMEM) supplemented with bovine fetal clone I serum (10%, v/v), streptomycin (100 μg/ml), and penicillin (100 units/ml) with 5% CO2at 37 °C. EL4 cells at 2 × 106 cells/ml were washed twice in neat DMEM and then incubated with or without 10 mmmethyl-β-cyclodextrin in DMEM for 30 min at 37 °C, rotating end over end. Following extraction, half of the cells were washed twice in DMEM and used in the cytotoxicity assay and for flow cytometry; the other half were washed twice in PBS, and then a cholesterol determination (Cholesterol 20; Sigma) was performed on them. Lymphocytes (1 ml of 2 × 107 cells/ml in DMEM, 0.5% bovine serum albumin) were incubated with 10−8m Y221G proaerolysin on ice for 1 h. We used the proaerolysin variant here because it can bind as well as native proaerolysin but does not cause cell death, since it cannot form functional channels. Following incubation, the cells were washed twice in PBS and then extracted with 500 μl of 25 mm Tris, mm mm Triton containing a (1 2 and 1 for 30 min on In some the extraction step was then 10−8m Y221G was to the and the was incubated for min at °C. were to and with ml of 10 mm Tris, by ml of 10 mm Tris, The were then in an at for at °C. of 1 ml were and was for at the of the was in The of lymphocyte Thy-1 was by after extraction J. Rietveld A. Ansorge I. Simons K. FEBS. 1999; 462: 47-50Crossref PubMed Scopus (77) Google Scholar). Cells were lysed by exposure to Triton in PBS with for min on ice as This was by in a at at for 30 was from the and the with (10%, and Thy-1 was by The of PLAP incorporated into liposomes was also by Liposomes (100 of were at at for min in a and the was in μl of Triton in and extracted on ice for at at for 30 the enzyme activity was in the and in the in the Lymphocytes at treated with or without 10 mmmethyl-β-cyclodextrin as were incubated with a of proaerolysin concentrations for 1 at 37 5% and were then to concentrations of and The were incubated at 37 with 5% for after which was as described (23.Nelson K.L. Brodsky R.A. Buckley J.T. Cell. Micro. 1999; 1: 69-74Crossref PubMed Scopus (65) Google Scholar). erythrocytes were treated with mmmethyl-β-cyclodextrin or PBS and then washed with aerolysin was to a concentration of in containing ml of washed cells in The rate of was by the decrease in density of the erythrocyte at and 37 as a of were made using a I as described R.A. Mukhina G. Nelson K.L. Lawrence T.S. Jones R.J. Buckley J.T. Blood. 1999; 93: 1749-1756Crossref PubMed Google Scholar). The aerolysin concentration of erythrocyte was as described D.B. Nelson K.L. Lawrence T.S. B. Buckley J.T. Molec. Microbiol. 1999; PubMed Scopus Google Scholar). cells and cells that had been treated with for 30 min at 37 were to 10−8m Alexa Y221G proaerolysin for 30 min on They were then washed twice with PBS and by flow of human PLAP were in ml of Triton in PBS containing 1 mm by for min on The was into and by the to 37 for 10 min and then in a for 10 min at and °C. The phase was and to Triton by mm Following and to the protein was from the phase by of at and on ice for 30 min and then at for 30 min at in a The was and the was for 2 The was in mm containing and to a in the The was with a gradient of in mm which was using a alkaline phosphatase assay at approximately after a for more than of The purified protein had a specific activity of approximately less than the specific activity for purified alkaline phosphatase the anchor S.M. Eur. J. Biochem. PubMed Scopus Google Scholar). containing of lipid in the and were were and then in 2 ml of mm Liposomes containing sphingomyelin were at °C, while the others were at Liposomes were and liposomes were a using a The sphingomyelin liposomes were at °C, and the others were at was to a procedure J. Biol. Chem. Full Text PDF PubMed Google Scholar). was using a determine concentrations for of GPI-anchored proteins into a was used Eur. J. Biochem. 1999; PubMed Scopus Google Scholar). The of liposomes at was while increasing the concentration of the at to This concentration mm for mm for and 30 mm for was for GPI-anchored protein of PLAP was incubated with 500 μl of mm This was against mm at and or to and Liposomes were then over a in mm to PLAP and were performed on liposomes of the was at 37 using a The was at and the was at was used for of of were to ml of mm aerolysin was to a concentration of at 1 were by J. Biol. Chem. Full Text PDF PubMed Google and were with proaerolysin, by and to our procedure K.L. Raja S.M. Buckley J.T. J. Biol. Chem. 1997; 272: 12170-12174Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). They were then by of the of lipid rafts is their in some nonionic detergents (5.Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar, 7.Schroeder R. London E. Brown D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar). The and proteins are thought to associated in Triton and the can be from other cell components by in density (5.Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar). proaerolysin binds with GPI-anchored proteins, which with the some proaerolysin be found in the fraction after extraction of cells that have been with the an was using cells and presented as evidence that rafts channel formation by aerolysin (25.Abrami L. van der Goot F.G. J. Cell Biol. 1999; 147: 175-184Crossref PubMed Scopus (138) Google Scholar). The in show that extraction of EL4 cells with proaerolysin also to a detergent-insoluble to rafts, that some of the all of the protein was with the Some at the of the indicating that it was extracted with and not similar of the GPI-anchored protein Thy-1 was not This is consistent with the that only a fraction of the plasma GPI-anchored proteins are localized in rafts at any It was not possible to a direct comparison of our with those obtained with since in that gradient were to the amount of aerolysin associated with the fraction in the (25.Abrami L. van der Goot F.G. J. Cell Biol. 1999; 147: 175-184Crossref PubMed Scopus (138) Google Scholar). The fact that extraction of cells that had been incubated with aerolysin in the of some of the toxin in a detergent-insoluble fraction is not evidence that there is a and significant of the toxin with rafts in Thus, when aerolysin was after the cells had been with Triton the amount in the low density detergent-insoluble fraction did not from the amount in this fraction when the cells had been with the toxin before extraction and is evidence that the detergent-insoluble or lipid rafts, can be by the cholesterol of the This can be by treating cells with the (27.Ilangumaran S. Hoessli D.C. Biochem. J. 1998; 335: 433-440Crossref PubMed Scopus (401) Google Scholar). of EL4 cells with 10 mm for 30 min to a in cholesterol levels and a change in the amount of Thy-1 in the detergent-insoluble fraction not evidence that rafts had been of the treated and cells using a labeled toxin that cholesterol extraction had or no effect on aerolysin binding This was not since there is no to that there be any in the binding of the protein to GPI-anchored proteins whether they are in rafts or on the of the membrane the in show that the containing less cholesterol and detergent-insoluble GPI-anchored were no less sensitive to the toxin than the does not affect the sensitivity of T lymphocytes to aerolysin. Cell was before and after by shown is the sensitivity of erythrocytes to aerolysin The of cell of erythrocytes was as described D.B. Nelson K.L. Lawrence T.S. B. Buckley J.T. Molec. Microbiol. 1999; PubMed Scopus Google Scholar). cell was as described We also studied the effect of cholesterol on the sensitivity of human are lysed by aerolysin at concentrations similar to those that EL4 cells they contain aerolysin-binding GPI-anchored This is not since aerolysin binds to the which has a from cell to cell and to Treatment with mm for min of the erythrocyte cholesterol, a higher than is from other cell because erythrocyte cholesterol is associated with the plasma the extraction of the from the cells did not lower the rate at which they were lysed by aerolysin the fact that the amount of raft-associated GPI-anchored if have been The no support to the that lipid rafts have an to in channel formation by aerolysin. However, it unlikely that the erythrocytes used to the in contain any significant after extraction of of their cholesterol, we could not the possibility that a In to membranes rafts with membranes that contain a we to containing a GPI-anchored protein that acts as an aerolysin receptor. We have shown that Thy-1 is an receptor for aerolysin when it is incorporated into however, for the present we wished to a protein that could be more so we if GPI-anchored placental alkaline phosphatase could for The in show that the of aerolysin to liposomes containing incorporated PLAP in whereas liposomes without PLAP were not by this concentration of the toxin. We obtained a similar with liposomes containing incorporated Thy-1 K.L. Raja S.M. Buckley J.T. J. Biol. Chem. 1997; 272: 12170-12174Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). shown that PLAP is an aerolysin receptor, we then incorporated the enzyme into liposomes of lipid of the PLAP in the was detergent-insoluble, evidence for the presence of rafts, whereas all of the enzyme in liposomes was in Triton made with half as had approximately half as detergent-insoluble PLAP not The ability to liposomes with and without rafts to determine whether or not these lipid have any effect on aerolysin The of channel formation by aerolysin were compared in liposomes raft-associated PLAP or containing or of the detergent-insoluble It may be that there were only in the rate of of the from the liposomes containing of PLAP was actually than from the liposomes sphingomyelin The similar of PLAP to the was were obtained when lower concentrations of aerolysin were used not Although the in this no direct on the or of lipid rafts in cell they show that if these they have or no influence on the rate of channel formation by aerolysin, in cells or in The is not for if rafts did the rate of aerolysin oligomerization, we would not to an effect on cells oligomerization the rate-limiting step in channel formation. binding is and there is no to that GPI-anchored proteins in rafts would bind the toxin any than when they are on the cell In the in 2 show that binding is not by it is not if oligomerization were the rate-limiting step in channel formation, it would more if the were in GPI-anchored proteins could be less when they in these because of the increased levels of saturated found there (7.Schroeder R. London E. Brown D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar). The mobility could lower the rate of oligomerization of the bound toxin. In any case, GPI-anchored proteins are also in the bulk of the lipid they are some have more than proteins K. Dietrich C. Trends Cell Biol. 1999; 9: 87-91Abstract Full Text Full Text PDF PubMed Scopus (378) Google M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (803) Google Scholar). They to in rafts as they the cell at any only a fraction is thought to be localized in rafts K. Dietrich C. Trends Cell Biol. 1999; 9: 87-91Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, R. K. Biochemistry. 1997; PubMed Scopus Google Scholar). The we obtained with erythrocytes extracted with to on the of cholesterol in channel formation by aerolysin. has been a recent that aerolysin is when with cholesterol, which was as evidence that the toxin can with the Infect. Immun. 1997; PubMed Google Scholar). we had shown that aerolysin is of channels in indicating that it has no for the Buckley J.T. Biochemistry. PubMed Scopus Google Scholar). we found that all of the cholesterol from the cell did not lower the rate of channel formation. Thus, it would that cholesterol no significant in the of aerolysin. In the ability of aerolysin to bind to GPI-anchored proteins may a to the and of lipid rafts, but there is no to that these are in the of channel formation by the toxin. We and for The of is

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