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The Lipids C2- and C16-Ceramide Form Large Stable Channels

2000; Elsevier BV; Volume: 275; Issue: 49 Linguagem: Inglês

10.1074/jbc.c000587200

1083-351X

Leah J. Siskind, Marco Colombini,

Neonatal Health and Biochemistry

We report that physiological concentrations of both short- and long-chain ceramides, despite being lipids, form large stable pores in membranes. Some of these pores should be large enough to allow cytochrome c to permeate. Dihydroceramide differs from ceramide by the reduction of one double bond, and yet both its apoptogenic and channel-forming activities are greatly reduced. A structural model provides insight into how ceramides might form pores. According to a mathematical model, both the individual conductance of the channels and the overall membrane conductance are directly related to the overall concentration of ceramide in the membrane. Slight changes in concentration have dramatic effects on the size of the channels formed, providing an easy way for rapidly altering membrane permeability by changing the activity of local synthetic and catabolic enzymes. A possible role for these channels in apoptosis is discussed. We report that physiological concentrations of both short- and long-chain ceramides, despite being lipids, form large stable pores in membranes. Some of these pores should be large enough to allow cytochrome c to permeate. Dihydroceramide differs from ceramide by the reduction of one double bond, and yet both its apoptogenic and channel-forming activities are greatly reduced. A structural model provides insight into how ceramides might form pores. According to a mathematical model, both the individual conductance of the channels and the overall membrane conductance are directly related to the overall concentration of ceramide in the membrane. Slight changes in concentration have dramatic effects on the size of the channels formed, providing an easy way for rapidly altering membrane permeability by changing the activity of local synthetic and catabolic enzymes. A possible role for these channels in apoptosis is discussed. N-acetyl-d-erythro-sphingosine N-hexadecyl-d-erythro-sphingosine N-octadecyl-d-erythro-sphingosine N-acetyl-d-erythro-sphinganine N-octadecyl-d- erythro-sphinganine 4-morpholineethanesulfonic acid nanosiemens Ceramide, a sphingosine-based lipid second messenger, is known to be involved in the regulation of several cellular responses to extracellular stimuli, including differentiation, growth suppression, cell senescence, and apoptosis (1Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (725) Google Scholar, 2Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar, 3Ariga T. Jarvis W.D. Yu R.K. J. Lipid Res. 1998; 39: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar). The role of ceramide specifically in apoptosis has attracted great attention in recent years. Ceramide is generated within the cell via the hydrolysis of sphingomyelin or de novo synthesis. A net increase in ceramide levels within the cell is observed in response to several inducers of cellular stress (1Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (725) Google Scholar, 2Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar, 3Ariga T. Jarvis W.D. Yu R.K. J. Lipid Res. 1998; 39: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar). The contents of ceramide in the membranes of cells undergoing apoptosis are thought to reach 10 mol % of the total phospholipid (2Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar). The wide range biological effects of ceramide, which depend on cell type, receptors involved, sub-cellular location, and concentration, suggest the existence of several downstream targets for distinct intracellular pathways.Mitochondria have been shown to play a major regulatory role in cell death via apoptosis (4Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (655) Google Scholar, 5Crompton M. Biochem. J. 1999; 342: 233-249Crossref Google Scholar, 6Kroemer G. Dallaporta B. Resche-Rigon M. Annu. Rev. Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1748) Google Scholar, 7Susin S.A. Zamzami N. Kroemer G. Biochim. Biophys. Acta. 1998; 1366: 151-165Crossref PubMed Scopus (755) Google Scholar, 8Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). A key feature is the release of mitochondrial proteins, including apoptosis-inducing factor, cytochrome c, procaspases, and heat shock proteins from the intermembrane space of mitochondria to the cytoplasm (4Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (655) Google Scholar, 5Crompton M. Biochem. J. 1999; 342: 233-249Crossref Google Scholar, 6Kroemer G. Dallaporta B. Resche-Rigon M. Annu. Rev. Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1748) Google Scholar, 7Susin S.A. Zamzami N. Kroemer G. Biochim. Biophys. Acta. 1998; 1366: 151-165Crossref PubMed Scopus (755) Google Scholar, 9Narula J. Pandey P. Arbustini E. Haider N. Narula N. Kolodgie F.D. Dal Bello B. Semigran M.J. Bielsa-Masdeu A. Dec G.W. Israels S. Ballester M. Virmani R. Saxna S. Kharbanda S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8144-8149Crossref PubMed Scopus (515) Google Scholar). The release of intermembrane space proteins into the cytoplasm is crucial for the activation of specific caspases and DNases with apoptosis as a final result. Increased ceramide levels and exogenously added cell-permeable ceramide analogues have been reported to have an effect on various aspects of mitochondrial function.N-Acetyl-d-erythro-sphingosine (C2-ceramide)1has been shown to induce cytochrome c release when added to whole cell cultures (10Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsh T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1420) Google Scholar, 11Castedo M. Hirsch T. Susin S.A. Zamzami N. Marchetti P. Macho A. Kroemer G. J. Immunol. 1996; 157: 512-521PubMed Google Scholar, 12Susin S.A. Zamizami N. Castedo M. Daugas E. Wang E.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 25-37Crossref PubMed Scopus (587) Google Scholar, 13Susin S.A. Zamzami N. Larochette N. Dallaporta B. Marzo I. Brenner C. Hirsch T. Petit P.X. Geuskens M. Kroemer G. Exp. Cell. Res. 1997; 236: 397-403Crossref PubMed Scopus (75) Google Scholar, 14DeMaria R. Lenti L. Malisan F. d'Agostino F. Tomasini B. Zeuner A. Rippo M.R. Testi R. Science. 1997; 277: 1652-1655Crossref PubMed Scopus (374) Google Scholar) and isolated mitochondria (15Arora A.S. Jones B.J. Patel T.C. Bronk S.F. Gores G.J. Hepatology. 1997; 25: 958-963Crossref PubMed Scopus (147) Google Scholar, 16Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (200) Google Scholar, 17Ghafourifar P. Klein S.D. Schucht O. Schenk U. Pruschy M. Rocha S. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). This cytochrome c release can be prevented by preincubation with or overexpression of the anti-death protein, Bcl-2 (18Zhang J. Alter N. Reed J.C. Borner C. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5325-5328Crossref PubMed Scopus (293) Google Scholar), or transfection of cells with Bcl-xL (19Gottschalk A. Boise L. Thompson C. Quintáns J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7350-7354Crossref PubMed Scopus (254) Google Scholar, 20Wiesner D.A. Kilkus J.P. Gottschalk A.R. Quintáns J. Dawson G. J. Biol. Chem. 1997; 272: 9868-9876Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In addition, solubilized long-chain C16-ceramide has been shown to induce a large release of cytochrome c when added to mitochondrial suspensions (16Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (200) Google Scholar). Additional effects of ceramide analogues include enhanced generation of reactive oxygen species (10Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsh T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1420) Google Scholar, 21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 22Quillet-Mary A. Jaffrézou J. Mansat V. Bordier C. Naval J. Laurent G. J. Biol. Chem. 1997; 272: 21388-21395Crossref PubMed Scopus (443) Google Scholar), alteration of mitochondrial calcium homeostasis (10Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsh T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1420) Google Scholar, 21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 22Quillet-Mary A. Jaffrézou J. Mansat V. Bordier C. Naval J. Laurent G. J. Biol. Chem. 1997; 272: 21388-21395Crossref PubMed Scopus (443) Google Scholar), and ATP depletion followed by collapse in the inner mitochondrial membrane potential (Δψm) (10Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsh T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1420) Google Scholar, 17Ghafourifar P. Klein S.D. Schucht O. Schenk U. Pruschy M. Rocha S. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). In addition, ceramide has been shown to destabilize membranes and cause leakage, fusion, and budding of vesicles (23Begona Ruiz-Arguello M. Basanez G. Goni F.M. Alonoso A. J. Biol. Chem. 1996; 271: 26616-26621Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 24Begona Ruiz-Arguello M. Goni F.M. Alonoso A. J. Biol. Chem. 1998; 273: 22977-22982Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 25Holopainen J.M. Angelova M.I. Kinnunen P.K.J. Biophys. J. 2000; 78: 830-838Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 26Veiga M.P. Arrondo J.L.R. Goni F.M. Alonso A. Biophys. J. 1999; 76: 342-350Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Ceramides have been reported to mix poorly with phospholipids in bilayers, form distinct ceramide-enriched microdomains, favor the stability of the gel over the lamellar phase, and facilitate the lamellar-hexagonal transition of phospholipids (26Veiga M.P. Arrondo J.L.R. Goni F.M. Alonso A. Biophys. J. 1999; 76: 342-350Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Di Paola et al. (16Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (200) Google Scholar) recently reported that C2-ceramide, when added to liposomes, causes a collapse in the membrane potential generated by reconstituted complex III of the mitochondrial electron transport chain. Their results indicate that C2-ceramide can somehow cause an increase in the permeability of membranes.Although ceramides have been demonstrated to increase the permeability of membranes, the means by which they do so remains unclear. We therefore tested the ability of C2- and C16-ceramides to form channels in membranes lacking proteins, utilizing the planar membrane technique (27Montal M. Mueller P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3561-3566Crossref PubMed Scopus (1579) Google Scholar) as modified (28Colombini M. J. Membr. Biol. 1989; 111: 103-111Crossref PubMed Scopus (253) Google Scholar). Here we show that both short- and long-chain ceramides can form large stable channels in membranes and have provided both structural and mathematical models to provide insight as to how they might do so. As far as we are aware, this is the first report of stable pore formation by a lipid in a membrane. The implication for a role of these ceramide channels in apoptosis is discussed.DISCUSSIONThe results of this paper provide evidence that both C2- and C16-ceramide form large stable pores in membranes, whereas the biologically inactive C2- and C18-dihydroceramides do not. The inability of other amphiphiles, including detergents, phospholipids, and products of phospholipase activity, to form stable channels may reside in their inability to form an extensive hydrogen-bonded network. In our model, hydrogen bonding stabilizes not only columns of ceramide molecules but also the polar ring that forms the wall of the aqueous pore.Channel formation by C2- and C16-ceramides explains their ability to increase the permeability of membranes. The pores formed by both short- and long-chain ceramides could be directly or indirectly related to their apoptotic activity. According to the bulk properties of water, the pores with conductances ranging from 1 to 200 nS would have estimated diameters ranging from 0.8 to 11 nm, respectively. The voltage-dependent anion channel, located in the outer mitochondrial membrane, is considered to be a relatively large pore. In the high conductance state, it only has a single channel conductance of 4 nS (in the equivalent solution) and cannot allow the passage of cytochrome c (30Colombini M. Methods Enzymol. 1987; 148: 465-475Crossref PubMed Scopus (52) Google Scholar). The larger ceramide pores, if formed in the outer mitochondrial membrane, would provide a pathway large enough for cytochrome c to pass. Alternatively, the ceramide pores could indirectly cause cytochrome c release through alterations in ion homeostasis (increased calcium levels alone are known to induce cytochrome c release) or through interactions with Bcl-2 family members. In addition, if ceramide pores formed in the inner mitochondrial membrane, they would dissipate Δψm and could accelerate electron transport, resulting in enhanced generation of reactive oxygen species. Results using C2- and C16-ceramides applied to mitochondrial suspensions indicated that only C2-ceramide was able to dissipate the inner mitochondrial membrane potential (16Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (200) Google Scholar). It has been reported that the relative proportion of ceramide and phospholipids plays a large role in the formation of ceramide-enriched microdomains and the way in which ceramides perturb the structure of bilayers (26Veiga M.P. Arrondo J.L.R. Goni F.M. Alonso A. Biophys. J. 1999; 76: 342-350Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 31Huang H. Goldberg E.M. Zidovetzki R. Eur. Biophys. J. 1998; 27: 361-366Crossref PubMed Scopus (47) Google Scholar). In addition, the 14 extra CH2 groups of C16-ceramide would make it much more difficult for the long-chain ceramides to move between the outer and inner mitochondrial membranes through the intervening aqueous phase. Using an estimated 3 kJ/mol/CH2 residue, the free energy difference between the hydrocarbon phase and water would increase by 42 kJ/mol, which translates to a change in partitioning of 3 × 107. Therefore, natural ceramides generated via de novo synthesis would exert their effects on local membrane fractions depending on the location of generation resulting in effects that would differ from those elicited by the more soluble ceramides.Ceramide channels consist of hundreds of ceramide molecules, hence their formation and size would be exceedingly sensitive to the local ceramide concentration. Garcı́a-Ruiz et al. (21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar) reported that mitochondria isolated from cells treated with tumor necrosis factor showed a 2–3-fold increase in mitochondrial ceramide concentrations as compared with control cells (21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). They showed that this ceramide is not locally produced within mitochondria by action of sphingomyelinases acting on sphingomyelin, as incubation of isolated mitochondria with sphingomyelinases did not result in an increase in mitochondrial ceramide levels (21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). Enzymes capable of both de novo synthesis (ceramide synthase) and hydrolysis (ceramidase) have been found in mitochondria (32Shimeno H. Soeda S. Sakamoto M. Kouchi T. Kowakame T. Kihara T. Lipids. 1998; 33: 601-605Crossref PubMed Scopus (85) Google Scholar, 33El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Thus the dynamic channel formation and breakdown seen in vitro should also occurin vivo. These could increase or decrease local levels of ceramide, and even small changes could alter mitochondrial membrane permeability by channel formation or dissolution. Overall ceramide levels are known to increase during apoptosis, and thus regulation of the enzymes in the ceramide pathway is somehow involved in the apoptotic process. Further characterization of the pores formed by ceramide is underway and will hopefully provide answers to some of these questions. Ceramide, a sphingosine-based lipid second messenger, is known to be involved in the regulation of several cellular responses to extracellular stimuli, including differentiation, growth suppression, cell senescence, and apoptosis (1Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (725) Google Scholar, 2Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar, 3Ariga T. Jarvis W.D. Yu R.K. J. Lipid Res. 1998; 39: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar). The role of ceramide specifically in apoptosis has attracted great attention in recent years. Ceramide is generated within the cell via the hydrolysis of sphingomyelin or de novo synthesis. A net increase in ceramide levels within the cell is observed in response to several inducers of cellular stress (1Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (725) Google Scholar, 2Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar, 3Ariga T. Jarvis W.D. Yu R.K. J. Lipid Res. 1998; 39: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar). The contents of ceramide in the membranes of cells undergoing apoptosis are thought to reach 10 mol % of the total phospholipid (2Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar). The wide range biological effects of ceramide, which depend on cell type, receptors involved, sub-cellular location, and concentration, suggest the existence of several downstream targets for distinct intracellular pathways. Mitochondria have been shown to play a major regulatory role in cell death via apoptosis (4Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (655) Google Scholar, 5Crompton M. Biochem. J. 1999; 342: 233-249Crossref Google Scholar, 6Kroemer G. Dallaporta B. Resche-Rigon M. Annu. Rev. Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1748) Google Scholar, 7Susin S.A. Zamzami N. Kroemer G. Biochim. Biophys. Acta. 1998; 1366: 151-165Crossref PubMed Scopus (755) Google Scholar, 8Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). A key feature is the release of mitochondrial proteins, including apoptosis-inducing factor, cytochrome c, procaspases, and heat shock proteins from the intermembrane space of mitochondria to the cytoplasm (4Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (655) Google Scholar, 5Crompton M. Biochem. J. 1999; 342: 233-249Crossref Google Scholar, 6Kroemer G. Dallaporta B. Resche-Rigon M. Annu. Rev. Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1748) Google Scholar, 7Susin S.A. Zamzami N. Kroemer G. Biochim. Biophys. Acta. 1998; 1366: 151-165Crossref PubMed Scopus (755) Google Scholar, 9Narula J. Pandey P. Arbustini E. Haider N. Narula N. Kolodgie F.D. Dal Bello B. Semigran M.J. Bielsa-Masdeu A. Dec G.W. Israels S. Ballester M. Virmani R. Saxna S. Kharbanda S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8144-8149Crossref PubMed Scopus (515) Google Scholar). The release of intermembrane space proteins into the cytoplasm is crucial for the activation of specific caspases and DNases with apoptosis as a final result. Increased ceramide levels and exogenously added cell-permeable ceramide analogues have been reported to have an effect on various aspects of mitochondrial function.N-Acetyl-d-erythro-sphingosine (C2-ceramide)1has been shown to induce cytochrome c release when added to whole cell cultures (10Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsh T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1420) Google Scholar, 11Castedo M. Hirsch T. Susin S.A. Zamzami N. Marchetti P. Macho A. Kroemer G. J. Immunol. 1996; 157: 512-521PubMed Google Scholar, 12Susin S.A. Zamizami N. Castedo M. Daugas E. Wang E.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 25-37Crossref PubMed Scopus (587) Google Scholar, 13Susin S.A. Zamzami N. Larochette N. Dallaporta B. Marzo I. Brenner C. Hirsch T. Petit P.X. Geuskens M. Kroemer G. Exp. Cell. Res. 1997; 236: 397-403Crossref PubMed Scopus (75) Google Scholar, 14DeMaria R. Lenti L. Malisan F. d'Agostino F. Tomasini B. Zeuner A. Rippo M.R. Testi R. Science. 1997; 277: 1652-1655Crossref PubMed Scopus (374) Google Scholar) and isolated mitochondria (15Arora A.S. Jones B.J. Patel T.C. Bronk S.F. Gores G.J. Hepatology. 1997; 25: 958-963Crossref PubMed Scopus (147) Google Scholar, 16Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (200) Google Scholar, 17Ghafourifar P. Klein S.D. Schucht O. Schenk U. Pruschy M. Rocha S. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). This cytochrome c release can be prevented by preincubation with or overexpression of the anti-death protein, Bcl-2 (18Zhang J. Alter N. Reed J.C. Borner C. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5325-5328Crossref PubMed Scopus (293) Google Scholar), or transfection of cells with Bcl-xL (19Gottschalk A. Boise L. Thompson C. Quintáns J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7350-7354Crossref PubMed Scopus (254) Google Scholar, 20Wiesner D.A. Kilkus J.P. Gottschalk A.R. Quintáns J. Dawson G. J. Biol. Chem. 1997; 272: 9868-9876Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In addition, solubilized long-chain C16-ceramide has been shown to induce a large release of cytochrome c when added to mitochondrial suspensions (16Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (200) Google Scholar). Additional effects of ceramide analogues include enhanced generation of reactive oxygen species (10Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsh T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1420) Google Scholar, 21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 22Quillet-Mary A. Jaffrézou J. Mansat V. Bordier C. Naval J. Laurent G. J. Biol. Chem. 1997; 272: 21388-21395Crossref PubMed Scopus (443) Google Scholar), alteration of mitochondrial calcium homeostasis (10Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsh T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1420) Google Scholar, 21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 22Quillet-Mary A. Jaffrézou J. Mansat V. Bordier C. Naval J. Laurent G. J. Biol. Chem. 1997; 272: 21388-21395Crossref PubMed Scopus (443) Google Scholar), and ATP depletion followed by collapse in the inner mitochondrial membrane potential (Δψm) (10Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsh T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1420) Google Scholar, 17Ghafourifar P. Klein S.D. Schucht O. Schenk U. Pruschy M. Rocha S. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). In addition, ceramide has been shown to destabilize membranes and cause leakage, fusion, and budding of vesicles (23Begona Ruiz-Arguello M. Basanez G. Goni F.M. Alonoso A. J. Biol. Chem. 1996; 271: 26616-26621Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 24Begona Ruiz-Arguello M. Goni F.M. Alonoso A. J. Biol. Chem. 1998; 273: 22977-22982Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 25Holopainen J.M. Angelova M.I. Kinnunen P.K.J. Biophys. J. 2000; 78: 830-838Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 26Veiga M.P. Arrondo J.L.R. Goni F.M. Alonso A. Biophys. J. 1999; 76: 342-350Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Ceramides have been reported to mix poorly with phospholipids in bilayers, form distinct ceramide-enriched microdomains, favor the stability of the gel over the lamellar phase, and facilitate the lamellar-hexagonal transition of phospholipids (26Veiga M.P. Arrondo J.L.R. Goni F.M. Alonso A. Biophys. J. 1999; 76: 342-350Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Di Paola et al. (16Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (200) Google Scholar) recently reported that C2-ceramide, when added to liposomes, causes a collapse in the membrane potential generated by reconstituted complex III of the mitochondrial electron transport chain. Their results indicate that C2-ceramide can somehow cause an increase in the permeability of membranes. Although ceramides have been demonstrated to increase the permeability of membranes, the means by which they do so remains unclear. We therefore tested the ability of C2- and C16-ceramides to form channels in membranes lacking proteins, utilizing the planar membrane technique (27Montal M. Mueller P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3561-3566Crossref PubMed Scopus (1579) Google Scholar) as modified (28Colombini M. J. Membr. Biol. 1989; 111: 103-111Crossref PubMed Scopus (253) Google Scholar). Here we show that both short- and long-chain ceramides can form large stable channels in membranes and have provided both structural and mathematical models to provide insight as to how they might do so. As far as we are aware, this is the first report of stable pore formation by a lipid in a membrane. The implication for a role of these ceramide channels in apoptosis is discussed. DISCUSSIONThe results of this paper provide evidence that both C2- and C16-ceramide form large stable pores in membranes, whereas the biologically inactive C2- and C18-dihydroceramides do not. The inability of other amphiphiles, including detergents, phospholipids, and products of phospholipase activity, to form stable channels may reside in their inability to form an extensive hydrogen-bonded network. In our model, hydrogen bonding stabilizes not only columns of ceramide molecules but also the polar ring that forms the wall of the aqueous pore.Channel formation by C2- and C16-ceramides explains their ability to increase the permeability of membranes. The pores formed by both short- and long-chain ceramides could be directly or indirectly related to their apoptotic activity. According to the bulk properties of water, the pores with conductances ranging from 1 to 200 nS would have estimated diameters ranging from 0.8 to 11 nm, respectively. The voltage-dependent anion channel, located in the outer mitochondrial membrane, is considered to be a relatively large pore. In the high conductance state, it only has a single channel conductance of 4 nS (in the equivalent solution) and cannot allow the passage of cytochrome c (30Colombini M. Methods Enzymol. 1987; 148: 465-475Crossref PubMed Scopus (52) Google Scholar). The larger ceramide pores, if formed in the outer mitochondrial membrane, would provide a pathway large enough for cytochrome c to pass. Alternatively, the ceramide pores could indirectly cause cytochrome c release through alterations in ion homeostasis (increased calcium levels alone are known to induce cytochrome c release) or through interactions with Bcl-2 family members. In addition, if ceramide pores formed in the inner mitochondrial membrane, they would dissipate Δψm and could accelerate electron transport, resulting in enhanced generation of reactive oxygen species. Results using C2- and C16-ceramides applied to mitochondrial suspensions indicated that only C2-ceramide was able to dissipate the inner mitochondrial membrane potential (16Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (200) Google Scholar). It has been reported that the relative proportion of ceramide and phospholipids plays a large role in the formation of ceramide-enriched microdomains and the way in which ceramides perturb the structure of bilayers (26Veiga M.P. Arrondo J.L.R. Goni F.M. Alonso A. Biophys. J. 1999; 76: 342-350Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 31Huang H. Goldberg E.M. Zidovetzki R. Eur. Biophys. J. 1998; 27: 361-366Crossref PubMed Scopus (47) Google Scholar). In addition, the 14 extra CH2 groups of C16-ceramide would make it much more difficult for the long-chain ceramides to move between the outer and inner mitochondrial membranes through the intervening aqueous phase. Using an estimated 3 kJ/mol/CH2 residue, the free energy difference between the hydrocarbon phase and water would increase by 42 kJ/mol, which translates to a change in partitioning of 3 × 107. Therefore, natural ceramides generated via de novo synthesis would exert their effects on local membrane fractions depending on the location of generation resulting in effects that would differ from those elicited by the more soluble ceramides.Ceramide channels consist of hundreds of ceramide molecules, hence their formation and size would be exceedingly sensitive to the local ceramide concentration. Garcı́a-Ruiz et al. (21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar) reported that mitochondria isolated from cells treated with tumor necrosis factor showed a 2–3-fold increase in mitochondrial ceramide concentrations as compared with control cells (21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). They showed that this ceramide is not locally produced within mitochondria by action of sphingomyelinases acting on sphingomyelin, as incubation of isolated mitochondria with sphingomyelinases did not result in an increase in mitochondrial ceramide levels (21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). Enzymes capable of both de novo synthesis (ceramide synthase) and hydrolysis (ceramidase) have been found in mitochondria (32Shimeno H. Soeda S. Sakamoto M. Kouchi T. Kowakame T. Kihara T. Lipids. 1998; 33: 601-605Crossref PubMed Scopus (85) Google Scholar, 33El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Thus the dynamic channel formation and breakdown seen in vitro should also occurin vivo. These could increase or decrease local levels of ceramide, and even small changes could alter mitochondrial membrane permeability by channel formation or dissolution. Overall ceramide levels are known to increase during apoptosis, and thus regulation of the enzymes in the ceramide pathway is somehow involved in the apoptotic process. Further characterization of the pores formed by ceramide is underway and will hopefully provide answers to some of these questions. The results of this paper provide evidence that both C2- and C16-ceramide form large stable pores in membranes, whereas the biologically inactive C2- and C18-dihydroceramides do not. The inability of other amphiphiles, including detergents, phospholipids, and products of phospholipase activity, to form stable channels may reside in their inability to form an extensive hydrogen-bonded network. In our model, hydrogen bonding stabilizes not only columns of ceramide molecules but also the polar ring that forms the wall of the aqueous pore. Channel formation by C2- and C16-ceramides explains their ability to increase the permeability of membranes. The pores formed by both short- and long-chain ceramides could be directly or indirectly related to their apoptotic activity. According to the bulk properties of water, the pores with conductances ranging from 1 to 200 nS would have estimated diameters ranging from 0.8 to 11 nm, respectively. The voltage-dependent anion channel, located in the outer mitochondrial membrane, is considered to be a relatively large pore. In the high conductance state, it only has a single channel conductance of 4 nS (in the equivalent solution) and cannot allow the passage of cytochrome c (30Colombini M. Methods Enzymol. 1987; 148: 465-475Crossref PubMed Scopus (52) Google Scholar). The larger ceramide pores, if formed in the outer mitochondrial membrane, would provide a pathway large enough for cytochrome c to pass. Alternatively, the ceramide pores could indirectly cause cytochrome c release through alterations in ion homeostasis (increased calcium levels alone are known to induce cytochrome c release) or through interactions with Bcl-2 family members. In addition, if ceramide pores formed in the inner mitochondrial membrane, they would dissipate Δψm and could accelerate electron transport, resulting in enhanced generation of reactive oxygen species. Results using C2- and C16-ceramides applied to mitochondrial suspensions indicated that only C2-ceramide was able to dissipate the inner mitochondrial membrane potential (16Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (200) Google Scholar). It has been reported that the relative proportion of ceramide and phospholipids plays a large role in the formation of ceramide-enriched microdomains and the way in which ceramides perturb the structure of bilayers (26Veiga M.P. Arrondo J.L.R. Goni F.M. Alonso A. Biophys. J. 1999; 76: 342-350Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 31Huang H. Goldberg E.M. Zidovetzki R. Eur. Biophys. J. 1998; 27: 361-366Crossref PubMed Scopus (47) Google Scholar). In addition, the 14 extra CH2 groups of C16-ceramide would make it much more difficult for the long-chain ceramides to move between the outer and inner mitochondrial membranes through the intervening aqueous phase. Using an estimated 3 kJ/mol/CH2 residue, the free energy difference between the hydrocarbon phase and water would increase by 42 kJ/mol, which translates to a change in partitioning of 3 × 107. Therefore, natural ceramides generated via de novo synthesis would exert their effects on local membrane fractions depending on the location of generation resulting in effects that would differ from those elicited by the more soluble ceramides. Ceramide channels consist of hundreds of ceramide molecules, hence their formation and size would be exceedingly sensitive to the local ceramide concentration. Garcı́a-Ruiz et al. (21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar) reported that mitochondria isolated from cells treated with tumor necrosis factor showed a 2–3-fold increase in mitochondrial ceramide concentrations as compared with control cells (21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). They showed that this ceramide is not locally produced within mitochondria by action of sphingomyelinases acting on sphingomyelin, as incubation of isolated mitochondria with sphingomyelinases did not result in an increase in mitochondrial ceramide levels (21Garcı́a-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). Enzymes capable of both de novo synthesis (ceramide synthase) and hydrolysis (ceramidase) have been found in mitochondria (32Shimeno H. Soeda S. Sakamoto M. Kouchi T. Kowakame T. Kihara T. Lipids. 1998; 33: 601-605Crossref PubMed Scopus (85) Google Scholar, 33El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Thus the dynamic channel formation and breakdown seen in vitro should also occurin vivo. These could increase or decrease local levels of ceramide, and even small changes could alter mitochondrial membrane permeability by channel formation or dissolution. Overall ceramide levels are known to increase during apoptosis, and thus regulation of the enzymes in the ceramide pathway is somehow involved in the apoptotic process. Further characterization of the pores formed by ceramide is underway and will hopefully provide answers to some of these questions. We thank Dr. Jason Kahn for computer assistance with the structural models and Dr. Stephen Wolniak for reading of the manuscript.