1.D.10 The Ceramide-forming Channel (Ceramide) Family
Ceramide, a sphingosine-based lipid second messenger in higher animals, is involved in differentiation, growth suppression, senescence and apoptosis. It arises by hydrolysis of sphingomyelin or by de novo synthesis. It is known to increase the permeability of biological membranes. Using the planar bilayer technique in the absense of proteins, it was shown that physiological concentrations of both long and short chain ceramides can form large stable channels, large enough for passage of small proteins. This is a unique case of a lipid that forms channels in a membrane. Ceramide self assembles into transmembrane ion channels in response to dynamic treatments of small ions (La3+) and proteins (Bcl-xL mutant) (Shao et al., 2011). Ceramide can be made from glucosylceramide (Liu et al. 2022).
Ceramides play a role in apoptosis in animals by inducing cytochrome c release from mitochondria. Other proteins are also released (Siskind et al., 2002). C2 and C16-ceramide but not dihydroceramide form these channels in the outer mitochondrial membrane.
Ceramide induces structural rearrangements in membrane bilayers that allow transmembrane lipid movement. This effect has been demonstrated in erythrocyte ghosts and large unilamellar vesicles (Contreras et al., 2003). In these studies, ceramide was generated in situ by adding external sphingomyelinase which hydrolyzed gangliosides. Ceramide-induced transmembrane lipid movement may be due to lamellar to non-lamellar lipid-phase transitions. Thus, ceramide on one side of the membrane generated with sphingomyelinase induces the transient formation of non-lamellar structure which causes the loss of bilayer lipid asymmetry. Both ceramides and other lipids then cross the membrane. This process may be related to transmembrane signaling (as in the sphingomyelin pathway), to specific types of bacterial internalization, to membrane fusion and to vesicle budding (Contreras et al., 2003). Bcl-2 family proteins (TC# 1.A.20) have been reported to regulate ceramide channel formation which then may mediate protein release as a prelude to apoptosis (Ganesan and Colombini, 2010).
Early in mitochondria-mediated apoptosis, the mitochondrial outer membrane becomes permeable to proteins that, when released into the cytosol, initiate the execution phase of apoptosis. Proteins in the Bcl-2 family regulate this permeabilization, but the molecular composition of the mitochondrial outer membrane pore is under debate. Ceramides form stable channels (1.D.10) in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis (Siskind et al., 2006) Mitochondrion 6, 118-125)). Bcl-2 proteins are not required for ceramide to form protein-permeable channels, but both recombinant human Bcl-x(L) and CED-9, the Caenorhabditis elegans Bcl-2 homologue, disassemble ceramide channels in the mitochondrial outer membranes. Bcl-x(L) and CED-9 disassemble ceramide channels in solvent-free planar phospholipid membranes. Thus, ceramide channel disassembly may result from direct interaction with these anti-apoptotic proteins (Siskind et al., 2008). Thus, ceramide channels may be one mechanism for releasing proteins from mitochondria during the induction phase of apoptosis.
A key, decision-making step in apoptosis is the release of proteins from the mitochondrial intermembrane space. Ceramides self-assemble in the mitochondrial outer membrane to form large stable channels capable of releasing proteins. Ceramide levels measured in mitochondria early in apoptosis are sufficient to form ceramide channels which are in dynamic equilibrium with non-conducting forms of ceramide. This equilibrium can be strongly influenced by other sphingolipids and Bcl-2 family proteins. Proteins are not required for channel formation. The channels are barrel-like structures whose staves are ceramide columns that span the membrane (Colombini, 2010).Anishkin et al. 2006 analyzed molecular models of ceramide channels and found that the structural units are columns of four to six ceramides H-bonded via amide groups and arranged as staves in either a parallel or antiparallel manner. Two cylindrical assemblies of 14 columns (four or six molecules per column) were embedded in a fully hydrated palmitoyloleoyl-phosphatidylcholine phospholipid bilayer, and the water-filled pore adopted an hourglass-like shape as headgroups of ceramides and phospholipids formed a smooth continuous interface. The structure-stabilizing interactions were both hydrogen bonds between the headgroups (including water-mediated interactions) and packing of the hydrocarbon tails. Ceramide's essential double bond reduced the mobility of the hydrocarbon tails and stabilized their packing (Anishkin et al. 2006).
The transport reactions facilitated by ceramide include:
ceramides (out) ceramides (in)
lipids (out) lipids (in)
solutes (out) solutes (in)
macromolecules (out) macromolecules (in)