Marco Colombini

THE MOLECULAR BASIS FOR THE STRUCTURE AND DYNAMICS OF MEMBRANE CHANNELS

We are studying the unique ability of a lipid, ceramide, to form large stable channels in phospholipid membranes and in the outer membrane of mitochondria. The channels are tens on nanometers in diameter and only 5 nm thick. They are capable of allowing proteins to cross membranes. They are implicated in a key decision-making step in a process of programmed cell death, apoptosis. This key step is the release of proteins from the mitochondrial intermembrane space, an irreversible step that initiates the execution phase of apoptosis.
Biophysical Questions: Ceramide channels are in dynamic equilibrium with ceramide monomers or aggregates in the membrane. Hundreds of monomers self-assemble into a stable channel (Fig. 1). What features of the ceramide molecules are required for this self-assembly? How can one probe the structure of a channel in dynamic equilibrium? Ceramide channels form easily in the mitochondrial outer membrane but not in the plasma membrane of cells. What determines this specificity? Proteins that regulate apoptosis (the Bcl-2 family) can also favor or disfavor the formation of ceramide channels. Anti-apoptotic proteins (inhibit apoptosis) favor ceramide channel disassembly. Evidence indicates that they do so by forming a 1:1 complex with the channel. They apparently shift the thermodynamic equilibrium between ceramides in the channel and ceramide monomers or aggregates. How can a small protein (20 kDa) shift this equilibrium? Does if confer on the channel a structural stress that is propagated throughout the structure in an allosteric manner? Pro-apoptotic proteins work synergistically with ceramide to form large channels. What does this combined channel look like?
Experimental Approaches: Channel formation and dynamics is studied at the single-channel level by means of electrophysiological recordings. Ceramide channels are allowed to form and grow in planar phospholipid membranes. Changes in conductance are monitored continuously allowing the experimenter to observe the smallest changes in structure. Channel formation in isolated mitochondria is monitored by the rate at which exogenously-added cytochrome c is oxidized by cytochrome oxidase located in the outer surface of the inner membrane. The initial rate of oxidation of cytochrome c is a measure of the permeability of the outer membrane to this protein. Alternatively, the release of proteins from the intermembrane space is measured. Ceramide-induced permeabilization of liposomes is monitored by a fluorescence dequenching assay. Ceramide channels formed in liposomes can be observed by electron microscopy. Changes in the size of liposomes resulting from the insertion of ceramide can be monitored by dynamic light scattering.

Some Relevant Publications:
Stiban, J., Fistere Jr., D., and Colombini, M. 2006. Dihydroceramide hinders ceramide channel formation: implications on apoptosis. Apoptosis 11:773-780.
Elrick, M.J., Fluss, S. and Colombini, M. 2006. Sphingosine, a product of ceramide hydrolysis by ceramidase, disassembles ceramide channels. Biophysical Journal 91: 1749-1756.
Stiban J., Caputo L. and Colombini M. 2008. Ceramide synthesis in the endoplasmic reticulum can permeabilize mitochondria to pro-apoptotic proteins. Journal of Lipid Research 49: 625-634.
Siskind L.J., Feinstein, L., Yu, T., Davis, J.S., Jones, D., Choi, J., Zuckerman, J.E., Tan, W., Hill, R.B., Hardwick, J.M. and Colombini, M. 2008. Anti-apoptotic Bcl-2 family proteins disassemble ceramide channels. Journal of Biological Chemistry 283: 6622-6630.
Figure Legends:

Figure 1: A model of a typical ceramide channel composed of 48 columns. The crystal structure of cytochrome c is shown in the center of the aqueous pore.