The Lipids C2- and C16-Ceramide Form Large Stable Channels: IMPLICATIONS FOR APOPTOSIS

Leah J Siskind,Marco Colombini

doi:10.1074/jbc.c000587200

Leah J Siskind, Marco Colombini

Open Access

https://doi.org/10.1074/jbc.c000587200

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Abstract

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.

Highlights

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 [1,2,3]
C2-Ceramide Increases Membrane Conductance by Forming Large Stable Channels—The effect of C2-ceramide on membrane conductance was monitored by utilizing the planar membrane technique [27] as modified [28]. 5 ␮M C2-ceramide, a concentration less than or equal to those used in whole cell and isolated mitochondrial suspension experiments, when added to the aqueous phase, resulted in stable pore formation in the planar phospholipid membrane
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

Summary

EXPERIMENTAL PROCEDURES

Drugs and Reagents—The following reagents were purchased from Avanti Polar Lipids: C2-ceramide, C2-dihydroceramide, C16-ceramide, C18-dihydroceramide, and asolectin (soybean phospholipids). Electrophysiological Recordings—Planar membranes were formed by the monolayer method [27], as modified [28], across a 100-␮m-diameter hole in a Saran partition using 1% (w/v) asolectin (soybean phospholipids), 0.2% (w/v) cholesterol in hexane solution. The aqueous solution contained 1 M KCl, 1 mM MgCl2, 5 mM MES (pH 6.0). The applied voltage was held constant at 9 mV. Me2SO was used as the vehicle for C2-ceramide and dihydroceramide experiments such that it was no more than 0.5% of the total volume of the aqueous solution.

The abbreviations used are

RESULTS

DISCUSSION