Membrane permeabilization induced by sphingosine: effect of negatively charged lipids - PubMed (original) (raw)

Membrane permeabilization induced by sphingosine: effect of negatively charged lipids

Noemi Jiménez-Rojo et al. Biophys J. 2014.

Abstract

Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease.

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Figures

Figure 1

Effect of vesicle aqueous contents efflux induced by sphingosine on electrically neutral and negatively charged LUV. (A) Time course of 15 mol % sphingosine-induced vesicle leakage. Vesicle composition was SM/PE/Ch (2:1:1) with (black trace) or without (gray trace) 5 mol % PA. (B) Extent of leakage under steady-state conditions. ( formula image ) SM/PE/Ch (2:1:1) (▪) SM/PE/Ch (2:1:1) + 5 mol % PA. (C) Time course of leakage. Early stages of sphingosine-induced efflux of vesicular contents. Vesicle efflux induced by 15 mol % sphingosine. (D) Rate constants of vesicle efflux kinetics induced by increasing sphingosine concentrations. The PA line was fitted to a monoexponential curve (r2 = 0.984). Inset: rates in the absence of PA, _Y_-axis expanded.

Figure 2

Fluorescence lifetime data of sphingosine-induced membrane leakage. 30 _μ_M LUV, 2 h incubation. The data correspond to the lifetime of free calcein (≈4 ns) and entrapped calcein (≈0.4 ns) (A) LUVs composed of SM/PE/Ch (2:1:1). (B) LUVs composed of SM/PE/Ch (2:1:1) + 5 mol % PA.

Figure 3

Confocal fluorescence microscopy images of GUVs after 2 h incubation in buffer containing BODIPY 492/515. (A) SM/PE/Ch (2:1:1) + buffer. (B) SM/PE/Ch (2:1:1) + 1.4 _μ_M sphingosine. (C) Assay of sphingosine-induced influx of BODIPY into GUV (t = 2 h). Black bars: SM/PE/Ch (2:1:1), gray bars: SM/PE/Ch (2:1:1) + 5 mol % PA. Average values ±S.D. of 90 vesicles. To see this figure in color, go online.

Figure 4

The distribution of GUVs as a function of the degree of filling after 2 h incubation with sphingosine is shown in GUVs made of (A) SM/PE/Ch (2:1:1) and (B) SM/PE/Ch (2:1:1) + 5% PA. Sphingosine concentration: ( formula image ) 0 _μ_M, (X) 0.2 _μ_M, (♦) 0.6 _μ_M, (✚) 1 _μ_M, (×) 1.3 _μ_M. Average values ± SD of 90 vesicles.

Figure 5

31P-NMR spectra as a function of temperature of the mixture SM/PE/Ch (2:1:1) + 20 mol % sphingosine. (A) Without PA and (B) with 20 mol % PA.

Figure 6

31P-RMN spectra as a function of temperature of the mixture SM/PE/Ch (2:1:1) + 20 mol % sphingosine. (A) Without PS and (B) with 20 mol % PS.

Figure 7

Small angle x-ray scattering of aqueous dispersions of SM/PE/Ch (2:1:1) + 20 mol % sphingosine, (A) without PA and (B) with 20 mol % PA.

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Membrane permeabilization induced by sphingosine: effect of negatively charged lipids - PubMed (original) (raw)