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Phases and domains in sphingomyelin–cholesterol membranes: structure and properties using EPR spin-labeling methods

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Abstract

EPR spin-labeling methods were used to investigate the order and fluidity of alkyl chains, the hydrophobicity of the membrane interior, and the order and motion of cholesterol molecules in coexisting phases and domains, or in a single phase of fluid-phase cholesterol/egg-sphingomyelin (Chol/ESM) membranes with a Chol/ESM mixing ratio from 0 to 3. A complete set of profiles for these properties was obtained for the liquid-disordered (l d) phase without cholesterol, for the liquid-ordered (l o) phase for the entire region of cholesterol solubility in this phase (from 33 to 66 mol%), and for the l o-phase domain that coexists with the cholesterol bilayer domain (CBD). Alkyl chains in the l o phase are more ordered than in the l d pure ESM membrane. However, fluidity in the membrane center is greater. Also, the profile of hydrophobicity changed from a bell to a rectangular shape. These differences are enhanced when the cholesterol content of the l o phase is increased from 33 to 66 mol%, with clear brake-points between the C9 and C10 positions (approximately where the steroid-ring structure of cholesterol reaches into the membrane). The organization and motion of cholesterol molecules in the CBD are similar to those in the l o-phase domain that coexists with the CBD.

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Notes

  1. A reviewer pointed out that our conclusion is in contrast with the commonly accepted statement that membrane saturation reduces membrane fluidity. This difference is clearly seen for lens lipid membranes, where the phospholipid composition changes drastically with age and with a preferential increase in saturated phospholipids, for example sphingomyelin and dihydrosphingomyelin, which should increase the lipid hydrocarbon chain order. Huang et al. (2005) showed that the structural order determined by the static measure of the trans/gauche rotamer ratio in the hydrocarbon chains increases linearly with the sphingolipid content of the lens lipid membrane (see also the review by Borchman and Yappert 2010). Thus, the properties of lens lipid membranes, including membrane order (fluidity), should change with the age of the donor, between species, and between regions of the lens. However, molecular order, measured with lipid spin labels in saturated membranes, strongly increases with an increase in cholesterol concentration up to ~30 mol%. Further increase of cholesterol concentration, up to 50 mol%, causes a decrease in the molecular order (Kusumi et al. 1986; Sankaram and Thompson 1990; Wisniewska and Subczynski 2008). In unsaturated membranes, the molecular order increases only weakly with an increase in cholesterol concentration up to 50 mol% (Kusumi et al. 1986). Thus, at saturation, both orders are very close. Similar effects were reported by Borchman et al. (1996) using the structural order parameter as a measure of membrane fluidity. The structural order parameter measured in membranes made from bovine nuclear phospholipids (more saturated membranes) increased with cholesterol concentration up to 20–30 mol%, which was then followed by a rapid decrease in the structural order parameter up to ~70 mol% cholesterol. In cortical phospholipid membranes (less saturated membranes), an increase in the structural order parameter induced by cholesterol was significantly weaker. A maximum was reached at ~40 mol% cholesterol. Further increase in cholesterol content also induced a decrease in the structural order parameter. As a result, at cholesterol saturation, the structural order parameters in nuclear and cortical membranes were very close. We should again note that the phospholipid compositions of these two membranes differ substantially. Borchman et al. concluded that the physiological role of cholesterol is to increase the structural order of cortical membrane lipids and reduce the order of nuclear lipids so that the two membranes have a similar order, which is in agreement with our main conclusion.

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Acknowledgments

This work was supported by NIH grants EY015526, TW008052, EB002052, and EB001980.

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  1. Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA

    Laxman Mainali, Marija Raguz & Witold K. Subczynski

  2. Department of Medical Physics and Biophysics, School of Medicine, University of Split, Split, Croatia

    Marija Raguz

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  1. Laxman Mainali
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Correspondence to Witold K. Subczynski.

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Mainali, L., Raguz, M. & Subczynski, W.K. Phases and domains in sphingomyelin–cholesterol membranes: structure and properties using EPR spin-labeling methods. Eur Biophys J 41, 147–159 (2012). https://doi.org/10.1007/s00249-011-0766-4

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