Interaction of 3β-amino-5-cholestene with phospholipids in binary and ternary bilayer membranes

Abstract

3β-Amino-5-cholestene (aminocholesterol) is a synthetic sterol whose properties in bilayer membranes have been examined. In fluid palmitoyl sphingomyelin (PSM) bilayers, aminocholesterol and cholesterol were equally effective in increasing acyl chain order, based on changes in diphenylhexatriene (DPH) anisotropy. In fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, aminocholesterol ordered acyl chains, but slightly less efficiently than cholesterol. Aminocholesterol eliminated the PSM and DPPC gel-to-liquid crystalline phase transition enthalpy linearly with concentration, and the enthalpy approached zero at 30 mol % sterol. Whereas cholesterol was able to increase the thermostability of ordered PSM domains in a fluid bilayer, aminocholesterol under equal conditions failed to do this, suggesting that its interaction with PSM was not as favorable as cholesterols. In ternary mixed bilayers, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), PSM or DPPC, and cholesterol at proportions to contain a liquid-ordered phase (60:40 by mol of POPC and PSM or DPPC, and 30 mol % cholesterol), the average lifetime of trans-parinaric acid (tPA) was close to 20 ns. When cholesterol was replaced with aminocholesterol in such mixed bilayers, the average lifetime of tPA was only marginally shorter (about 18 ns). This observation, together with acyl chain ordering data, clearly shows that aminocholesterol was able to form a liquid-ordered phase with saturated PSM or DPPC. We conclude that aminocholesterol should be a good sterol replacement in model membrane systems for which a partial positive charge is deemed beneficial.

Figure 1

Steady-state DPH anisotropy in PSM bilayers with increasing amounts of either cholesterol or aminocholesterol. The final phospholipid lipid concentration was 50 μM with DPH present at 1 mol%. Sterol concentration is indicated (panel A for cholesterol and panel B for aminocholesterol). Temperature was ramped at 5°C/min. Curves are representative from three similar curves.

Figure 2

Steady-state DPH anisotropy in DPPC bilayers with increasing amounts of either cholesterol or aminocholesterol. The final phospholipid lipid concentration was 50 μM with DPH present at 1 mol%. Sterol concentration is indicated (panel A for cholesterol and panel B for aminocholesterol). Temperature was ramped at 5°C/min. Curves are representative from three similar curves.

Figure 3

DSC thermograms of PSM or DPPC in the presence of different concentration of sterol. The phospholipid concentration was 1 mM, and aminocholesterol was included at indicated concentrations (mol%) in PSM (left panel) or DPPC (right panel) multilamellar vesicles. The temperature scanning rate was 1°C/min. Peak temperature or T_m is indicated in the figure above each relevant trace. The insert plot shows calculated molar enthalpies as a sterol function for each of the two phospholipids. Values are averages from integration of at least two endotherms for each composition, with estimated S.E.M being less than ±5 %.

Figure 4

c-Laurdan emission from PSM or DPPC multilamellar vesicles containing increasing amounts of sterol. The calculated GP (I₄₄₀-I₄₈₀ nm/I₄₄₀ + I₄₈₀ nm) is shown as a function of sterol concentration for indicated samples. Panel A shows data for 36°C (gel phase), whereas panel B shows data for 46°C (liquid-crystalline phase).

Figure 5

Solubilization of bilayer membranes with Triton X-100 at 23°C. Unilamellar vesicles were prepared by extrusion through 200 nm filters to a final concentration of 0.25 mM. Triton X-100 was added from a 5 mM solution in 5 μl aliquots to the vesicle solution with constant stirring, and the light scattering from vesicles was followed at 300 nm. Scattering intensity is plotted against the detergent/lipid molar ratio for PSM (left panel) or DPPC (right panel) vesicles. The sterol concentration was 30 mol%. Curves are representative from at least two different solubilization experiments for each composition

Figure 6

Quenching of DPH fluorescence in ternary bilayers. The fluid lipid was POPC (60 mol%) and the ordered lipid either PSM (30 mol%; left panel) or DPPC (30 mol%; right panel). The sterol (10 mol%) was either cholesterol or aminocholesterol. For other details, see materials and methods. Curves are representative from at least three different experiments for each bilayer composition.

Figure 7

Life-time analysis of tPA in ternary bilayers. The lipid mixtures contained a 60:40 molar ratio of POPC/PSM or POPC/DPPC with sterol added to different concentrations. tPA was added to 1 mol%. For other details, see materials and methods. Each value is the average of three determinations ± SEM.

Scheme 1

Molecular structures of some relevant sterols. Cholesterol (cholest-5-en-3β-ol), ergosterol (ergosta-5,7,22-trien-3β-ol), sitosterol (stigmast-5-en-3β-ol), cholesterol sulfate (cholest-5-en-3β-sulfate), aminocholesterol (cholest-5-en-3β-amine).