Phase diagram of ternary cholesterol/palmitoylsphingomyelin/palmitoyloleoyl-phosphatidylcholine mixtures: spin-label EPR study of lipid-raft formation

Abstract

For canonical lipid raft mixtures of cholesterol (chol), N-palmitoylsphingomyelin (PSM), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), electron paramagnetic resonance (EPR) of spin-labeled phospholipids--which is insensitive to domain size--is used to determine the ternary phase diagram at 23°C. No phase boundaries are found for binary POPC/chol mixtures, nor for ternary mixtures with PSM content <24 mol %. EPR lineshapes indicate that conversion from the liquid-disordered (L(α)) to liquid-ordered (L(o)) phase occurs continuously in this region. Two-component EPR spectra and several tie lines attributable to coexistence of gel (L(β)) and fluid phases are found for ternary mixtures with low cholesterol or low POPC content. For PSM/POPC alone, coexistence of L(α) and L(β) phases occurs over the range 50-95.5 mol % PSM. A further tie line is found at 3 mol % chol with endpoints at 50 and ≥77 mol % PSM. For PSM/chol, L(β)-L(o) coexistence occurs over the range 10-38 mol % chol and further tie lines are found at 4.5 and 7 mol % POPC. Two-component EPR spectra indicative of fluid-fluid (L(α)-L(o)) phase separation are found for lipid compositions: 25%<PSM<65%, 5%<chol<30-35%, 65%>POPC>10%, and confirmed by nonlinear EPR. Tie lines are identified in the L(α)-L(o) coexistence region, indicating that the fluid domains are of sufficient size to obey the phase rule. The three-phase triangle is bounded approximately by the compositions 40 and 75 mol % PSM with 10 mol % chol, and 60 mol % PSM with 25 mol % chol. These studies define the compositions of raft-like L(o) phases for a minimal realistic biological lipid mixture.

Figure 1

Top: Conventional V₁-EPR spectra of the 14-PCSL spin label in POPC/chol binary mixtures (0 mol % PSM), for different cholesterol contents as indicated. Bottom: Dependence on cholesterol content of the hyperfine anisotropy, 2〈ΔA〉 = 2(〈A_max〉 − 〈A_min〉) (solid squares), and outer hyperfine splitting, 2〈A_max〉 (open circles), for the 14-PCSL spin label in POPC/chol membranes. T = 23°C.

Figure 2

First-derivative V₁-EPR spectra of the 14-PCSL spin label in POPC/PSM binary mixtures (0 mol % chol). With decreasing line height, the spectra correspond to 32.5, 40, 50, 60, 66, 70, 80, 90, and 95.5 mol % PSM contents. Spectra are normalized to constant second integral. In the range 40−95.5 mol % PSM, the EPR spectra form an isosbestic set; I indicates isosbestic points. w and s indicate the outer hyperfine lines of the coexisting weakly and more strongly immobilized components, respectively. T = 23°C.

Figure 3

Top: First-derivative V₁-EPR spectra of the 14-PCSL spin label in PSM/chol binary mixtures (0 mol % POPC). With decreasing line height, the spectra correspond to 38, 30, 20, and 10 mol % cholesterol contents. Spectra are normalized to a constant second integral. In the range 10−38 mol % chol, the EPR spectra form an isosbestic set. Bottom: Dependence of the normalized line height, I_o, of the central peak in the 14-PCSL EPR spectrum (see top panel) on cholesterol content for PSM/chol membranes. T = 23°C.

Figure 4

Ternary phase diagram for hydrated POPC/PSM/chol mixtures at 23°C constructed from the EPR results of this study. Symbols: , , , , , , and refer to L_α, L_o, L_β, L_α+L_o, L_α+L_β, L_o+L_β, and L_α+L_o+L_β phases, respectively. The star indicates the approximate location of a putative critical point. Heavy straight lines within the L_α+L_β, L_o+L_β, and L_α+L_o coexistence regions are tie lines. The three sides of the three-phase triangle are simultaneously tie lines in the neighboring two-phase regions.

Figure 5

Isosbestic sets of first-derivative V₁-EPR spectra from the 16-PCSL spin label in POPC/PSM/chol membranes corresponding to tie lines in the central (top panel) and low (bottom panel) parts of the L_α+L_o coexistence region. With decreasing line height, the spectra correspond to the following POPC/PSM/chol compositions: 60:30:10, 50:35:15, 40:40:20, 30:45:25, and 25:48:27 mol/mol/mol (top); and 50:40:10, 40:46:14, 30:52:18, 24:55.5:20.5, and 20:58:22 mol/mol/mol (bottom). Spectra are normalized to constant second integral. T = 23°C. The fine-line spectrum in the top panel is obtained by weighted addition of spectra for POPC/PSM/chol compositions 60:30:10 and 30:45:25 mol/mol/mol, in the ratio 1:0.47. It is almost indistinguishable from the spectrum for POPC/PSM/chol composition 40:40:20 mol/mol/mol. The inset defines the parameters used in the subtraction procedure to establish the endpoint spectra; see text for details.

Figure 6

Ternary phase diagrams of hydrated POPC/PSM/chol mixtures at 23°C. Top: from spin-label EPR; this study (see Fig. 4). Middle: From fluorescence microscopy of giant vesicles (11). Open circles, single fluid phase; solid circles, coexisting fluid phases; open squares, absence of fluid phase; solid squares, presence of gel and fluid phases. Bottom: from fluorescence polarization of diphenylhexatriene and fluorescence lifetimes of trans-parinaric acid (25). Solid squares represent suggested phase boundaries. Phase diagrams are presented as described in reference (8).