n-Alcohol Length Governs Shift in Lo-Ld Mixing Temperatures in Synthetic and Cell-Derived Membranes

Abstract

A persistent challenge in membrane biophysics has been to quantitatively predict how membrane physical properties change upon addition of new amphiphiles (e.g., lipids, alcohols, peptides, or proteins) in order to assess whether the changes are large enough to plausibly result in biological ramifications. Because of their roles as general anesthetics, n-alcohols are perhaps the best-studied amphiphiles of this class. When n-alcohols are added to model and cell membranes, changes in membrane parameters tend to be modest. One striking exception is found in the large decrease in liquid-liquid miscibility transition temperatures (T_mix) observed when short-chain n-alcohols are incorporated into giant plasma membrane vesicles (GPMVs). Coexisting liquid-ordered and liquid-disordered phases are observed at temperatures below T_mix in GPMVs as well as in giant unilamellar vesicles (GUVs) composed of ternary mixtures of a lipid with a low melting temperature, a lipid with a high melting temperature, and cholesterol. Here, we find that when GUVs of canonical ternary mixtures are formed in aqueous solutions of short-chain n-alcohols (n ≤ 10), T_mix increases relative to GUVs in water. This shift is in the opposite direction from that reported for cell-derived GPMVs. The increase in T_mix is robust across GUVs of several types of lipids, ratios of lipids, types of short-chain n-alcohols, and concentrations of n-alcohols. However, as chain lengths of n-alcohols increase, nonmonotonic shifts in T_mix are observed. Alcohols with chain lengths of 10-14 carbons decrease T_mix in ternary GUVs of dioleoyl-PC/dipalmitoyl-PC/cholesterol, whereas 16 carbons increase T_mix again. Gray et al. observed a similar influence of the length of n-alcohols on the direction of the shift in T_mix. These results are consistent with a scenario in which the relative partitioning of n-alcohols between liquid-ordered and liquid-disordered phases evolves as the chain length of the n-alcohol increases.

Figure 1

(A) Left: Below T_mix, 35/35/30 DOPC/DPPC/cholesterol vesicles exhibit domains of L_o (dark) and L_d (bright) phases. In taut vesicles, domains merge until only one domain of each phase remains (34). Right: Above T_mix, all lipids mix uniformly. Scale bars represent 20 μm. (B) T_mix is higher for 35/35/30 DOPC/DPPC/cholesterol vesicles in 100 mM butanol (squares, T_mix = 32.3 ± 0.19°C) than in pure water (circles, T_mix = 30.4 ± 0.29°C). Each point records the percent of vesicles with coexisting L_o and L_d phases at a given temperature. Dashed lines are 95% confidence intervals (13). The arrow points to T_mix for the left curve; the width of the arrow’s base is the uncertainty. To see this figure in color, go online.

Figure 2

(A) Increases in miscibility transition temperatures of 35/35/30 DOPC/DPPC/cholesterol vesicles scale with the concentration of n-alcohol in the aqueous solution. Lines are least square fits. (B) Data from the shaded region in (A) are rescaled by AC50 values from (14). Because AC50 values are reported to be proportional to partition coefficients of n-alcohols from water into PC bilayers (39), concentration/AC50 should be proportional to the concentration of n-alcohol in the membrane. Each point represents a single experiment for which symbols are typically larger than uncertainties, determined as in Fig. 1. SDs for repeated experiments are typically ± 0.5°C. Fig. 1 data are not replotted here.

Figure 3

Miscibility transition temperatures of GUVs of five different ratios of DOPC/DPPC/cholesterol increase with the concentration of butanol in solution. Colors within circles on the Gibbs triangle record T_mix in the absence of butanol; corresponding T_mix values are plotted in (A) and (B). The gray region denotes compositions over which a transition from one uniform phase to coexisting L_o and L_d phases is observed at a temperature between 15 and 40°C, from (29). GUVs in (A) contain increasing cholesterol fractions (following the vertical arrow on the triangle). GUVs in (B) maintain a constant fraction of cholesterol (following the horizontal arrow). Each point represents a single experiment for which uncertainties are smaller than symbols. To see this figure in color, go online.

Figure 4

Miscibility transition temperatures of GUVs composed of 17.5/17.5/35/30 lyso(18:0)-PC/DOPC/DPPC/cholesterol (green, top) and composed of 35/35/30 DOPC/DPPC/cholesterol (gray, bottom) increase with the concentration of butanol in solution (increasing darkness of the points). Each point represents a single experiment for which uncertainties are smaller than symbols. ΔT_mix is with respect to vesicles with no butanol. To see this figure in color, go online.

Figure 5

Miscibility transition temperatures for GUVs composed of POPC/PSM/cholesterol increase with the concentration of butanol in solution. (A) The molar ratio of POPC to PSM is held constant at 1:1 while the fraction of cholesterol is increased. (B) The fraction of cholesterol is held constant at 30 mol% while the ratio of PSM to POPC is increased. Each point represents a single experiment for which uncertainties are smaller than symbols.

Figure 6

Two scenarios of how miscibility transition temperatures (the curved surfaces) may shift in response to changes in the composition of a membrane. The Gibbs phase triangle in the x-y plane contains all possible ratios of the three components of the ternary membrane. (A) shows an increase in T_mix over all lipid ratios, upon moving from the control to the new system. (B) shows an increase in T_mix for some lipid ratios (upward arrow) and a decrease for others (downward arrow). To see this figure in color, go online.

Figure 7

Differences between laurdan GP values in L_o and L_d phases in GUVs increase with butanol concentration in solution (A and B) and remain roughly constant in GPMVs (C and D). GUVs were composed of 35/35/30 DOPC/DPPC/cholesterol, and GPMVs were derived from RBL cells. Points represent average GP values for batches of 20–30 vesicles on different days. The slope of each line arises from a linear regression with fixed intercepts to offset untreated batch differences from day to day. Shaded areas are 95% confidence intervals of the fit. Slopes of the lines in (B and D) are 4.80 × 10⁻³ ± 1.37 × 10⁻³ and −6.81 × 10⁻⁴ ± 1.58 × 10⁻³, in normalized units of ΔGP/[(butanol concentration)(butanol’s membrane-water partition coefficient from (14))]. Fig. S5 and Table S1 contain corresponding data for GUVs and GPMVs with tetradecanol and hexadecanol.

Figure 8

Increasing the number of carbons in n-alcohol solutions results in nonmonotonic shifts in T_mix for GUVs composed of 35/35/30 DOPC/DPPC/cholesterol. ΔT_mix is with respect to GUVs in water. Concentrations of n-alcohol solutions correspond to three times the AC50 in (14). AC50 values for tetradecanol and hexadecanol were estimated at 5 μM. Each point represents a single experiment with uncertainties as in Fig. 1. Fig. S3 contains corresponding data at lower n-alcohol concentrations.

Figure 9

Shift in miscibility transition temperatures for GUVs composed of 35/35/30 mol% mixtures of an unsaturated lipid, DPPC, and cholesterol. Four types of unsaturated lipids were used. For all four, incorporation of an n-alcohol with two carbons into the membrane produces a positive ΔT_mix. A significant decrease in T_mix (> 1°C, shown by arrows) occurs at a crossover alkanol length characteristic of each unsaturated lipid. Labeled right to left in the figure, with symbols from darkest to lightest, the unsaturated lipid is either DOPC (18:1 cisΔ9 PC), a DOPC analog with the double bond in a different position (18:1 cisΔ6 PC), or DOPC analogs with shorter acyl chains (16:1 cisΔ9 PC or 14:1 cisΔ9 PC). All GUVs were in n-alcohol solutions at three times the AC50 concentrations in (14). ΔT_mix is with respect to vesicles in water. Each point represents a single experiment. Symbols are larger than uncertainties determined as in Fig. 1. To see this figure in color, go online.

Figure 10

35/35/30 DOPC/DPPC/cholesterol GUVs produced in 3 μM DHM or 100 nM Ro15-4513 (white bars) have higher miscibility transition temperatures than control GUVs in water (ΔT_mix). Similarly, GUVs produced in 120 mM ethanol (EtOH, black bars) have higher miscibility temperatures under all conditions in the figure. Each bar represents a single experiment for which uncertainties are calculated as in Fig. 1.

Figure 11

Propofol, a general anesthetic, increases T_mix in 35/35/30 DOPC/DPPC/cholesterol GUVs. In contrast, 2,6-di-tert-butylphenol, which is structurally similar but anesthetically inactive, does not increase T_mix. Each point represents a single experiment. In all cases, symbols are larger than uncertainties determined as in Fig. 1.

Figure 12

Miscibility transition pressures for GUVs of 35/35/30 DOPC/DPPC/cholesterol decrease with increasing concentration of butanol in solution. Values of ΔP_mix are relative to control vesicles in water. Each point represents a single experiment for which uncertainties derive from fits to sigmoidal curves of % vesicles separated versus hydrostatic pressure, in analogy to Fig. 1.