n-Alcohol Length Governs Shift in L_o-L_d Mixing Temperatures in Synthetic and Cell-Derived Membranes

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.