Chemically induced lipid phase separation in model membranes containing charged lipids: A spin label study

Abstract

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca 2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, W ex. By measuring W ex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithinphosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8·10 −8 cm 2/s at 59°C. Addition of Ca 2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca 2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of W ex on the Ca 2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca 2+ concentration clusters containing about 30 mol% lecithin are formed. At high lecithin or Ca 2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn 2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca 2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from T t = 47.5° to T t = 62°C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.