Calcium Induced Nonideal Mixing in Liquid-Crystalline Phosphatidylcholine−Phosphatidic Acid Bilayer Membranes

The influence of increasing concentrations of calcium ions on the mixing properties of 1,2-dimyristoyl- sn -glycero-3-phosphatidic acid (DMPA) with 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) at pH 7 was investigated using differential scanning calorimetry (DSC). The composition of the mixture as well as the DMPA to calcium molar ratio (DMPA/Ca 2+ ) was systematically changed. We particularly focused our attention on the question of binding of Ca 2+ to fluid bilayers and whether low amounts of Ca 2+ (the physiological relevant concentration is between 10 and 250 μM) can induce fluid−fluid immiscibility in liquid-crystalline bilayers. Therefore, we investigated mixtures with low calcium content, i.e., with DMPA/Ca 2+ ratios in the range of 1:0.01−1:0.1. The analysis of the DSC curves for DMPA/DMPC + Ca 2+ (DMPA/Ca 2+ > 1:0.1) mixtures lead to the conclusion that with increasing Ca 2+ content increased nonideal mixing in the liquid-crystalline phase with the tendency of domain formation for like lipids occurs. In this concentration regime Ca 2+, binds to DMPA in a stoichiometry 2:1, the 2:1 complex being miscible with the remaining DMPC/DMPA mixture in the fluid bilayers. With higher Ca 2+ content, the apparent p K of DMPA shifts to lower values and doubly charged DMPA is formed which forms a 1:1 complex with Ca 2+ which is quasi-crystalline and no longer miscible with the remaining DMPC/DMPA mixture. A phase separation of a solidlike DMPA/Ca 2+ complex is observed. The bilayers then consist of DMPA−Ca 2+ rich domains with tightly packed DMPA hydrocarbon chains in a liquid-crystalline like DMPC rich phase.