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/Ca2+) was systematically changed. We particularly focused our attention on the question of binding of Ca2+ to fluid bilayers and whether low amounts of Ca2+ (the physiological relevant concentration is between 10 and 250 mu M) can induce fluid-fluid immiscibility in liquid-crystalline bilayers. Therefore, we investigated mixtures with low calcium content, i.e., with DMPA/Ca2+ ratios in the range of 1:0.01-1:0.1. The analysis of the DSC curves for DMPA/DMPC + Ca2+ (DMPA/Ca2+ > 1:0.1) mixtures lead to the conclusion that with increasing Ca2+ content increased nonideal mixing in the liquid-crystalline phase with the tendency of domain formation for like lipids occurs. In this concentration regime Ca2+, 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 Ca2+ content, the apparent pK of DMPA shifts to lower values and doubly charged DMPA is formed which forms a 1:1 complex with Ca2+ which is quasi-crystalline and no longer miscible with the remaining DMPC/DMPA mixture. A phase separation of a solidlike DMPA/Ca2+ complex is observed. The bilayers then consist of DMPA-Ca2+ rich domains with tightly packed DMPA hydrocarbon chains in a liquid-crystalline like DMPC rich phase.