It should be noted that the Ca 2+ distribution can often be asymmetrical on the two sides of membranes. For example, the plasma membrane often faces a much higher Ca 2+ concentration on the outer lealet than on the inner lealet, while the SR membrane usually experiences a higher Ca 2+ concentration on the inner lealet than on the outer lealet. In fact, membranes themselves are often asymmetric. For example, the inner lealet of plasma membranes contains much more negatively charged lipid molecules than the outer lealet. Therefore, studying the effect of asymmetrical Ca 2+ on asymmetric membranes is of particular interest. In line with previous indings, our atomistic simulations showed that the Ca 2+ -exposed lealet of membranes becomes more packed. One would expect such a scenario to generate imbalanced stress in the two lealets of membranes. Indeed, our pressure proiles analysis showed that the addition of Ca 2+ to the negatively charged lealet leads to a signiicant imbalance of the lateral pressure proiles across the membrane Fig. , while the effect is much less pronounced in the case of the Ca 2+ ions added to the lealet with only zwitterionic lipids.