To study the effect of asymmetrical Ca 2+ on the lateral stress of asymmetric membranes, doublemembrane systems were generated by duplicating a single-membrane system along the z direction (Fig. ). The outer lealet and inner lealet of each asymmetric lipid bilayer consisted of POPC (64) and POPC:POPS (48:16), respectively. In these double-membrane systems, K + ions were added to neutralize the negative charges of PS lipids. Ca 2+ ions were added to only one water compartment.