Membrane phospholipid organization in calcium-loaded human erythrocytes

Abstract

Intracellular Ca²⁺ levels in human erythrocytes were increased by incubating them with variable concentrations of Ca²⁺ in the presence of ionophore A23187. Experiments were done to confirm that the Ca²⁺ loading did induce changes in the cell shape and membrane protein composition. The effect of the increased cytoplasmic Ca²⁺ levels on the membrane phospholipid organization was analysed using bee venom and pancreatic phospholipases A₂, Merocyanine 540 and fluorescamine as the external membrane probes. About 20% phosphatidylethanolamine (PE) and 0% phosphatidylserine (PS) were hydrolysed by the phospholipases in intact control cells, whereas in identical conditions these enzymes readily degraded, 20-30% PE and 7-30% PS, in Ca²⁺-loaded erythrocytes, depending on the cytoplasmic Ca²⁺ concentration. Also, Merocyanine 540 failed to stain the fresh or control erythrocytes, but it labeled the cells loaded with Ca²⁺. Furthermore, fluorescamine labeled approx. 20% PE in fresh or control erythrocytes while in identical conditions, significantly higher amounts of PE were modified in intact Ca²⁺-loaded cells. These results demonstrate that Ca²⁺ loading in human erythrocytes leads to loss of the transbilayer phospholipid asymmetry, and suggest that, together with spectrin, polypeptides 2.1 and 4.1 may also play an important role in maintaining the asymmetric distribution of various phospholipids across the erythrocyte membrane bilayer.