A characteristic feature of all cell plasma membranes is the asymmetric transbilayer distribution of phospholipids. The choline-containing phospholipids, phosphatidylcholine and sphingomyelin are enriched in the outer monolayer, while the aminophospholipids, particularly phosphatidylserine (PS), are enriched on the cytoplasmic surface. The dissipation of this gradient and exposure of PS on the surface of the cell induces the recognition and destruction of the cell by macrophages and, in blood cells, the activation of plasma clotting factors. The action of several lipid-specific transporters, included an inwardly-directed, aminophospholipid-specific, ATPase or �flippase,� outwardly-directed 'floppases,' and a bi-directlional, non-specific 'scramblase', play important roles in the generation, maintenance and regulated dissipation of plasma membrane phospholipid asymmetry. The goal of our studies is to identify and characterize these lipid transporters, using substrate specificity and biochemical properties as criteria. We have purified and characterized two candidate flippases: a PS-stimulated ATPase from human erythrocytes and a P4-ATPase (ATP8A1) heterologously expressed in insect cells. These enzymes share an exquisite structural specificity for PS that is distinct from other PS-binding proteins and support the hypothesis that they are PS-selective flippases.In separate experiments, we have addressed the role of plasma membrane asymmetry in the cardiovascular complications associated with diabetes. Erythrocytes from poorly controlled diabetics express PS on the cell surface, which stimulates blood clotting factors and may promote unwanted thromboses. Using a combination of in vitro hyperglycemia, animal models of diabetes, and erythrocytes from human diabetics, we have determined that hyperglycemia induces oxidative damage of the erythrocyte membrane, increases passive phospholipid flip-flop, increases PS externalization, and stimulates blood clotting factors, all of which are suppressible with antioxidants. These studies indicate that lipid transporters are critical for the maintenance of plasma membrane phospholipid asymmetry and that they may be potential therapeutic targets for the treatment or prevention of cardiovascular disease in diabetes.

Generation and maintenance of plasma membrane transbilayer phospholipid asymmetry in health and disease: Role of flippases, floppases, and scramblases

247th National Meeting (2014)

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