Annexins are a family of structurally related proteins which share the property of Ca2+-dependent binding to aminophospholipids. They have been extensively studied in non-erythroid cells where they have been proposed to perturb the cell membrane lipid asymmetry and cation permeability, and to induce membrane bridging. Our recent studies revealed accumulation annexins in membranes of dense sickle cells with particular enrichment in Heinz bodies. In this proposal, we postulate that annexins are responsible, at least in part, for the reported sickle cell membrane alterations including loss of lipid asymmetry, increase of cation permeability and attachment of Heinz bodies to the membrane. This work has the following specific aims.
Aim (1) Annexin distribution and characterization in red cells. Employing immunolabeling, immunoblotting and electron microscopic techniques, we will examine the distribution of annexins between the cytosol, plasma membrane, Heinz bodies and membrane spicules and the effect of Ca2+, ATP depletion and sickling on annexin distribution in the cell.
Aim (2) Identification of types of annexins in red cells. To identify annexin species in red cells, we plan to clone the red cell annexins by screening reticulocyte cDNA library with a human cDNA probe, isolate positive clones and sequence the red cell annexin cDNA by dideoxy chain termination method.
Aim (3) Effect of annexins on phospholipid scrambling. To assess the role of annexins by immunoabsorption or enriched by introducing exogenous annexins, and examine changes of phospholipid asymmetry induced by ionophore/Ca2+ or by sickling.
Aim (4) Effect of annexins on cation permeability, in collaboration with Dr. Brugnara, we will analyze the effect of annexins on cation permeability in annexin-depleted versus annexin-containing reconstituted sickle cells. In collaboration with Dr. Joiner from Cincinnati Sickle Cell Center, we plan to study the effect of annexin incorporation into planar lipid bilayers on their ion channel properties. In addition, we will compare the cation channels derived from membrane spicules of annexin-depleted versus annexin-containing, sickle cells.
Aim (5) Role of annexins in Heinz body membrane attachment. We plan to examine the effect of annexin and calcium on binding of hemichromes to inside-out membrane vesicles and on Heinz body-membrane attachment in annexin- depleted or annexin-enriched reconstituted sickle cells. Taken together, we believe that the proposed studies will aid or elucidate the molecular basis or membrane abnormalities of sickle cells.
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