SPECTRIN ASSEMBLY IN IRREVERSIBLY SICKLED CELL MEMBRANES: ROLE OF CALCIUM AND ATP

Abstract

Irreversibly sickled cell (ISC) shape results from a permanent membrane deformation which may be related to a reorganization of a spectrin-actin cytoskeleton. We have evaluated the role of decreased red cell (rbc) ATP and increased Ca in the ISC shape lesion by comparing membrane protein arrangement of ISCs and ATP depleted-Ca enriched rbcs. We employed cross-linking by a spontaneous or catalytic oxidation (CuSO 4 + o-phenanthroline) of nearest membrane protein neighbor SH-groups and a two dimensional polyacrylamide gel electrophoresis. ATP depleted or Ca enriched normal rbcs subjected to crosslinking contained a >1,000,000 daltons spectrin rich polymer which was cleavable by dithiothreitol (DTT) reduction. This suggested spectrin rearrangement into closer contacts or aggregation. Another >1,000,000 daltons complex was produced in fresh rbcs by high (>0.5 mM) Ca concentration and ionophore A23187; this was not cleavable with DTT. This was associated with a decrease of both spectrin and band 3, the major integral membrane protein, and was presumably due to crosslinking of these proteins catalyzed by an endogenous transglutaminase. ISCs exhibiting a 3–4 fold increase in Ca were separated on a discontinuous stractan gradient into 3 fractions (>80% ISCs) of increasing densities which all failed to exhibit such polymers. However, ISCs had an increased propensity to form a spectrin rich, cleavable polymer during a subsequent ATP depletion and Ca accumulation and this was associated with a transformation of ISCs into spheroechinocytes. We conclude that neither Ca, ATP dependent spectrin aggregation nor an irreversible cross-linking underlie the membrane lesion of ISCs. However, accelerated calcium gain and ATP depletion in ISCs subsequently leads to spectrin rearrangement and transformation of ISCs into spheroechinocytes which presumably may represent an end stage ISC lesion resulting in an ISC removal from circulation.