Regulation of red cell membrane deformability and stability by skeletal protein network

Abstract

The skeletal protein network of the red blood cell is thought to be important in regulating such membrane functions as deformability and stability. In the present study, we measured membrane deformability and stability of the resealed ghosts using an ektacytometer, a laser diffraction method, and identified the functional role of protein 4.1 and that of Ca²⁺ and calmodulin in maintaining membrane stability. To obtain direct evidence for a crucial role of protein 4.1 in maintaining membrane stability, we reconstituted protein 4.1-deficient membranes with purified protein 4.1. Although native membranes deficient in protein 4.1 had marked reduction in membrane stability, reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability, providing direct evidence that protein 4.1 is essential for normal membrane stability. To determine if Ca²⁺ and calmodulin could modulate membrane properties, we measured membrane stability and deformability of resealed ghosts prepared in the presence of varying concentrations of Ca²⁺ and physiologic concentrations of calmodulin. Our data show that Ca²⁺ concentrations in the range of 1 to 100 μM can markedly decrease membrane stability only in the presence of calmodulin, but not in its absence. In contrast, deformability decreased only at Ca²⁺ concentrations higher than 100 μM, and calmodulin had no effect. Examination of the the effects of Ca²⁺ and calmodulin on various membrane protein interactions has enabled us to suggest that the observed changes in membrane stability may be partly related to the effects of Ca²⁺ and calmodulin on spectrin-protein 4.1-actin interaction.