Hereditary ovalocytic red cells are characterized by a marked increase in membrane rigidity and resistance to invasion by malarial parasites. The underlying molecular defect in ovalocytes remained a mystery until Liu and colleagues (N. Engli J. Med. 1990Med. . 323:1530 made the surprising observation that the ovalocytic phenotype was linked to a structural polymorphism in band 3, the anion transporter. We have now defined the mutation in band 3 gene and established the biophysical sequelae of this mutation. This mutation involves the deletion of amino-acids 400-408 in the boundary between the cytoplasmic and the first transmembrane domains of band 3. The biophysical consequences of this mutation are a marked decrease in lateral mobility of band 3 and an increase in membrane rigidity. Based on these findings, we propose the following model for increased membrane rigidity. The mutation induces a conformational change in the cytoplasmic domain of band 3, leading to its entanglement in the skeletal protein network. This entanglement inhibits the normal unwinding and stretching of the spectrin tetramers necessary for membrane extension, leading to increased rigidity. These findings imply that the cytoplasmic domain of an integral membrane protein can have profound effects on membrane material behavior. (J. Clin. Invest. 1992.

Abstract. Mechanical strength Of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB peptides were engineered to define the sequences involved in spectrin-actin interactions, and also membrane strength. Analysis of in vitro supramolecular assemblies showed that gelation activity of SAB peptides correlates with their ability to recruit a critical amount of spectrin into the complex to cross-link actin filaments. Also, several SAB peptides appeared to exhibit a weak, cooperative actin-binding activity which mapped to the first 26 residues of the constitutive 59 aa. Fluorescence-imaged microdeformation was used to show SAB peptide integration into the elastic skeletal network of spectrin, actin, and protein 4.1. In situ membrane-binding and membrane-strengthening abilities of the SAB peptides correlated with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21-aa cassette and a conserved region near the middle of the 59 aa. However, it is shown that only weak SAB affinity is necessary for physiologically relevant action. Alternatively spliced exons can thus translate into strong modulation of specific protein interactions, economizing protein function in the cell without, in and of themselves, imparting unique function.T HE erythrocyte is among the simplest and best characterized of cellular systems within which the chemistry of composition can be intricately related to structural function. Indeed, most of the red cell's mechanical behavior, except for limited aspects of cell shape, appears to depend little on the complexities of intracellular metabolism (14). However, to withstand the forces of blood flow over a several-month lifespan, the erythrocyte's membrane components must assemble into a highly durable structure. Physical limits to this durability are set by the system's mechanochemistry, which is defined here as those molecular interactions that permit a pliable, yet resilient structure.Spectrin, actin, and protein 4.1 are the principal structural proteins of the thin, cohesive skeletal network that underlies the red cell's lipid bilayer (3,5,19). The network formed by these highly conserved and pervasive proteins confers resilience and durability to the red cell's composite membrane (14,16,36,44,46). Mixtures of these network proteins also show that they can form very stable complexes in vitro (37) which at approximately micromolar protein concentrations can undergo a phase transition to a three-dimensional gel (7,15,49). In the formation of the basic ternary complex, protein 4.1 appears to play a crucial role in stabilizing spectrin cross-links between actin filaments. Consistent with cross-link reinforcement, red cells deficient in protein 4.1 possess membranes that fragment more...

No abstract