Analysis of Integral Membrane Protein Contributions to the Deformability and Stability of the Human Erythrocyte Membrane

Abstract

Three major hypotheses have been proposed to explain the role of membrane-spanning proteins in establishing/maintaining membrane stability. These hypotheses ascribe the essential contribution of integral membrane proteins to (i) their ability to anchor the membrane skeleton to the lipid bilayer, (ii) their capacity to bind and stabilize membrane lipids, and (iii) their ability to influence and regulate local membrane curvature. In an effort to test these hypotheses in greater detail, we have modified both the membrane skeletal and lipid binding interactions of band 3 (the major membrane-spanning and skeletal binding protein of the human erythrocyte membrane) and have examined the impact of these modifications on erythrocyte membrane morphology, deformability, and stability. The desired changes in membrane skeletal and protein-lipid interactions were induced by 1) reaction of the cells with 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS), an inhibitor of band 3-mediated anion transport that dissociates band 3 into dimers (increasing its surface area in contact with lipid) and severs band 3 linkages to the membrane skeleton; 2) a fragment of ankyrin that ruptures the same ankyrin-band 3 bridge to the membrane skeleton, but drives the band 3 subunit equilibrium toward the tetramer (i.e. decreasing the band 3 surface area in contact with lipid); and 3) an antibody to the ankyrin-binding site on band 3 that promotes the same changes in band 3 skeletal and lipid interactions as the ankyrin fragment. We observed that although DIDS induced echinocytic morphological changes in the treated erythrocytes, it had little impact on either membrane deformability or stability. In contrast, resealing of either the ankyrin fragment or anti-band 3 IgG into erythrocytes caused spontaneous membrane fragmentation and loss of deformability/stability. Because these and other new observations cannot all be reconciled with any single hypothesis on membrane stability, we suggest that more than one hypothesis may be operative and provide an explanation of how each might individually contribute to net membrane stability.In mechanistic models, the erythrocyte membrane is often depicted as a composite material with two coupled components surrounding a protein-rich cytoplasm. In this representation, the outer component is composed of a lipid bilayer traversed by multiple transport and structural proteins, predominantly the glycophorins and band 3. In contrast, the inner component consists of a two-dimensional scaffold of interconnecting skeletal proteins, primarily spectrin, protein 4.1R, and actin. Connections between the two components occur at regular intervals and are mainly established by bridging proteins. Prominent among the bridging proteins are ankyrin, which links the ␤-subunit of spectrin to the cytoplasmic domain of band 3, and protein 4.1R, which connects a spectrin-protein 4.1R-actin junctional complex to the cytoplasmic tail of glycophorin C (1). Although other proteins such as protein 4.2, dematin, p55, and adducin undoubtedly...

Rate of Rupture and Reattachment of the Band 3-Ankyrin Bridge on the Human Erythrocyte Membrane

“…4 and 5, substitution of cdb3-(His) 6 into the endogenous band 3-ankyrin bridge not only renders the cell less stable during exposure to shear stress but also leads to an altered morphology and subsequent vesiculation under non-stressed conditions. These results are consistent with earlier observations of Van Dort et al (4) showing that rupture of the band 3-ankyrin bridge upon addition of either a band 3-binding domain of ankyrin or an antibody to cdb3 also yields mechanically weakened cells. The data also support the contention that the dimer of band 3 can bind ankyrin with moderate affinity.…”

“…Importantly, the rate of dissociation of this critical linkage was found to occur on an intermediate time scale, permitting some rearrangement of the membrane spectrin skeleton during periods of extended deformation but protecting the cell from plastic deformation during normal transit through the vasculature. Unfortunately, although the major bridge between the lipid bilayer and the spectrin skeleton, mediated by the band 3-ankyrin-spectrin linkage, is also known to be critical for cell morphology and stability (4), no information is available on its association dynamics. In this report, we have undertaken to characterize the rate of dissociation of this membrane-to-skeleton bridge in both inside-out membrane vesicles and resealed erythrocytes.…”

“…Several lines of evidence suggest that the protein-protein interactions within the membrane skeleton are essential for membrane stability, including data demonstrating that rupture of individual protein linkages can lead to membrane instability (2)(3)(4)(5) and deficiencies in specific skeletal components can yield misshapen and poorly deformable cells (6 -10). Similar evidence supporting the importance of membrane-to-skeleton bridges derives from mechanical studies showing that deletion, reduction, or mutation of major bridging components can result in mechanically and morphologically compromised cells (2,4). In virtually all well characterized membrane mechanical defects, a reduction in either the number or affinity of critical protein-protein associations has been documented.…”

Monomers of the NhaA Na+/H+ Antiporter of Escherichia coli Are Fully Functional yet Dimers Are Beneficial under Extreme Stress Conditions at Alkaline pH in the Presence of Na+ or Li+

“…Multimerization of membrane proteins provides possibilities for scaffolding, interfaces, and allostery (63). Yet, the role of oligomers in the function of secondary transporters is still an open question in most cases.…”

An 11-amino acid β-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

“…Thus, interactions between the cell membrane and the cytoskeleton play a fundamental role in cell membrane structure, function, and other cellular processes in all cells. In the erythrocyte, rupture of the ankyrin-cdb3-spectrin linkage with antibodies against cdb3 leads to loss of membrane stability and spontaneous erythrocyte fragmentation (2).…”