Influence of temperature and cholesterol on the rotational diffusion of band 3 in the human erythrocyte membrane

Abstract

Band 3 rotation in the human erythrocyte membrane is measured by observing flash-induced dichroism of eosin probes. The decay of the absorption anisotropy is found to be strongly dependent on temperature. The results are analyzed on the assumption that rotation of band 3 only occurs about the membrane normal. It is deduced that both fast and slowly rotating forms of band 3 coexist in the membrane. The equilibrium between these forms is temperature dependent, the slowly rotating species becoming increasingly dominant as the temperature is reduced. Plots of the fractional distribution of the different species against temperature show a marked change of slope at around 37--40 degrees C. The effects are essentially reversible over the range 1--45 degrees C and independent of the presence of the spectrin--actin network. The results could be due to temperature-dependent protein--protein associations mediated either by a protein conformational change or by lipid phase segregation. In further experiments, the cholesterol content of the erythrocyte membrane is varied by incubation with lipid vesicles. No significant changes in the rotational diffusion of band 3 are observed following variation of membrane cholesterol/phospholipid mole ratios over the range 0.34--1.66. This is a surprising result in view of the well-known effects of cholesterol on lipid fluidity.

Band 3 and glycophorin are progressively aggregated in density-fractionated sickle and normal red blood cells. Evidence from rotational and lateral mobility studies.

“…The consistent observation of distinct, well-defined rotational populations suggests that equilibria exist among the various molecular states of band 3 in the membrane. Others have estimated that, at 37°C, 25-35% of band 3 molecules rotate with correlation times < 150 ,us, 30-50% rotate with correlation times -3 ms, and 25-35% are rotationally immobile on the time scale ofthe experiment (67,69). Recent measurements with improved instrumentation indicate that about one-half of the rapidly rotating fraction of band 3 molecules rotate with correlation times of 25 to 30 As (67) (81,82), abnormally exposed galactosyl residues (46,83,84), and clustered band 3 molecules (27,48,50).…”

“…EMA labels band 3 specifically at the external anion transport site of this protein (68,69). EMA bound to band 3 in intact cells has multiple phosphorescence (triplet) lifetimes of 2.3, 0.4, and 0.04 ms (67), and a single fluorescence (singlet) lifetime of 2.99 ns (68).…”

Molecular basis for membrane rigidity of hereditary ovalocytosis. A novel mechanism involving the cytoplasmic domain of band 3.

“…with eosin-5-maleimide, as originally described by Nigg and Cherry (17), with slight modifications. In brief, 20 Ml of whole blood was washed three times in 1 ml of phosphate-buffered saline (290 mosM, pH 7.4) with 0.05 g% human serum albumin (PBS/HSA).…”

Effect of cholesterol on phosphate uptake by human red blood cells

“…This may lead to trapping of lipids, including cholesterol. Varying the cholesterol content of the erythrocyte membrane, on the other hand, had no effect on the rotational diffusion of Band 3 [48]. In order to study the specificity of the effect of cholesterol on Band 3 and anion transport, the effect of a cholesterol derivative, cholestenone, on anion transport was determined.…”

“…It is unlikely that the increased cholesterol level in the membrane influences the oligomeric structure of Band 3. Cholesterol has been shown not to affect the rotation of Band 3 in the membrane [48] suggesting that a dramatic rearrangement of the protein oligomers does not occur.…”