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