Kinetics of the protein 4.1-erythrocyte membrane interaction studied by total internal reflection fluorescence recovery after photobleaching.

Abstract

Protein 4.1 is a major component of the membrane skeleton, mediating the spectrin-actin interaction as well as the attachment of the spectrin network to the red cell membrane. Using the technique of TIR-FRAP microscopy, we have studied the kinetics of the interaction of carboxyfluorescein-labeled 4.1 with the inside surfaces of individual human red cell membranes adhered to polylysinated glass. The specificity of the observed CF-4.1-membrane binding was evaluated by measuring its concentration dependence and by comparison of surface fluorescence among various membrane treatments. The binding of CF-4.1 to the stripped membranes as indicated by fluorescence emission under TIR illumination appeared to have a K$\sb{\rm D}$ of 150 $\pm$ 50 nM. Analysis of the TIR-FRAP data reveals that reversibly-bound 4.1 accounts for, on average, 35% of the total population of bound 4.1, and it exhibits a range of relatively slow off-rates extending from about 0.01 sec$\sp{-1}$ to 1.0 sec$\sp{-1}$. The remaining $\sim$65% of bound 4.1 does not leave the membrane during the time spans measurable in these experiments. Both mild trypsinization (to remove the band 3 binding site) and leaving native 4.1 and ankyrin on the membranes resulted in a roughly 45% reduction in surface fluorescence, most of which appears to correspond to decreases in the number of sites which bind 4.1 "irreversibly"; the total number of reversibly-adsorbing sites was decreased only slightly by each treatment. Attempts to block another high-affinity 4.1-binding site by exposure to polyclonal antibodies to the cytoplasmic portion of glycophorin C resulted in a more dramatic reduction in surface fluorescence and almost complete removal of irreversible binding. Together, these results indicate that most reversible binding of 4.1 to red cell membranes could be due to interactions with phospholipids, which were unaffected by any of these treatments. The proteinaceous binding sites, band 3 and glycophorin C, would appear to bind 4.1 in a more irreversible manner. The possibility that surface diffusion along the reversibly-adsorbing surface could serve to enhance the probability of 4.1 binding to higher-affinity sites was investigated through application of two distinct models. This examination of the behavior of a single component of the membrane skeleton could aid in understanding how relatively permanent shape changes take place in the erythrocyte, thus contributing to a picture of this and other cytoskeletal structures as dynamic systems capable of dramatic rearrangements.